Expression induction of P450 genes by imidacloprid in Nilaparvata lugens: A genome-scale analysis.

PubMed

Zhang, Jianhua; Zhang, Yixi; Wang, Yunchao; Yang, Yuanxue; Cang, Xinzhu; Liu, Zewen

2016-09-01

The overexpression of P450 monooxygenase genes is a main mechanism for the resistance to imidacloprid, a representative neonicotinoid insecticide, in Nilaparvata lugens (brown planthopper, BPH). However, only two P450 genes (CYP6AY1 and CYP6ER1), among fifty-four P450 genes identified from BPH genome database, have been reported to play important roles in imidacloprid resistance until now. In this study, after the confirmation of important roles of P450s in imidacloprid resistance by the synergism analysis, the expression induction by imidacloprid was determined for all P450 genes. In the susceptible (Sus) strain, eight P450 genes in Clade4, eight in Clade3 and two in Clade2 were up-regulated by imidacloprid, among which three genes (CYP6CS1, CYP6CW1 and CYP6ER1, all in Clade3) were increased to above 4.0-fold and eight genes to above 2.0-fold. In contrast, no P450 genes were induced in Mito clade. Eight genes induced to above 2.0-fold were selected to determine their expression and induced levels in Huzhou population, in which piperonyl butoxide showed the biggest effects on imidacloprid toxicity among eight field populations. The expression levels of seven P450 genes were higher in Huzhou population than that in Sus strain, with the biggest differences for CYP6CS1 (9.8-fold), CYP6ER1 (7.7-fold) and CYP6AY1 (5.1-fold). The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1. Screening the induction of P450 genes by imidacloprid in the genome-scale will provide an overall view on the possible metabolic factors in the resistance to neonicotinoid insecticides. The further work, such as the functional study of recombinant proteins, will be performed to validate the roles of these P450s in imidacloprid resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

PubMed Central

Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

2014-01-01

ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies.

PubMed

Liu, G; Gelboin, H V; Myers, M J

1991-02-01

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.

Human kidney methoxyflurane and sevoflurane metabolism. Intrarenal fluoride production as a possible mechanism of methoxyflurane nephrotoxicity.

PubMed

Kharasch, E D; Hankins, D C; Thummel, K E

1995-03-01

Methoxyflurane nephrotoxicity is mediated by cytochrome P450-catalyzed metabolism to toxic metabolites. It is historically accepted that one of the metabolites, fluoride, is the nephrotoxin, and that methoxyflurane nephrotoxicity is caused by plasma fluoride concentrations in excess of 50 microM. Sevoflurane also is metabolized to fluoride ion, and plasma concentrations may exceed 50 microM, yet sevoflurane nephrotoxicity has not been observed. It is possible that in situ renal metabolism of methoxyflurane, rather than hepatic metabolism, is a critical event leading to nephrotoxicity. We tested whether there was a metabolic basis for this hypothesis by examining the relative rates of methoxyflurane and sevoflurane defluorination by human kidney microsomes. Microsomes and cytosol were prepared from kidneys of organ donors. Methoxyflurane and sevoflurane metabolism were measured with a fluoride-selective electrode. Human cytochrome P450 isoforms contributing to renal anesthetic metabolism were identified by using isoform-selective inhibitors and by Western blot analysis of renal P450s in conjunction with metabolism by individual P450s expressed from a human hepatic complementary deoxyribonucleic acid library. Sevoflurane and methoxyflurane did undergo defluorination by human kidney microsomes. Fluoride production was dependent on time, reduced nicotinamide adenine dinucleotide phosphate, protein concentration, and anesthetic concentration. In seven human kidneys studied, enzymatic sevoflurane defluorination was minima, whereas methoxyflurane defluorination rates were substantially greater and exhibited large interindividual variability. Kidney cytosol did not catalyze anesthetic defluorination. Chemical inhibitors of the P450 isoforms 2E1, 2A6, and 3A diminished methoxyflurane and sevoflurane defluorination. Complementary deoxyribonucleic acid-expressed P450s 2E1, 2A6, and 3A4 catalyzed methoxyflurane and sevoflurane metabolism, in diminishing order of activity. These three P450s catalyzed the defluorination of methoxyflurane three to ten times faster than they did that of sevoflurane. Expressed P450 2B6 also catalyzed methoxyflurane defluorination, but 2B6 appeared not to contribute to renal microsomal methoxyflurane defluorination because the P450 2B6-selective inhibitor had no effect. Human kidney microsomes metabolize methoxyflurane, and to a much lesser extent sevoflurane, to fluoride ion. P450s 2E1 and/or 2A6 and P450 3A are implicated in the defluorination. If intrarenally generated fluoride or other metabolites are nephrotoxic, then renal metabolism may contribute to methoxyflurane nephrotoxicity. The relative paucity of renal sevoflurane defluorination may explain the absence of clinical sevoflurane nephrotoxicity to date, despite plasma fluoride concentrations that may exceed 50 microM.

Expression of Cytochrome P450s in the Liver of Rats Administered with Socheongryong-tang, a Traditional Herbal Formula

PubMed Central

Jin, Seong Eun; Ha, Hyekyung; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

2016-01-01

Objective: The purpose of this study was to investigate the potential influences of Socheongryong-tang (SCRT) on the messenger ribonucleic acid (mRNA) and protein expression of cytochrome P450 (CYP450) in vivo. Materials and Methods: SCRT was orally administered to either male or female Sprague-Dawley rats once daily at doses of 0, 1000, 2000, or 5000 mg/kg/day for 13 weeks. The mRNA expression of CYP450s (CYP1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) in liver tissues was measured by reverse transcription polymerase chain reaction. And then, the protein expression of CYP1A1 and CYP2B1/2 in liver tissues was analyzed by the Western blot. Results: We found no significant influence in the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1 after repeated administration of SCRT for 13 weeks. By contrast, the mRNA and protein expression of hepatic CYP1A1 was increased by repeated SCRT treatment in male rats, but not in female rats. The mRNA and protein expression of hepatic CYP2B1/2 in both genders was increased by administration of SCRT. Conclusion: A caution is needed when SCRT is co-administered with substrates of CYP2B1/2 for clinical usage. In case of male, an attention is also required when SCRT and drugs metabolized by CYP1A1 are taken together. Our findings provide information regarding the safety and effectiveness of SCRT when combined with conventional drugs. SUMMARY Oral administration of Socheongryong-tang for 13 weeks did not affect the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1In male rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP1A1 and CYP2B1/2In female rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP2B1/2. Abbreviations used: SCRT: Socheongryong-tang, CYP450: Cytochrome P450, HPLC: High performance liquid chromatography, RT-PCR: Reverse transcription polymerase chain reaction. PMID:27601852

Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2.

PubMed

Wang, R; Zhu, Y; Deng, L; Zhang, H; Wang, Q; Yin, M; Song, P; Elzaki, M E A; Han, Z; Wu, M

2017-10-01

Laodelphax striatellus (Fallén) is one of the most destructive pests of rice, and has developed high resistance to imidacloprid. Our previous work indicated a strong association between imidacloprid resistance and the overexpression of a cytochrome P450 gene CYP6AY3v2 in a L. striatellus imidacloprid resistant strain (Imid-R). In this study, a transgenic Drosophila melanogaster line that overexpressed the L. striatellus CYP6AY3v2 gene was established and was found to confer increased levels of imidacloprid resistance. Furthermore, CYP6AY3v2 was co-expressed with D. melanogaster cytochrome P450 reductase (CPR) in Spodoptera frugiperda 9 (SF9) cells. A carbon monoxide difference spectra analysis indicated that CYP6AY3v2 was expressed predominately in its cytochrome P450 (P450) form, which is indicative of a good-quality functional enzyme. The recombinant CYP6AY3v2 protein efficiently catalysed the model substrate P-nitroanisole to p-nitrophenol with a maximum velocity (V max ) of 60.78 ± 3.93 optical density (mOD)/min/mg protein. In addition, imidacloprid itself was metabolized by the recombinant CYP6AY3v2/nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) CPR microsomes in in vitro assays (catalytic constant (K cat ) = 0.34 pmol/min/pmol P450, michaelis constant (K m ) = 41.98 μM), and imidacloprid depletion and metabolite peak formation were with a time dependence. The data provided direct evidence that CYP6AY3v2 is capable of hydroxylation of imidacloprid and conferring metabolic resistance in L. striatellus. © 2017 The Royal Entomological Society.

Characterization of human cytochrome P450s involved in the bioactivation of tri-ortho-cresyl phosphate (ToCP).

PubMed

Reinen, Jelle; Nematollahi, Leyla; Fidder, Alex; Vermeulen, Nico P E; Noort, Daan; Commandeur, Jan N M

2015-04-20

Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP.

Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants.

PubMed

Ohkawa, Hideo; Inui, Hideyuki

2015-06-01

A yeast gene expression system originally established for mammalian cytochrome P450 monooxygenase cDNAs was applied to functional analysis of a number of mammalian and plant P450 species, including 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). The human P450 species CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 were identified as P450 species metabolising various agrochemicals and environmental chemicals. CYP2C9 and CYP2E1 specifically metabolised sulfonylurea herbicides and halogenated hydrocarbons respectively. Plant P450 species metabolising phenylurea and sulfonylurea herbicides were also identified mainly as the CYP71 family, although CYP76B1, CYP81B1 and CYP81B2 metabolised phenylurea herbicides. The transgenic plants expressing these mammalian and plant P450 species were applied to herbicide tolerance as well as phytoremediation of agrochemical and environmental chemical residues. The combined use of CYP1A1, CYP2B6 and CYP2C19 belonging to two families and three subfamilies covered a wide variety of herbicide tolerance and phytoremediation of these residues. The use of 2,4-D-and bromoxynil-induced CYP71AH11 in tobacco seemed to enhance herbicide tolerance and selectivity. © 2014 Society of Chemical Industry.

Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

PubMed

Ram, P A; Waxman, D J

1992-02-15

The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major increase in P450 reductase protein or activity was detectable over a 3-day period. Together, these studies establish that thyroid hormone regulates P450 reductase expression by pretranslational mechanisms. They also suggest that other regulatory mechanisms, which may involve changes in P450 reductase protein stability and/or changes in the translational efficiency of its mRNA, are likely to occur.

Impacts of Blast-Induced Traumatic Brain Injury on Expressions of Hepatic Cytochrome P450 1A2, 2B1, 2D1, and 3A2 in Rats.

PubMed

Ma, Jie; Wang, Junrui; Cheng, Jingmin; Xiao, Wenjing; Fan, Kaihua; Gu, Jianwen; Yu, Botao; Yin, Guangfu; Wu, Juan; Ren, Jiandong; Hou, Jun; Jiang, Yan; Tan, Yonghong; Jin, Weihua

2017-01-01

The hepatic cytochrome P450 (CYP450) enzyme superfamily is one of the most important drug-metabolizing enzyme systems, which is responsible for the metabolism of a large number of clinically relevant medications used in traumatic brain injury (TBI) therapy. Modification of CYP450 expression may have important influences on drug metabolism and lead to untoward effects on those with narrow therapeutic windows. However, the impact of blast-induced TBI (bTBI) on the expression of CYP450 has received little attention. The subfamilies of CYP1A, 2B, 2D, and 3A account for about 85 % of all human drug metabolism of clinical significance. Therefore, in this study, we investigated the expressions of hepatic CYP1A2, CYP2B1, CYP2D1, and CYP3A2 in rats suffering bTBI. Meanwhile, we also measured some important cytokines in serum after injury, and calculated the correlation between these cytokines and the expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2. The results showed that bTBI could significantly reduce mRNA expressions of CYP1A2, CYP2D1, and CYP3A2 at the early stage and induce the expressions from 48 h to 1 week after injury. The protein expressions of these CYP450s had all been downregulated from 24 to 48 h post- injury, and then began to elevate at 48 h after bTBI. The cytokines, IL-1β, IL-2, IL-6, and TNF-α, increased significantly in the early phase, and began to reduce at the delayed phase of bTBI. The serum levels of IL-1β, IL-6, and TNF-α but not IL-2 were significantly negative correlated with the mRNA expressions of CYP2B1 and CYP2D1 and the proteins expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2. In conclusion, our work has, for the first time, indicated that bTBI has significant impact on the expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2, which may be related to the cytokines induced by the injury.

[Effect of Transcutaneuos Acupoint Electrostimulation on Serum Sex Hormone Levels and Expression of Ovarian Steroid Hormone Metabolic Enzymes in Polycystic Ovary Syndrome Rats].

PubMed

Zhou, Jian-yong; Zhang, Xiao-yue; Yu, Mei-ling; Lu, Sheng-feng; Chen, Xia

2016-02-01

To observe the effect of transcutaneuos acupoint electrostimulation(TAES) on ovarian serum sex hormone levels and ovarian follicle granular cell aromatase cytochrome P 450 (P 450 arom) protein and follicle theca cell cytochrome P 450 17 α-hydroxylase/c 17-20 lyase cytochrome P 450 (P 450 c 17 α) protein expression in polycystic ovary syndrome (PCOS)rats, so as to explore its mechanisms underlying improvement of PCOS. METHODS Forty SD rats were randomly divided into four groups: normal control, model, medication and TAES (10 rats/group). The PCOS model was established by giving (gavage) the animals with letrozole solution (1.0 mg/kg, once daily for 21 consecutive days). Rats of the medication group were treated with Clomiphene (1 mg/kg) once daily for 7 days, and those of the TAES group were treated with electrical stimulation (2 Hz, 3 mA) of "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) areas for 30 min, once daily for 7 consecutive days. The rats body weight and bilateral ovarian weight were detected, and the ovarian structure and follicular development degree were observed under light microscope after H. E. stain, and the serum testosterone (T), estradiol (E2), luteotrophic hormone (LH) and follicle-stimulating hormone (FSH) contents were detected using radioimmunoassay. The expression of ovarian P 450 arom (for production of estrogen)protein and P 450 c 17 α (for production of androgen) protein was detected by using immunohistochemical stain and Western blot, respectively. The body weight, bilateral ovary weight, serum T and LH contents, and ratio of LH/FSH, and ovarian P 450 c 17 α immunoactivity and protein expression levels in the model group were all significantly increased compared with the normal control group (P < 0.01), and the levels of serum E2 and ovarian P 450 arom immunoactivity and protein expression were significantly decreased after modeling (P < 0.01). Following the treatment, the increased body weight, ovary weight, serum T and LH contents, ratio of LH/FSH, and ovarian P 450 c 17 α immunoactivity and protein expression levels, and the decreased ovarian P 450 arom immunoactivity and protein expression levels were reversed in the TAES group (P < 0.01, P < 0.05) rather than in the medication group, except serum T and ratio of LH/FSH in the medication group. No significant differences were found between the medication and TAES groups in the serum T and ratio of LH/FSH (P > 0.05). In addition, the increased vesicular follicle number, the decreased corepus luteum number and the thickness of granular cell layer were markedly improved in the TAES group. TAES intervention can reduce both body weight and ovarian weight and regulate the levels of serum sex hormones and ovarian P 450 c 17 α and P 450 arom protein expression levels in PCOS rats, which may contribute to its effect in improving PCOS.

Activation of amino-alpha-carboline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a copper phthalocyanine cellulose extract of cigarette smoke condensate by cytochrome P-450 enzymes in rat and human liver microsomes.

PubMed

Shimada, T; Guengerich, F P

1991-10-01

The ability of cigarette smoke condensate to induce a genotoxic response has been measured in liver microsomal and reconstituted monooxygenase systems containing rat and human cytochrome P-450 (P-450) enzymes, as determined by umu gene expression in Salmonella typhimurium TA1535/pSK1002. The reactivities of amino-alpha-carboline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), two compounds known to be present at considerable levels in cigarette smoke condensate, were also determined and compared with regard to genotoxicity. Amino-alpha-carboline and PhIP are activated principally by P-450 1A2 enzymes in human and rat liver microsomes: (a) activation of both compounds was catalyzed efficiently by liver microsomes prepared from rats treated with 5,6-benzoflavone, isosafrole, or the commercial polychlorinated biphenyl mixture Aroclor 1254, and the activities could be considerably inhibited by antibodies raised against P-450 1A1 or 1A2; (b) the rates of activation of these compounds were correlated with the amount of human P-450 1A2 and of phenacetin O-deethylation activity in different human liver microsomal preparations, and these activities were inhibited by anti-P-450 1A2; (c) reconstituted enzyme systems containing P-450 1A enzymes isolated from rats and humans showed the highest rates of activation of amino-alpha-carboline and PhIP. In rat liver microsomes PhIP may also be activated by P-450 3A enzymes; activity was induced in rats treated with pregnenolone 16 alpha-carbonitrile and was inhibited by anti-human P-450 3A4. However, in humans the contribution of P-450 3A enzymes could be excluded as judged by the very low effects of anti-P-450 3A4 on the microsomal activities and poor correlation with P-450 3A4-catalyzed activities in various liver samples. Cigarette smoke condensate strongly inhibited the activation of several potent procarcinogens by human liver microsomes, particularly the reactions catalyzed by P-450 1A2, but was not so inhibitory of the activation reactions catalyzed by P-450 3A4 and of P-450 2D6-catalyzed bufuralol 1'-hydroxylation. Genotoxic components of the cigarette smoke condensate were extracted by using copper phthalocyanine cellulose (blue cotton). Genotoxicity of this extract was observed only after activation by P-450, and the inhibition of P-450 1A2 activities by these extracts was slight.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxidation of Methyl and Ethyl Nitrosamines by Cytochromes P450 2E1 and 2B1

PubMed Central

Chowdhury, Goutam; Calcutt, M. Wade; Nagy, Leslie D.; Guengerich, F. Peter

2012-01-01

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine (N,N-dimethylnitrosamine, DMN), a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥ 8. The KIE was not attenuated in non-competitive intermolecular experiments with rat liver microsomes (DV 12.5, D(V/K) 10.9, nomenclature of Northrop, D.B. (1982) Methods Enzymol. 87, 607–625) but was with purified human P450 2E1 (DV 3.3, D(V/K) 3.7), indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine (N,N-diethylnitrosamine, DEN), the intrinsic KIE was slightly lower and was not expressed (e.g., D(V/K) 1.2) in non-competitive intermolecular experiments. The same general pattern of KIEs was also seen in the D(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH3NH2, CH3CH2NH2, and NO2−). Experiments with deuterated N-nitroso-N-methyl,N-ethylamine demonstrated that the lower KIEs associated for ethyl compared to methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 (Chowdhury, G. et al. (2010) J. Biol. Chem. 285, 8031–8044). These same features (no lag phase for HCO2H formation, lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkylnitrosamine oxidation appears to be shared by a number of P450s. PMID:23186213

Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment

PubMed Central

Cheung, Connie; Gonzalez, Frank J

2008-01-01

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating “humanized” transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, PXR, PPARα were generated and characterized. These humanized mouse models offers a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs. PMID:18682571

Differential Expression of P450 Genes and nAChR Subunits Associated With Imidacloprid Resistance in Laodelphax striatellus (Hemiptera: Delphacidae).

PubMed

Zhang, Yueliang; Liu, Baosheng; Zhang, Zhichun; Wang, Lihua; Guo, Huifang; Li, Zhong; He, Peng; Liu, Zewen; Fang, Jichao

2018-05-28

Imidacloprid is a key insecticide used for controlling sucking insect pests, including the small brown planthopper (Laodelphax striatellus, Fallén) (Hemiptera: Delphacidae), an important agricultural pest of rice. A strain of L. striatellus (YN-ILR) developed 21-fold resistance when selected with imidacloprid on a susceptible YN strain. An in vitro study on piperonyl butoxide synergism indicated that enhanced detoxification mediated by cytochrome P450s contributed to imidacloprid resistance to some extent, and multiple P450 genes showed altered expression in the imidacloprid-resistant YN-ILR strain compared with the susceptible YN strain (CYP425B1-CYP6BD10 had 1.51- to 11.45-fold higher expression, CYP4CE2-CYP4DD1V2 had 0.12- to 0.57-fold lower expression). While there were no mutations in target nicotinic acetylcholine receptor (nAChR) genes, subunits of Lsα1, Lsβ1, and Lsβ3 in the YN-ILR strain showed 3.86-, 4.39-, and 2.59-fold higher expression and Lsa8 displayed 0.38-fold lower expression than the YN strain. Moreover, 21-fold moderate imidacloprid resistance in individuals of L. striatellus did not produce a fitness cost. The findings suggest that L. striatellus has the capacity to develop resistance to imidacloprid through P450 detoxification and potential target nAChR expression changes, and moderate imidacloprid resistance was not associated with a fitness cost.

A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains.

PubMed

Højland, Dorte H; Vagn Jensen, Karl-Martin; Kristensen, Michael

2014-08-01

The housefly is a global pest that has developed resistance to most insecticides applied for its control. Resistance has been associated with cytochrome P450 monooxygenases (P450s). The authors compare the expression of six genes possibly associated with insecticide resistance in three unselected strains: a multiresistant strain (791a), a neonicotinoid-resistant strain (766b) and a new field strain (845b). CYP4G2 was highly expressed throughout the range of strains and proved to be the one of the most interesting expression profiles of all P450s analysed. CYP6G4 was expressed up to 11-fold higher in 766b than in WHO-SRS. Significant differences between expression of P450 genes between F1 flies from 845b and established laboratory strains were shown. In general, P450 gene expression in 845b was 2-14-fold higher than in the reference strain (P < 0.0101) and 2-23-fold higher than in the multiresistant strain (P < 0.0110). The newly collected field strain 845b had significantly higher constitutive gene expression than both WHO-SRS and 791a. High constitutive expression of CYP4G2 in houseflies indicates a possible role of this gene in metabolic resistance. There is a strong indication that CYP6G4 is a major insecticide resistance gene involved in neonicotinoid resistance. © 2013 Society of Chemical Industry.

[Effects of electroacupuncture of "Guanyuan" (CV 4)-"Zhongji" (CV 3) on ovarian P450 arom and P450c 17alpha expression and relevant sex hormone levels in rats with polycystic ovary syndrome].

PubMed

Sun, Jie; Zhao, Ji-meng; Ji, Rong; Liu, Hui-rong; Shi, Yin; Jin, Chun-lan

2013-12-01

To observe the effect of electroacupuncture (EA) on ovarian P 450 arom and P 450 c 17 alpha (aromatases) expression and related sex hormone levels in polycystic ovary syndrome (PCOS) rats. Thirty SD rats were randomly divided into normal control group, model group and EA group (10 rats/group). PCOS model was made by intragastric administration of letrozole at 1 mg/kg per day for consecutive 21 days. "Guanyuan" (CV 4) and "Zhongji" (CV 3) acupoints were stimulated 20 min by EA (2 mA, 2 Hz), once daily for consecutive 14 days. The damp ovarian weight was weighed and the pathological changes of the ovarian tissue were observed after H. E. staining. Ultrastructural changes of the ovarian tissue were observed by transmission electron microscope. Immunohistochemical staining was adopted to detect ovarian follicle granulosa cell P 450 arom and follicle membrane cell P 450 c 17 alpha expression. The contents of estradiol (E 2), estrone (E 1), androstenedione (ASD), testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the ovarian tissue were measured by ELISA. Compared with the normal group, there was a significant increase in the damp weight of both left and right ovarian tissues in the model group (P < 0.01). After EA, the ovarian weight was remarkably reduced (P < 0.01). Pathological changes of the ovarian tissue such as thickening of the superficial albugineous coat of the ovary, thinning of the granular cell layer, and disappearance of the intraovular oocytes and coronaradiata under light microscope, and mitochondrion swelling, fracture or disappearance of mitochondrial cristae, and enlargement of the endoplasmic reticulum, etc. after modeling were obviously improved in the EA group. In comparison to the control group, the expression of the follicle granulosa cell P450 arom was significantly down-regulated and that of follicle membrane cell P 450 c 17 alpha was significantly upregulated in the model group (P < 0.01). After EA intervention these changes were obviously reversed (P < 0.05, P < 0.01). In the model group, there was a significant increase in the levels of ASD, T and LH in the ovarian tissues (P < 0.01) and a marked decrease in the contents of ovarian E 1 and E2 (P < 0.01) in comparison to the control group. After EA, the ovarian ASD, T and LH levels were notably decreased (P < 0.05, P < 0.01) and the E 1 and E 2 levels apparently increased (P < 0.01) compared with the model group. EA can improve letrozole-induced pathological changes of ovarian morphology and ultrastructure and obviously promote P 450 arom expression in the follicle granulosa cell layer and inhibit P 450 c 17 alpha expression in the follicle membrane cell layer, as well as regulate sex hormone levels in PCOS rats, facilitating the normal transformation of ovarian androgen to estrogen and restoring the local endocrine disorders.

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines.

PubMed

Macé, K; Aguilar, F; Wang, J S; Vautravers, P; Gómez-Lechón, M; Gonzalez, F J; Groopman, J; Harris, C C; Pfeifer, A M

1997-07-01

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

PubMed Central

Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

2008-01-01

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

PubMed

Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

2002-01-01

A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.

Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

PubMed

Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

2015-11-01

The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. Copyright © 2015 Elsevier Inc. All rights reserved.

Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver

PubMed Central

Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao

2015-01-01

Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

The Caffeine Cytochrome P450 1A2 Metabolic Phenotype Does Not Predict the Metabolism of Heterocyclic Aromatic Amines in Humans

PubMed Central

Turesky, Robert J.; White, Kami K.; Wilkens, Lynne R.; Marchand, Loïc Le

2015-01-01

2-Amino-1-methylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are carcinogenic heterocyclic aromatic amines (HAAs) formed in well-done cooked meats. Chemicals that induce cytochrome P450 (P450) 1A2, a major enzyme involved in the bioactivation of HAAs, also form in cooked meat. Therefore, well-done cooked meat may pose an increase in cancer risk because it contains both inducers of P450 1A2 and procarcinogenic HAAs. We examined the influence of components in meat to modulate P450 1A2 activity and the metabolism of PhIP and MeIQx in volunteers during a 4 week feeding study of well-done cooked beef. The mean P450 1A2 activity, assessed by caffeine metabolic phenotyping, ranged from 6.3 to 7.1 before the feeding study commenced and from 9.6 to 10.4 during the meat feeding period: the difference in means was significant (P < 0.001). Unaltered PhIP, MeIQx, and their P450 1A2 metabolites, N2-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N2-Gl); N3-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N3-Gl); 2-amino-3-methylimidazo-[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH); and 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-IQx) were measured in urine during days 2, 14, and 28 days of the meat diet. Significant correlations were observed on these days between the levels of the unaltered HAAs and their oxidized metabolites, when expressed as percent of dose ingested or as metabolic ratios. However, there was no statistically significant correlation between the caffeine P450 1A2 phenotype and any urinary HAA biomarker. Although the P450 1A2 activity varied by greater than 20-fold among the subjects, there was a large intra-individual variation of the P450 1A2 phenotype and inconsistent responses to inducers of P450 1A2. The coefficient of variation of the P450 1A2 phenotype within-individual ranged between 1 to 112% (median=40%) during the entire course of the study. The caffeine metabolic phenotype for P450 1A2 was a poor predictor of oxidative urinary metabolites of PhIP and MeIQx and may not be a reliable measure to assess the role of HAAs in cancer risk. PMID:26203673

Cytochrome P450 2J2, a new key enzyme in cyclophosphamide bioactivation and a potential biomarker for hematological malignancies.

PubMed

El-Serafi, I; Fares, M; Abedi-Valugerdi, M; Afsharian, P; Moshfegh, A; Terelius, Y; Potácová, Z; Hassan, M

2015-10-01

The role of cytochrome P450 2J2 (CYP2J2) in cyclophosphamide (Cy) bioactivation was investigated in patients, cells and microsomes. Gene expression analysis showed that CYP2J2 mRNA expression was significantly (P<0.01) higher in 20 patients with hematological malignancies compared with healthy controls. CYP2J2 expression showed significant upregulation (P<0.05) during Cy treatment before stem cell transplantation. Cy bioactivation was significantly correlated to CYP2J2 expression. Studies in HL-60 cells expressing CYP2J2 showed reduced cell viability when incubated with Cy (half maximal inhibitory concentration=3.6 mM). Inhibition of CYP2J2 using telmisartan reduced Cy bioactivation by 50% and improved cell survival. Cy incubated with recombinant CYP2J2 microsomes has resulted in apparent Km and Vmax values of 3.7-6.6 mM and 2.9-10.3 pmol/(min·pmol) CYP, respectively. This is the first study demonstrating that CYP2J2 is equally important to CYP2B6 in Cy metabolism. The heart, intestine and urinary bladder express high levels of CYP2J2; local Cy bioactivation may explain Cy-treatment-related toxicities in these organs.

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

PubMed

Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

Permethrin induction of multiple cytochrome P450 genes in insecticide resistant mosquitoes, Culex quinquefasciatus.

PubMed

Gong, Youhui; Li, Ting; Zhang, Lee; Gao, Xiwu; Liu, Nannan

2013-01-01

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.

Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California Poppy) and Its Alkaloids.

PubMed

Manda, Vamshi K; Ibrahim, Mohamed A; Dale, Olivia R; Kumarihamy, Mallika; Cutler, Stephen J; Khan, Ikhlas A; Walker, Larry A; Muhammad, Ilias; Khan, Shabana I

2016-04-01

Eschscholzia californica, a native US plant, is traditionally used as a sedative, analgesic, and anxiolytic herb. With the rapid rise in the use of herbal supplements together with over-the-counter and prescription drugs, the risk for potential herb-drug interactions is also increasing. Most of the clinically relevant pharmacokinetic drug interactions occur due to modulation of cytochrome P450 enzymes (CYPs), P-glycoprotein, and the pregnane X receptor by concomitantly used herbs. This study aimed to determine the effects of an EtOH extract, aqueous extract (tea), basic CHCl3 fractions, and isolated major alkaloids, namely protopine (1), escholtzine (2), allocryptopine (3), and californidine (4), of E. californica on the activity of cytochrome P450s, P-glycoprotein and the pregnane X receptor. The EtOH extract and fractions showed strong time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19, and reversible inhibition of CYP 2D6. Among the alkaloids, escholtzine (2) and allocryptopine (3) exhibited time-dependent inhibition of CYP 3A4, CYP 2C9, and CYP 2C19 (IC50 shift ratio > 2), while protopine (1) and allocryptopine (3) showed reversible inhibition of CYP 2D6 enzyme. A significant activation of the pregnane X receptor (> 2-fold) was observed with the EtOH extract, basic CHCl3 fraction, and alkaloids (except protopine), which resulted into an increased expression of mRNA and the activity of CYP 3A4 and CYP 1A2. The expression of P-glycoprotein was unaffected. However, aqueous extract (tea) and its main alkaloid californidine (4) did not affect cytochrome P450s, P-glycoprotein, or the pregnane X receptor. This data suggests that EtOH extract of E. californica and its major alkaloids have a potential of causing interactions with drugs that are metabolized by cytochrome P450s, while the tea seems to be safer. Georg Thieme Verlag KG Stuttgart · New York.

Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo.

PubMed

Donato, M Teresa; Hallifax, David; Picazo, Laura; Castell, José V; Houston, J Brian; Gomez-Lechón, M José; Lahoz, Agustin

2010-09-01

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.

The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdulla, Dalya; Goralski, Kerry B.; College of Pharmacy, Burbidge Building, Dalhousie University, Halifax, Nova Scotia, B3H 3J5

2006-10-01

It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo,more » an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.« less

Retinoblastoma protein (pRB) was significantly phosphorylated through a Ras-to-MAPK pathway in mutant K-ras stably transfected human adrenocortical cells.

PubMed

Chen, Y-F; Chiu, H-H; Wu, C-H; Wang, J-Y; Chen, F-M; Tzou, W-H; Shin, S-J; Lin, S-R

2003-10-01

Our previous studies have shown that the cell proliferation rate, mRNA levels of p450scc, p450c17, and 3betaHSD, and secretion of cortisol were significantly increased in human adrenocortical cells stably transfected with mutated K-ras expression plasmid "pK568MRSV" after being inducted with IPTG. In addition, the increased level was a time-dependent manner. However, the levels of p450, p450scc, p450c17, 3betaHSD, cortisol, and cell proliferation rate were inhibited by a MEK phospholation inhibitor, PD098059. The above results prove that mutated K-ras oncogene is able to regulate tumorigenesis and steroidogenesis through a Ras-RAF-MEK-MAPK signal transduction pathway. The aim of this study was to investigate regulated factors in this pathway and also examine whether the other signal transduction pathways or other moles involved in tumorigenesis or steroidogenesis. In the first year, we analyzed gene profiles of mutant K-ras-transfected adrenocortical cells by DNA microarray to determine the gene expression related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tag. After being affected by the K-ras mutant, gene expression was significantly increased in some upregulated genes. Human zinc-finger protein 22 increased by 28.5 times, Osteopontin increased by 5.8 times, LIM domain Kinase 2 (LIMK2) increased by 3.3 times, Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated Kinase 2 (DYRK2) increased by 2.2 times, and human syntaxin 3 increased by two times. On the other hand, significant decreases in gene expression were also observed in some downregulated genes. Retinoblastoma binding protein 1 (RBBP1) decreased by four times, Homo sapiens craniofacial development protein 1 (CFDP1) decreased by 2.4 times, DAP Kinase-related apoptosis-inducing protein Kinase 1 (DRAK1) decreased by 2.3 times, SKI-interacting protein (SKIP) decreased by 2.2 times, and human poly(A)-Binding protein (PABP) decreased by 2.1 times. In all significant differentially expressed genes, preliminary analysis by bioinformatics revealed that after induced K-ras mutant expression by isopropyl thiogalctoside (IPTG), the downregulation of RBBP1 gene was most correlated to cell proliferation. RBBP1 can bind with RB/E2F to form a mSIN3-HDAC complex, which induces cell cycle arrest in the G1/G0 stage by repressing transcription of E2F-regulated genes. The result of a Northern blot showed that RBBP1 were inhibited after an induction of IPTG for 36 h. Another Northern blot analysis proved that mRNA levels of cyclin D1 and c-myc increased in proportion to K-ras expression. Finally, Western blot was carried out, and the results showed that phosphorylated pRB also increased. Taken together, we infer that the mutant K-ras oncogene promoted the cells to proceed to the G1/S stage by the inhibiting the formation of RB/RBBP1-dependent repressor complex from binding with the SIN3-HDAC complex, which resulted in the acetylation of histone to active transcription of E2F-regulated genes. However, the roles of the other differentially expressed genes involved in cell proliferation, cell morphologic change, tumorigenesis, or steroidogenesis still need further investigation.

Cytochrome P-450 isoforms involved in carboxylic acid ester cleavage of Hantzsch pyridine ester of pranidipine.

PubMed

Kudo, S; Okumura, H; Miyamoto, G; Ishizaki, T

1999-02-01

Cytochrome P-450 (CYP) isoforms responsible for the cleavage of Hantzsch pyridine ester at the 3-position of pranidipine were studied in vitro using cDNA-expressed human CYP enzymes. CYP1A1, 1A2, 2D6, and 3A4 cleaved the ester with a catalytic activity of 5.5, 0. 93, 13.1, and 22.4 nmol/30 min/nmol P-450, respectively. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2E1 were not involved in the de-esterification. The Km and Vmax values for the de-esterification were 11.8 microM and 0.47 nmol/min/nmol P-450 in the CYP2D6-catalyzed reaction and 8. 7 microM and 0.84 nmol/min/nmol P-450 in the CYP3A4-catalyzed reaction. The intrinsic clearance (Vmax/Km) of the de-esterification by CYP3A4 was 2-fold greater than that by CYP2D6. Quinidine almost completely inhibited the CYP2D6-mediated de-esterification at the concentration of 1 x 10(-6) M. Ketoconazole and troleandomycin inhibited the CYP3A4-mediated reaction in a dose-related manner. The results indicate that although the multiple CYP isoforms can catalyze the de-esterification, CYP3A4 and 2D6 are the major isoforms.

Cytochrome P450 Activity in Ex Vivo Cornea Models and a Human Cornea Construct.

PubMed

Kölln, Christian; Reichl, Stephan

2016-07-01

The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

PubMed

Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

2015-03-10

Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.)

PubMed Central

Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W.; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

2015-01-01

Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification. PMID:25752830

Genome-wide and expression-profiling analyses suggest the main cytochrome P450 genes related to pyrethroid resistance in the malaria vector, Anopheles sinensis (Diptera Culicidae).

PubMed

Yan, Zheng-Wen; He, Zheng-Bo; Yan, Zhen-Tian; Si, Feng-Ling; Zhou, Yong; Chen, Bin

2018-02-02

Anopheles sinensis is one of the major malaria vectors. However, pyrethroid resistance in An. sinensis is threatening malaria control. Cytochrome P450-mediated detoxification is an important pyrethroid resistance mechanism that has been unexplored in An. sinensis. In this study, we performed a comprehensive analysis of the An. sinensis P450 gene superfamily with special attention to their role in pyrethroid resistance using bioinformatics and molecular approaches. Our data revealed the presence of 112 individual P450 genes in An. sinensis, which were classified into four major clans (mitochondrial, CYP2, CYP3 and CYP4), 18 families and 50 subfamilies. Sixty-seven genes formed nine gene clusters, and genes within the same cluster and the same gene family had a similar gene structure. Phylogenetic analysis showed that most of An. sinensis P450s (82/112) had very close 1: 1 orthology with Anopheles gambiae P450s. Five genes (AsCYP6Z2, AsCYP6P3v1, AsCYP6P3v2, AsCYP9J5 and AsCYP306A1) were significantly upregulated in three pyrethroid-resistant populations in both RNA-seq and RT-qPCR analyses, suggesting that they could be the most important P450 genes involved in pyrethroid resistance in An. sinensis. Our study provides insight on the diversity of An. sinensis P450 superfamily and basis for further elucidating pyrethroid resistance mechanism in this mosquito species. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Molecular cloning and expression of CYP9A61: a chlorpyrifos-ethyl and lambda-cyhalothrin-inducible cytochrome P450 cDNA from Cydia pomonella.

PubMed

Yang, Xueqing; Li, Xianchun; Zhang, Yalin

2013-12-13

Cytochrome P450 monooxygenases (CYPs or P450s) play paramount roles in detoxification of insecticides in a number of insect pests. However, little is known about the roles of P450s and their responses to insecticide exposure in the codling moth Cydia pomonella (L.), an economically important fruit pest. Here we report the characterization and expression analysis of the first P450 gene, designated as CYP9A61, from this pest. The full-length cDNA sequence of CYP9A61 is 2071 bp long and its open reading frame (ORF) encodes 538 amino acids. Sequence analysis shows that CYP9A61 shares 51%-60% identity with other known CYP9s and contains the highly conserved substrate recognition site SRS1, SRS4 and SRS5. Quantitative real-time PCR showed that CYP9A61 were 67-fold higher in the fifth instar larvae than in the first instar, and more abundant in the silk gland and fat body than other tissues. Exposure of the 3rd instar larvae to 12.5 mg L(-1) of chlorpyrifos-ethyl for 60 h and 0.19 mg L(-1) of lambda-cyhalothrin for 36 h resulted in 2.20- and 3.47-fold induction of CYP9A61, respectively. Exposure of the 3rd instar larvae to these two insecticides also significantly enhanced the total P450 activity. The results suggested that CYP9A61 is an insecticide-detoxifying P450.

Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

PubMed Central

Yang, Xueqing; Li, Xianchun; Zhang, Yalin

2013-01-01

Cytochrome P450 monooxygenases (CYPs or P450s) play paramount roles in detoxification of insecticides in a number of insect pests. However, little is known about the roles of P450s and their responses to insecticide exposure in the codling moth Cydia pomonella (L.), an economically important fruit pest. Here we report the characterization and expression analysis of the first P450 gene, designated as CYP9A61, from this pest. The full-length cDNA sequence of CYP9A61 is 2071 bp long and its open reading frame (ORF) encodes 538 amino acids. Sequence analysis shows that CYP9A61 shares 51%–60% identity with other known CYP9s and contains the highly conserved substrate recognition site SRS1, SRS4 and SRS5. Quantitative real-time PCR showed that CYP9A61 were 67-fold higher in the fifth instar larvae than in the first instar, and more abundant in the silk gland and fat body than other tissues. Exposure of the 3rd instar larvae to 12.5 mg L−1 of chlorpyrifos-ethyl for 60 h and 0.19 mg L−1 of lambda-cyhalothrin for 36 h resulted in 2.20-and 3.47-fold induction of CYP9A61, respectively. Exposure of the 3rd instar larvae to these two insecticides also significantly enhanced the total P450 activity. The results suggested that CYP9A61 is an insecticide-detoxifying P450. PMID:24351812

Regulation of cytochrome P450 mRNA expression in primary porcine hepatocytes by selected secondary plant metabolites from chicory (Cichorium intybus L.).

PubMed

Rasmussen, Martin Krøyer; Klausen, Christina Lindgaard; Ekstrand, Bo

2014-03-01

Chicory (Cichorium intybus) has been shown to induce enzymes of pharmacokinetic relevance (cytochrome P450; CYP). The aim of this study was to investigate the effects of selected secondary plant metabolites with a global extract of chicory root, on the expression of hepatic CYP mRNA (1A2, 2A19, 2C33, 2D25, 2E1 and 3A29), using primary porcine hepatocytes. Of the tested secondary plant metabolites, artemisinin, scoparone, lactucin and esculetin all induced increased expression of specific CYPs, while esculin showed no effect. In contrast, a global extract of chicory root decreased the expression of CYP1A2, 2C33, 2D25 and 3A29 at high concentrations. The results suggest that purified secondary metabolites from chicory affect CYP expression and thereby might affect detoxification in general, and that global extracts of plants can have effects different from individual components. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

PubMed

Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

2017-09-01

Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

PubMed Central

Waxman, D J; Morrissey, J J; Naik, S; Jauregui, H O

1990-01-01

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones. Images Fig. 3. Fig. 5. PMID:2222405

Effects of Electro-Acupuncture on Ovarian P450arom, P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

PubMed Central

Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

2013-01-01

Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg−1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and endocrine metabolic disorders in PCOS. PMID:24260211

Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

PubMed

Sun, Jie; Jin, Chunlan; Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

2013-01-01

Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1) of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and endocrine metabolic disorders in PCOS.

The roles of CYP6AY1 and CYP6ER1 in imidacloprid resistance in the brown planthopper: Expression levels and detoxification efficiency.

PubMed

Bao, Haibo; Gao, Hongli; Zhang, Yixi; Fan, Dongzhe; Fang, Jichao; Liu, Zewen

2016-05-01

Two P450 monooxygenase genes, CYP6AY1 and CYP6ER1, were reported to contribute importantly to imidacloprid resistance in the brown planthopper, Nilaparvata lugens. Although recombinant CYP6AY1 could metabolize imidacloprid efficiently, the expression levels of CYP6ER1 gene were higher in most resistant populations. In the present study, three field populations were collected from different countries, and the bioassay, RNAi and imidacloprid metabolism were performed to evaluate the importance of two P450s in imidacloprid resistance. All three populations, DOT (Dongtai) from China, CNA (Chainat) from Thailand and HCM (Ho Chi Minh) from Vietnam, showed high resistance to imidacloprid (57.0-, 102.9- and 89.0-fold). CYP6AY1 and CYP6ER1 were both over expressed in three populations, with highest ratio of 13.2-fold for CYP6ER1 in HCM population. Synergism test and RNAi analysis confirmed the roles of both P450 genes in imidacloprid resistance. However, CYP6AY1 was indicated more important in CNA population, and CYP6AY1 and CYP6ER1 were equal in HCM population, although the expression level of CYP6ER1 (13.2-fold) was much higher than that of CYP6AY1 (4.11-fold) in HCM population. Although the recombinant proteins of both P450 genes could metabolize imidacloprid efficiently, the catalytic activity of CYP6AY1 (Kcat=3.627 pmol/min/pmol P450) was significantly higher than that of CYP6ER1 (Kcat=2.785 pmol/min/pmol P450). It was supposed that both P450 proteins were important for imidacloprid resistance, in which CYP6AY1 metabolized imidacloprid more efficiently and CYP6ER1 gene could be regulated by imidacloprid to a higher level. Copyright © 2015 Elsevier B.V. All rights reserved.

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chunxue; Pallan, Pradeep S.; Zhang, Wei

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12more » variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37–13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C–H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.« less

Effect of chondroitin sulfate on turpentine-induced down-regulation of CYP1A2 and CYP3A6.

PubMed

Iovu, Mirela-Onita; Héroux, Lucie; Vergés, Josep; Montell, Eulália; Paiement, Jacques; du Souich, Patrick

2012-07-01

This study aimed to assess whether chronic administration of chondroitin sulfate (CS) affects baseline expression of cytochrome P450 isoforms and impedes the decrease in expression and activity of CYP1A2 and CYP3A6 in rabbits with a turpentine-induced inflammatory reaction (TIIR). Seven groups of 5 rabbits, 3 control groups and 4 receiving 20 mg/kg/day of CS for 20 and 30 days, were used. The rabbits of 1 control group and 2 groups receiving CS had a TIIR; finally, the rabbits of one of the control groups remained in the animal facilities for 30 days to assess the effect of time and environment on cytochrome P450. In control rabbits, intake of CS for 20 and 30 days did not affect CYP3A6, CYP1A2 and NADPH cytochrome P450 reductase (CPR) mRNA, protein expression and activity. Compared with control rabbits, the TIIR not only reduced mRNA, protein expression and activity of CYP3A6 and CYP1A2 but also that of CPR. In rabbits with TIIR, CS prevented the decrease of CYP3A6 expression but not the reduction in activity. CS did not impede TIIR-induced down-regulation of CYP1A2. Hepatic NO() concentrations and NF-κB nuclear translocation were increased by the TIIR, effect reversed by CS. In vitro, in hepatocytes, CS did not alter the expression and activity of CYP3A6, CYP1A2, and CPR. In conclusion, oral CS elicits a systemic effect but does not affect CYP1A2, CYP3A6, and CPR in control rabbits, although in rabbits with TIIR, CS prevents CYP3A6 protein down-regulation but not that of CYP1A2. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver.

PubMed

Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao; Zhong, Xiao-bo

2015-12-01

Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

An in vitro evaluation of cytochrome P450 inhibition and P-glycoprotein interaction with goldenseal, Ginkgo biloba, grape seed, milk thistle, and ginseng extracts and their constituents.

PubMed

Etheridge, Amy S; Black, Sherry R; Patel, Purvi R; So, James; Mathews, James M

2007-07-01

Drug-herb interactions can result from the modulation of the activities of cytochrome P450 (P450) and/or drug transporters. The effect of extracts and individual constituents of goldenseal, Ginkgo biloba (and its hydrolyzate), grape seed, milk thistle, and ginseng on the activities of cytochrome P450 enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes were determined using enzyme-selective probe substrates, and their effect on human P-glycoprotein (Pgp) was determined using a baculovirus expression system by measuring the verapamil-stimulated, vanadate-sensitive ATPase activity. Extracts were analyzed by HPLC to standardize their concentration(s) of constituents associated with the pharmacological activity, and to allow comparison of their effects on P450 and Pgp with literature values. Many of the extracts/constituents exerted > or = 50 % inhibition of P450 activity. These include those from goldenseal (normalized to alkaloid content) inhibiting CYP2C8, CYP2D6, and CYP3A4 at 20 microM, ginkgo inhibiting CYP2C8 at 10 microM, grape seed inhibiting CYP2C9 and CYP3A4 at 10 microM, milk thistle inhibiting CYP2C8 at 10 microM, and ginsenosides F1 and Rh1 (but not ginseng extract) inhibiting CYP3A4 at 10 microM. Goldenseal extracts/constituents (20 microM, particularly hydrastine) and ginsenoside Rh1 stimulated ATPase at about half of the activity of the model substrate, verapamil (20 microM). The data suggest that the clearance of a variety of drugs may be diminished by concomitant use of these herbs via inhibition of P450 enzymes, but less so by Pgp-mediated effects.

Examination of Zolpidem effects on AhR- and PXR-dependent expression of drug-metabolizing cytochromes P450 in primary cultures of human hepatocytes.

PubMed

Bachleda, Petr; Vrzal, Radim; Pivnicka, Jakub; Cvek, Boris; Dvorak, Zdenek

2009-12-01

A hypnotic drug Zolpidem is used in clinical practice for more than 25 years. Surprisingly, the effects of Zolpidem on the expression of drug-metabolizing cytochromes P450 (CYPs) were not examined yet. Recently, the unexpected capacity of several "old drugs", such as valproic acid or azoles, to induce CYPs was reported. Therefore, we tested whether Zolpidem induces the expression of important CYPs in primary cultures of human hepatocytes. Cells were treated for 24h with Zolpidem in therapeutic (0.1mg/L) and toxic (1mg/L) concentrations. The levels of CYP1A1, CYP1A2, CY2C9 and CYP3A4 mRNAs were not altered by Zolpidem, whereas model inducers dioxin and rifampicin significantly induced CYP1A and CYP2/3 gene expression, respectively. Consistently, Zolpidem did not activate aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), the key regulators of cytochromes P450s, as revealed by transient transfection gene reporter assays using HepG2 cells. We conclude Zolpidem be considered a safe drug with respect to the possible interactions through AhR- and PXR-dependent induction of drug-metabolizing CYPs.

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

PubMed Central

Unterweger, Birgit; Bulach, Dieter M.; Scoble, Judith; Midgley, David J.; Greenfield, Paul; Lyras, Dena; Johanesen, Priscilla

2016-01-01

ABSTRACT We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling. PMID:27590809

CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

PubMed

Davies, Benjamin J; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

2007-01-01

The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.

Cytochrome P450 humanised mice

PubMed Central

2004-01-01

Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

Expression of paclitaxel-inactivating CYP3A activity in human colorectal cancer: implications for drug therapy

PubMed Central

Martínez, C; García-Martín, E; Pizarro, R M; García-Gamito, F J; Agúndez, J A G

2002-01-01

Cytochrome P450 3A is a drug-metabolising enzyme activity due to CYP3A4 and CYP3A5 gene products, that is involved in the inactivation of anticancer drugs. This study analyses the potential of cytochrome P450 3A enzyme in human colorectal cancer to impact anticancer therapy with drugs that are cytochrome P450 3A substrates. Enzyme activity, variability and properties, and the ability to inactivate paclitaxel (taxol) were analysed in human colorectal cancer and healthy colorectal epithelium. Cytochrome P450 3A enzyme activity is present in healthy and tumoral samples, with a nearly 10-fold interindividual variability. Nifedipine oxidation activity±s.d. for colorectal cancer microsomes was 67.8±36.6 pmol min−1 mg−1. The Km of the tumoral enzyme (42±8 μM) is similar to that in healthy colorectal epithelium (36±8 μM) and the human liver enzyme. Colorectal cancer microsomes metabolised the anticancer drug paclitaxel with a mean activity was 3.1±1.2 pmol min−1 mg−1. The main metabolic pathway is carried out by cytochrome P450 3A, and it is inhibited by the cytochrome P450 3A-specific inhibitor ketoconazole with a KI value of 31 nM. This study demonstrates the occurrence of cytochrome P450 3A-dependent metabolism in colorectal cancer tissue. The metabolic activity confers to cancer cells the ability to inactivate cytochrome P450 3A substrates and may modulate tumour sensitivity to anticancer drugs. British Journal of Cancer (2002) 87, 681–686. doi:10.1038/sj.bjc.6600494 www.bjcancer.com © 2002 Cancer Research UK PMID:12237780

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

PubMed

Akazawa, Takanori; Uchida, Yasuo; Miyauchi, Eisuke; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

2018-01-02

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

Hepatic cytochrome P450 activity, abundance, and expression throughout human development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.

Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomicmore » analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.« less

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity.

PubMed

Sata, F; Sapone, A; Elizondo, G; Stocker, P; Miller, V P; Zheng, W; Raunio, H; Crespi, C L; Gonzalez, F J

2000-01-01

To determine the existence of mutant and variant CgammaP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression. A bacterial artificial chromosome that contains the complete CgammaP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation. To investigate the existence of mutant and variant CgammaP3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human CgammaP3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human CgammaP3A4 gene, designated CgammaP3A4*1B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CgammaP3A4*1B allele was not found in Chinese subjects. A second variant allele, designated CgammaP3A4*2, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A4*2 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6beta-hydroxylation. Another rare allele, designated CgammaP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. These are the first examples of potential function polymorphisms resulting from missense mutations in the CgammaP3A4 gene. The CgammaP3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.

In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4

PubMed Central

Lv, Qiao-Li; Wang, Gui-Hua; Chen, Shu-Hui; Hu, Lei; Zhang, Xue; Ying, Guo; Qin, Chong-Zhen; Zhou, Hong-Hao

2015-01-01

Glycyrrhetinic acid (GA) has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450) cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs) and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4. PMID:26712778

In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4.

PubMed

Lv, Qiao-Li; Wang, Gui-Hua; Chen, Shu-Hui; Hu, Lei; Zhang, Xue; Ying, Guo; Qin, Chong-Zhen; Zhou, Hong-Hao

2015-12-25

Glycyrrhetinic acid (GA) has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450) cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs) and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver-Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.

Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

PubMed

Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

2009-09-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half themore » drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.« less

Stereoselective metabolism of endosulfan by human liver microsomes and human cytochrome P450 isoforms.

PubMed

Lee, Hwa-Kyung; Moon, Joon-Kwan; Chang, Chul-Hee; Choi, Hoon; Park, Hee-Won; Park, Byeoung-Soo; Lee, Hye-Suk; Hwang, Eul-Chul; Lee, Young-Deuk; Liu, Kwang-Hyeon; Kim, Jeong-Han

2006-07-01

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo(e)dioxathiepin-3-oxide) is a broad-spectrum chlorinated cyclodiene insecticide. This study was performed to elucidate the stereoselective metabolism of endosulfan in human liver microsomes and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of endosulfan. Human liver microsomal incubation of endosulfan in the presence of NADPH resulted in the formation of the toxic metabolite, endosulfan sulfate. The intrinsic clearances (CL(int)) of endosulfan sulfate from beta-endosulfan were 3.5-fold higher than those from alpha-endosulfan, suggesting that beta-endosulfan would be cleared more rapidly than alpha-endosulfan. Correlation analysis between the known P450 enzyme activities and the rate of the formation of endosulfan sulfate in the 14 human liver microsomes showed that alpha-endosulfan metabolism is significantly correlated with CYP2B6-mediated bupropion hydroxylation and CYP3A-mediated midazolam hydroxylation, and that beta-endosulfan metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in human liver microsomes and the incubation study of cDNA-expressed enzymes also demonstrated that the stereoselective sulfonation of alpha-endosulfan is mediated by CYP2B6, CYP3A4, and CYP3A5, and that that of beta-endosulfan is transformed by CYP3A4 and CYP3A5. The total CL(int) values of endosulfan sulfate formation catalyzed by CYP3A4 and CYP3A5 were consistently higher for beta-endosulfan than for the alpha-form (CL(int) of 0.67 versus 10.46 microl/min/pmol P450, respectively). CYP2B6 enantioselectively metabolizes alpha-endosulfan, but not beta-endosulfan. These findings suggest that the CYP2B6 and CYP3A enzymes are major enzymes contributing to the stereoselective disposition of endosulfan.

Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison’s Disease)

PubMed Central

Boag, Alisdair M.; Christie, Michael R.; McLaughlin, Kerry A.; Syme, Harriet M.; Graham, Peter; Catchpole, Brian

2015-01-01

Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison’s disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism. PMID:26618927

Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison's Disease).

PubMed

Boag, Alisdair M; Christie, Michael R; McLaughlin, Kerry A; Syme, Harriet M; Graham, Peter; Catchpole, Brian

2015-01-01

Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.

Application of HC-AFW1 Hepatocarcinoma Cells for Mechanistic Studies: Regulation of Cytochrome P450 2B6 Expression by Dimethyl Sulfoxide and Early Growth Response 1.

PubMed

Petzuch, Barbara; Groll, Nicola; Schwarz, Michael; Braeuning, Albert

2015-11-01

Various exogenous compounds, for example, the drugs bupropione and propofol, but also various cytostatics, are metabolized in the liver by the enzyme cytochrome P450 (P450) CYP2B6. Transcription from the CYP2B6 gene is regulated mainly via the transcription factors constitutive androstane receptor (CAR) and pregnane-X-receptor (PXR). Most hepatic cell lines express no or only low levels of CYP2B6 because of loss of these two regulators. Dimethyl sulfoxide (DMSO) is frequently used in liver cell cultivation and is thought to affect the expression of various P450 isoforms by inducing or preserving cellular differentiation. We studied the effects of up to 1.5% of DMSO as cell culture medium supplement on P450 expression in hepatocarcinoma cells from line HC-AFW1. DMSO did not induce differentiation of the HC-AFW1 cell line, as demonstrated by unaltered levels of selected mRNA markers important for hepatocyte differentiation, and also by the lack of a DMSO effect on a broader spectrum of P450s. By contrast, CYP2B6 mRNA was strongly induced by DMSO. This process was independent of CAR or PXR activation. Interestingly, elevated transcription of CYP2B6 was accompanied by a simultaneous induction of early growth response 1 (EGR1), a transcription factor known to influence the expression of CYP2B6. Expression of wild-type EGR1 or of a truncated, dominant-negative EGR1 mutant was able to mimic or attenuate the DMSO effect, respectively. These findings demonstrate that EGR1 is involved in the regulation of CYP2B6 by DMSO in HC-AFW1 cells. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

2000-02-25

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.

Changes in the immunolocalization of steroidogenic enzymes and the androgen receptor in raccoon (Procyon lotor) testes in association with the seasons and spermatogenesis.

PubMed

Okuyama, Minami W; Shimozuru, Michito; Yanagawa, Yojiro; Tsubota, Toshio

2014-04-24

The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3βHSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3βHSD expression, suggesting that 3βHSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.

Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes, caffeine, warfarin, omeprazole, metoprolol and midazolam, in common marmosets (Callithrix jacchus).

PubMed

Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Toda, Akiko; Shimizu, Makiko; Uno, Yasuhiro; Sasaki, Erika; Yamazaki, Hiroshi

2016-01-01

1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, to four male common marmosets were investigated. 2. The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans. 3. Bioavailabilities were ∼100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1'-hydroxylation as evident in the recombinant system. 4. These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.

Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

PubMed

Naderi-Kalali, B; Allameh, A; Rasaee, M J; Bach, H-J; Behechti, A; Doods, K; Kettrup, A; Schramm, K-W

2005-04-01

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

CYP6 P450 enzymes and ACE-1 duplication produce extreme and multiple insecticide resistance in the malaria mosquito Anopheles gambiae.

PubMed

Edi, Constant V; Djogbénou, Luc; Jenkins, Adam M; Regna, Kimberly; Muskavitch, Marc A T; Poupardin, Rodolphe; Jones, Christopher M; Essandoh, John; Kétoh, Guillaume K; Paine, Mark J I; Koudou, Benjamin G; Donnelly, Martin J; Ranson, Hilary; Weetman, David

2014-03-01

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

[Effect of clinical doses of Realgar-Indigo Naturalis formula and large-dose of realgar on CYP450s of rat liver].

PubMed

Xu, Huan-Hua; Wang, Mei-Xi; Tan, Hong-Ling; Wang, Yu-Guang; Tang, Xiang-Lin; Xiao, Cheng-Rong; Li, Hua; Gao, Yue; Ma, Zeng-Chun

2017-02-01

To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application. Copyright© by the Chinese Pharmaceutical Association.

Differential expression of steroidogenic factors 1 and 2, cytochrome p450scc, and steroidogenic acute regulatory protein in human pancreas.

PubMed

Morales, Angélica; Vilchis, Felipe; Chávez, Bertha; Morimoto, Sumiko; Chan, Carlos; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

2008-08-01

The aim of this study was to investigate the expression of the 4 gene transcripts, steroidogenic factors 1 (SF-1) and 2 (SF-2), steroidogenic acute regulatory (StAR), and cytochrome P450 11A1, involved in the synthesis of steroid hormones in normal human pancreas. Total RNA was extracted from normal male (n = 5) and female (n = 5) samples, obtained from the organ donor program. The expression levels of SF-1, SF-2, StAR protein, and P450scc were assessed by reverse transcription-polymerase chain reaction and complemented with immunohistochemistry analysis. Polymerase chain reaction products amplification for all genes was present in both male and female samples, although differential expression was observed. The signals detected were much more evident in male than in female messenger RNA isolates for SF-1, SF-2, and StAR protein. The expression for P450scc was more intense in female samples. A similar pattern was observed in the immunohistochemical studies. Normal human pancreas expresses 4 gene transcripts involved in steroid synthesis similarly to steroidogenic organs. A distinctive characteristic is the sexually dimorphic expression of these factors. These data provide further evidence to support that the pancreas is a truly steroidogenic tissue, highlighting the presence of sex- and location-related differences in the expression of steroidogenic factors.

NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells.

PubMed

Gray, Joshua P; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A

2016-11-16

NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H 2 O 2 . Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

NAD(P)H-dependent Quinone Oxidoreductase 1 (NQO1) and Cytochrome P450 Oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells

PubMed Central

Gray, Joshua P.; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A.

2017-01-01

NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H2O2. Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. PMID:27558805

Identification and analysis of CYP450 genes from transcriptome of Lonicera japonica and expression analysis of chlorogenic acid biosynthesis related CYP450s.

PubMed

Qi, Xiwu; Yu, Xu; Xu, Daohua; Fang, Hailing; Dong, Ke; Li, Weilin; Liang, Chengyuan

2017-01-01

Lonicera japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of L. japonica are mainly due to its rich natural active ingredients, most of which are secondary metabolites. CYP450s are a large, complex, and widespread superfamily of proteins that participate in many endogenous and exogenous metabolic reactions, especially secondary metabolism. Here, we identified CYP450s in L. japonica transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis. The recent availability of L. japonica transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in L. japonica transcriptome. Then, phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation analyses were conducted to characterize the identified CYP450s. qRT-PCR was used to explore expression patterns of five CGA biosynthesis related CYP450s. In this study, 151 putative CYP450s with complete cytochrome P450 domain, which belonged to 10 clans, 45 families and 76 subfamilies, were identified in L. japonica transcriptome. Phylogenetic analysis classified these CYP450s into two major branches, A-type (47%) and non-A type (53%). Both types of CYP450s had conserved motifs in L. japonica . The differences of typical motif sequences between A-type and non-A type CYP450s in L. japonica were similar with other plants. GO classification indicated that non-A type CYP450s participated in more molecular functions and biological processes than A-type. KEGG pathway annotation totally assigned 47 CYP450s to 25 KEGG pathways. From these data, we cloned two LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of L. japonica . qRT-PCR results indicated that two LjC3Hs exhibited oppositing expression patterns during the flower development and LjC3H2 exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three LjC4Hs were quite different and the expression pattern of LjC4H3 was quite similar with that of LjC3H1 . Our results provide a comprehensive identification and characterization of CYP450s in L. japonica . Five CGA biosynthesis related CYP450s were cloned and their expression patterns were explored. The different expression patterns of two LjC3Hs and three LjC4Hs may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjC3H1 and LjC4H3 strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica .

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

PubMed Central

Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

1988-01-01

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

Effects of 17alpha-methyltestosterone exposure on steroidogenesis and cyclin-B mRNA expression in previtellogenic oocytes of Atlantic cod (Gadus morhua).

PubMed

Kortner, Trond M; Arukwe, Augustine

2007-11-01

Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

PubMed Central

Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

2016-01-01

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

PubMed

Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

2006-10-01

The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

Dietary Omega-3 Polyunsaturated Fatty Acids Prevent Vascular Dysfunction and Attenuate Cytochrome P4501A1 Expression by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin.

PubMed

Wiest, Elani F; Walsh-Wilcox, Mary T; Rothe, Michael; Schunck, Wolf-Hagen; Walker, Mary K

2016-11-01

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) found in fish protect against cardiovascular morbidity and mortality; however, many individuals avoid fish consumption due to concerns about pollutants. We tested the hypothesis that n-3 PUFAs would prevent vascular dysfunction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). C57Bl/6 male mice were fed a chow or n-3 PUFA diet for 10 weeks and were exposed to vehicle or 300 ng/kg/d TCDD during the final 2 weeks on each diet. Aortic vasoconstriction mediated by arachidonic acid (AA) ± SKF525 (P450 inhibitor) or SQ29548 (thromboxane/prostanoid [TP] receptor antagonist) was assessed. RBC fatty acids and expression of n-3 and n-6 PUFA metabolites were analyzed. Cytochrome P4501A1 (CYP1A1), CYP1B1, and aryl hydrocarbon receptor (AHR) expression was measured. TCDD significantly increased AA-mediated vasoconstriction on a chow diet by increasing the contribution of P450s and TP receptor to the constriction response. In contrast, the n-3 PUFA diet prevented the TCDD-induced increase in AA vasoconstriction and normalized the contribution of P450s and TP receptor. Although TCDD increased the levels of AA vasoconstrictors on the chow diet, this increase was prevent by the n-3 PUFA diet. Additionally, the n-3 PUFA diet significantly increased the levels of n-3 PUFA-derived vasodilators and TCDD increased these levels further. Interestingly, the n-3 PUFA diet significantly attenuated CYP1A1 induction by TCDD without a significant effect on AHR expression. These data suggest that n-3 PUFAs can prevent TCDD-induced vascular dysfunction by decreasing vasoconstrictors, increasing vasodilators, and attenuating CYP1A1 induction, which has been shown previously to contribute to TCDD-induced vascular dysfunction. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimizu, Takashi, E-mail: shimizut@obihiro.ac.jp; Hirai, Yuko; Murayama, Chiaki

2011-08-19

Highlights: {yields} Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression. {yields}Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom. {yields} Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. {yields}Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. {yields} The expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. -- Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number ofmore » granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.« less

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

PubMed

Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko

2012-03-01

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.

Tissue Specific Modulation of cyp2c and cyp3a mRNA Levels and Activities by Diet-Induced Obesity in Mice: The Impact of Type 2 Diabetes on Drug Metabolizing Enzymes in Liver and Extra-Hepatic Tissues

PubMed Central

Chamoun, Michel; Gravel, Sophie; Turgeon, Jacques; Michaud, Veronique

2017-01-01

Various diseases such as type 2 diabetes (T2D) may alter drug clearance. The objective of this study was to evaluate the effects of T2D on CYP450 expressions and activities using high-fat diet (HFD) as a model of obesity-dependent diabetes in C57BL6 mice. The cyp450 mRNA expression levels for 15 different isoforms were determined in the liver and extra-hepatic tissues (kidneys, lungs and heart) of HFD-treated animals (n = 45). Modulation of cyp450 metabolic activities by HFD was assessed using eight known substrates for specific human ortholog CYP450 isoforms: in vitro incubations were conducted with liver and extra-hepatic microsomes. Expression levels of cyp3a11 and cyp3a25 mRNA were decreased in the liver (>2–14-fold) and kidneys (>2-fold) of HFD groups which correlated with a significant reduction in midazolam metabolism (by 21- and 5-fold in hepatic and kidney microsomes, respectively, p < 0.001). HFD was associated with decreased activities of cyp2b and cyp2c subfamilies in all organs tested except in the kidneys (for tolbutamide). Other cyp450 hepatic activities were minimally or not affected by HFD. Taken together, our data suggest that substrate-dependent and tissue-dependent modulation of cyp450 metabolic capacities by early phases of T2D are observed, which could modulate drug disposition and pharmacological effects in various tissues. PMID:28954402

Water Oxidation by a Cytochrome P450: Mechanism and Function of the Reaction

PubMed Central

Prasad, Brinda; Mah, Derrick J.; Lewis, Andrew R.; Plettner, Erika

2013-01-01

P450cam (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O2). We report that P450cam catalysis is controlled by oxygen levels: at high O2 concentration, P450cam catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using 17O and 2H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450cam, and we present a plausible mechanism that accounts for the 1∶1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450cam and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce. PMID:23634216

Transcriptome analysis and identification of P450 genes relevant to imidacloprid detoxification in Bradysia odoriphaga.

PubMed

Chen, Chengyu; Wang, Cuicui; Liu, Ying; Shi, Xueyan; Gao, Xiwu

2018-02-07

Pesticide tolerance poses many challenges for pest control, particularly for destructive pests such as Bradysia odoriphaga. Imidacloprid has been used to control B. odoriphaga since 2013, however, imidacloprid resistance in B. odoriphaga has developed in recent years. Identifying actual and potential genes involved in detoxification metabolism of imidacloprid could offer solutions for controlling this insect. In this study, RNA-seq was used to explore differentially expressed genes in B. odoriphaga that respond to imidacloprid treatment. Differential expression data between imidacloprid treatment and the control revealed 281 transcripts (176 with annotations) showing upregulation and 394 transcripts (235 with annotations) showing downregulation. Among them, differential expression levels of seven P450 unigenes were associated with imidacloprid detoxification mechanism, with 4 unigenes that were upregulated and 3 unigenes that were downregulated. The qRT-PCR results of the seven differential expression P450 unigenes after imidacloprid treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of these four upregulated P450 unigenes followed by an insecticide bioassay significantly increased the mortality of imidacloprid-treated B. odoriphaga. This result indicated that the four upregulated P450s are involved in detoxification of imidacloprid. This study provides a genetic basis for further exploring P450 genes for imidacloprid detoxification in B. odoriphaga.

Cytochrome P450IA mRNA expression in feral Hudson River tomcod

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kreamer, G.L.; Squibb, K.; Gioeli, D.

1991-06-01

The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached bymore » 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.« less

Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

PubMed Central

2010-01-01

Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development. Conclusions The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction. PMID:21062474

Constitutive Androgen Receptor-Null Mice Are Sensitive to the Toxic Effects of Parathion: Association with Reduced Cytochrome P450-Mediated Parathion MetabolismS⃞

PubMed Central

Mota, Linda C.; Hernandez, Juan P.

2010-01-01

Constitutive androgen receptor (CAR) is activated by several chemicals and in turn regulates multiple detoxification genes. Our research demonstrates that parathion is one of the most potent, environmentally relevant CAR activators with an EC50 of 1.43 μM. Therefore, animal studies were conducted to determine whether CAR was activated by parathion in vivo. Surprisingly, CAR-null mice, but not wild-type (WT) mice, showed significant parathion-induced toxicity. However, parathion did not induce Cyp2b expression, suggesting that parathion is not a CAR activator in vivo, presumably because of its short half-life. CAR expression is also associated with the expression of several drug-metabolizing cytochromes P450 (P450). CAR-null mice demonstrate lower expression of Cyp2b9, Cyp2b10, Cyp2c29, and Cyp3a11 primarily, but not exclusively in males. Therefore, we incubated microsomes from untreated WT and CAR-null mice with parathion in the presence of esterase inhibitors to determine whether CAR-null mice show perturbed P450-mediated parathion metabolism compared with that in WT mice. The metabolism of parathion to paraoxon and p-nitrophenol (PNP) was reduced in CAR-null mice with male CAR-null mice showing reduced production of both paraoxon and PNP, and female CAR-null mice showing reduced production of only PNP. Overall, the data indicate that CAR-null mice metabolize parathion slower than WT mice. These results provide a potential mechanism for increased sensitivity of individuals with lower CAR activity such as newborns to parathion and potentially other chemicals due to decreased metabolic capacity. PMID:20573718

Glucocorticoid receptor contributes to the altered expression of hepatic cytochrome P450 upon cigarette smoking.

PubMed

Li, Xue; Yan, Zhongfang; Wu, Qi; Sun, Xin; Li, Fan; Zhang, Subei; Li, Kuan; Li, Li; Wu, Junping; Xu, Long; Feng, Jing; Ning, Wen; Liu, Zhixue; Chen, Huaiyong

2016-12-01

Cigarette smoking has been shown to cause pathological alterations in the liver. However, how hepatic metabolism is altered during cigarette smoking‑induced inflammation remains to be fully elucidated. In the present study, a rat model of smoking was established to examine the effects of cigarette smoking on inflammation, autophagy activity, and the expression of nuclear receptor and CYP in the liver. Elevated expression of interleukin 1β and activation of autophagy in the liver were observed upon smoking exposure in rats. Cigarette smoking induced a significant reduction in the mRNA expression levels of cytochromes, including cytochrome P450 (Cyp)1A2, Cyp2D4 and Cyp3A2. Accordingly, a decrease was also observed in glucocorticoid receptor (GR), a regulator of the expression of Cyp. Activation of the GR signal in human hepatic LO2 cells did not affect autophagic genes, however, it led to the upregulation of hCYP1A2, hCYP2C19 and hCYP3A4, and the downregulation of hCYP2C9. The GR antagonist, RU486, eliminated this effect, suggesting the importance of GR in liver metabolism upon cigarette smoking.

Effects of chronic exposure to tributyltin on tissue-specific cytochrome P450 1 regulation in juvenile common carp.

PubMed

Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

2016-01-01

1. The purpose of this study was to compare tributyltin (TBT)-induced cytochrome P450 1 (CYP450 1) regulation in liver, gills and muscle of juvenile common carp (Cyprinus carpio). 2. Fish were exposed to sublethal concentrations of TBT (75, 0.75 and 7.5 μg/L) for 60 days. CYP450 1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, as well as the mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) in fish tissues. 3. Based on the results, the liver displayed the highest absolute levels of EROD activity, both under nonexposed and exposed conditions. Additional, EROD activities and CYP1A gene levels showed a good correlation in all three organs. According to the mRNA expression of CYP450 1 family genes, it suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. 4. Overall, the study revealed both similarities and differences in the concentration-dependent CYP450 1 responses of the three target organs, which could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity.

Evaluation of Memory Enhancing Clinically Available Standardized Extract of Bacopa monniera on P-Glycoprotein and Cytochrome P450 3A in Sprague-Dawley Rats

PubMed Central

Singh, Rajbir; Panduri, Jagadeesh; Kumar, Devendra; Kumar, Deepak; Chandsana, Hardik; Ramakrishna, Rachumallu; Bhatta, Rabi Sankar

2013-01-01

Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp. PMID:24015255

Evaluation of memory enhancing clinically available standardized extract of Bacopa monniera on P-glycoprotein and cytochrome P450 3A in Sprague-Dawley rats.

PubMed

Singh, Rajbir; Panduri, Jagadeesh; Kumar, Devendra; Kumar, Deepak; Chandsana, Hardik; Ramakrishna, Rachumallu; Bhatta, Rabi Sankar

2013-01-01

Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp.

The Cytochrome P450 Enzyme Responsible for the Production of (Z)-Norendoxifen in vitro.

PubMed

Ma, Jianli; Chu, Zhong; Lu, Jessica Bo Li; Liu, Jinzhong; Zhang, Qingyuan; Liu, Zhaoliang; Tang, Dabei

2018-01-01

Norendoxifen, an active metabolite of tamoxifen, is a potent aromatase inhibitor. Little information is available regarding production of norendoxifen in vitro. Here, we conducted a series of kinetic and inhibition studies in human liver microsomes (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLMs incubates of endoxifen were measured using a HPLC/MS/MS method. To further probe the specific isoforms involved in the metabolic route, endoxifen was incubated with recombinant P450s (CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and CYP4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLMs. The peak of norendoxifen was found in the incubations consisting of endoxifen, HLMs, and cofactors. The retention times of norendoxifen, endoxifen, and the internal standard (diphenhydramine) were 7.81, 7.97, and 5.86 min, respectively. The K m (app) and V max (app) values of norendoxifen formation from endoxifen in HLM was 47.8 μm and 35.39 pmol min -1 mg -1 . The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 μl mg -1 min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol -1 P450 × min. CYP1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6-fold lower. One micromolar ketoconazole (CYP3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 μm quinidine (CYP2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

Transcriptional analysis of four family 4 P450s in a Puerto Rico strain of Aedes aegypti (Diptera: Culicidae) compared with an Orlando strain and their possible functional roles in permethrin resistance.

PubMed

Reid, William R; Thornton, Anne; Pridgeon, Julia W; Becnel, James J; Tang, Fang; Estep, Alden; Clark, Gary G; Allan, Sandra; Liu, Nannan

2014-05-01

A field strain of Aedes aegypti (L.) was collected from Puerto Rico in October 2008. Based on LD50 values by topical application, the Puerto Rico strain was 73-fold resistant to permethrin compared with a susceptible Orlando strain. In the presence of piperonyl butoxide, the resistance of Puerto Rico strain of Ae. aegypti was reduced to 15-fold, suggesting that cytochrome P450-mediated detoxification is involved in the resistance of the Puerto Rico strain to permethrin. To determine the cytochrome P450s that might play a role in the resistance to permethrin, the transcriptional levels of 164 cytochrome P450 genes in the Puerto Rico strain were compared with that in the Orlando strain. Of the 164 cytochrome P450s, 33 were significantly (P < 0.05) up-regulated, including cytochrome P450s in families four, six, and nine. Multiple studies have investigated the functionality of family six and nine cytochrome P450s, therefore, we focused on the up-regulated family 4 cytochrome P450s. To determine whether up-regulation of the four cytochrome P450s had any functional role in permethrin resistance, transgenic Drosophila melanogaster Meigen lines overexpressing the four family 4 P450 genes were generated, and their ability to survive exposure to permethrin was evaluated. When exposed to 5 microg per vial permethrin, transgenic D. melanogaster expressing CYP4D24, CYP4H29, CYP4J15v1, and CYP4H33 had a survival rate of 60.0 +/- 6.7, 29.0 +/- 4.4, 64.4 +/- 9.7, and 11.0 +/- 4.4%, respectively. However, none of the control flies survived the permethrin exposure at the same concentration. Similarly, none of the transgenic D. melanogaster expressing CYP4J15v1 or CYP4H33 ?5 survived when they were exposed to permethrin at 10 microg per vial. However, transgenic D. melanogaster expressing CYP4D24 and CYP4H29 had a survival rate of 37.8 +/- 4.4 and 2.2 +/- 2.2%, respectively. Taken together, our results suggest that CYP4D24 might play an important role in cytochrome P450-mediated resistance to permethrin.

Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation

PubMed Central

2016-01-01

Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of standardization of botanical extracts to multiple chemical markers for both safety and desired bioactivity. PMID:27269377

Molecular modeling of cytochrome P450 3A4

NASA Astrophysics Data System (ADS)

Szklarz, Grazyna D.; Halpert, James R.

1997-05-01

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6β-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.

Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

PubMed

Lee, Hwayoun; Park, Hyoung-Goo; Lim, Young-Ran; Lee, Im-Soon; Kim, Beom Joon; Seong, Cheul-Hun; Chun, Young-Jin; Kim, Donghak

2012-01-01

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

Expression and localization of aromatase P450AROM, estrogen receptor-α, and estrogen receptor-β in the developing fetal bovine frontal cortex.

PubMed

Peruffo, A; Giacomello, M; Montelli, S; Corain, L; Cozzi, B

2011-06-01

The enzyme aromatase (P450(AROM)) converts testosterone (T) into 17-β estradiol (E(2)) and is crucial for the control of development of the central nervous system during ontogenesis. The effects of E(2) in various brain areas are mediated by the estrogen receptor alpha (ER-α) and the estrogen receptor beta (ER-β). During fetal development, steroids are responsible for the sexual differentiation of the hypothalamus. Estrogens are also able to exert effects in other brain areas of the fetus including the frontal cortex, where they act through estrogen receptors (ERs) modulating cognitive function and affective behaviors. In this study we have determined the expression profiles of P450(AROM) and ERs in the fetal bovine frontal cortex by quantitative Real-Time PCR (qRT-PCR) throughout the prenatal development. The data show that the patterns of expression of both ERs are strongly correlated during pregnancy and increase in the last stage of gestation. On the contrary, the expression of P450(AROM) has no correlation with ERs expression and is not developmentally regulated. Moreover, we performed immunochemical studies showing that fetal neurons express P450(AROM) and the ERs. P450(AROM) is localized in the cytoplasm and only seldom present in the fine extensions of the cells; ER-α is detected predominantly in the soma whereas ER-β is only present in the nucleus of a few cells. This study provides new data on the development of the frontal cortex in a long gestation mammal with a large convoluted brain. Copyright © 2011 Elsevier Inc. All rights reserved.

Regulation of cytochrome P-450 4A activity by peroxisome proliferator-activated receptors in the rat kidney.

PubMed

Ishizuka, Tsuneo; Ito, Osamu; Tan, Liping; Ogawa, Susumu; Kohzuki, Masahiro; Omata, Ken; Takeuchi, Kazuhisa; Ito, Sadayoshi

2003-11-01

The localization of cytochrome P-450 4A, peroxisome proliferator-activated receptor (PPAR) alpha, and PPARgamma proteins, and the inducibility of P-450 4A expression and activity by PPAR agonists were determined in the rat kidney. The expressions of these proteins in isolated nephron segments were evaluated by immunoblot analysis, and the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was measured as P-450 4A activity. P-450 4A proteins were expressed predominantly in the proximal tubule (PT), with lower expression in the preglomerular arteriole (Art), glomerulus (Glm), and medullary thick ascending limb (mTAL), but their expression was not detected in the inner medullary collecting duct (IMCD). PPARalpha protein was expressed in the PT and mTAL, and PPARgamma protein was expressed in the IMCD and mTAL. Treatment with clofibrate, the PPARalpha agonist, increased P-450 4A protein levels and the production of 20-HETE in microsomes prepared from the renal cortex, whereas treatment with pioglitazone, the PPARgamma agonist, affected neither of them. These results indicate that PPARalpha and PPARgamma proteins are localized in different nephron segments and the inducibility of P-450 4A expression and activity by the PPAR agonists correlates with the nephron-specific localization of the respective PPAR isoforms.

GmCYP82A3, a Soybean Cytochrome P450 Family Gene Involved in the Jasmonic Acid and Ethylene Signaling Pathway, Enhances Plant Resistance to Biotic and Abiotic Stresses

PubMed Central

Yan, Qiang; Cui, Xiaoxia; Lin, Shuai; Gan, Shuping; Xing, Han; Dou, Daolong

2016-01-01

The cytochrome P450 monooxygenases (P450s) represent a large and important enzyme superfamily in plants. They catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways, P450s are involved in a variety of metabolic pathways and participate in the homeostasis of phytohormones. The CYP82 family genes specifically reside in dicots and are usually induced by distinct environmental stresses. However, their functions are largely unknown, especially in soybean (Glycine max L.). Here, we report the function of GmCYP82A3, a gene from soybean CYP82 family. Its expression was induced by Phytophthora sojae infection, salinity and drought stresses, and treatment with methyl jasmonate (MeJA) or ethephon (ETH). Its expression levels were consistently high in resistant cultivars. Transgenic Nicotiana benthamiana plants overexpressing GmCYP82A3 exhibited strong resistance to Botrytis cinerea and Phytophthora parasitica, and enhanced tolerance to salinity and drought stresses. Furthermore, transgenic plants were less sensitive to jasmonic acid (JA), and the enhanced resistance was accompanied with increased expression of the JA/ET signaling pathway-related genes. PMID:27588421

Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA 1535/pSK1002.

PubMed

Shimada, T; Iwasaki, M; Martin, M V; Guengerich, F P

1989-06-15

A total of 57 procarcinogens was examined for induction of umu gene response in the chimeric plasmid pSK1002, carried in Salmonella typhimurium TA 1535, after incubation with a series of human liver microsomal preparations which had been selected on the basis of characteristic levels of individual cytochrome P-450 (P-450) enzymes. The 18 most active compounds were selected and further analyzed using the umu gene response and correlative studies with a larger number of microsomal preparations, enzyme reconstitution studies involving purified enzymes, immunochemical inhibition, and patterns of stimulation and inhibition of catalytic activity by 7,8-benzoflavone. The results collectively indicate that 16 of these 18 most potent genotoxins examined are activated primarily either by P-450NF (the nifedipine oxidase) or P-450PA (the phenacetin O-deethylase). P-450NF appears to be the major enzyme involved in the bioactivation of aflatoxin B1, aflatoxin G1, sterigmatocystin, trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, 6-aminochrysene, and tris-(2,3-dibromopropyl)phosphate in human liver. P-450PA appears to be the major enzyme involved in the bioactivation of 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl, 3-amino-1-methyl-5H-pyrido[4,3-b] indole, and 2-aminodipyrido[1,2-a:3',2'-d]imidazole. More than one enzyme appears to contribute significantly to the bioactivation of the other two compounds examined, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole and 6-nitrochrysene. The literature suggests that the two human liver P-450s involved in activation of these 16 procarcinogens are highly inducible by barbiturates, macrolide antibodies, and certain steroids (P-450NF) and by smoking and ingestion of charcoal-containing food (P-450PA); noninvasive assays are available to monitor the function of both P-450NF and P-450PA.

An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae.

PubMed

Fasullo, Michael; Freedland, Julian; St John, Nicholas; Cera, Cinzia; Egner, Patricia; Hartog, Matthew; Ding, Xinxin

2017-05-01

P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB 1 ). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB 1 , the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB 1 , and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217-227, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides.

PubMed

Giraudo, M; Hilliou, F; Fricaux, T; Audant, P; Feyereisen, R; Le Goff, G

2015-02-01

Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment. © 2014 The Royal Entomological Society.

Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

PubMed Central

Højland, Dorte H.; Jensen, Karl-Martin Vagn; Kristensen, Michael

2014-01-01

Background The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain. Results We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F1, F13 and F29 to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F29 than in F1. For males, about half of the genes accessed in the global gene expression were up-regulated in F13 and F29 in comparison with the F1 population. In females, 60% of the genes were up-regulated in F13 in comparison with F1, while 33% were up-regulated in F29. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F13 flies in comparison with F1. Gene expression then increased from F13 to F29 in males and decreased further in females. Conclusion The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains. PMID:24489682

A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells.

PubMed

Chen, Liming; Bao, Yifan; Piekos, Stephanie C; Zhu, Kexin; Zhang, Lirong; Zhong, Xiao-Bo

2018-07-01

Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1 α (HNF1 α ), hepatocyte nuclear factor 4 α (HNF4 α ), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1 α antisense RNA 1 (HNF1 α -AS1) and HNF4 α antisense RNA 1 (HNF4 α -AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1 α and HNF4 α affected expression of a wide range of P450s as well as other transcription factors. HNF1 α and HNF4 α controlled the expression of their neighborhood lncRNAs, HNF1 α -AS1 and HNF4 α -AS1, respectively. HNF1 α -AS1 and HNF4 α -AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Cytochrome P450s and molecular epidemiology

NASA Astrophysics Data System (ADS)

Gonzalez, Frank J.; Gelboin, Harry V.

1993-03-01

Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals

PubMed Central

2015-01-01

Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in the metabolism of all chemicals (general chemicals, natural, and physiological compounds, and drugs) are rather consistent in the findings that >90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs. PMID:25485457

Effects of fluoride and aluminum on expressions of StAR and P450scc of related steroidogenesis in guinea pigs' testis.

PubMed

Dong, Chunguang; Cao, Jinling; Cao, Chunfang; Han, Yichao; Wu, Shouyan; Wang, Shaolin; Wang, Jundong

2016-03-01

A lot of studies have shown that fluoride and aluminum have toxic effect on male reproductive system, but the mechanism of which and the interaction between fluoride and aluminum is still unknown. This study investigated the effects of fluoride (NaF) or/and aluminum (AlCl3) on serum testosterone level, gene and protein expression levels of Steroidogenic Acute Regulatory Protein (StAR) and Cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in the testes of guinea pigs. Fifty-two guinea pigs were divided randomly into four groups (Control, HiF, HiAl and HiF + HiAl). Fluoride (150 mg NaF/L) or/and aluminum (300 mg AlCl3/L) were orally administrated to male guinea pigs for 13 weeks. The results showed that F and Al reduced number and elevated abnormal ratio of sperm. Meanwhile, the concentrations of serum testosterone in all experimental groups were decreased. P450scc protein expression was significantly reduced in all treatment groups, and StAR expression was decreased remarkably in HiF group and HiF + HiAl group. The levels of StAR mRNA in three groups were reduced by 53.9%, 21.4% and 33.4%, respectively, while the expressions of P450scc mRNA were reduced by 67.8%, 17.0% and 47.8%. Therefore, we concluded that F induced the reduction in testosterone and sperm amount, and thus in lower fertility, which might occur as a consequence of depressed StAR and P450scc mRNA expression. There were no synergistic effects between F and Al, instead, Al weakened the toxicity of F to some extents. The results indicated that Al had antagonism effects on F. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pregnane X receptor-dependent induction of the CYP3A4 gene by o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane.

PubMed

Medina-Díaz, Irma M; Arteaga-Illán, Georgina; de León, Mario Bermudez; Cisneros, Bulmaro; Sierra-Santoyo, Adolfo; Vega, Libia; Gonzalez, Frank J; Elizondo, Guillermo

2007-01-01

CYP3A4, the predominant cytochrome P450 (P450) expressed in human liver and intestine, contributes to the metabolism of approximately half the drugs in clinical use today. CYP3A4 catalyzes the 6beta-hydroxylation of a number of steroid hormones and is involved in the bioactivation of environmental procarcinogens. The expression of CYP3A4 is affected by several stimuli, including environmental factors such as insecticides and pesticides. The o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT) isomer of DDT comprises approximately 20% of technical grade DDT, which is an organochloride pesticide. We have recently shown that o,p'-DDT exposure increases CYP3A4 mRNA levels in HepG2 cells. To determine the mechanism by which o,p'-DDT induces CYP3A4 expression, transactivation and electrophoretic mobility shift assays were carried out, revealing that o,p'-DDT activates the CYP3A4 gene promoter through the pregnane X receptor (PXR). CYP3A4 gene promoter activation resulted in both an increase in CYP3A4 mRNA levels and an increase in the total CYP3A4 activity in HepG2 cells. We also observed induction of CYP3A4 and mouse Cyp3a11 mRNA in the intestine of CYP3A4-transgenic mice after exposure to 1 mg/kg o,p'-DDT. At higher doses, a decrease of CYP3A4 inducibility was observed together with an increase in levels of interleukin 6 mRNA, a proinflammatory cytokine that strongly represses CYP3A4 transcription. The present study indicates that regulation of other genes under PXR control may be altered by o,p'-DDT exposure.

Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium.

PubMed

Abdulmughni, Ammar; Jóźwik, Ilona K; Brill, Elisa; Hannemann, Frank; Thunnissen, Andy-Mark W H; Bernhardt, Rita

2017-11-01

Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D 3 (VD 3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD 3 deficiency and other disorders. However, the chemical synthesis of VD 3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD 3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD 3 . Product yield amounted to 54.9 mg·L -1 ·day -1 , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for VD 3 binding as the related VD 3 hydroxylases CYP109E1 from B. megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of VD 3 hydroxylation. The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2). Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1, UniProt ID: D5DF88, Ferredoxin, UniProt ID: D5DFQ0, cytochrome P450 reductase, EC 1.8.1.2, UniProt ID: D5DGX1. © 2017 Federation of European Biochemical Societies.

YouScript IMPACT Registry

ClinicalTrials.gov

2017-02-27

Adverse Drug Events; Adverse Drug Reactions; Drug Interaction Potentiation; Drug Metabolism, Poor, CYP2D6-RELATED; Drug Metabolism, Poor, CYP2C19-RELATED; Cytochrome P450 Enzyme Deficiency; Cytochrome P450 CYP2D6 Enzyme Deficiency; Cytochrome P450 CYP2C9 Enzyme Deficiency; Cytochrome P450 CYP2C19 Enzyme Deficiency; Cytochrome P450 CYP3A Enzyme Deficiency; Poor Metabolizer Due to Cytochrome P450 CYP2C9 Variant; Poor Metabolizer Due to Cytochrome P450 CYP2C19 Variant; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant

Pharmacogenetic Testing Among Home Health Patients

ClinicalTrials.gov

2016-09-20

Adverse Drug Events; Adverse Drug Reactions; Drug Interaction Potentiation; Drug Metabolism, Poor, CYP2D6-RELATED; Drug Metabolism, Poor, CYP2C19-RELATED; Cytochrome P450 Enzyme Deficiency; Cytochrome P450 CYP2D6 Enzyme Deficiency; Cytochrome P450 CYP2C9 Enzyme Deficiency; Cytochrome P450 CYP2C19 Enzyme Deficiency; Cytochrome P450 CYP3A Enzyme Deficiency; Poor Metabolizer Due to Cytochrome P450 CYP2C9 Variant; Poor Metabolizer Due to Cytochrome p450 CYP2C19 Variant; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant

Cytochrome p450 architecture and cysteine nucleophile placement impact raloxifene-mediated mechanism-based inactivation.

PubMed

VandenBrink, Brooke M; Davis, John A; Pearson, Josh T; Foti, Robert S; Wienkers, Larry C; Rock, Dan A

2012-11-01

The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 μM and 0.10 min(-1) and 0.81 μM and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.

Binding of Diverse Environmental Chemicals with Human Cytochromes P450 2A13, 2A6, and 1B1 and Enzyme Inhibition

PubMed Central

Shimada, Tsutomu; Kim, Donghak; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Nagy, Leslie D.; Folkman, Lindsay M.; Foroozesh, Maryam K.; Komori, Masayuki; Yamazaki, Hiroshi; Guengerich, F. Peter

2014-01-01

A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e. the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2’,5’-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2’-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2’-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450 enzymes, is able to catalyze many detoxication and activation reactions with chemicals of environmental interest. PMID:23432429

Splenda alters gut microflora and increases intestinal p-glycoprotein and cytochrome p-450 in male rats.

PubMed

Abou-Donia, Mohamed B; El-Masry, Eman M; Abdel-Rahman, Ali A; McLendon, Roger E; Schiffman, Susan S

2008-01-01

Splenda is comprised of the high-potency artificial sweetener sucralose (1.1%) and the fillers maltodextrin and glucose. Splenda was administered by oral gavage at 100, 300, 500, or 1000 mg/kg to male Sprague-Dawley rats for 12-wk, during which fecal samples were collected weekly for bacterial analysis and measurement of fecal pH. After 12-wk, half of the animals from each treatment group were sacrificed to determine the intestinal expression of the membrane efflux transporter P-glycoprotein (P-gp) and the cytochrome P-450 (CYP) metabolism system by Western blot. The remaining animals were allowed to recover for an additional 12-wk, and further assessments of fecal microflora, fecal pH, and expression of P-gp and CYP were determined. At the end of the 12-wk treatment period, the numbers of total anaerobes, bifidobacteria, lactobacilli, Bacteroides, clostridia, and total aerobic bacteria were significantly decreased; however, there was no significant treatment effect on enterobacteria. Splenda also increased fecal pH and enhanced the expression of P-gp by 2.43-fold, CYP3A4 by 2.51-fold, and CYP2D1 by 3.49-fold. Following the 12-wk recovery period, only the total anaerobes and bifidobacteria remained significantly depressed, whereas pH values, P-gp, and CYP3A4 and CYP2D1 remained elevated. These changes occurred at Splenda dosages that contained sucralose at 1.1-11 mg/kg (the US FDA Acceptable Daily Intake for sucralose is 5 mg/kg). Evidence indicates that a 12-wk administration of Splenda exerted numerous adverse effects, including (1) reduction in beneficial fecal microflora, (2) increased fecal pH, and (3) enhanced expression levels of P-gp, CYP3A4, and CYP2D1, which are known to limit the bioavailability of orally administered drugs.

Investigations on the human hepatic cytochrome P450 isozymes involved in the metabolism of 3,4-methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) enantiomers.

PubMed

Meyer, Markus R; Peters, Frank T; Maurer, Hans H

2009-10-08

3,4-Methylenedioxy-amphetamine (MDA) and benzodioxolyl-butanamine (BDB) are chiral designer drugs distributed on the illicit drug market and they are also N-dealkyl metabolites of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam), 3,4-methylenedioxyethylamphetamine (MDEA, Eve), and N-methyl-benzodioxolyl-butanamine (MBDB, Eden), respectively. MDA and BDB are mainly metabolized via demethylenation to the corresponding catecholamines. The aim of the present work was to elucidate the contribution of the relevant human P450s in the demethylenation of the MDA and BDB enantiomers. They were incubated using heterologously expressed human P450s and the corresponding metabolites dihydroxyamphetamine and 1,2-dihydroxy-4-[2-amino-butyl]benzene were determined. Highest contributions to the demethylenation as calculated from the enzyme kinetic data were obtained for CYP2D6 (MDA and BDB) and additionally CYP3A4 in the case of BDB at substrate concentrations corresponding to plasma concentrations of recreational users. A preferred transformation of the S-enantiomer could be observed for the CYP2D6- and CYP3A4-catalyzed reactions.

Differential effects of Glycyrrhiza species on genotoxic estrogen metabolism: licochalcone A downregulates P450 1B1 whereas isoliquiritigenin stimulates

PubMed Central

Dunlap, Tareisha L.; Wang, Shuai; Simmler, Charlotte; Chen, Shao-Nong; Pauli, Guido F.; Dietz, Birgit M.; Bolton, Judy L.

2015-01-01

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by up-regulating P450 1B1. The present study tested the three authenticated medicinal species of licorice, [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)], used by women as dietary supplements, for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI > GG > GU and LigC ≅ LicA > LigF. The Michael acceptor chalcone LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as non-toxic and 4-MeOE1 as genotoxic biomarkers in the non-tumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1 μM, 10 μM) decreased cytokine- and TCDD-induced, P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50=12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women’s health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds. PMID:26134484

Protective effect of tea polyphenols against paracetamol-induced hepatotoxicity in mice is significantly correlated with cytochrome P450 suppression.

PubMed

Chen, Xia; Sun, Chang-Kai; Han, Guo-Zhu; Peng, Jin-Yong; Li, Ying; Liu, Yan-Xia; Lv, Yuan-Yuan; Liu, Ke-Xin; Zhou, Qin; Sun, Hui-Jun

2009-04-21

To investigate the hepatoprotective activity of tea polyphenols (TP) and its relation with cytochrome P450 (CYP450) expression in mice. Hepatic CYP450 and CYPb(5) levels were measured by UV-spectrophotometry in mice 2 d after intraperitoneal TP (25, 50 and 100 mg/kg per day). Then the mice were intragastricly pre-treated with TP (100, 200 and 400 mg/kg per day) for six days before paracetamol (1000 mg/kg) was given. Their acute mortality was compared with that of control mice. The mice were pre-treated with TP (100, 200, and 400 mg/kg per day) for five days before paracetamol (500 mg/kg) was given. Hepatic CYP2E1 and CYP1A2 protein and mRNA expression levels were evaluated by Western blotting, immunohistochemical staining and transcriptase-polymerase chain reaction. The hepatic CYP450 and CYPb(5) levels in mice of TP-treated groups (100, 200 and 400 mg/kg per day) were decreased in a dose-dependent manner compared with those in the negative control mice. TP significantly attenuated the paracetamol-induced hepatic injury and dramatically reduced the mortality of paracetamol-treated mice. Furthermore, TP reduced CYP2E1 and CYP1A2 expression at both protein and mRNA levels in a dose-dependent manner. TP possess potential hepatoprotective properties and can suppress CYP450 expression.

Expression and regulation of aromatase cytochrome P450 in THP 1 human myeloid leukaemia cells.

PubMed

Jakob, F; Homann, D; Seufert, J; Schneider, D; Köhrle, J

1995-04-28

Aromatase cytochrome P450 mRNA and activity was strongly expressed in THP 1 myeloid leukaemia cells after treatment with phorbol-myristate-acetate (PMA) and dexamethasone, low level expression was caused by calcitriol. mRNA species of 4.0, 3.0, 2.4 and 1.1 kb size were differentially stimulated. After calcitriol-mediated differentiation (72 h, measured by CD 14 expression) mRNA expression was further enhanced by PMA (45-fold), dexamethasone (15-fold), oestradiol (3.7-fold), testosterone (2.5-fold) and androstenedione (3.5-fold). Forskolin, cAMP and follicle stimulating hormone had no stimulatory effect. Oestradiol formation from testosterone (oestradiol radioimmunoassay in culture supernatants) increased to > 2000 pg/ml/10(6) cells/24 h after PMA-stimulation, mirrored mRNA expression and was suppressed below 10% of original values in the presence of 4-OH-androstenedione. Exons I.2 and I.4 were expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. A new splicing variant was expressed after calcitriol-stimulation, which did not hybridize to an exon II-derived oligonucleotide but to an exon III-derived one. Local aromatisation of androgens into oestradiol may be important in the concerted crosstalk of cells of the monocyte/macrophage lineage with their respective tissues in inflammation and bone metabolism.

Point mutation of Arg440 to his in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fardella, C.E.; Hum, D.W.; Miller, W.L.

Genetic disorders in the gene encoding P450c17 cause 17{alpha}-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralorcorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1more » cells showed that the mutant P450c17 protein was produced, but it lacked both 17{alpha}-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17{alpha}-hydroxylase deficiency, indicating that mutations in this gene are due to random events. 36 refs., 3 figs., 2 tabs.« less

Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

PubMed Central

Rasmussen, Martin Krøyer; Zamaratskaia, Galia

2014-01-01

Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint. PMID:25408844

Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

PubMed Central

Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

2016-01-01

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

PubMed Central

Amunom, Immaculate; Dieter, Laura J.; Tamasi, Viola; Cai, Jan; Conklin, Daniel J.; Srivastava, Sanjay; Martin, Martha V.; Guengerich, F. Peter; Prough, Russell A.

2011-01-01

The metabolism of α,β-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of α,β-unsaturated aldehydes in liver. PMID:21766881

Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts.

PubMed

Ding, Xinxin; Kaminsky, Laurence S

2003-01-01

Cytochrome P450 (CYP) enzymes in extrahepatic tissues often play a dominant role in target tissue metabolic activation of xenobiotic compounds. They may also determine drug efficacy and influence the tissue burden of foreign chemicals or bioavailability of therapeutic agents. This review focuses on xenobiotic-metabolizing CYPs of the human respiratory and gastrointestinal tracts, including the lung, trachea, nasal respiratory and olfactory mucosa, esophagus, stomach, small intestine, and colon. Many CYPs are expressed in one or more of these organs, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2S1, CYP3A4, CYP3A5, and CYP4B1. Of particular interest are the preferential expression of certain CYPs in the respiratory tract and the regional differences in CYP expression profile in different parts of the gastrointestinal tract. Current research activities on the characterization of CYP expression, function, and regulation in these tissues, as well as future research needs, are discussed.

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

PubMed Central

Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

2013-01-01

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

PubMed

Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

2013-05-01

The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined. Copyright © 2012 John Wiley & Sons, Ltd.

Summary of Information on the Effects of Ionizing and Non-ionizing Radiation on Cytochrome P450 and Other Drug Metabolizing Enzymes and Transporters

PubMed Central

Rendic, Slobodan; Guengerich, F. Peter

2014-01-01

The present paper is an update of data on the effects of ionizing radiation (γ-rays, X-rays, high energy UV, fast neutron) caused by environmental pollution or clinical treatments and the effects of non-ionizing radiation (low energy UV) on the expression and/or activity of drug metabolism (e.g., cytochrome P450,, glutathione transferase), enzymes involved in oxidative stress (e.g., peroxidases, catalase,, aconitase, superoxide dismutase), and transporters. The data are presented in tabular form (Tables 1–3) and are a continuation of previously published summaries on the effects of drugs and other chemicals on cytochrome P450 enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29 (1–2), 413–580, Rendic, S. Drug Metab. Rev., 2002, 34 (1–2), 83–448) and of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 enzymes and transporters (Guengerich, F.P.; Rendic, S. Curr. Drug Metab., 2010, 11(1), 1–3, Rendic, S.; Guengerich, F.P. Curr. Drug Metab., 2010, 11 (1), 4–84). The collective information is as presented by the cited author(s) in cases where several references are cited the latest published information is included. Remarks and conclusions suggesting clinically important impacts are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file (for information about file availability contact the corresponding author). PMID:22571481

Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

PubMed

Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

1998-12-08

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle

Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. Themore » reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.« less

Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

PubMed

Tanino, Tadatoshi; Komada, Akira; Ueda, Koji; Bando, Toru; Nojiri, Yukie; Ueda, Yukari; Sakurai, Eiichi

2016-12-01

Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Expression of a ripening-related cytochrome P450 cDNA in Cavendish banana (Musa acuminata cv. Williams).

PubMed

Pua, Eng-Chong; Lee, Yi-Chuan

2003-02-13

As part of a study to understand the molecular basis of fruit ripening, this study reports the isolation and characterization of a banana cytochrome P450 (P450) cDNA, designated as MAP450-1, which was associated with fruit ripening of banana. MAP450-1 encoded a single polypeptide of 507 amino acid residues that shared an overall identity of 27-45% with that of several plant P450s, among which MAP450-1 was most related phylogenetically to the avocado P450 CYP71A1. The polypeptide that possessed residue domains conserved in all P450s was classified as CYP71N1. Expression of CYP71N1 varied greatly between banana organs. Transcripts were detected only in peel and pulp of the ripening fruit and not in unripe fruit tissues at all developmental stages or other organs (root, leaf, ovary and flower). During ripening, transcripts were barely detectable in pre-climacteric and climacteric fruits but, as ripening progressed, they began to accumulate and reached a maximum in post-climacteric fruits. CYP71N1 expression in pre-climacteric fruit could be upregulated by exogenous application of ethylene (1-5 ppm) and treatment of overripe fruit with exogenous sucrose (50-300 mM) but not glucose downregulated the expression. These results indicate that P450s may not play a role in fruit development and its expression is associated with ripening, which may be regulated, in part, by ethylene and/or sucrose, at the transcript level.

Monocrotophos induced apoptosis in PC12 cells: role of xenobiotic metabolizing cytochrome P450s.

PubMed

Kashyap, Mahendra Pratap; Singh, Abhishek Kumar; Kumar, Vivek; Tripathi, Vinay Kumar; Srivastava, Ritesh Kumar; Agrawal, Megha; Khanna, Vinay Kumar; Yadav, Sanjay; Jain, Swatantra Kumar; Pant, Aditya Bhushan

2011-03-21

Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10(-5) M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl(2), P(53), P(21), GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10(-5) M), whereas induction of CYPs was insignificant in cells exposed to 10(-6) M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells.

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Dominant role of cytochrome P-450 2E1 in human hepatic microsomal oxidation of the CFC-substitute 1,1,1,2-tetrafluoroethane.

PubMed

Surbrook, S E; Olson, M J

1992-01-01

The chlorofluorocarbon substitute 1,1,1,2-tetrafluoroethane (HFC-134a) is subject to metabolism by cytochrome P-450 in hepatic microsomes from rat, rabbit, and human. In rat and rabbit, the P-450 form 2E1 is a predominant low-KM, high-rate catalyst of HFC-134a biotransformation and is prominently involved in the metabolism of other tetrahaloalkanes of greater toxicity than HFC-134a [e.g. 1,2-dichloro-1,1-difluoroethane (HCFC-132b)]. In this study, we determined that the human ortholog of P-450 2E1 plays a role of similar importance in the metabolism of HFC-134a. In human hepatic microsomes from 12 individuals, preparations from subjects with relatively high P-450 2E1 levels were shown to metabolize HFC-134a at rates 5- to 10-fold greater than microsomes of individuals with lower levels of this enzyme; the increased rate of metabolism of HFC-134a was specifically linked to increased expression of P-450 2E1. The primary evidence for this conclusion is drawn from studies using mechanism-based inactivation of P-450 2E1 by diethyldithiocarbamate, competitive inhibition of HFC-134a oxidation by p-nitrophenol (a high-affinity substrate for P-450 2E1), strong positive correlation of rates of HFC-134a defluorination with p-nitrophenol hydroxylation in the study population, and correlation of P-450 2E1 levels with rates of halocarbon oxidation. Thus, our findings support the conclusion that human metabolism of HFC-134a is qualitatively similar to that of the species (rat and rabbit) used for toxicological assessment of this halocarbon.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of an estrogen-related receptor γ modulator, GSK5182, by human liver microsomes and recombinant cytochrome P450s.

PubMed

Joo, Jeongmin; Wu, Zhexue; Lee, Boram; Shon, Jong Cheol; Lee, Taeho; Lee, In-Kyu; Sim, Taebo; Kim, Kyung-Hee; Kim, Nam Doo; Kim, Seong Heon; Liu, Kwang-Hyeon

2015-04-01

GSK5182 (4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-hydroxy-2-phenylpent-1-enyl]phenol) is a specific inverse agonist for estrogen-related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N-desmethyl-GSK5182 and GSK5182 N-oxide, respectively, on the basis of liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. M2 was suggested to be hydroxy-GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N-demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N-oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.

Transcriptome Analysis of an Insecticide Resistant Housefly Strain: Insights about SNPs and Regulatory Elements in Cytochrome P450 Genes.

PubMed

Mahmood, Khalid; Højland, Dorte H; Asp, Torben; Kristensen, Michael

2016-01-01

Insecticide resistance in the housefly, Musca domestica, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes. The de novo assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in cyp6d1. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools. Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s in xenobiotic detoxification.

Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.

PubMed

Stenger, Benjamin; Gerber, Adrian; Bernhardt, Rita; Hannemann, Frank

2018-01-01

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

Regio- and Stereo-Selective Oxidation of a Cardiovascular Drug, Metoprolol, Mediated by Cytochrome P450 2D and 3A Enzymes in Marmoset Livers.

PubMed

Uehara, Shotaro; Ishii, Sakura; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

2017-08-01

A β -blocker, metoprolol, is one of the in vivo probes for human cytochrome P450 (P450) 2D6. Investigation of nonhuman primate P450 enzymes helps to improve the accuracy of the extrapolation of pharmacokinetic data from animals into humans. Common marmosets ( Callithrix jacchus ) are a potential primate model for preclinical research, but the detailed roles of marmoset P450 enzymes in metoprolol oxidation remain unknown. In this study, regio- and stereo-selectivity of metoprolol oxidations by a variety of P450 enzymes in marmoset and human livers were investigated in vitro. Although liver microsomes from cynomolgus monkeys and rats preferentially mediated S -metoprolol O -demethylation and R -metoprolol α -hydroxylation, respectively, those from humans, marmosets, minipigs, and dogs preferentially mediated R -metoprolol O -demethylation, in contrast to the slow rates of R - and S -metoprolol oxidation in mouse liver microsomes. R - and S -metoprolol O -demethylation activities in marmoset livers were strongly inhibited by quinidine and ketoconazole, and were significantly correlated with bufuralol 1'-hydroxylation and midazolam 1'-hydroxylation activities and also with P450 2D and 3A4 contents, which is different from the case in human livers that did not have any correlations with P450 3A-mediated midazolam 1'-hydroxylation. Recombinant human P450 2D6 enzyme and marmoset P450 2D6/3A4 enzymes effectively catalyzed R -metoprolol O -demethylation, comparable to the activities of human and marmoset liver microsomes, respectively. These results indicated that the major roles of P450 2D enzymes for the regio- and stereo-selectivity of metoprolol oxidation were similar between human and marmoset livers, but the minor roles of P450 3A enzymes were unique to marmosets. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Enhanced expression of rat hepatic CYP2B1/2B2 and 2E1 by pyridine: differential induction kinetics and molecular basis of expression.

PubMed

Kim, H; Putt, D; Reddy, S; Hollenberg, P F; Novak, R F

1993-11-01

Expression of the cytochrome P450 (CYP) 2B subfamily in rat and rabbit hepatic tissues after pyridine (PY) treatment has been examined, and the molecular basis for enhanced 2B1/2B2 expression has been determined. P450 expression was monitored using metabolic activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses, and the identity of the proteins was confirmed through N-terminus microsequence analysis. PY caused a dose-dependent elevation of hepatic CYP2B1/B2B levels in rats, which ranged from 4- to 22-fold over the dosing regimen of 100 to 400 mg PY/kg/day, for 3 days, respectively. PY at low dose failed to induce CYP2B in rabbit hepatic tissue, suggesting a species-dependent response in 2B expression. Anti-2B1 IgG addition to PY-induced microsomes inhibited benzphetamine N-demethylase activity by only approximately 15%, in sharp contrast to the approximately 73% inhibition observed for phenobarbital-induced microsomes, suggesting the induction of other form(s) of P450 having benzphetamine N-demethylase activity. Northern blot analysis revealed that PY treatment increased 2B1 and 2B2 poly(A)+ RNA levels approximately 69- and approximately 34-fold, respectively, whereas the 2E1 poly(A)+ RNA levels failed to increase. The results of this study show that PY induces CYP2B1/2B2 and that induction is species-dependent and kinetically distinguishable from 2E1 induction. Moreover, 2B1/2B2 induction occurs as a result of elevated mRNA levels associated with either transcriptional activation or mRNA stabilization, and it differs from the mechanism of hepatic 2E1 induction by PY.

Time course for the modulation of hepatic cytochrome P450 after administration of ethylbenzene and its correlation with toluene metabolism.

PubMed

Yuan, W; Sequeira, D J; Cawley, G F; Eyer, C S; Backes, W L

1997-03-01

The goal of the present study was to examine the time course for changes in P450 expression and hydrocarbon metabolism after acute treatment with the simple aromatic hydrocarbon ethylbenzene (EB) and to correlate these alterations with the changes observed in alkylbenzene metabolism. Male Holtzman rats were treated with a single intraperitoneal injection of EB, and the effects on specific P450-dependent activities, immunoreactive P450 isozyme levels, and RNA levels were measured at various times after injection. Toluene was used as the test alkylbenzene for examination of the EB-mediated changes on in vitro hydrocarbon metabolism. In untreated rats, toluene was metabolized almost entirely by aliphatic hydroxylation (to benzyl alcohol); however, in EB-treated rats, significant quantities of benzyl alcohol, o-cresol, and p-cresol were produced. Interestingly, 5-10 h after EB treatment, there was a 40% decrease in benzyl alcohol production. By 24 h, rates of benzyl alcohol formation returned to control levels, whereas there was a 7-fold increase in o-cresol and a greater that 50-fold increase in p-cresol production. The changes in the disposition of toluene were then correlated with changes in particular P450 isozymes. Several P450 isozymes were induced after EB administration. P450 2B1/2-dependent testosterone 16 beta-hydroxylation and P450 2B1/2-immunoreactive protein were elevated 30-fold after EB administration, reaching maxima by 24 h and remaining elevated 48 h after exposure. Changes in P450 2B1 and 2B2 RNA preceded those of the proteins. Similar results were observed with P450 1A1. P450 2E1 RNA levels were elevated after a single EB injection. However, the elevation in P450 2E1-dependent activities and immunoreactive protein levels preceded the changes in RNA, suggesting that multiple steps are affected by EB exposure. In contrast to the increases in some isozymes, P450 2C11 protein was rapidly suppressed (within the first 2-10 h) after hydrocarbon exposure, suggestive of a destabilization of the protein. When comparing the changes in P450 isozymes to alterations in toluene metabolism, the immediate suppression in aliphatic hydroxylation of toluene (in the first 5-10 h) was consistent with the decrease in P450 2C11. Subsequent to this effect, P450 2B1/2 and 2E1 were induced, which elevated production of this metabolite to control levels. The increase in the aromatic hydroxylation of toluene to both o, and p-cresol was consistent with the induction of P450s 2B1/2, 2E1, and 1A1.

Relative contribution of rat cytochrome P450 isoforms to the metabolism of caffeine: the pathway and concentration dependence.

PubMed

Kot, Marta; Daniel, Władysława A

2008-04-01

The aim of the present study was to estimate the relative contribution of rat P450 isoforms to the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance for estimating the activity of P450 in rat liver and its potential for pharmacokinetic interactions in pharmacological experiments. The results obtained using rat cDNA-expressed P450s indicated that 8-hydroxylation was the main oxidation pathway of caffeine (70%) in the rat. CYP1A2 was found to be a key enzyme catalyzing 8-hydroxylation (72%) and substantially contributing to 3-N-demethylation (47%) and 1-N-demethylation (37.5%) at a caffeine concentration of 0.1mM (relevant to "the maximum therapeutic concentration in humans"). Furthermore, CYP2C11 considerably contributed to 3-N-demethylation (31%). The CYP2C subfamily (66%) - mainly CYP2C6 (27%) and CYP2C11 (29%) - played a major role in catalyzing 7-N-demethylation. At higher substrate concentrations, the contribution of CYP1A2 to the metabolism of caffeine decreased in favor of CYP2C11 (N-demethylations) and CYP3A2 (mainly 8-hydroxylation). The obtained results were confirmed with liver microsomes (inhibition and correlation studies). Therefore, caffeine may be used as a marker substance for assessing the activity of CYP1A2 in rats, using 8-hydroxylation (but not 3-N-demethylation-like in humans); moreover, caffeine may also be used to simultaneously, preliminarily estimate the activity of CYP2C using 7-N-demethylation as a marker reaction. Hence caffeine pharmacokinetics in rats may be changed by drugs affecting the activity of CYP1A2 and/or CYP2C, e.g. by some antidepressants.

Mammalian cytochrome CYP2E1 triggered differential gene regulation in response to trichloroethylene (TCE) in a transgenic poplar.

PubMed

Kang, Jun Won; Wilkerson, Hui-Wen; Farin, Federico M; Bammler, Theo K; Beyer, Richard P; Strand, Stuart E; Doty, Sharon L

2010-08-01

Trichloroethylene (TCE) is an important environmental contaminant of soil, groundwater, and air. Studies of the metabolism of TCE by poplar trees suggest that cytochrome P450 enzymes are involved. Using poplar genome microarrays, we report a number of putative genes that are differentially expressed in response to TCE. In a previous study, transgenic hybrid poplar plants expressing mammalian cytochrome P450 2E1 (CYP2E1) had increased metabolism of TCE. In the vector control plants for this construct, 24 h following TCE exposure, 517 genes were upregulated and 650 genes were downregulated over 2-fold when compared with the non-exposed vector control plants. However, in the transgenic CYP2E1 plant, line 78, 1,601 genes were upregulated and 1,705 genes were downregulated over 2-fold when compared with the non-exposed transgenic CYP2E1 plant. It appeared that the CYP2E1 transgenic hybrid poplar plants overexpressing mammalian CYP2E1 showed a larger number of differentially expressed transcripts, suggesting a metabolic pathway for TCE to metabolites had been initiated by activity of CYP2E1 on TCE. These results suggest that either the over-expression of the CYP2E1 gene or the abundance of TCE metabolites from CYP450 2E1 activity triggered a strong genetic response to TCE. Particularly, cytochrome p450s, glutathione S-transferases, glucosyltransferases, and ABC transporters in the CYP2E1 transgenic hybrid poplar plants were highly expressed compared with in vector controls.

Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.

PubMed

Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Valetti, Francesca; Gilardi, Gianfranco

2015-10-01

This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs.

PubMed

Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Di Nardo, Giovanna; Gilardi, Gianfranco

2012-12-01

In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. Copyright © 2012 Elsevier Inc. All rights reserved.

A novel P450-initiated biphasic process for sustainable biodegradation of benzo[a]pyrene in soil under nutrient-sufficient conditions by the white rot fungus Phanerochaete chrysosporium

PubMed Central

Bhattacharya, Sukanta S.; Syed, Khajamohiddin; Shann, Jodi; Yadav, Jagjit S.

2013-01-01

High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) such as benzo[a]pyrene (BaP) are resistant to biodegradation in soil. Conventionally, white rot fungus Phanerochaete chrysosporium has been investigated for HMW-PAH degradation in soil primarily using nutrient-deficient (ligninolytic) conditions, albeit with limited and non-sustainable biodegradation outcomes. In this study, we report development of an alternative novel biphasic process initiated under nutrient-sufficient (non-ligninolytic) culture conditions, by employing an advanced experimental design strategy. During the initial nutrient-sufficient non-ligninolytic phase (16 days), the process showed upregulation (3.6-and 22.3-fold, respectively) of two key PAH-oxidizing P450 monooxygenases pc2 (CYP63A2) and pah4 (CYP5136A3) and formation of typical P450-hydroxylated metabolite. This along with abrogation (84.9%) of BaP degradation activity in response to a P450-specific inhibitor implied key role of these monooxygenases. The subsequent phase triggered on continued incubation (to 25 days) switched the process from non-ligninolytic to ligninolytic resulting in a significantly higher net degradation (91.6% as against 67.4% in the control nutrient-limited set) of BaP with concomitant de novo ligninolytic enzyme expression making it a biphasic process yielding improved sustainable bioremediation of PAH-contaminated soil. To our knowledge this is the first report on development of such biphasic process for bioremediation application of a white rot fungus. PMID:24051002

Multiple Cytochrome P450 genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes, Culex quinquefasciatus.

PubMed

Liu, Nannan; Li, Ting; Reid, William R; Yang, Ting; Zhang, Lee

2011-01-01

Four cytochrome P450 cDNAs, CYP6AA7, CYP9J40, CYP9J34, and CYP9M10, were isolated from mosquitoes, Culex quinquefasciatus. The P450 gene expression and induction by permethrin were compared for three different mosquito populations bearing different resistance phenotypes, ranging from susceptible (S-Lab), through intermediate (HAmCq(G0), the field parental population) to highly resistant (HAmCq(G8), the 8(th) generation of permethrin selected offspring of HAmCq(G0)). A strong correlation was found for P450 gene expression with the levels of resistance and following permethrin selection at the larval stage of mosquitoes, with the highest expression levels identified in HAmCq(G8), suggesting the importance of CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 in the permethrin resistance of larva mosquitoes. Only CYP6AA7 showed a significant overexpression in HAmCq(G8) adult mosquitoes. Other P450 genes had similar expression levels among the mosquito populations tested, suggesting different P450 genes may be involved in the response to insecticide pressure in different developmental stages. The expression of CYP6AA7, CYP9J34, and CYP9M10 was further induced by permethrin in resistant mosquitoes. Taken together, these results indicate that multiple P450 genes are up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, thus increasing the overall expression levels of P450 genes.

Detoxification of ivermectin by ATP binding cassette transporter C4 and cytochrome P450 monooxygenase 6CJ1 in the human body louse, Pediculus humanus humanus.

PubMed

Kim, J H; Gellatly, K J; Lueke, B; Kohler, M; Nauen, R; Murenzi, E; Yoon, K S; Clark, J M

2018-02-01

We previously observed that ivermectin-induced detoxification genes, including ATP binding cassette transporter C4 (PhABCC4) and cytochrome P450 6CJ1 (CYP6CJ1) were identified from body lice following a brief exposure to a sublethal dose of ivermectin using a non-invasive induction assay. In this current study, the functional properties of PhABCC4 and CYP6CJ1 were investigated after expression in either X. laevis oocytes or using a baculovirus expression system, respectively. Efflux of [ 3 H]-9-(2-phosphonomethoxyethyl) adenine ([ 3 H]-PMEA), a known ABCC4 substrate in humans, was detected from PhABCC4 cRNA-injected oocytes by liquid scintillation spectrophotometric analysis and PhABCC4 expression in oocytes was confirmed using ABC transporter inhibitors. Efflux was also determined to be ATP-dependent. Using a variety of insecticides in a competition assay, only co-injection of ivermectin and dichlorodiphenyltrichloroethane led to decreased efflux of [ 3 H]-PMEA. PhABCC4-expressing oocytes also directly effluxed [ 3 H]-ivermectin, which increased over time. In addition, ivermectin appeared to be oxidatively metabolized and/or sequestered, although at low levels, following functional expression of CYP6CJ1 along with cytochrome P450 reductase in Sf9 cells. Our study suggests that PhABCC4 and perhaps CYP6CJ1 are involved in the Phase III and Phase I xenobiotic metabolism of ivermectin, respectively, and may play an important role in the evolution of ivermectin resistance in lice and other insects as field selection occurs. © 2017 The Royal Entomological Society.

Cytochrome P450 2D6 enzyme neuroprotects against 1-methyl-4-phenylpyridinium toxicity in SH-SY5Y neuronal cells

PubMed Central

Mann, Amandeep; Tyndale, Rachel F.

2016-01-01

Cytochrome P450 (CYP) 2D6 is an enzyme that is expressed in liver and brain. It can inactivate neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3,4-tetrahydroisoquinoline and β-carbolines. Genetically slow CYP2D6 metabolizers are at higher risk for developing Parkinson’s disease, a risk that increases with exposure to pesticides. The goal of this study was to investigate the neuroprotective role of CYP2D6 in an in-vitro neurotoxicity model. SH-SY5Y human neuroblastoma cells express CYP2D6 as determined by western blotting, immunocytochemistry and enzymatic activity. CYP2D6 metabolized 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin and the CYP2D6-specific inhibitor quinidine (1 μM) blocked 96 ± 1% of this metabolism, indicating that CYP2D6 is functional in this cell line. Treatment of cells with CYP2D6 inhibitors (quinidine, propanolol, metoprolol or timolol) at varying concentrations significantly increased the neurotoxicity caused by 1-methyl-4-phenylpyridinium (MPP+) at 10 and 25 μM by between 9 ± 1 and 22 ± 5% (P < 0.01). We found that CYP3A is also expressed in SH-SY5Y cells and inhibiting CYP3A with ketoconazole significantly increased the cell death caused by 10 and 25 μM of MPP+ by between 8 ± 1 and 30 ± 3% (P < 0.001). Inhibiting both CYP2D6 and CYP3A showed an additive effect on MPP+ neurotoxicity. These data further support a possible role for CYP2D6 in neuroprotection from Parkinson’s disease-causing neurotoxins, especially in the human brain where expression of CYP2D6 is high in some regions (e.g. substantia nigra). PMID:20345925

CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes.

PubMed

Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai; Grimmett, Paul E; Yadav, Jagjit S

2013-04-01

Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes

PubMed Central

Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai; Grimmett, Paul E.

2013-01-01

Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills). PMID:23416995

Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego.

PubMed

Fairhead, Michael; Giannini, Silva; Gillam, Elizabeth M J; Gilardi, Gianfranco

2005-12-01

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

Aromatic Hydroxylation of Salicylic Acid and Aspirin by Human Cytochromes P450

PubMed Central

Bojić, Mirza; Sedgeman, Carl A.; Nagy, Leslie D.; Guengerich, F. Peter

2015-01-01

Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1. PMID:25840124

The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

PubMed

Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

2013-10-01

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling

PubMed Central

Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

2013-01-01

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies. PMID:23844938

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats.

PubMed

Martínez, María-Aránzazu; Ares, Irma; Rodríguez, José-Luis; Martínez, Marta; Roura-Martínez, David; Castellano, Victor; Lopez-Torres, Bernardo; Martínez-Larrañaga, María-Rosa; Anadón, Arturo

2018-08-01

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury. Copyright © 2018. Published by Elsevier B.V.

Adult granulosa cell tumors of the ovary: a retrospective study of 30 cases with respect to the expression of steroid synthesis enzymes.

PubMed

Kitamura, Sachiko; Abiko, Kaoru; Matsumura, Noriomi; Nakai, Hidekatsu; Akimoto, Yumiko; Tanimoto, Hirotoshi; Konishi, Ikuo

2017-07-01

Some, but not all, granulosa cell tumors are characterized by estrogen production. This study was designed to determine whether there are clinical or pathological variations in granulosa cell tumors in relation to the expression of sex steroid synthesis enzymes. Clinical symptoms, serum hormonal values, and histology of 30 granulosa cell tumor patients who underwent surgery between 2002 and 2014 were retrospectively reviewed. Most patients presented with abnormal genital bleeding including abnormal menstrual cycles. Eight of 16 patients older than 50 years had endometrial hyperplasia and one had endometrial cancer. Serum 17β-estradiol (E₂) levels tended to be higher in patients over 50 years of age (p=0.081). Serum follicle-stimulating hormone (FSH) levels were low in all patients irrespective of serum E₂ levels. Magnetic resonance imaging revealed a thicker endometrium in older as compared to younger patients (p<0.05). Tumor cells in the majority of cases were positive for inhibin α and P450 aromatase, irrespective of age and serum E₂ levels. P450 17α-hydroxylase (P450c17) expression varied among cases. P450c17 was strongly positive in luteinized tumor cells and weakly positive in theca cells and fibroblasts. High E₂ levels were associated with P450c17-positive cells in the tumor (p<0.05). The expression of hormone-synthesizing enzymes divides granulosa cell tumors into 2 distinct types; tumors with P450c17-positive cells show elevated serum E₂ and related clinical symptoms, while tumors without these cells show symptoms related to FSH suppression by inhibin. Copyright © 2017. Asian Society of Gynecologic Oncology, Korean Society of Gynecologic Oncology

Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

PubMed

Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

2016-11-01

The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

PubMed Central

Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

2014-01-01

Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

Identification, Characterization, and Expression of a Novel P450 Gene Encoding CYP6AE25 from the Asian Corn Borer, Ostrinia furnacalis

PubMed Central

Zhang, Yu-liang; Kulye, Mahesh; Yang, Feng-shan; Xiao, Luo; Zhang, Yi-tong; Zeng, Hongmei; Wang, Jian-hua; Liu, Zhi-xin

2011-01-01

An allele of the cytochrome P450 gene, CYP6AE14, named CYP6AE25 (GenBank accession no. EU807990) was isolated from the Asian com borer, Ostrinia fumacalis (Guenée) (Lepidoptera: Pyralidae) by RT-PCR. The cDNA sequence of CYP6AE25 is 2315 bp in length and contains a 1569 nucleotides open reading frame encoding a putative protein with 523 amino acid residues and a predicted molecular weight of 59.95 kDa and a theoretical pI of 8.31. The putative protein contains the classic heme-binding sequence motif F××G×××C×G (residues 451–460) conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 52% identity with the previously published sequence of CYP6AE14 (GenBank accession no. DQ986461) from Helicoverpa armigera. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that CYP6AE25 has a closer phylogenetic relationship with CYP6AE14 and CYP6B1 that are related to metabolism of plant allelochemicals, CYP6D1 which is related to pyrethroid resistance and has a more distant relationship to CYP302A1 and CYP307A1 which are related to synthesis of the insect molting hormones. The expression level of the gene in the adults and immature stages of O. furnacalis by quantitative real-time PCR revealed that CYP6AE25 was expressed in all life stages investigated. The mRNA expression level in 3rd instar larvae was 12.8- and 2.97-fold higher than those in pupae and adults, respectively. The tissue specific expression level of CYP6AE25 was in the order of midgut, malpighian tube and fatty body from high to low but was absent in ovary and brain. The analysis of the CYP6AB25 gene using bioinformatic software is discussed. PMID:21529257

Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples.

PubMed

Al Ali, Ahmad; Touboul, David; Le Caër, Jean-Pierre; Schmitz-Afonso, Isabelle; Flinois, Jean-Pierre; Marchetti, Catherine; De Waziers, Isabelle; Brunelle, Alain; Laprévote, Olivier; Beaune, Philippe

2014-08-01

Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.

The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

PubMed Central

Cohen-Wolkowiez, Michael; Sampson, Mario R.; Kearns, Gregory L.

2013-01-01

Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a “therapeutic concentration” of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo. PMID:23813797

Isoform-specific regulation of cytochrome P450 expression and activity by estradiol in female rats

PubMed Central

Choi, Su-Young; Fischer, Liam; Yang, Kyunghee; Chung, Hyejin; Jeong, Hyunyoung

2011-01-01

Estradiol (E2) is the major endogenous estrogen, and its plasma concentration increases up to 100-fold during pregnancy in humans. Accumulating evidence suggests that an elevated level of E2 may influence hepatic drug metabolism, potentially being responsible for altered drug metabolism during pregnancy. We characterized effects of E2 on expression and activities of cytochrome P450 enzymes (CYPs) in an in vivo system using rats. To this end, female rats were treated with estradiol benzoate (EB) or known CYP inducers. Liver tissues were obtained after 5 days of treatment, and mRNA and protein expression levels as well as activities of major hepatic CYPs were determined by qRT-PCR, immunoblot, and microsomal assay. E2 increased CYP1A2 expression and activity to a smaller extent than β-naphthoflavone did. E2 also enhanced CYP2C expression (CYP2C6, CYP2C7, and CYP2C12) to levels comparable to those observed by phenobarbital. E2 upregulated CYP3A9 expression, while expression of CYP3A1 was downregulated. Expression of hepatic nuclear receptors (PXR and CAR) and the obligate redox partner of CYPs (POR) was downregulated in EB-treated rats, suggesting their potential involvement in regulation of CYP expression and activity by E2. In summary, in female rats E2 regulates expression of hepatic CYPs in a CYP isoform-specific manner although the directional changes are different from those clinically observed during human pregnancy. Further study is warranted to determine whether the changes in drug metabolism during human pregnancy are attributable to involvement of hormones other than E2. PMID:21219883

Identification of a cytochrome P450 gene in the earthworm Eisenia fetida and its mRNA expression under enrofloxacin stress.

PubMed

Li, Yinsheng; Zhao, Chun; Lu, Xiaoxu; Ai, Xiaojie; Qiu, Jiangping

2018-04-15

Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. Earthworms have been used as a bioindicator of soil pollution in numerous studies, but no CYP450 gene has so far been cloned. RT-PCR and RACE-PCR were employed to construct and sequence the CYP450 gene DNA from the extracted mRNA in the earthworm Eisenia fetida. The cloned gene (EW1) has an open reading frame of 477bp. The 3'-terminal region contained both the consensus and the signature sequences characteristic of CYP450. It was closely related to the CYP450 gene from the flatworm genus Opisthorchis felineus with 87% homology. The predicted structure of the putative protein was 97% homologous to human CYP450 family 27. This gene has been deposited in GenBank (accession no. KM881474). Earthworms (E. fetida) were then exposed to 1, 10, 100, and 500mgkg -1 enrofloxacin in soils to explore the mRNA expression by real time qPCR. The effect of enrofloxacin on mRNA expression levels of EW1 exhibited a marked hormesis pattern across the enrofloxacin dose range tested. This is believed to be the first reported CYP450 gene in earthworms, with reference value for molecular studies on detoxification processes in earthworms. Copyright © 2017 Elsevier Inc. All rights reserved.

The Cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdr-free Malaysian populations of the dengue vector Aedes albopictus.

PubMed

Ishak, Intan H; Riveron, Jacob M; Ibrahim, Sulaiman S; Stott, Rob; Longbottom, Joshua; Irving, Helen; Wondji, Charles S

2016-04-20

Control of Aedes albopictus, major dengue and chikungunya vector, is threatened by growing cases of insecticide resistance. The mechanisms driving this resistance remain poorly characterised. This study investigated the molecular basis of insecticide resistance in Malaysian populations of Ae. albopictus. Microarray-based transcription profiling revealed that metabolic resistance (cytochrome P450 up-regulation) and possibly a reduced penetration mechanism (consistent over-expression of cuticular protein genes) were associated with pyrethroid resistance. CYP6P12 over-expression was strongly associated with pyrethroid resistance whereas CYP6N3 was rather consistently over-expressed across carbamate and DDT resistant populations. Other detoxification genes also up-regulated in permethrin resistant mosquitoes included a glucuronosyltransferase (AAEL014279-RA) and the glutathione-S transferases GSTS1 and GSTT3. Functional analyses further supported that CYP6P12 contributes to pyrethroid resistance in Ae. albopictus as transgenic expression of CYP6P12 in Drosophila was sufficient to confer pyrethroid resistance in these flies. Furthermore, molecular docking simulations predicted CYP6P12 possessing enzymatic activity towards pyrethroids. Patterns of polymorphism suggested early sign of selection acting on CYP6P12 but not on CYP6N3. The major role played by P450 in the absence of kdr mutations suggests that addition of the synergist PBO to pyrethroids could improve the efficacy of this insecticide class and overcome resistance in field populations of Ae. albopictus.

Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.

PubMed Central

Ingelman-Sundberg, M; Kaur, H; Terelius, Y; Persson, J O; Halliwell, B

1991-01-01

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation. PMID:2064611

Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

PubMed

Xu, Xiao-Lu; Wu, Xiao-Qin; Ye, Jian-Ren; Huang, Lin

2015-03-06

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

The mRNA expression of P450 aromatase, gonadotropin beta-subunits and FTZ-F1 in the orange-spotted grouper (Epinephelus Coioides) during 17alpha-methyltestosterone-induced precocious sex change.

PubMed

Zhang, Weimin; Zhang, Yong; Zhang, Lihong; Zhao, Huihong; Li, Xin; Huang, He; Lin, Haoran

2007-06-01

The orange-spotted grouper Epinephelus coioides is a protogynous hermaphroditic fish, but the physiological basis of its sex change remains largely unknown. In the present study, the 2-year-old orange-spotted grouper was induced to change sex precociously by oral administration of 17alpha-methyltestosterone (MT, 50 mg/Kg diet, twice a day at daily ration of 5% bodyweight) for 60 days. The serum testosterone levels were significantly elevated after MT treatment for 20 and 40 days as compared to control, but the levels of serum estradiol (E(2)) remained unchanged. The expression of P450aromA in the gonad significantly decreased after MT treatment for 20, 40, and 60 days. Accordingly, the enzyme activity of gonadal aromatase was also lower. The expression of FSHbeta subunit in the pituitary was significantly decreased after MT treatment for 20 days, but returned to the control levels after 40 and 60 days; however, the expression of LHbeta subunit was not altered significantly by MT treatment. The expression of FTZ-F1 in the gonad also decreased significantly in response to MT treatment for 40 and 60 days, but its expression in the pituitary was not altered significantly. Interestingly, when tested in vitro on ovarian fragments, MT had no direct effect on the expression of P450aromA and FTZ-F1 as well as the activity of gonadal aromatase, suggesting that the inhibition of gonadal P450aromatase and FTZ-F1 by MT may be mediated at upper levels of the brain-pituitary-gonadal axis. Taken together, these results indicated that FSH, P450aromA, FTZ-F1, and serum testosterone are associated with the MT-induced sex change of the orange-spotted grouper, but the cause-effect relationship between these factors and sex change in this species remains to be characterized. (c) 2006 Wiley-Liss, Inc.

Drug Metabolism in Human Brain: High Levels of Cytochrome P4503A43 in Brain and Metabolism of Anti-Anxiety Drug Alprazolam to Its Active Metabolite

PubMed Central

Agarwal, Varsha; Kommaddi, Reddy P.; Valli, Khader; Ryder, Daniel; Hyde, Thomas M.; Kleinman, Joel E.; Strobel, Henry W.; Ravindranath, Vijayalakshmi

2008-01-01

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more α-hydroxy alprazolam (α-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both α-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of α-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of α-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action. PMID:18545703

Differential effects of traumatic brain injury on the cytochrome p450 system: a perspective into hepatic and renal drug metabolism.

PubMed

Kalsotra, Auinash; Turman, Cheri M; Dash, Pramod K; Strobel, Henry W

2003-12-01

Traumatic brain injury is known to cause several secondary effects, one of which is altered drug clearance. Given the fact that patients who sustain TBI are subsequently treated with a variety of pharmacological agents for the purpose of either neuroprotection or physiological support, it is imperative to clarify changes in expression and/or activities of enzymes involved in clearing drugs. The mixed function oxidase system, which consists of cytochrome P450 and cytochrome P450 reductase, plays a vital role in phase I drug metabolism. This paper addresses the issue as to what extent TBI affects the levels and activity of various rat CYP450 subfamilies. Our results show that TBI induces tissue-specific and time-dependent alterations. Total hepatic CYP450 content showed a biphasic response with a decrease seen at 24 h followed by an increase at 2 weeks. CYP450 reductase, in contrast, showed an opposite temporal profile. Immunoblot analyses and marker substrate metabolism demonstrated a clear decrease in hepatic CYP1A levels while a significant increase in kidney was seen at both 24 h and 2 weeks. A dramatic induction of CYP3A was evident at 2 weeks in liver, while no changes were noticed in CYP2B or CYP2D subfamilies. CYP4F subfamily showed induction in kidney only. Collectively, the data reveal the differential effects of TBI on hepatic and renal drug metabolism.

Comparative transcriptome analysis of Sogatella furcifera (Horváth) exposed to different insecticides.

PubMed

Zhou, Cao; Yang, Hong; Wang, Zhao; Long, Gui-Yun; Jin, Dao-Chao

2018-06-08

White-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae), one of the main agricultural insect pests in China, is resistant to a wide variety of insecticides. We used transcriptome analysis to compare the expression patterns of resistance- and stress-response genes in S. furcifera subjected to imidacloprid, deltamethrin, and triazophos stress, to determine the molecular mechanisms of resistance to these insecticides. A comparative analysis of gene expression under imidacloprid, deltamethrin, and triazophos stress revealed 1,123, 841, and 316 upregulated unigenes, respectively, compared to the control. These upregulated genes included seven P450s (two CYP2 clade, three CYP3 clade, and two CYP4 clade), one GST, one ABC transporter (ABCF), and seven Hsps (one 90 and six Hsp70s) under imidacloprid stress; one P450 (CYP3 clade), two ABC transporters (one ABCF and one ABCD), and one Hsp (Hsp90) under deltamethrin stress; one P450 (CYP3 clade) and one ABC transporter (ABCF) under triazophos stress. In addition, 80 genes were commonly upregulated in response to the three insecticide treatments, including laminin, larval cuticle protein, and fasciclin, which are associated with epidermal formation. These results provide a valuable resource for the molecular characterisation of insecticide action in S. furcifera, especially the molecular characteristics of insecticide cross resistance.

[The role of cytochrome P450 in nonalcoholic fatty liver induced by high-fat diet: a gene expression profile analysis].

PubMed

Liu, Y; Cheng, F; Luo, Y X; Hu, P; Ren, H; Peng, M L

2017-04-20

Objective: To clarify the role of cytochrome P450 in nonalcoholic fatty liver disease (NAFLD) by RNA-Seq and bioinformatics analysis. Methods: A total of 20 male C57BL/6 mice were used. Ten mice were fed with high-fat diet (D12492, 60% kcal fat) for 16 weeks to establish a mouse model of NAFLD, and the other 10 mice were fed with low-fat diet (D12450B, 10% kcal fat) as control group. At the end of the experiment, the body weight, liver weight, and hepatic triglyceride (TG) content were measured. Meanwhile, HE staining and RNA-Seq analysis were performed for the liver tissues. The differentially expressed genes were screened out and subjected to bioinformatics analysis, including KEGG and GO BP enrichment analyses and interaction network analysis. Comparison of means between the two groups was made using t-test. Results: Compared with the control group, the mice in the model group were obviously obese, with significantly increased body weight (41.41 ± 6.01 g vs 28.78 ± 1.79 g, t = 6.04, P < 0.01) and liver weight (1.38 ± 0.30 g vs 1.08 ± 0.10 g, t = 2.89, P < 0.01). The mice in the model group showed obvious steatosis, accompanied by a small amount of inflammatory cell infiltration, but with no obvious fibrosis, according to the results of HE staining. In addition, the hepatic TG content in the model group was significantly increased compared with that in the control group (0.64 ± 0.01 mg/mg vs 0.29 ± 0.06 mg/mg, t = 10.11, P = 0.04). Compared with the control group, a total of 367 differentially expressed genes, including 211 down-regulated and 156 up-regulated ones, were identified in the model group according to the RNA-seq results. Meanwhile, 19 CYP450 subtypes, accounting for 5% of the differentially expressed genes, were identified, and CYP2E1, CYP2C70, CYP3A11, CYP3A25, CYP2D26, CYP4A10, CYP17A1, CYP2B10, and CYP2C38 were involved in oxidative stress, steroid hormone metabolism, fatty acid metabolism, arachidonic acid metabolism, and the PPAR signaling pathway. An interaction network was constructed with 30 nodes, and CYP2E1 and CYP2C70 were identified as key nodes. RT-PCR validation results showed that the expression changes of CYP450 subtypes and lipid metabolism-related genes were consistent with the findings of sequencing. Conclusion: The CYP450 family plays a vital role in the pathogenesis of fatty liver by regulating lipid metabolism-related pathways, including oxidative stress, arachidonic acid metabolism, steroid hormone metabolism , and fatty acid metabolism.

Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

PubMed Central

Kashyap, Mahendra Pratap; Singh, Abhishek Kumar; Kumar, Vivek; Tripathi, Vinay Kumar; Srivastava, Ritesh Kumar; Agrawal, Megha; Khanna, Vinay Kumar; Yadav, Sanjay; Jain, Swatantra Kumar; Pant, Aditya Bhushan

2011-01-01

Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10−5 M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl2, P53, P21, GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10−5 M), whereas induction of CYPs was insignificant in cells exposed to 10−6 M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells. PMID:21445290

Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

PubMed

Li, Ting; Liu, Nannan

2017-12-01

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

Genome-wide identification of 52 cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus and their B[α]P-induced expression patterns.

PubMed

Han, Jeonghoon; Kim, Duck-Hyun; Kim, Hui-Su; Nelson, David R; Lee, Jae-Seong

2017-09-01

Cytochrome P450s (CYPs) are enzymes with a heme-binding domain that are found in all living organisms. CYP enzymes have important roles associated with detoxification of xenobiotics and endogenous compounds (e.g. steroids, fatty acids, and hormones). Although CYP enzymes have been reported in several invertebrates, including insects, little is known about copepod CYPs. Here, we identified the entire repertoire of CYP genes (n=52) from whole genome and transcriptome sequences of the benthic copepod Tigriopus japonicus, including a tandem duplication (CYP3026A3, CYP3026A4, CYP3026A5), and examined patterns of gene expression over various developmental stages and in response to benzo[α]pyrene (B[α]P) exposure. Through phylogenetic analysis, the 52 T. japonicus CYP genes were assigned to five distinct clans: CYP2 (22 genes), CYP3 (19 genes), CYP4 (two genes), CYP20 (one gene), and mitochondrial (eight genes). Developmental stage and gender-specific expression patterns of the 52 T. japonicus CYPs were analyzed. CYP3022A1 was constitutively expressed during all developmental stages. CYP genes in clans 2 and 3 were induced in response to B[α]P, suggesting that these differentially modulated CYP transcripts are likely involved in defense against exposure to B[α]P and other pollutants. This study enhances our understanding of the repertoire of CYP genes in copepods and of their potential role in development and detoxification in copepods. Copyright © 2017 Elsevier Inc. All rights reserved.

Progesterone production requires activation of caspase-3 in preovulatory granulosa cells in a serum starvation model.

PubMed

An, Li-Sha; Yuan, Xiao-Hua; Hu, Ying; Shi, Zi-Yun; Liu, Xiao-Qin; Qin, Li; Wu, Gui-Qing; Han, Wei; Wang, Ya-Qin; Ma, Xu

2012-11-01

Granulosa cells proliferate, differentiate, and undergo apoptosis throughout follicular development. Previous studies have demonstrated that stimulation of progesterone production is accompanied by caspase-3 activation. Moreover, we previously reported that arsenic enhanced caspase-3 activity coupled with progesterone production. Inhibition of caspase-3 activity can significantly inhibit progesterone production induced by arsenic or follicle-stimulating hormone (FSH). Here, we report that serum starvation induces caspase-3 activation coupled with augmentation of progesterone production. Serum starvation also increased the levels of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein, both of which may contribute to progesterone synthesis in preovulatory granulosa cells. Inhibition of caspase-3 activity resulted in a decrease in progesterone production. Deactivation of caspase-3 activity by caspase-3 specific inhibitor also resulted in decreases in P450scc and StAR expression, which may partly contribute to the observed decrease in progesterone production. Our study demonstrates for the first time that progesterone production in preovulatory granulosa cells is required for caspase-3 activation in a serum starvation model. Inhibition of caspase-3 activity can result in decreased expression of the steroidogenic proteins P450scc and StAR. Our work provides further details on the relationship between caspase-3 activation and steroidogenesis and indicates that caspase-3 plays a critical role in progesterone production by granulosa cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Cytochrome P450 CYP716A254 catalyzes the formation of oleanolic acid from β-amyrin during oleanane-type triterpenoid saponins biosynthesis in Anemone flaccida.

PubMed

Zhan, Chuansong; Ahmed, Shakeel; Hu, Sheng; Dong, Shuang; Cai, Qian; Yang, Tewu; Wang, Xuekui; Li, Xiaohua; Hu, Xuebo

2018-01-01

Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the β-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of β-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida. Copyright © 2017 Elsevier Inc. All rights reserved.

Metronidazole reduces the expression of cytochrome P450 enzymes in HepaRG cells and cryopreserved human hepatocytes.

PubMed

Kudo, Toshiyuki; Endo, Yumiko; Taguchi, Rina; Yatsu, Masami; Ito, Kiyomi

2015-05-01

1. Blood levels of S-warfarin have been reported to be increased by concomitant administration of metronidazole (MTZ), an antiprotozoal imidazole derivative. 2. To elucidate the mechanism of this interaction and to identify other possible drug-drug interactions, we conducted an in vitro study with the human hepatoma HepaRG cells and cryopreserved human hepatocytes on the ability of MTZ to reduce the expression of cytochrome P450 (CYP) as well as nuclear receptors that regulate the expression of these enzymes. 3. HepaRG cells and cryopreserved human hepatocytes were treated with MTZ (20 to 500 µM) and were then analyzed by real-time RT-PCR to determine mRNA levels of drug-metabolizing enzymes and nuclear receptors. 4. In both cells, the expressions of CYP2C8, CYP2C9, CYP3A4 and constitutive androstane receptor (CAR) were decreased by MTZ treatment. Particularly, in HepaRG cells, their mRNA levels were decreased by MTZ treatment in a concentration-dependent manner. 5. Our findings suggest that the interaction between MTZ and S-warfarin may be due to the MTZ-induced down-regulation of CYP2C9, the primary enzyme responsible for S-warfarin hydroxylation, and CAR, which regulates CYP2C9 expression. We also found that MTZ use may alter the disposition of drugs metabolized by the CYP isozymes investigated.

Varied clinical presentations of seven patients with mutations in CYP11A1 encoding the cholesterol side-chain cleavage enzyme, P450scc.

PubMed

Tee, Meng Kian; Abramsohn, Michal; Loewenthal, Neta; Harris, Mark; Siwach, Sudeep; Kaplinsky, Ana; Markus, Barak; Birk, Ohad; Sheffield, Val C; Parvari, Ruti; Pavari, Ruti; Hershkovitz, Eli; Miller, Walter L

2013-02-01

The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously. We sought to expand clinical and genetic experience with P450scc deficiency. We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel. Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity. Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

Evolutionary history and functional divergence of the cytochrome P450 gene superfamily between Arabidopsis thaliana and Brassica species uncover effects of whole genome and tandem duplications.

PubMed

Yu, Jingyin; Tehrim, Sadia; Wang, Linhai; Dossa, Komivi; Zhang, Xiurong; Ke, Tao; Liao, Boshou

2017-09-18

The cytochrome P450 monooxygenase (P450) superfamily is involved in the biosynthesis of various primary and secondary metabolites. However, little is known about the effects of whole genome duplication (WGD) and tandem duplication (TD) events on the evolutionary history and functional divergence of P450s in Brassica after splitting from a common ancestor with Arabidopsis thaliana. Using Hidden Markov Model search and manual curation, we detected that Brassica species have nearly 1.4-fold as many P450 members as A. thaliana. Most P450s in A. thaliana and Brassica species were located on pseudo-chromosomes. The inferred phylogeny indicated that all P450s were clustered into two different subgroups. Analysis of WGD event revealed that different P450 gene families had appeared after evolutionary events of species. For the TD event analyses, the P450s from TD events in Brassica species can be divided into ancient and recent parts. Our comparison of influence of WGD and TD events on the P450 gene superfamily between A. thaliana and Brassica species indicated that the family-specific evolution in the Brassica lineage can be attributed to both WGD and TD, whereas WGD was recognized as the major mechanism for the recent evolution of the P450 super gene family. Expression analysis of P450s from A. thaliana and Brassica species indicated that WGD-type P450s showed the same expression pattern but completely different expression with TD-type P450s across different tissues in Brassica species. Selection force analysis suggested that P450 orthologous gene pairs between A. thaliana and Brassica species underwent negative selection, but no significant differences were found between P450 orthologous gene pairs in A. thaliana-B. rapa and A. thaliana-B. oleracea lineages, as well as in different subgenomes in B. rapa or B. oleracea compared with A. thaliana. This study is the first to investigate the effects of WGD and TD on the evolutionary history and functional divergence of P450 gene families in A. thaliana and Brassica species. This study provides a biology model to study the mechanism of gene family formation, particularly in the context of the evolutionary history of angiosperms, and offers novel insights for the study of angiosperm genomes.

Utility of Nicotiana tabacum cell suspension cultures expressing human CYP1A1, CYP1A2 and CYP3A4 to study the oxidative metabolism of the herbicide 14C-fluometuron.

PubMed

Breuer, Maren Anne; Schmidt, Burkhard; Schuphan, Ingolf

2009-01-01

The metabolism and biotransformation of the (14)C-labeled phenylurea herbicide fluometuron was examined using tobacco cell suspension cultures transformed separately with human cyp1a1, cyp1a2 and cyp3a4, and corresponding non-transformed cultures in order to screen and predict metabolic patterns. Experimental parameters modified were concentration of (14)C-fluometuron, incubation period, and additional application of inhibitor carbaryl. Media and cell extracts were analyzed by radio-TLC and radio-HPLC, isolated metabolites by LC-MS, and non-extractable residues by combustion. During 48 hours, the CYP1A1 expressing cultures metabolized 90.0 % of applied fluometuron, while the non-transgenic controls transformed 67.0 %. The CYP1A2 expressing cultures exhibited highest rates (95.1 %), CYP3A4 expressing cultures lowest rates (43.0 %). The primary metabolites identified were mono-demethyl (main metabolite in controls) and di-demethyl fluometuron (mainly in CYP1A2 cultures), besides a non-identified primary product (mainly in CYP1A1 cultures); metabolic profiles differed distinctly among cultures. After addition of carbaryl, rates of fluometuron decreased noticeably in controls and not in CYP3A4 expressing cultures. This may indicate inhibition of endogenous tobacco P450s involved in fluometuron metabolism but not of CYP3A4. Additionally, the P450-transgenic cultures proved to be valuable tools to produce large amounts of metabolites for thorough identification.

Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans.

PubMed

Inokuchi, Ayako; Yamamoto, Ryoko; Morita, Fumiyo; Takumi, Shota; Matsusaki, Hiromi; Ishibashi, Hiroshi; Tominaga, Nobuaki; Arizono, Koji

2015-09-01

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000 µm, respectively, for LiCl and 750, 750 and 3000 µm, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78 µm LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375 µm Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans. Copyright © 2015 John Wiley & Sons, Ltd.

Transcriptome Analysis of an Insecticide Resistant Housefly Strain: Insights about SNPs and Regulatory Elements in Cytochrome P450 Genes

PubMed Central

Asp, Torben; Kristensen, Michael

2016-01-01

Background Insecticide resistance in the housefly, Musca domestica, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes. Results The de novo assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in cyp6d1. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools. Conclusion Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s in xenobiotic detoxification. PMID:27019205

Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R

PubMed Central

Gravells, P; Hoh, L; Canovas, D; Rennie, I G; Sisley, K; Bryant, H E

2011-01-01

Background: Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU). Methods: Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation. Results: Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM. Conclusion: We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R. PMID:21386838

Mining of the Uncharacterized Cytochrome P450 Genes Involved in Alkaloid Biosynthesis in California Poppy Using a Draft Genome Sequence

PubMed Central

Hori, Kentaro; Yamada, Yasuyuki; Purwanto, Ratmoyo; Minakuchi, Yohei; Toyoda, Atsushi; Hirakawa, Hideki

2018-01-01

Abstract Land plants produce specialized low molecular weight metabolites to adapt to various environmental stressors, such as UV radiation, pathogen infection, wounding and animal feeding damage. Due to the large variety of stresses, plants produce various chemicals, particularly plant species-specific alkaloids, through specialized biosynthetic pathways. In this study, using a draft genome sequence and querying known biosynthetic cytochrome P450 (P450) enzyme-encoding genes, we characterized the P450 genes involved in benzylisoquinoline alkaloid (BIA) biosynthesis in California poppy (Eschscholzia californica), as P450s are key enzymes involved in the diversification of specialized metabolism. Our in silico studies showed that all identified enzyme-encoding genes involved in BIA biosynthesis were found in the draft genome sequence of approximately 489 Mb, which covered approximately 97% of the whole genome (502 Mb). Further analyses showed that some P450 families involved in BIA biosynthesis, i.e. the CYP80, CYP82 and CYP719 families, were more enriched in the genome of E. californica than in the genome of Arabidopsis thaliana, a plant that does not produce BIAs. CYP82 family genes were highly abundant, so we measured the expression of CYP82 genes with respect to alkaloid accumulation in different plant tissues and two cell lines whose BIA production differs to estimate the functions of the genes. Further characterization revealed two highly homologous P450s (CYP82P2 and CYP82P3) that exhibited 10-hydroxylase activities with different substrate specificities. Here, we discuss the evolution of the P450 genes and the potential for further genome mining of the genes encoding the enzymes involved in BIA biosynthesis. PMID:29301019

The effects of in utero bisphenol A exposure on ovarian follicle numbers and steroidogenesis in the F1 and F2 generations of mice.

PubMed

Mahalingam, Sharada; Ther, Laura; Gao, Liying; Wang, Wei; Ziv-Gal, Ayelet; Flaws, Jodi A

2017-12-01

Bisphenol A (BPA) is a commonly used plasticizer. Previous studies show that in utero exposure to BPA affects reproductive outcomes in the F1-F3 generations of mice. However, its multigenerational effects on ovarian histology and steroidogenesis over the reproductive lifespan are unknown. Thus, we tested the hypothesis that BPA has multigenerational effects on follicle numbers and steroidogenesis. Mice were exposed in utero to vehicle control or BPA (0.5, 20, and 50μg/kg/day). Ovaries were collected for histological and gene expression analyses and sera were collected for hormone assays. In utero BPA exposure decreased preantral follicle numbers, cytochrome P450 aromatase mRNA levels, and estradiol levels in the F1 generation, whereas it decreased testosterone levels and altered steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase 1, and cytochrome P450 aromatase mRNA levels in the F2 generation. These data suggest that BPA has multigenerational effects on the ovary in mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of Gestational Exposure of Cypermethrin on Postnatal Development of Brain Cytochrome P450 2D1 and 3A1 and Neurotransmitter Receptors.

PubMed

Singh, Anshuman; Mudawal, Anubha; Shukla, Rajendra K; Yadav, Sanjay; Khanna, Vinay K; Sethumadhavan, Rao; Parmar, Devendra

2015-08-01

Oral administration of low doses (1.25, 2.5, or 5 mg/kg) of cypermethrin to pregnant Wistar rats from gestation days 5 to 21 led to dose-dependent differences in the induction of cytochrome P450 2D1 (CYP2D1) and 3A1 messenger RNA (mRNA) and protein in brain regions isolated from the offsprings postnatally at 3 weeks that persisted up to adulthood (12 weeks). Similar alterations were observed in the expression of GABAergic, muscarinic, dopaminergic, and serotonergic neurotransmitter receptors in brain regions of rat offsprings. Rechallenge of the prenatally exposed offsprings at adulthood (12 weeks old) with cypermethrin (p.o., 10 mg/kg for 6 days) led to a greater magnitude of alterations in the expression of CYPs, neurotransmitter receptors, and neurotransmitter receptor binding in the brain regions when compared to the control offsprings treated at adulthood with cypermethrin or prenatally exposed offsprings. A greater magnitude of decrease was also observed in the spontaneous locomotor activity (SLA) in prenatally exposed offsprings rechallenged with cypermethrin. The present data indicating similarities in the alterations in the expression of CYPs (2D1 and 3A1) and neurotransmitter receptors in brain has led us to suggest that endogenous function regulating CYPs is possibly associated with neurotransmission processes. A greater magnitude of alterations in CYP2D1, 3A1, neurotransmitter receptors, and SLA in rechallenged animals has further provided evidence that alterations in CYPs are possibly linked with neurotransmission processes.

Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.

PubMed

Takemura, Tomoya; Ikezawa, Nobuhiro; Iwasa, Kinuko; Sato, Fumihiko

2013-07-01

Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction. Copyright © 2012 Elsevier Ltd. All rights reserved.

The WRKY transcription factor HpWRKY44 regulates CytP450-like1 expression in red pitaya fruit (Hylocereus polyrhizus).

PubMed

Cheng, Mei-Nv; Huang, Zi-Juan; Hua, Qing-Zhu; Shan, Wei; Kuang, Jian-Fei; Lu, Wang-Jin; Qin, Yong-Hua; Chen, Jian-Ye

2017-01-01

Red pitaya ( Hylocereus polyrhizus ) fruit is a high-value, functional food, containing a high level of betalains. Several genes potentially related to betalain biosynthesis, such as cytochrome P450-like ( CytP450-like ), have been identified in pitaya fruit, while their transcriptional regulation remains unclear. In this work, the potential involvement of a WRKY transcription factor, HpWRKY44, in regulating CytP450-like1 expression in pitaya fruit was examined. HpWRKY44, a member of the Group 1 WRKY family, contains two conserved WRKY motifs and is localized in the nucleus. HpWRKY44 also exhibits trans-activation ability. Gene expression analysis showed that the expression of HpCytP450-like1 and HpWRKY44 increased steadily during pitaya fruit coloration, which corresponded with the production of elevated betalain levels in the fruit. HpWRKY44 was also demonstrated to directly bind to and activate the HpCytP450-like1 promoter via the recognition of the W-box element present in the promoter. Collectively, our findings indicate that HpWRKY44 transcriptionally activates HpCytP450-like1 , which perhaps, at least in part, contributes to betalain biosynthesis in pitaya fruit. The information provided in the current study provides novel insights into the regulatory network associated with betalain biosynthesis during pitaya fruit coloration.

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

PubMed

Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

2008-03-01

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Intestinal and Hepatic Expression of Cytochrome P450s and mdr1a in Rats with Indomethacin-Induced Small Intestinal Ulcers

PubMed Central

Kawauchi, Shoji; Nakamura, Tsutomu; Yasui, Hiroyuki; Nishikawa, Chikako; Miki, Ikuya; Inoue, Jun; Horibe, Sayo; Hamaguchi, Tsuneo; Tanahashi, Toshihito; Mizuno, Shigeto

2014-01-01

Background: Non-steroidal anti-inflammatory drugs induce the serious side effect of small intestinal ulcerations (SIUs), but little information is available regarding the consequences to drug metabolism and absorption. Aim: We examined the existence of secondary hepatic inflammation in rats with indomethacin (INM)-induced SIUs and assessed its relationship to the cytochrome P450 (CYP) and P-glycoprotein (mdr1a), the major drug-metabolizing factors in the small intestine and the liver. Methods: Gene expression of the CYP family of enzymes and mdr1a was measured with quantitative real-time polymerase chain reaction (qPCR). Vancomycin (VCM), a poorly absorbed drug, was administered intraduodenally to rats with SIUs. Results: INM induced SIUs predominantly in the lower region of the small intestine with high expression of inflammatory markers. Liver dysfunction was also observed, which suggested a secondary inflammatory response in rats with SIUs. In the liver of rats with SIUs, the expression of CYP2C11, CYP2E1, and CYP3A1 was significantly decreased, and loss of CYP3A protein was observed. Although previous studies have shown a direct effect of INM on CYP3A activity, we could not confirm any change in hepatic CY3A4 expression (major isoform of human CYP3A) in vitro. The plasma VCM concentration was increased in rats with SIUs due to partial absorption from the mucosal injury, but not in normal mucosa. Conclusions: INM-induced SIUs had a subtle effect on intestinal CYP expression, but had an apparent action on hepatic CYP, which was influenced, at least in part, by the secondary inflammation. Furthermore, drug absorption was increased in rats with SIUs. PMID:25317066

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

PubMed

Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

2013-01-01

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

PubMed

Puinean, Alin M; Foster, Stephen P; Oliphant, Linda; Denholm, Ian; Field, Linda M; Millar, Neil S; Williamson, Martin S; Bass, Chris

2010-06-24

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2-16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.

Arabidopsis Cytochrome P450 Monooxygenase 71A13 Catalyzes the Conversion of Indole-3-Acetaldoxime in Camalexin Synthesis[W

PubMed Central

Nafisi, Majse; Goregaoker, Sameer; Botanga, Christopher J.; Glawischnig, Erich; Olsen, Carl E.; Halkier, Barbara A.; Glazebrook, Jane

2007-01-01

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola. PMID:17573535

Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane, and methoxyflurane.

PubMed

Kharasch, E D; Thummel, K E

1993-10-01

Renal and hepatic toxicity of the fluorinated ether volatile anesthetics is caused by biotransformation to toxic metabolites. Metabolism also contributes significantly to the elimination pharmacokinetics of some volatile agents. Although innumerable studies have explored anesthetic metabolism in animals, there is little information on human volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The first purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second purpose was to test the hypothesis that cytochrome P450 2E1 is the specific P450 isoform responsible for volatile anesthetic defluorination in humans. Microsomes were prepared from human livers. Anesthetic metabolism in microsomal incubations was measured by fluoride production. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human livers, correlation of microsomal defluorination rate with microsomal P450 2E1 content (measured by Western blot analysis), correlation of defluorination rate with microsomal P450 2E1 catalytic activity using marker substrates (para-nitrophenol hydroxylation and chlorzoxazone 6-hydroxylation), and chemical inhibition by P450 isoform-selective inhibitors. The rank order of anesthetic metabolism, assessed by fluoride production at saturating substrate concentrations, was methoxyflurane > sevoflurane > enflurane > isoflurane > desflurane > 0. There was a significant linear correlation of sevoflurane and methoxyflurane defluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, respectively), but not with either P450 1A2 or P450 3A3/4. Comparison of anesthetic defluorination with either para-nitrophenol or chlorzoxazone hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0.66). Sevoflurane defluorination was also highly correlated with that of enflurane (r = 0.93), which is known to be metabolized by human P450 2E1. Diethyldithiocarbamate, a selective inhibitor of P450 2E1, produced a concentration-dependent inhibition of sevoflurane, methoxyflurane, and isoflurane defluorination. No other isoform-selective inhibitor diminished the defluorination of sevoflurane, whereas methoxyflurane defluorination was inhibited by the selective P450 inhibitors furafylline (P450 1A2), sulfaphenazole (P450 2C9/10), and quinidine (P450 2D6) but to a much lesser extent than by diethyldithiocarbamate. These results demonstrate that cytochrome P450 2E1 is the principal, if not sole human liver microsomal enzyme catalyzing the defluorination of sevoflurane. P450 2E1 is the principal, but not exclusive enzyme responsible for the metabolism of methoxyflurane, which also appears to be catalyzed by P450s 1A2, 2C9/10, and 2D6. The data also suggest that P450 2E1 is responsible for a significant fraction of isoflurane metabolism. Identification of P450 2E1 as the major anesthetic metabolizing enzyme in humans provides a mechanistic understanding of clinical fluorinated ether anesthetic metabolism and toxicity.

CYP450s analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development.

PubMed

Ventura, Tomer; Bose, Utpal; Fitzgibbon, Quinn P; Smith, Gregory G; Shaw, P Nicholas; Cummins, Scott F; Elizur, Abigail

2017-07-01

Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Mechanisms of olfactory toxicity of the herbicide 2,6-dichlorobenzonitrile: Essential roles of CYP2A5 and target-tissue metabolic activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie Fang; Zhou Xin; Behr, Melissa

The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450more » enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.« less

Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

PubMed Central

2012-01-01

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes. PMID:22876969

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bell, Stephen G.; Hoskins, Nicola; Xu Feng

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to {approx}2.0 A have been obtained.

Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

PubMed

Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

2016-01-01

The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants.

Variation in P450-mediated fenvalerate resistance levels is not correlated with CYP337B3 genotype in Chinese populations of Helicoverpa armigera.

PubMed

Han, Yangchun; Yu, Wanting; Zhang, Weiqing; Yang, Yihua; Walsh, Tom; Oakeshott, John G; Wu, Yidong

2015-06-01

Metabolic resistance to synthetic pyrethroids in Helicoverpa   armigera has recently been associated with the chimeric cytochrome P450 enyzme CYP337B3. One variant of the latter, CYP337B3v1, accounts for 40-50 fold resistance to fenvalerate in an Australian population while a second variant, CYP337B3v2, has been associated with ~7 fold resistance to cypermethrin in a Pakistani population. Here we show that ~250-1200 fold resistance to fenvalerate in populations of the species from northern and northwestern China is largely due to P450-based metabolism, and that CYP337B3v2 is also at high frequency in these populations but absent in a susceptible control strain. However we find little correlation between the level of resistance and CYP337B3v2 frequency, either across the resistant populations studied, or over time within them. While there is variation between populations in the levels of CYP337B3v2 expression, this is not correlated with the level of resistance either. These data suggest that much of the variation in the level of fenvalerate resistance in China is explained by P450s other than CYP337B3. We also find that both the level of resistance and CYP337B3v2 frequency decline in field populations transferred to the laboratory and remained there without fenvalerate exposure, suggesting a fitness cost to both characters in the absence of the pesticide pressure. However the declines in the two characters are not well correlated across populations, again consistent with a large contribution to the variation in resistance levels from factors other than CYP337B3. Copyright © 2014 Elsevier Inc. All rights reserved.

Cytochrome P450 3A expression and activity in the rabbit lacrimal gland: glucocorticoid modulation and the impact on androgen metabolism.

PubMed

Attar, Mayssa; Ling, Kah-Hiing John; Tang-Liu, Diane D-S; Neamati, Nouri; Lee, Vincent H L

2005-12-01

Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naïve untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1% or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [3H]testosterone. Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naïve rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naïve rabbit lacrimal gland at a rate of 14 +/- 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [3H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly prescribed glucocorticoid and can be inhibited by known CYP3A inhibitors.

Over-expression of multiple cytochrome P450 genes in fenvalerate-resistant field strains of Helicoverpa armigera from north of China.

PubMed

Xu, Li; Li, Dongzhi; Qin, Jianying; Zhao, Weisong; Qiu, Lihong

2016-09-01

Pyrethroid resistance was one of the main reasons for control failure of cotton bollworm Helicoverpa armigera (Hübner) in China. The promotion of Bt crops decreased the application of chemical insecticides in controlling H.armigera. However, the cotton bollworm still kept high levels of resistance to fenvalerate. In this study, the resistance levels of 8 field-collected strains of H. armigera from north of China to 4 insecticides, as well as the expression levels of related P450 genes were investigated. The results of bioassay indicated that the resistance levels to fenvalerate in the field strains varied from 5.4- to 114.7-fold, while the resistance levels to lambda-cyhalothrin, phoxim and methomyl were low, which were ranged from 1.5- to 5.2-, 0.2- to 1.6-, and 2.9- to 8.3- fold, respectively, compared to a susceptible strain. Synergistic experiment showed that PBO was the most effective synergist in increasing the sensitivity of H. armigera to fenvalerate, suggesting that P450 enzymes were involved in the pyrethroid resistance in the field strains. The results of quantitative RT-PCR indicated that eight P450 genes (CYP332A1, CYP4L11, CYP4L5, CYP4M6, CYP4M7, CYP6B7, CYP9A12, CYP9A14) were all significantly overexpressed in Hejian1 and Xiajin1 strains of H. armigera collected in 2013, and CYP4L5 was significantly overexpressed in all the 6 field strains collected in 2014. CYP332A1, CYP6B7 and CYP9A12 had very high overexpression levels in all the field strains, indicating their important roles in fenvalerate resistance. The results suggested that multiple P450 genes were involved in the high-level fenvalerate-resistance in different field strains of H. armigera collected from north of China. Copyright © 2016 Elsevier B.V. All rights reserved.

[Effects of vitamins deficiency on the cytochrome P450 inducibility in rats].

PubMed

Trusov, N V; Guseva, G V; Beketova, N A; Aksenov, I V; Avrent'eva, L I; Kravchenko, L V

2014-01-01

The purpose of the study was to determine multi-vitamin deficiency effects on the inducibility of main isoforms of cytochrome P450 in the rat liver. The study was carried out on 4 groups of Wistar rats. Rats of the 1st and 3rd group received semi-synthetic diets containing adequate (100% of recommended vitamin level) level of vitamins, the 2nd and 4th--the semi-synthetic diet containing vitamins in the amount of 20% from adequate level. The duration of the experiment was 4 weeks. During the last week indole-3-carbinol (I-3-C) in dose of 20 mg/kg body weight was added to the diet of the 3rd and 4th group of rats. Vitamin E content in liver and blood serum declined by 59 and 34%, respectively in rats which were fed vitamin-deficient diet (2nd group); vitamin A level decreased by 5 times in the liver, but was not changed in blood serum. Multi-vitamin deficiency in the diet led to the increase in the liver ethoxyresorufin O-dealkylase (EROD) activity of CYP1A1, methoxyresorufin O-dealkylase (MROD) activity of CYP1A2 and testosteron 6beta-hydroxylase (6beta-TG) activity of CYP3A by 11, 80 and 53%, respectively, and gene expression of CYP1A1, CYP1A2, CYP3A and AhR by 8,5; 1,6; 2,4 and 3,6 fold. In rats fed diet with adequate levels of vitamins (3rd group) I-3-C increased activity of EROD and MROD by 4,4 and 5,5 fold, and the expression of CYP1A1, CYP1A2 and AhR genes by 148; 3 and 3,5 fold compared to the parameters of the 1st group (without I-3-C). Multi-vitamin deficiency increased I-3-C-related induction of EROD activity and expression of CYP1A1 and CYP1A2 genes, but decreased I-3-C-related induction of the MROD activity. Thus, 5-fold reducing of vitamin content in rat diet lead to significant changes in activity and inducibility of cytochrome P450 of CYP1A and 3A family, which play a key role in the detoxification and metabolism of drugs.

Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats

PubMed Central

Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

2016-01-01

The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases. PMID:27882225

Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon

PubMed Central

Tian, Yuan; Knaak, James B.; Kostyniak, Paul J.; Olson, James R.

2012-01-01

Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (Km and Vmax) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (Km = 1.25 μM; Vmax = 9.78 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 1.03 μM; Vmax = 4.67 nmol · min−1 · nmol P450−1), and CYP1A2 (Km = 1.96 μM; Vmax = 5.14 nmol · min−1 · nmol P450−1), and the bioactivation of diazinon was mediated primarily by CYP1A1 (Km = 3.05 μM; Vmax = 2.35 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 7.74 μM; Vmax = 4.14 nmol · min−1 · nmol P450−1), and CYP2B6 (Km = 14.83 μM; Vmax = 5.44 nmol · min−1 · nmol P450−1). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (Km = 16.8 μM; Vmax = 1.38 nmol · min−1 · nmol P450−1) and 3A4 (Km = 104 μM; Vmax = 5.15 nmol · min−1 · nmol P450−1), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (Km = 5.04 μM; Vmax = 5.58 nmol · min−1 · nmol P450−1). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure. PMID:21969518

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura).

PubMed

Scaia, María Florencia; Volonteri, María Clara; Czuchlej, Silvia Cristina; Ceballos, Nora Raquel

2015-09-15

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder's organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E2). The purpose of this study is to analyze the effect of E2 on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24h with or without 2 or 20nM of E2. After treatments, the activities of cytochrome P450 17α-hydroxylase-C17-20 lyase (CypP450c17) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3β-HSD/I activity during the post reproductive season. Furthermore, although E2 has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E2 has no effect on testicular proliferation all year long. Taken together, these results suggest that E2 is involved in the regulation of testicular steroidogenesis and spermatogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

PubMed Central

2011-01-01

Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450. PMID:21961524

Impact of Fusarium mycotoxins on hepatic and intestinal mRNA expression of cytochrome P450 enzymes and drug transporters, and on the pharmacokinetics of oral enrofloxacin in broiler chickens.

PubMed

Antonissen, Gunther; Devreese, Mathias; De Baere, Siegrid; Martel, An; Van Immerseel, Filip; Croubels, Siska

2017-03-01

Cytochrome P450 (CYP450) drug biotransformation enzymes and multidrug resistance (MDR) proteins may influence drug disposition processes. The first part of the study aimed to evaluate the effect of mycotoxins deoxynivalenol (DON) and/or fumonisins (FBs), at contamination levels approaching European Union guidance levels, on intestinal and hepatic CYP450 enzymes and MDR proteins gene expression in broiler chickens. mRNA expression of genes encoding CYP450 enzymes (CYP3A37, CYP1A4 and CYP1A5) and drug transporters (MDR1/ABCB1 and MRP2/ABCC2) was determined using qRT-PCR. A significant up-regulation of CYP1A4 (P = 0.037) and MDR1 (P = 0.036) was observed in the jejunum of chickens fed a diet contaminated with FBs. The second part of this study aimed to investigate the impact of feeding a FBs contaminated diet on the oral absorption of enrofloxacin (10 mg/kg BW), a MDR1 substrate. A significant (P = 0.045), however small, decreased area under the plasma concentration-time curve (AUC 0-48  h, mean ± SD) was observed for enrofloxacin in chickens fed the FBs contaminated diet compared to the control group, 16.28 ± 1.82 h μg/mL versus 18.27 ± 1.79 h μg/mL. These findings suggest that concurrent administration of drugs with FBs contaminated feed might alter the pharmacokinetic characteristics of CYP1A4 substrate drugs and MDR1 substrates, such as enrofloxacin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of thalidomide on endotoxin-induced decreases in activity and expression of hepatic cytochrome P450 3A2.

PubMed

Ueyama, Jun; Nadai, Masayuki; Zhao, Ying Lan; Kanazawa, Hiroaki; Takagi, Kenji; Kondo, Takaaki; Takagi, Kenzo; Wakusawa, Shinya; Abe, Fumie; Saito, Hiroko; Miyamoto, Ken-Ichi; Hasegawa, Takaaki

2008-08-01

Thalidomide has been reported to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) that are involved in the down-regulation of hepatic cytochrome P450 (CYP) induced by endotoxin. In the present study, we investigated the effects of thalidomide on endotoxin-induced decreases in the activity and expression of hepatic CYP3A2 in rats. Thalidomide (50 mg/kg) was administered orally 22 h and 2 h before intraperitoneal injection of endotoxin (1 mg/kg). Twenty-four hours after the injection of endotoxin, antipyrine clearance experiments were conducted, in which the rats were sacrificed and protein levels of hepatic CYP3A2 were measured. There were no significant differences in the histopathological changes in the liver between the endotoxin-treated and endotoxin plus thalidomide-treated rats. Thalidomide had no effect on the systemic clearance of antipyrine, which is a proper indicator for hepatic CYP3A2 activity, whereas it enhanced endotoxin-induced decrease in the systemic clearance of antipyrine. Western blot analysis revealed that thalidomide had no effect on the protein levels of hepatic CYP3A2, whereas it enhanced the down-regulation of hepatic CYP3A2 by endotoxin. However, there were no significant differences in the concentrations of TNF-alpha and NO in plasma between the endotoxin-treated and endotoxin plus thalidomide-treated rats. The present findings suggest that thalidomide enhances endotoxin-induced decreases in the activity and expression of hepatic CYP3A2.

Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B

PubMed Central

Jackson, Rosamond G.; Rylott, Elizabeth L.; Fournier, Diane; Hawari, Jalal; Bruce, Neil C.

2007-01-01

Widespread contamination of land and groundwater has resulted from the use, manufacture, and storage of the military explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX). This contamination has led to a requirement for a sustainable, low-cost method to remediate this problem. Here, we present the characterization of an unusual microbial P450 system able to degrade RDX, consisting of flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA. The affinity of XplA for the xenobiotic compound RDX is high (Kd = 58 μM) and comparable with the Km of other P450s toward their natural substrates (ranging from 1 to 500 μM). The maximum turnover (kcat) is 4.44 per s, only 10-fold less than the fastest self-sufficient P450 reported, BM3. Interestingly, the presence of oxygen determines the final products of RDX degradation, demonstrating that the degradation chemistry is flexible, but both pathways result in ring cleavage and release of nitrite. Carbon monoxide inhibition is weak and yet the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a potent inhibitor. To test the efficacy of this system for the remediation of groundwater, transgenic Arabidopsis plants expressing both xplA and xplB were generated. They are able to remove saturating levels of RDX from liquid culture and soil leachate at rates significantly faster than those of untransformed plants and xplA-only transgenic lines, demonstrating the applicability of this system for the phytoremediation of RDX-contaminated sites. PMID:17940033

Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs

PubMed Central

LYTTON, S D; BERG, U; NEMETH, A; INGELMAN-SUNDBERG, M

2002-01-01

Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato-and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8·5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection. PMID:11876753

Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs.

PubMed

Lytton, S D; Berg, U; Nemeth, A; Ingelman-Sundberg, M

2002-02-01

Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria.

PubMed

Guo, Yanqiong; Zhang, Jianzhen; Yu, Rongrong; Zhu, Kun Yan; Guo, Yaping; Ma, Enbo

2012-05-01

Cytochrome P450 monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. We have previously evaluated the responses of the oriental migratory locust (Locusta migratoria) to the pyrethroid insecticide deltamethrin and revealed that increased cytochrome P450 enzyme activity was due to increased transcription of multiple cytochrome P450 genes. In this study, we identified for the first time two new cytochrome P450 genes, which belong to two novel cytochrome P450 gene families. CYP409A1 belongs to CYP409 family whereas CYP408B1 belongs to CYP408 family. Our molecular analysis indicated that CYP409A1 was mainly expressed in fatbodies, midgut, gastric caecum, foregut and Malpighian tubules of the third- and fourth-instar nymphs, whereas CYP408B1 was mainly expressed in foregut, hindgut and muscle of the insects at all developmental stages examined. The expression of these two cytochrome P450 genes were differentially affected by three representative insecticides, including carbaryl (carbamate), malathion (organophosphate) and deltamethrin (pyrethroid). The exposure of the locust to carbaryl, malathion and deltamethrin resulted in reduced, moderately increased and significantly increased transcript levels, respectively, of the two cytochrome P450 genes. Our further analysis of their detoxification roles by using RNA interference followed by deltamethrin bioassay showed increased nymph mortalities by 21.1% and 16.7%, respectively, after CYP409A1 and CYP408B1 were silenced. These results strongly support our notion that these two new cytochrome P450 genes play an important role in deltamethrin detoxification in the locust. Copyright © 2011 Elsevier Ltd. All rights reserved.

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish.

PubMed

Callard, G V; Tchoudakova, A V; Kishida, M; Wood, E

2001-12-01

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromB>A) and ovary (P450aromA>B) and have a different developmental program (B>A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24-48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (alpha, beta, and gamma). The 5'-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a>b) are opposite to fish pituitary (b>a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30-48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.

Differential protein expression and localization of CYP450 enzymes in three species of earthworm; is this a reflection of environmental adaptation?

PubMed

Lu, Xiaoxu; Li, Yinsheng; Thunders, Michelle; Cavanagh, Jo; Matthew, Cory; Wang, Xiuhong; Zhou, Xinchu; Qiu, Jiangping

2017-03-01

Cytochrome P450 (CYP450) is a hemoprotein superfamily, among which CYP1, CYP2 and CYP3 play a major role in the metabolism of vast array of xenobiotics and endobiotics. This paper reports on three CYP enzyme variants (CYP1A2, CYP2E1 and CYP3A4) in three species of earthworm (Eisenia fetida, Metaphire guillelmi and Amynthas carnosus). The relative expression levels and localization of the three associated proteins were investigated at three life-cycle points (juvenile, sub-adult and adult), through comparison of anterior and posterior body tissue and between specific organs (body wall, intestine and reproductive tissues) using western blot analysis. This study confirmed the presence of CYP3A4, CYP1A2 and CYP2E1 in all three species of earthworm tested. The levels of expression varied with earthworm species, age, and body location. These differences in occurrence of the three CYP enzymes appeared to reflect the ecological niche (the spatial and temporal location and functional relationship of each individual or population in populations or communities), and the likelihood of contact with soil contaminants of the respective species. These results may help to explain why earthworms are capable of adapting to very different and extensively polluted soil environments and provide important data for subsequent ecotoxicology and ecological adaptability studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals.

PubMed

Usmani, Khawja A; Cho, Taehyeon M; Rose, Randy L; Hodgson, Ernest

2006-09-01

Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.

Cloning of three genes involved in the flavonoid metabolic pathway and their expression during insect resistance in Pinus massoniana Lamb.

PubMed

Yang, Z Q; Chen, H; Tan, J H; Xu, H L; Jia, J; Feng, Y H

2016-12-23

Pinus massoniana Lamb. is an important timber and turpentine-producing tree species in China. Dendrolimus punctatus and Dasychira axutha are leaf-eating pests that have harmful effects on P. massoniana production. Few studies have focused on the molecular mechanisms underlying pest resistance in P. massoniana. Based on sequencing analysis of the transcriptomes of insect-resistant P. massoniana, three key genes involved in the flavonoid metabolic pathway were identified in the present study (PmF3H, PmF3'5'H, and PmC4H). Structural domain analysis showed that the PmF3H gene contains typical binding sites for the 2OG-Fe (II) oxygenase superfamily, while PmF3'5'H and PmC4H both contain the cytochrome P450 structural domain, which is specific for P450 enzymes. Phylogenetic analysis showed that each of the three P. massoniana genes, and the homologous genes in gymnosperms, clustered into a group. Expression of these three genes was highest in the stems, and was higher in the insect-resistant P. massoniana varieties than in the controls. The extent of the increased expression in the insect-resistant P. massoniana varieties indicated that these three genes are involved in defense mechanisms against pests in this species. In the insect-resistant varieties, rapid induction of PmF3H increased the levels of PmF3'5'H and PmC4H expression. The enhanced anti-pest capability of the insect-resistant varieties could be related to temperature and humidity. In addition, these results suggest that these three genes maycontribute to the change in flower color during female cone development.

Phytomonitoring and phytoremediation of agrochemicals and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs).

PubMed

Shimazu, Sayuri; Inui, Hideyuki; Ohkawa, Hideo

2011-04-13

Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

In vitro characterization of sarizotan metabolism: hepatic clearance, identification and characterization of metabolites, drug-metabolizing enzyme identification, and evaluation of cytochrome p450 inhibition.

PubMed

Gallemann, Dieter; Wimmer, Elmar; Höfer, Constance C; Freisleben, Achim; Fluck, Markus; Ladstetter, Bernhard; Dolgos, Hugues

2010-06-01

In vitro biotransformation studies of sarizotan using human liver microsomes (HLM) showed aromatic and aliphatic monohydroxylation and dealkylation. Recombinant cytochromes P450 (P450) together with P450-selective inhibitors in HLM/hepatocyte cultures were used to evaluate the relative contribution of different P450s and revealed major involvement of CYP3A4, CYP2C9, CYP2C8, and CYP1A2 in sarizotan metabolism. The apparent K(m, u) and V(max) of sarizotan clearance, as investigated in HLM, were 9 microM and 3280 pmol/mg/min, predicting in vivo hepatic clearance of 0.94 l/h, which indicates that sarizotan is a low-clearance compound in humans and suggests nonsaturable metabolism at the targeted plasma concentration (< or =1 microM). This finding is confirmed by the reported human clearance (CL/F of 3.6-4.4 l/h) and by the dose-linear area under the curve increase observed with doses up to 25 mg. The inhibitory effect of sarizotan toward six major P450s was evaluated using P450-specific marker reactions in pooled HLM. K(i, u) values of sarizotan against CYP2C8, CYP2C19, and CYP3A4 were >10 microM, whereas those against CYP2D6 and CYP1A2 were 0.43 and 8.7 microM, respectively. Based on the estimates of sarizotan concentrations at the enzyme active sites, no clinically significant drug-drug interactions (DDIs) due to P450 inhibition are expected. This result has been confirmed in human DDI studies in which no inhibition of five major P450s was observed in terms of marker metabolite formation.

Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon.

PubMed

Sun, G; Grindstaff, R D; Thai, S F; Lambert, G R; Tully, D B; Dix, D J; Nesnow, S

2007-02-01

This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon, on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague Dawley rats. Rats were dosed with the conazoles at three dose levels by gavage for 14 days: myclobutanil (150, 75, and 10mgkg(-1) body weight day(-1); triadimefon (115, 50, and 10 mg kg(-1) body weight day-'), which included their maximum tolerated dose levels (MTD). Both myclobutanil and triadimefon significantly induced pentoxyresorufin O-depentylase activities at their MTD levels: myclobutanil, 8.1-fold at 150mgkg(-1) body weight day- ; and triadimefon, 18.5-fold at 115mgkg(-1) body weight day-'. Benzyloxyresorufin O-debenzylase activities were similarly increased: myclobutanil, 13.3-fold; triadimefon, 27.7-fold. Quantitative real-time reverse-transcription polymerase chain reaction assays were used to characterize the mRNA expression of specific CYP genes induced by these two conazoles. Myclobutanil and triadimefon treatment at their MTD levels significantly increased rat hepatic mRNA expression of CYP2B1 (14.3- and 54.6-fold), CYP3A23/3A1 (2.2- and 7.3-fold), and CYP3A2 (1.5- and 1.7-fold). Western immunoblots of rat hepatic microsomal proteins identified significantly increased levels of CYP isoforms after myclobutanil or triadimefon treatment at their MTD levels: CYP2BI/2 (4.8- and 5.3-fold), and CYP3A1 (2.2- and 2.9-fold). Triadimefon also increased CYP3A2 immunoreactive protein levels 1.8-fold. These results indicate that triadimefon and myclobutanil, like other triazole-containing conazoles, induced CYP2B and CYP3A families of cytochromes in rat liver.

Effect of hypoxia alone or combined with inflammation and 3-methylcholanthrene on hepatic cytochrome P450 in conscious rabbits

PubMed Central

Kurdi, J; Maurice, H; El-Kadi, A O S; Ong, H; Dalkara, S; Bélanger, P M; du Souich, P

1999-01-01

To investigate the effect of moderate hypoxia alone or combined with an inflammatory reaction or after 3-methylcholanthrene (3MC) pre-treatment on cytochrome P450 (P450), conscious rabbits were exposed for 24 h to a fractional concentration of inspired O2 of 10% (mean PaO2 of 34 mmHg). Hypoxia decreased theophylline metabolic clearance (ClM) from 1.73±0.43 to 1.48±0.13 ml min−1 kg−1 (P<0.05), and reduced (P<0.05) the formation clearance of theophylline metabolites, 3-methylxanthine (3MX), 1-methyluric acid (1MU) and 1,3-dimethyluric acid (1,3DMU). Hypoxia reduced the amount of CYP1A1 and 1A2 but increased CYP3A6 proteins.Turpentine-induced inflammatory reaction reduced (P<0.05) the formation clearance of 3MX, 1MU, and 1,3DMU, and diminished the amount of CYP1A1, 1A2 and 3A6 proteins. However, when combined with hypoxia, inflammation partially prevented the decrease in ClM, especially by impeding the reduction of 1,3DMU. The amount of CYP1A1 and 1A2 remained reduced but the amount of CYP3A6 protein returned to normal values.Pre-treatment with 3MC augmented the ClM by 114% (P<0.05) due to the increase in the formation clearance of 3MX, 1MU and 1,3DMU. 3MC treatment increased the amount of CYP1A1 and 1A2 proteins. Pre-treatment with 3MC prevented the hypoxia-induced decrease in amount and activity of the P450.It is concluded that acute moderate hypoxia and an inflammatory reaction individually reduce the amount and activity of selected apoproteins of the P450. However, the combination of hypoxia and the inflammatory reaction restores P450 activity to near normal values. On the other hand, pre-treatment with 3MC prevents the hypoxia-induced depression of the P450. PMID:10510446

A new CYP3A5 variant, CYP3A5*11, is shown to be defective in nifedipine metabolism in a recombinant cDNA expression system

PubMed Central

Lee, Su-Jun; van der Heiden, Ilse P; Goldstein, Joyce A; van Schaik, Ron HN

2012-01-01

A new CYP3A5 variant, CYP3A5*11, was found in a single white European by DNA sequencing. The CYP3A5*11 allele contains a single nucleotide polymorphism (SNP) (g.3775 A>G) in exon 2 which results in a Tyr53Cys substitution and a g.6986A>G splice change, the latter SNP previously reported in the defective CYP3A5*3 allele. However, the CYP3A5*3 is not a null allele because this variant is associated with leaky splicing, resulting in small amounts of functional protein still being produced. We therefore constructed a cDNA coding for the newly identified CYP3A5.11 protein by site-directed mutagenesis, expressed it in Escherichia coli and partially purified it. While bacteria transformed with wild-type CYP3A5*1 cDNA expressed predominantly cytochrome P450, those transfected with CYP3A5*11 expressed a significant amount of denatured cytochrome P420 in addition to cytochrome P450, suggesting the protein to be unstable. CYP3A5.11 exhibited a 38% decrease in the Vmax for nifedipine metabolism, a 2.7-fold increase in the Km, and a 4.4-fold decrease in the CLint of nifedipine compared with CYP3A5.1. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping procedure was developed, and used to genotyping DNA of 500 white individuals for CYP3A5*11. No additional examples of this allele were identified. In summary, individuals carrying the rare CYP3A5*11 allele are predicted to have lower metabolism of CYP3A5 substrates than individuals expressing CYP3A5*3. PMID:17035598

Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis Flowers[W][OPEN

PubMed Central

Ginglinger, Jean-François; Boachon, Benoit; Höfer, René; Paetz, Christian; Köllner, Tobias G.; Miesch, Laurence; Lugan, Raphael; Baltenweck, Raymonde; Mutterer, Jérôme; Ullmann, Pascaline; Beran, Franziska; Claudel, Patricia; Verstappen, Francel; Fischer, Marc J.C.; Karst, Francis; Bouwmeester, Harro; Miesch, Michel; Schneider, Bernd; Gershenzon, Jonathan; Ehlting, Jürgen; Werck-Reichhart, Danièle

2013-01-01

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (−)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined. PMID:24285789

Immunolocalization of steroidogenic enzymes in the vaginal mucous of Galea spixii during the estrous cycle.

PubMed

Dos Santos, Amilton Cesar; Conley, Alan James; de Oliveira, Moacir Franco; Oliveira, Gleidson Benevides; Viana, Diego Carvalho; Assis Neto, Antônio Chaves de

2017-04-24

The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses. Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry. Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues. Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.

Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

PubMed

De Silva, Matharage S I; Dayton, Adam W; Rhoten, Lance R; Mallett, John W; Reese, Jared C; Squires, Mathieu D; Dalley, Andrew P; Porter, James P; Judd, Allan M

2018-06-01

In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1. Copyright © 2018 Elsevier Inc. All rights reserved.

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

PubMed Central

Helliwell, Chris A.; Chandler, Peter M.; Poole, Andrew; Dennis, Elizabeth S.; Peacock, W. James

2001-01-01

We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA12. A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA12. Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase. PMID:11172076

Endosulfan-alpha Induces CYP26 and CYP3A4 by Activating the Pregnane X Receptor But Not the Constitutive Androstane Receptor

DTIC Science & Technology

2006-01-01

CYP3A4 gene expression by organochlorine pesticides . Biochem Pharmacol 64:1513-1519. Dinham B (1993) The Pesticide Hazard. A Global Health and...Coumoul X, Diry M and Barouki R (2002) PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides . Biochem Pharmacol 64:1513...system: CYP3A4 and CYP2B6 induction by pesticides . Biochem Pharmacol 68:2347-2358. 71 Nelson D (2003) Cytochrome P450 Homepage (http

RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

PubMed Central

2012-01-01

Background Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius. PMID:22226239

The Potassium Channel, Kir3.4 Participates in Angiotensin II-Stimulated Aldosterone Production by a Human Adrenocortical Cell Line

PubMed Central

Oki, Kenji; Plonczynski, Maria W.; Lam, Milay Luis; Gomez-Sanchez, Elise P.

2012-01-01

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4. PMID:22798349

Cytochrome P450-mediated metabolism of vitamin D

PubMed Central

Jones, Glenville; Prosser, David E.; Kaufmann, Martin

2014-01-01

The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field. PMID:23564710

Chronic effects of ethanol and/or darunavir/ritonavir on U937 monocytic cells: Regulation of cytochrome P450 and antioxidant enzymes, oxidative stress, and cytotoxicity

PubMed Central

Rao, P.S.S.; Kumar, Santosh

2015-01-01

Background Our recent study has shown that acute treatment with ethanol increases oxidative stress and cytotoxicity through cytochrome P450 2E1 (CYP2E1)-mediated pathway in U937 monocytic cells. U937 cells are derived from blood monocytes and are considered as the model system for HIV-related study. Since the prevalence of alcohol use in HIV-infected population is high, and HIV+ patients are on antiretroviral therapy (ART) soon after they are diagnosed, it is important to study the interactions between ethanol and ART in monocytes. Methods This study examined the chronic effects of ethanol and ART (darunavir/ritonavir), alone and in combination, on expression/levels of cytochrome P450 enzymes (CYPs), antioxidant enzymes (AOEs), reactive oxygen species (ROS), and cytotoxicity in U937 cells. The mRNA and protein levels were measured using quantitative RTPCR and Western blot, respectively. ROS and cytotoxicity were measured using flow cytometry and XTT assay, respectively. Results While chronic ART treatment increased CYP2E1 protein expression by 2-fold, ethanol and ethanol+ART increased CYP2E1 by ~5-fold. In contrast, ART and ethanol treatments decreased CYP3A4 protein expression by 38±17% and 74±15%, respectively, and the combination additively decreased CYP3A4 level by 90±8%. Expressions of superoxide dismutase (SOD1) and peroxiredoxin (PRDX6) were decreased by both ethanol and ART, however, the expressions of SOD2 and catalase were unaltered. These results suggested increased ethanol metabolism, increased ART accumulation, and decreased defense against ROS. Therefore, we determined the effects of ethanol and ART on ROS and cytotoxicity. While ART showed a slight increase, ethanol and ethanol+ART displayed significant increase in ROS and cytotoxicity. Moreover, the combination showed additive effects on ROS and cytotoxicity. Conclusions These results suggest that chronic ethanol, in the absence and presence of ART, increases ROS and cytotoxicity in monocytes, perhaps via CYPs and AOEs mediated pathways. This study has clinical implications in HIV+ alcohol users who are on ART. PMID:26727525

Constitutive overexpression of cytochrome P450 associated with imidacloprid resistance in Laodelphax striatellus (Fallén).

PubMed

Elzaki, Mohammed Esmail Abdalla; Zhang, Wanfang; Feng, Ai; Qiou, Xiaoyan; Zhao, Wanxue; Han, Zhaojun

2016-05-01

Imidacloprid is a principal insecticide for controlling rice planthoppers worldwide. Resistance to imidacloprid has been reported in a field population of Laodelphax striatellus. The present work was conducted to study the molecular mechanisms of imidacloprid resistance. An imidacloprid-resistant strain was produced by selecting a field population with imidacloprid for 24 generations. Piperonyl butoxide (PBO) showed a 1.70-fold synergistic effect. Enzyme activity assays were conducted, and cytochrome P450 monooxygenase showed 1.88-fold activity. The mRNA expression levels of 57 P450 genes were compared. Four CYP genes were found to be overexpressed and significantly different to the susceptible strain. Four strains were selected with imidacloprid for a short period, and the expression levels of ten identified detoxification genes were then compared. Only CYP353D1v2 overexpressed and was significantly different to the susceptible strain. Strong correlation was found between CYP353D1v2 expression levels and imidacloprid treatments. Additionally, gene-silencing RNAi via dsRNA feeding showed that depressing the expression of CYP353D1v2 could significantly enhance the sensitivity of L. striatellus to imidacloprid. Constitutive overexpression of four CYP genes was associated with imidacloprid resistance in long-term selection, and expression of CYP353D1v2 with imidacloprid resistance in short-term selection in L. striatellus. © 2015 Society of Chemical Industry.

Flower colour and cytochromes P450†

PubMed Central

Tanaka, Yoshikazu; Brugliera, Filippa

2013-01-01

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

Flower colour and cytochromes P450.

PubMed

Tanaka, Yoshikazu; Brugliera, Filippa

2013-02-19

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer DrugS⃞

PubMed Central

Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Johnson, William W.

2011-01-01

An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b5 and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b5. The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a KI of 24 μM and a kinact of 0.04 min−1. This KI is significantly greater than the clinical OSI-930 Cmax of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site for oxidation of the thiophene sulfur to give the sulfoxide, which has previously been shown to be a significant metabolite of OSI-930. Because OSI-930 is an inactivator of P450 3A4 but does not exhibit any effect on P450 3A5 activity under the same conditions, it may be an appropriate probe for exploring unique aspects of these two very similar P450s. PMID:21068193

Ectopic expression of an apple cytochrome P450 gene MdCYPM1 negatively regulates plant photomorphogenesis and stress response in Arabidopsis.

PubMed

An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-01-29

Cytochrome P450s play an important role in plant growth and are involved in multiple stresses response. However, little is known about the functions of cytochrome P450s in apple. Here, a Malus × domestica cytochrome P450 monooxygenase 1 gene, MdCYPM1, was identified and subsequently cloned from apple 'Gala' (Malus × domestica). To verify the functions of MdCYPM1, we generated transgenic Arabidopsis plants expressing the apple MdCYPM1 gene under the control of the Cauliflower mosaic virus 35S promoter. Four transgenic lines (#3, #5, #7 and #8) were selected for further study. The transgenic plants exhibited a series of skotomorphogenesis phenotypes relative to wild-type controls, such as reduction of the chlorophyll, anthocyanins content and hypocotyls elongation. In addition, overexpression of MdCYPM1 influenced auxin transport and flowering time in transgenic Arabidopsis. Furthermore, MdCYPM1 expression was induced by salt and mannitol treatments, and the transgenic plants were negatively regulated by salinity and osmotic stresses during germination. These results suggest that MdCYPM1 plays a vital role in plant growth and development. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of three cytochrome P450 genes in the Chagas' disease vector Triatoma infestans: Expression analysis in deltamethrin susceptible and resistant populations.

PubMed

Grosso, Carla G; Blariza, María J; Mougabure-Cueto, Gastón; Picollo, María I; García, Beatriz A

2016-10-01

Cytochrome P450 monooxygenases play a predominant role in the metabolism of insecticides. Many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides. The increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects. As pyrethroid resistance has been detected in Triatoma infestans, it was of interest to analyze genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, the cDNA sequences of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6) were identified in this species. Primers and specific Taqman probes were designed from these sequences to determine their expression by quantitative PCR. The mRNA levels of the cytochrome P450 genes identified were determined from total RNA extracted from pools of fat body collected from individuals of different resistant and susceptible strains of T. infestans, and at different interval times after the topical application of the lethal doses 50% (LD50) of deltamethrin on the ventral abdomen of insects belonging to the different populations analyzed. It was detected overexpression of the CYP4EM7 gene in the most resistant strain of T. infestans and the expression of the three cytochrome P450 genes isolated was induced by deltamethrin in the susceptible and resistant populations included in this study. These results suggest that these genes would be involved in the detoxification of deltamethrin and support the hypothesis that considers to the cytochrome P450 genes of importance in the development of pyrethroid resistance. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4.

PubMed

Casabar, Richard C T; Wallace, Andrew D; Hodgson, Ernest; Rose, Randy L

2006-10-01

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.

A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance.

PubMed

Terhzaz, Selim; Cabrero, Pablo; Brinzer, Robert A; Halberg, Kenneth A; Dow, Julian A T; Davies, Shireen-A

2015-12-01

The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation.

PubMed

Chowdhury, Indrajit; Thomas, Kelwyn; Zeleznik, Anthony; Thompson, Winston E

2016-05-01

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs. © 2016 The authors.

Diet-induced non-alcoholic fatty liver disease affects expression of major cytochrome P450 genes in a mouse model.

PubMed

Chiba, Tsuyoshi; Noji, Keiko; Shinozaki, Shohei; Suzuki, Sachina; Umegaki, Keizo; Shimokado, Kentaro

2016-12-01

Non-alcoholic fatty liver disease (NAFLD) is associated with impaired liver function, and resveratrol could suppress NAFLD progression. This study examined the effects of NAFLD on the expression of major cytochrome P450 (CYP) subtypes in the liver and whether the expression could be attenuated by resveratrol. C57BL/6 mice (male, 10 weeks of age) were fed a high-fat and high-sucrose (HFHS) diet to induce NAFLD. Major Cyp subtype mRNA expression in the liver was measured by real-time RT-PCR. Body and liver weights at 4 and 12 weeks were significantly higher in mice fed the HFHS diet compared with control. The HFHS diet significantly increased the accumulation of cholesterol and triglycerides at 12 weeks. Under this condition, the HFHS diet increased the expression of Cyp1a2 and decreased that of Cyp3a11 at 1 week and thereafter. On the other hand, Cyp1a1, 2b10 and 2c29 mRNA expression levels in the liver were significantly increased at 12 weeks only. Resveratrol (0.05% (w/w) in diet) slightly suppressed lipid accumulation in the liver, but failed to recover impaired Cyp gene expression levels in NAFLD. Drug metabolism may be impaired in NAFLD, and each Cyp subtype is regulated in a different manner. © 2016 Royal Pharmaceutical Society.

Cytochrome P450 Initiates Degradation of cis-Dichloroethene by Polaromonas sp. Strain JS666

PubMed Central

Nishino, Shirley F.; Shin, Kwanghee A.; Gossett, James M.

2013-01-01

Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes. PMID:23354711

Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats.

PubMed

Liu, Aiming; Yang, Julin; Zhao, Xin; Jiao, Xiaolan; Zhao, Weihong; Ma, Qing; Tang, Zhiyuan; Dai, Renke

2011-01-01

Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2, 2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

Amplification of a Cytochrome P450 Gene Is Associated with Resistance to Neonicotinoid Insecticides in the Aphid Myzus persicae

PubMed Central

Puinean, Alin M.; Foster, Stephen P.; Oliphant, Linda; Denholm, Ian; Field, Linda M.; Millar, Neil S.; Williamson, Martin S.; Bass, Chris

2010-01-01

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2–16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae. PMID:20585623

Hepatotoxicity of Herbal Supplements Mediated by Modulation of Cytochrome P450

PubMed Central

Chen, Taosheng

2017-01-01

Herbal supplements are a significant source of drug-drug interactions (DDIs), herb-drug interactions, and hepatotoxicity. Cytochrome P450 (CYP450) enzymes metabolize a large number of FDA-approved pharmaceuticals and herbal supplements. This metabolism of pharmaceuticals and supplements can be augmented by concomitant use of either pharmaceuticals or supplements. The xenobiotic receptors constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) can respond to xenobiotics by increasing the expression of a large number of genes that are involved in the metabolism of xenobiotics, including CYP450s. Conversely, but not exclusively, many xenobiotics can inhibit the activity of CYP450s. Induction of the expression or inhibition of the activity of CYP450s can result in DDIs and toxicity. Currently, the United States (US) Food and Drug Administration does not require the investigation of the interactions of herbal supplements and CYP450s. This review provides a summary of herbal supplements that inhibit CYP450s, induce the expression of CYP450s, and/or whose toxicity is mediated by CYP450s. PMID:29117101

A novel cytochrome P450 CYP6AB14 gene in spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification

USDA-ARS?s Scientific Manuscript database

Cytochrome P450 monooxygenases (P450) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th...

A novel cytochrome P450 CYP6AB14 gene in Spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification

USDA-ARS?s Scientific Manuscript database

Cytochrome P450 monooxygenases (P450) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th...

Characterization of human liver cytochrome P-450 enzymes involved in the O-demethylation of a new P-glycoprotein inhibitor HM-30181.

PubMed

Paek, In Bok; Kim, Sung Yeon; Kim, Maeng Sup; Kim, John; Lee, Gwansun; Lee, Hye Suk

2007-08-01

HM-30181, 4-oxo-4H-chromene-2-carboxylic acid [2-(2-{4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-phenyl}-2H-tetrazol-5-yl)-4,5-dimethoxy-phenyl]-amide, is a new P-glycoprotein inhibitor with the potential to increase the cytotoxic activity of orally coadministered paclitaxel. This study was performed to characterize human cytochrome P-450 (CYP) enzymes involved in the metabolism of HM-30181 to 4- or 5-O-desmethyl-HM-30181 (M2) and 6- or 7-O-desmethyl-HM-30181 (M3) and to investigate the inhibitory potential of HM-30181 on CYP enzymes in human liver microsomes. CYP3A4 was identified as the major isozyme responsible for the O-demethylation of HM-30181 to M2 and M3 based on the correlation analysis, chemical inhibition and immuno-inhibition study and metabolism in cDNA-expressed human CYP isozymes. HM-30181 itself had no inhibitory effects on CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 in human liver microsomes, suggesting the possibility that the pharmacokinetics of HM-30181 could be changed with coadministration of known CYP3A4 inducers or inhibitors.

Enhanced phytoremediation of mixed heavy metal (mercury)-organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1.

PubMed

Zhang, Yuanyuan; Liu, Junhong; Zhou, Yuanming; Gong, Tingyun; Wang, Jing; Ge, Yinlin

2013-09-15

Soil contamination is a global environmental problem and many efforts have been made to find efficient remediation methods over the last decade. Moreover, remediation of mixed contaminated soils are more difficult. In the present study, transgenic alfalfa plants pKHCG co-expressing glutathione S-transferase (GST) and human P450 2E1 (CYP2E1) genes were used for phytoremediation of mixed mercury (Hg)-trichloroethylene (TCE) contaminants. Simultaneous expression of GST and CYP2E1 may produce a significant synergistic effect, and leads to improved resistance and accumulation to heavy metal-organic complex contaminants. Based on the tolerance and accumulation assays, pKHCG transgenic plants were more resistant to Hg/TCE complex pollutants and many folds higher in Hg/TCE-accumulation than the non-transgenic control plants in mixed contaminated soil. It is confirmed that GST and CYP2E1 co-expression may be a useful strategy to help achieve mixed heavy metal-organic pollutants phytoremediation. Copyright © 2013 Elsevier B.V. All rights reserved.

Ascorbic acid transported by sodium-dependent vitamin C transporter 2 stimulates steroidogenesis in human choriocarcinoma cells.

PubMed

Wu, Ximei; Iguchi, Takuma; Itoh, Norio; Okamoto, Kousuke; Takagi, Tatsuya; Tanaka, Keiichi; Nakanishi, Tsuyoshi

2008-01-01

Reduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48-h dose dependently increased progesterone and estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the maximal velocity of AA uptake by approximately 50%, but apparent affinity values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and estradiol production. In addition, inhibition of MAPK kinase-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MAPK kinase-ERK signaling is not involved in AA-induced steroidogenesis.

Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

PubMed

Hu, Z; Lin, Q; Chen, H; Li, Z; Yin, F; Feng, X

2014-12-01

Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24 h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24 h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes.

PubMed

Zhang, Yixi; Yang, Yuanxue; Sun, Huahua; Liu, Zewen

2016-12-01

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hepatic microsomal cytochromes P450 in mink fed Saginaw Bay carp (SBC)

USGS Publications Warehouse

Melancon, M.J.; LeCaptain, L.; Rattner, B.A.; Heaton, S.; Aulerich, R.; Tillitt, D.; Stegeman, John J.; Woodin, B.

1992-01-01

Livers from mink fed diets containing 0% (n = 12), 10% (n = 11), 20% (n = 12) and 40% (n = 10) SBC for 6 months contained 0.1, 2.2, 3.6, and 6.3 ug/g total PCBs, respectively. Hepatic microsomes were prepared and assayed for protein, arylhydrocarbon hydroxylase (AHH), benzyloxyresorufin-O-dealkylase (BROD), ethoxy-ROD (ER0D), pentoxy-ROD (PROD), and ethoxycoumarin-OD (ECOD). Mink fed SBC had increased AHH, EROD, and ECOD (group means 2.2-3.4 X control means), decreased BROD and unchanged PROD (the latter 2 assays indicators for phenobarbital-type induction in mammals). Three samples from each group were examined by western blot using a polyclonal anti-P450llB antibody and a monoclonal anti-P450lA antibody (MAb 1-12-3). Mink fed SBC showed induction of a protein recognized by anti-P450lA (8 X control), but had little protein recognized by anti-P450IlB. The monooxygenase activities and western blot data give a consistent picture of MC-type but not PB-type induction in mink fed SBC.

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice

PubMed Central

Zang, Zhi-Jun; Tang, Hong-Feng; Tuo, Ying; Xing, Wei-Jie; Ji, Su-Yun; Gao, Yong; Deng, Chun-Hua

2016-01-01

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg−1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3β-HSD) and the following promotion of testosterone synthesis in vivo. PMID:26608944

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice.

PubMed

Zang, Zhi-Jun; Tang, Hong-Feng; Tuo, Ying; Xing, Wei-Jie; Ji, Su-Yun; Gao, Yong; Deng, Chun-Hua

2016-01-01

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg-1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3β-HSD) and the following promotion of testosterone synthesis in vivo.

Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

PubMed Central

Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

2015-01-01

NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

The effect of ZnO nanoparticles on liver function in rats

PubMed Central

Tang, Hua-Qiao; Xu, Min; Rong, Qian; Jin, Ru-Wen; Liu, Qi-Ji; Li, Ying-Lun

2016-01-01

Zinc oxide (ZnO) is widely incorporated as a food additive in animal diets. In order to optimize the beneficial effects of ZnO and minimize any resultant environmental pollution, ZnO nanoparticles are often used for delivery of the zinc. However, the possible toxic effects of ZnO nanoparticles, including effects on cytochrome P450 (CYP450) enzymes, have not been evaluated. In this study, we investigated the effect of ZnO nanoparticles, in doses used in animal feeds, on CYP450 enzymes, liver and intestinal enzymes, liver and kidney histopathology, and hematologic indices in rats. We found that liver and kidney injury occurred when the concentrations of ZnO nanoparticles in feed were 300–600 mg/kg. Also, liver mRNA expression for constitutive androstane receptor was suppressed and mRNA expression for pregnane X receptor was induced when feed containing ZnO nanoparticles was given at a concentration of 600 mg/kg. Although the expression of mRNA for CYP 2C11 and 3A2 enzymes was induced by ZnO nanoparticles, the activities of CYP 2C11 and 3A2 were suppressed. While liver CYP 1A2 mRNA expression was suppressed, CYP 1A2 activity remained unchanged at all ZnO nanoparticle doses. Therefore, it has been concluded that ZnO nanoparticles, in the doses customarily added to animal feed, changed the indices of hematology and blood chemistry, altered the expression and activity of hepatic CYP enzymes, and induced pathological changes in liver and kidney tissues of rats. These findings suggest that greater attention needs to be paid to the toxic effects of ZnO nanoparticles in animal feed, with the possibility that the doses of ZnO should be reduced. PMID:27621621

Regulation of oocyte maturation in fish.

PubMed

Nagahama, Yoshitaka; Yamashita, Masakane

2008-06-01

A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17alpha, 20beta-dihydroxy-4-pregnen-3-one, 17alpha, 20beta-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17alpha,20beta-DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17alpha,20beta-DP in response to LH is correlated with decreases in P450c17 (P450c17-I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17-II) and 20beta-hydroxysteroid dehydrogenase (20beta-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17alpha, 20beta-DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH(2) terminus at lysine 57.

Betanin attenuates paraquat-induced liver toxicity through a mitochondrial pathway.

PubMed

Han, Junyan; Zhang, Zongju; Yang, Shaobin; Wang, Jun; Yang, Xuelian; Tan, Dehong

2014-08-01

We attempted to determine whether betanin (from natural pigments) that has anti-oxidant properties would be protective against paraquat-induced liver injury in Sprague-Dawley rats. Paraquat was injected intraperitoneally into rats to induce liver toxicity. The rats were randomly divided into four groups: a control group, a paraquat group, and two groups that received betanin at doses of 25 and 100mg/kg/day three days before and two days after they were administered paraquat. We evaluated liver histopathology, serum liver enzymatic activities, oxidative stress, cytochrome P450 (CYP) 3A2 mRNA expression, and mitochondrial damage. The rats that were injected with paraquat incurred liver injury, evidenced by histological changes and elevated serum aspartate aminotransferase and alanine aminotransferase levels; paraquat also led to oxidative stress, an increase of cytochrome P450 3A2 mRNA expression, and mitochondrial damage, indicated by mitochondrial membrane swelling, reduced mitochondrial cytochrome C, and apoptosis-inducing factor protein levels. Pathological damage and all of the above mentioned markers were lesser in the animals treated with betanin than in those who received paraquat alone. Betanin had a protective effect against paraquat-induced liver damage in rats. The mechanism of the protection appears to be the inhibition of CYP 3A2 expression and protection of mitochondria. Copyright © 2014 Elsevier Ltd. All rights reserved.

Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice

EPA Science Inventory

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we ...

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osawa, Y.; Coon, M.J.

1987-08-01

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, (1,2,6,7,16,17-3H)progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting (1,7,16-3H)progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of /sup 3/H/submore » 2/O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of (1,7,16-/sup 3/H)progesterone and (4-14C)progesterone was employed.« less

Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Young Joo; Seoul National University Bundang Hospital, Seoul; Lee, Eun Kyung

Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR{sup −/−} mice, but not in hypothyroid PXR{sup −/−} mice. In contrast, expression of Cyp2b10 was inducedmore » in both WT and PXR{sup −/−} hypothyroid mice, and this induction was abolished in CAR{sup −/−} mice and in and CAR{sup −/−} PXR{sup −/−} double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR{sup −/−} mice and lowest in WT and PXR{sup −/−} mice. Hypothyroid WT or PXR{sup −/−} mice survived chronic CBZ treatment, but all hypothyroid CAR{sup −/−} and CAR{sup −/−} PXR{sup −/−} mice died, with CAR{sup −/−}PXR{sup −/−} mice surviving longer than CAR{sup −/−} mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice. ► Hypothyroidism sensitizes CAR null mice to toxic effects of carbamazepine.« less

Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera

PubMed Central

Johnson, Reed M.; Mao, Wenfu; Pollock, Henry S.; Niu, Guodong; Schuler, Mary A.; Berenbaum, May R.

2012-01-01

Background Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. Methodology/Principal Findings We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. Conclusions/Significance These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey. PMID:22319603

Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

PubMed

Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

2017-07-01

Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y) and glial (U373-MG) cell lines following the exposure of MCP.

Expression patterns of bark beetle cytochromes P450 during host colonization: Likely physiological functions and potential targets for pest management

Treesearch

Dezene P. W. Huber; Melissa Erickson; Christian Leutenegger; Joerg Bohlmann; Steven J. Seybold

2007-01-01

Cytochromes P450 family genes (P450s) are found in a diverse array of organisms ranging from bacteria to mammals to plants to arthropods. Although there are exceptions to this rule, organisms generally contain a fairly large number of P450 genes and pseudogenes in their genomes. For instance, among arthropods whose genomes are well characterized, the mosquito,

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis.

PubMed

Matsuno, Michiyo; Nagatsu, Akito; Ogihara, Yukio; Ellis, Brian E; Mizukami, Hajime

2002-03-13

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.

Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities.

PubMed

Backes, W L; Sequeira, D J; Cawley, G F; Eyer, C S

1993-12-01

1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation); however, m-xylene treatment led to elevated P450 2B1/2B2 levels without significantly suppressing P450 2C11. m-Xylene produced significant increases in activities efficiently catalysed by both isozymes. Therefore, the unique induction pattern observed after m-xylene treatment can be accounted for by induction of P450 2B1/2B2 without concomitant suppression of P450 2C11.

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

PubMed Central

Yamamoto, A M; Mura, C; De Lemos-Chiarandini, C; Krishnamoorthy, R; Alvarez, F

1993-01-01

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:7685669

Generation of a mouse model with a reversible hypomorphic cytochrome P450 reductase gene: utility for tissue-specific rescue of the reductase expression, and insights from a resultant mouse model with global suppression of P450 reductase expression in extrahepatic tissues.

PubMed

Wei, Yuan; Zhou, Xin; Fang, Cheng; Li, Lei; Kluetzman, Kerri; Yang, Weizhu; Zhang, Qing-Yu; Ding, Xinxin

2010-07-01

A mouse model termed Cpr-low (CL) was recently generated, in which the expression of the cytochrome P450 reductase (Cpr) gene was globally down-regulated. The decreased CPR expression was accompanied by phenotypical changes, including reduced embryonic survival, decreases in circulating cholesterol, increases in hepatic P450 expression, and female infertility (accompanied by elevated serum testosterone and progesterone levels). In the present study, a complementary mouse model [named reversible-CL (r-CL)] was generated, in which the reduced CPR expression can be reversed in an organ-specific fashion. The neo cassette, which was inserted into the last Cpr intron in r-CL mice, can be deleted by Cre recombinase, thus returning the structure of the Cpr gene (and hence CPR expression) to normal in Cre-expressing cells. All previously identified phenotypes of the CL mice were preserved in the r-CL mice. As a first application of the r-CL model, we have generated an extrahepatic-CL (xh-CL) mouse for testing of the functions of CPR-dependent enzymes in all extrahepatic tissues. The xh-CL mice, generated by mating of r-CL mice with albumin-Cre mice, had normal CPR expression in hepatocytes but down-regulated CPR expression elsewhere. They were indistinguishable from wild-type mice in body and liver weights, circulating cholesterol levels, and hepatic microsomal P450 expression and activities; however, they still showed elevated serum testosterone and progesterone levels and sterility in females. Embryonic lethality was prevented in males, but apparently not in females, indicating a critical role for fetal hepatic CPR-dependent enzymes in embryonic development, at least in males.

Crude oil exposure results in oxidative stress-mediated dysfunctional development and reproduction in the copepod Tigriopus japonicus and modulates expression of cytochrome P450 (CYP) genes.

PubMed

Han, Jeonghoon; Won, Eun-Ji; Hwang, Dae-Sik; Shin, Kyung-Hoon; Lee, Yong Sung; Leung, Kenneth Mei-Yee; Lee, Su-Jae; Lee, Jae-Seong

2014-07-01

In this study, we investigated the effects of the water-accommodated fraction (WAF) of crude oil on the development and reproduction of the intertidal copepod Tigriopus japonicus through life-cycle experiments. Furthermore, we investigated the mechanisms underlying the toxic effects of WAF on this benthic organism by studying expression patterns of cytochrome P450 (CYP) genes. Development of T. japonicus was delayed and molting was interrupted in response to WAF exposure. Hatching rate was also significantly reduced in response to WAF exposure. Activities of antioxidant enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), and catalase (CAT) were increased by WAF exposure in a concentration-dependent manner. These results indicated that WAF exposure resulted in oxidative stress, which in turn was associated with dysfunctional development and reproduction. To evaluate the involvement of cytochrome P450 (CYP) genes, we cloned the entire repertoire of CYP genes in T. japonicus (n=52) and found that the CYP genes belonged to five different clans (i.e., Clans 2, 3, 4, mitochondrial, and 20). We then examined expression patterns of these 52 CYP genes in response to WAF exposure. Three TJ-CYP genes (CYP3024A2, CYP3024A3, and CYP3027C2) belonging to CYP clan 3 were significantly induced by WAF exposure in a time- and concentration-dependent manner. We identified aryl hydrocarbon responsive elements (AhRE), xenobiotic responsive elements (XREs), and metal response elements (MRE) in the promoter regions of these three CYP genes, suggesting that these genes are involved in detoxification of toxicants. Overall, our results indicate that WAF can trigger oxidative stress and thus induce dysfunctional development and reproduction in the copepod T. japonicus. Furthermore, we identified three TJ-CYP genes that represent potential biomarkers of oil pollution. Copyright © 2014 Elsevier B.V. All rights reserved.

Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptotic cell death in the embryonic vasculature with embryotoxicity

USGS Publications Warehouse

Cantrell, Susannah M.; Joy-Schlezinger, Jennifer; Stegeman, John J.; Tillitt, Donald E.; Hannington, Mark D.

1998-01-01

Vertebrate embryos are particularly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Identification of tissues that are susceptible to the adverse effects of TCDD is requisite for understanding the embryo toxic effects of TCDD. The objective of the present study was to quantitate the temporal appearance of and dose dependence of apoptosis in TCDD-exposed medaka embryos (Oryzias latipes). A fluorescent-based DNA end-labeling assay provided a sensitive method for detection of TCDD-induced apoptosis in tissue sections of medaka embryos. Apoptotic cells were readily apparent in the medial yolk vein at all observed embryonic stages in TCDD-exposed embryos. Slope-comparison analysis indicated that TCDD-induced programmed cell death in the embryonic medial yolk vein was mechanistically linked to embryo mortality. These data are consistent with the hypothesis that vascular damage contributes to the acute embryo toxic effects of TCDD. However, as sublethal concentrations of dioxin-like compounds are more typical of environmental exposures, tissue damage was also assessed in medaka fry that were exposed to low doses of TCDD during embryonic development. Cell death was detected in gill and digestive tissues in visibly healthy medaka fry that had been exposed to low doses of TCDD during embryonic development. Increased expression of cytochrome P450 1A is a major biochemical consequence of TCDD exposure and is often used as a biomarker for exposure to dioxin-like compounds. Therefore, we compared the tissue distribution of TCDD-induced P450 1A expression and TCDD-induced programmed cell death. TCDD-induced programmed cell death co-localized with TCDD-induced P450 1A expression in both embryos and in visibly healthy post-hatch fry. Our results suggest that aberrant programmed cell death may be a suitable marker for exposure of feral organisms to dioxin-like compounds.

Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker ( sup 3 H)GBR-12935

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalow, W.; Tyndale, R.F.; Niznik, H.B.

1990-02-26

P450IID6 (human sparteine monooxygenase) metabolizes many drugs including neuroleptics, antidepressants, and beta-blockers. The P450IID6 exists in human, bovine, rat and canine brains, but in very low quantities causing methodological difficulties in its assessment. Work with ({sup 3}H)GBR-12935; 1-(2-(diphenylmethoxy) ethyl)-4-(3-phenyl propyl) piperazine has shown that it binds a neuronal/hepatic protein with high affinity ({approximately}7nM) and a rank order of inhibitory potency suggesting that the binding protein is cytochrome P450IID6. The binding was used to predict that d-amphetamine and methamphetamine would interact with P450IID6. Inhibition studies indicated that these compounds were competitive inhibitors of P450IID6. Haloperidol (HAL) and it's metabolite hydroxy-haloperidol (RHAL)more » are both competitive inhibitors of P450IID6 activity and were found to inhibit ({sup 3}H)GBR-12935 binding. K{sub i} values of twelve compounds (known to interact with the DA transporter or P450IID6) for ({sup 3}H)GRB-12935 binding and P450IID6 activity. The techniques are now available for measurements of cytochrome P450IID6 in healthy and diseased brain/liver tissue using radio-receptor binding assay techniques with ({sup 3}H)GBR-12935.« less

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

PubMed

Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

PubMed

Estabrook, R W; Shet, M S; Faulkner, K; Fisher, C W

1996-11-01

A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

Alteration of cytochrome P450 1 regulation and HSP 70 level in brain of juvenile common carp (Cyprinus carpio) after chronic exposure to tributyltin.

PubMed

Li, Zhi-Hua; Zhong, Li-Qiao; Wu, Yan-Hua; Mu, Wei-Na

2016-02-01

Tributyltin (TBT), a toxic contaminant in aquatic environments, has bio-accumulated in aquatic food webs throughout the world and can be found at toxic levels in some biota. However, the molecular mechanisms and effects of TBT are not fully understood. The aim of the present study was to investigate the effect of long-term exposure of TBT on cytochrome P450 (CYP450) 1 regulation and heat-shock proteins (HSPs) profiling in brain of freshwater teleost. The effects of long-term exposure to TBT on mRNA expression of cytochrome P450 (CYP450) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in the brain of common carp were evaluated, as well as HSP 70 level. Fish were exposed to sublethal concentrations of TBT (75 ng/L, 0.75 μg/L and 7.5 μg/L) for 15, 30, and 60 days. Based on the results, long-term exposure (more than 15 days) to TBT could lead to obvious physiological-biochemical responses (based on EROD activity, HSP 70 level and CYP450 1 family genes expression). The mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. Thus, the measured physiological responses in fish brain could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity and could be used as potential biomarkers for monitoring the TBT pollution in the field.

Abnormal regulation for progesterone production in placenta with prenatal cocaine exposure in rats.

PubMed

Wu, L; Yan, J; Qu, S C; Feng, Y Q; Jiang, X L

2012-12-01

Cocaine abuse in pregnant women is currently a significant public hygiene problem and is tightly associated with elevated risk for preterm delivery. Placental steroidogenesis especially progesterone production was essential for success and maintenance of pregnancy in humans and rodents. In the present study, we determined the impact of prenatal cocaine exposure on pathways of placental progesterone synthesis in rats. Pregnant rats were treated cocaine twice daily (15 mg/kg/day) during the third trimester, and the maternal and fetal plasma progesterone and pregnenolone concentrations were detected. We also examined both the protein and mRNA expression of some key enzymes and regulators for progesterone production in placenta. Results showed that, after maternal cocaine use during pregnancy, progesterone and pregnenolone concentrations in both maternal and fetal rats were significantly decreased. Although prenatal cocaine exposure had no effects on placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) expression, protein and mRNA expression of the cholesterol side-chain cleavage enzyme (P450scc/CYP11a) in placenta was significantly inhibited. Moreover, protein and mRNA expressions of MLN64 that regulating cholesterol transport and activating protein 2γ (AP2γ/Tfap2c) that controlling P450scc/CYP11a gene expression in placenta were both decreased following maternal cocaine use in pregnancy. Collectively, this study suggested that prenatal cocaine exposure could insult the placental progesterone production in rats possibly associated with the high risk for preterm delivery. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization and expression profile of the ovarian cytochrome P-450 aromatase (cyp19A1) gene during thermolabile sex determination in Pejerrey, Odontesthes bonariensis

USGS Publications Warehouse

Karube, M.; Fernandino, J.I.; Strobl-Mazzulla, P.; Strussmann, C.A.; Yoshizaki, G.; Somoza, G.M.; Patino, R.

2007-01-01

Cytochrome P450 aromatase (cyp19) is an enzyme that catalyzes the conversion of androgens to estrogens and may play a role in temperature- dependent sex determination (TSD) of reptiles, amphibians, and fishes. In this study, the ovarian P450 aromatase form (cyp19A1) of pejerrey Odontesthes bonariensis, a teleost with marked TSD, was cloned and its expression profile evaluated during gonadal differentiation at feminizing (17??C, 100% females), mixed-sex producing (24 and 25??C, 73.3 and 26.7% females, respectively), and masculinizing (29??C, 0% females) temperatures. The deduced cyp19A1 amino acid sequence shared high identity (>77.8%) with that from other teleosts but had low identity (<61.8%) with brain forms (cyp19A2), including that of pejerrey itself. The tissue distribution analysis of cyp19A1 mRNA in adult fish revealed high expression in the ovary. Semi-quantitative reverse transcription polymerase chain reaction analysis of the bodies of larvae revealed that cyp19A1 expression increased before the appearance of the first histological signs of ovarian differentiation at the feminizing temperature but remained low at the masculinizing temperature. The expression levels at mixed-sex producing temperatures were bimodal rather than intermediate, showing low and high modal values similar to those at the feminizing and masculinizing temperatures, respectively. The population percentages of high and low expression levels at intermediate temperatures were proportional to the percentage of females and males, respectively, and high levels were first observed at about the time of sex differentiation of females. These results suggest that cyp19A1 is involved in the process of ovarian formation and possibly also in the TSD of pejerrey. ?? 2007 Wiley-Liss, Inc.

Identification and Functional Analysis of a Novel Cytochrome P450 Gene CYP9A105 Associated with Pyrethroid Detoxification in Spodoptera exigua Hübner

PubMed Central

Wang, Rui-Long; Liu, Shi-Wei; Baerson, Scott R.; Qin, Zhong; Ma, Zhi-Hui; Su, Yi-Juan; Zhang, Jia-En

2018-01-01

In insects, cytochrome P450 monooxygenases (P450s or CYPs) are known to be involved in the detoxification and metabolism of insecticides, leading to increased resistance in insect populations. Spodoptera exigua is a serious polyphagous insect pest worldwide and has developed resistance to various insecticides. In this study, a novel CYP3 clan P450 gene CYP9A105 was identified and characterized from S. exigua. The cDNAs of CYP9A105 encoded 530 amino acid proteins, respectively. Quantitative real-time PCR analyses showed that CYP9A105 was expressed at all developmental stages, with maximal expression observed in fifth instar stage larvae, and in dissected fifth instar larvae the highest transcript levels were found in midguts and fat bodies. The expression of CYP9A105 in midguts was upregulated by treatments with the insecticides α-cypermethrin, deltamethrin and fenvalerate at both LC15 concentrations (0.10, 0.20 and 5.0 mg/L, respectively) and LC50 concentrations (0.25, 0.40 and 10.00 mg/L, respectively). RNA interference (RNAi) mediated silencing of CYP9A105 led to increased mortalities of insecticide-treated 4th instar S. exigua larvae. Our results suggest that CYP9A105 might play an important role in α-cypermethrin, deltamethrin and fenvalerate detoxification in S. exigua. PMID:29510578

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu

2013-02-15

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomicmore » approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more diverse spectrum of CYP isozymes than male. ► First data showing expression of 2F1, 2S1, 2W1, 4A22, 4X1, 26A1 on a protein level.« less

Albendazole sulfonation by rat liver cytochrome P-450c.

PubMed

Souhaili-El Amri, H; Mothe, O; Totis, M; Masson, C; Batt, A M; Delatour, P; Siest, G

1988-08-01

The metabolism of albendazole (ABZ) was studied in perfused livers from control and ABZ-treated rats (10.6 mg/kg, per os, each day for 10 days). In the perfusion fluid, the concentration of ABZ-sulfoxide (SO-ABZ) remained unchanged in treated, as compared to control animals, whereas ABZ-sulfone (SO2-ABZ) was increased in treated animals. In bile, only SO-ABZ was present. The transformation kinetics of SO-ABZ to SO2-ABZ in microsomes from rats treated with ABZ, 3-methylcholanthrene, Aroclor and isosafrole were biphasic. This suggests that enzyme activity was a consequence of two enzyme systems, one characterized by low affinity and high capacity, the other by high affinity and low capacity, the latter could be induced by 3-methylcholanthrene, ABZ, Aroclor and isosafrole. Cytochrome P-450c was induced potently in vivo by ABZ as proven by increased monooxygenase (7-ethoxyresorufin and 7-ethoxycoumarin-O-deethylase) activities and by Elisa test (a 5-fold increase in hemoprotein concentration was observed). Purified and reconstituted cytochrome P-450c from 3-methylcholanthrene or ABZ-treated rat liver were able to produce SO2-ABZ (2.01 and 1.70 nmol/mg/15 min, respectively, whereas cytochrome P-450b produced 10 times less SO2-ABZ). Immunological assays, as well as activity measurements showed a relationship between cytochrome P-450c-3-methylcholanthrene and cytochrome P-450c-ABZ. We conclude that induction of cytochrome P-450c by ABZ is the probable explanation for the enhanced formation of SO2-ABZ in vivo.

Inhibitory effects of spirulina platensis on carcinogen-activating cytochrome P450 isozymes and potential for drug interactions.

PubMed

Savranoglu, Seda; Tumer, Tugba Boyunegmez

2013-01-01

Spirulina platensis (SP) has been considered as potential food source of 21st century due to its remarkable nutrient profile and therapeutic benefits. However, the cytochrome P450 (CYP)-mediated drug/chemical interaction potential of SP has not yet been pursued. We investigated the effects of SP on the expressions and enzymatic activities of main CYP isozymes. After the rats were orally administered with SP daily for 5 consecutive weeks, there were significant downregulations in hepatic expression levels and inhibition in enzymatic activities of CYP1A2 and CYP2E1 compared to controls. In addition, a significant decrease was observed in CYP2C6-associated enzyme activity with no remarkable changes in messenger RNA (mRNA)/protein levels. The SP application resulted in significant increases in mRNA/protein levels of both CYP2B1 and CYP3A1 without a significant change in enzyme activities. These findings partly explain the chemopreventive properties of SP toward various organ toxicities, mutagenesis, and carcinogenesis; however, its coadministration with some CYP substrates may lead to undesirable drug interactions.

Metabolic resistance in Nilaparvata lugens to etofenprox, a non-ester pyrethroid insecticide.

PubMed

Sun, Huahua; Yang, Baojun; Zhang, Yixi; Liu, Zewen

2017-03-01

Etofenprox, a non-ester pyrethroid insecticide, will be registered to control rice pests such as the brown planthopper (BPH, Nilaparvata lugens Stål) in mainland China. Insecticide resistance is always a problem to the effective control of N. lugens by chemical insecticides. An etofenprox resistance selection of N. lugens was performed in order to understand the related mechanisms. Through successive selection by etofenprox for 16 generations in the laboratory, an etofenprox-resistant strain (G16) with the resistance ratio (RR) of 422.3-fold was obtained. The resistance was partly synergised (2.68-fold) with the metabolic inhibitor PBO, suggesting a role for P450 monooxygenases. In this study, 11 P450 genes were significantly up-regulated in G16, among which eight genes was above 2.0-fold higher than that in US16, a population with the same origin of G16 but without contacting any insecticide in the laboratory. The expression level of four genes (CYP6AY1, CYP6FU1 and CYP408A1 from Clade 3, and CYP425A1 from Clade 4) were above 4.0-fold when compared to US16. RNA interference (RNAi) was performed to evaluate the importance of the selected P450s in etofenprox resistance. When CYP6FU1, CYP425A1 or CYP6AY1 was interfered, the susceptibility was significantly recovered in both G16 and US16, while the knockdown of CYP408A1 or CYP353D1 did not cause significant changes in etofenprox susceptibility. We supposed that CYP6FU1 was the most important P450 member for etofenprox resistance because of the highest expression level and the most noticeable effects on resistance ratios following RNAi. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

PubMed

Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

2017-05-28

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Differences in the expression of xenobiotic-metabolizing enzymes between islets derived from the ventral and dorsal anlage of the pancreas.

PubMed

Standop, Jens; Ulrich, Alexis B; Schneider, Matthias B; Büchler, Markus W; Pour, Parviz M

2002-01-01

Chronic pancreatitis and pancreatic cancer have been linked to the exposure of environmental chemicals (xenobiotics), which generally require metabolic activation to highly reactive toxic or carcinogenic intermediates. The primary enzyme system involved is made up of numerous cytochrome P450 mono-oxygenases (CYP). Glutathione S-transferases (GST) belong to the enzyme systems that catalyze the conjugation of the reactive intermediates produced by CYPs to less toxic or readily excretable metabolites. Because the majority of chronic pancreatitis and pancreatic cancers develop in the organ's head, we compared the expression of selected CYP and GST enzymes between the tissues deriving from the ventral anlage (head) and dorsal anlage (corpus, tail). A total of 20 normal pancreatic tissue specimen from organ donors and early autopsy cases were processed immunohistochemically by using antibodies to CYP 1A1, 1A2, 2B6, 2C8/9/19, 2D6, 2E1, 3A1, 3A2 and 3A4, GST-alpha, GST-mu and GST-pi, and the NADPH cytochrome P450 oxido-reductase (NA-OR), the specificity of which has been verified in our previous study by Western blot and RT-PCR analyses. In all pancreatic regions, most of the enzymes were expressed in islet cells. However, more islets in the head region expressed CYP 2B6, 2C8/9/19, 2E1 and the NA-OR, than those in the body and tail. Moreover, the expression of CYP 2B6 and 2E1 was restricted to the pancreatic polypeptide (PP) cells, and the concentration of CYP 3A1 and 3A4 was stronger in PP cells than in other islet cells. On the other hand, GST-mu and GST-pi were expressed primarily in islet cells of the body and tail. The greater content of xenobiotic-metabolizing and carcinogen-activating CYP enzymes and a lower expression of detoxifying GST enzymes in the head of the pancreas could be one reason for the greater susceptibility of this region for inflammatory and malignant diseases. Copyright 2002 S. Karger AG, Basel and IAP

Fructose intake during gestation and lactation differentially affects the expression of hippocampal neurosteroidogenic enzymes in rat offspring.

PubMed

Mizuno, Genki; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Murase, Yuri; Kondo, Kanako; Ishikawa, Hiroaki; Teradaira, Ryoji; Suzuki, Koji; Ohashi, Koji

2017-02-01

Neurosteroids, steroidal hormones synthesized de novo from cholesterol within the brain, stimulate hippocampal functions such as neuron protection and synapse formation. Previously, we examined the effect of maternal fructose on the transcriptional regulation of neurosteroidogenic enzymes. We found that the mRNA expression level of the steroidogenic acute regulatory protein (StAR), peripheral benzodiazepine receptor (PBR), cytochrome P450(11β), 11β-hydroxysteroid dehydrogenase (HSD), and 17β-HSD was altered. However, we could not determine whether maternal fructose intake played a role in the gestation or lactation period because the dam rats were fed fructose solution during both periods. Thus, in this study, we analyzed the hippocampi of the offspring of dams fed fructose during the gestation or lactation period. Maternal fructose consumption during either the gestation or lactation period did not affect the mRNA levels of StAR, P450(17α), 11β-HSD-2, and 17β-HSD-1. PBR expression was down-regulated, even when rats consumed fructose during the lactation period only, while fructose consumption during gestation tended to activate the expression of P450(11β)-2. We found that maternal fructose intake during gestation and lactation differentially affected the expression of hippocampal neurosteroidogenic enzymes in the offspring.

Genetic basis of pyrethroid resistance in a population of Anopheles arabiensis, the primary malaria vector in Lower Moshi, north-eastern Tanzania.

PubMed

Matowo, Johnson; Jones, Christopher M; Kabula, Bilali; Ranson, Hilary; Steen, Keith; Mosha, Franklin; Rowland, Mark; Weetman, David

2014-06-19

Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and β-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population. Microarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR). Microarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC '2060'), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC '2060'and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC '2060' was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC '2060' was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC '2060' are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Increased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation.

Gravity Persistent Signal 1 (GPS1) reveals novel cytochrome P450s involved in gravitropism.

PubMed

Withers, John C; Shipp, Matthew J; Rupasinghe, Sanjeewa G; Sukumar, Poornima; Schuler, Mary A; Muday, Gloria K; Wyatt, Sarah E

2013-01-01

Gravity is an important environmental factor that affects growth and development of plants. In response to changes in gravity, directional growth occurs along the major axes and lateral branches of both shoots and roots. The gravity persistent signal (gps) mutants of Arabidopsis thaliana were previously identified as having an altered response to gravity when reoriented relative to the gravity vector in the cold, with the gps1 mutant exhibiting a complete loss of tropic response under these conditions. Thermal asymmetric interlaced (TAIL) PCR was used to identify the gene defective in gps1. Gene expression data, molecular modeling and computational substrate dockings, quantitative RT-PCR analyses, reporter gene fusions, and physiological analyses of knockout mutants were used to characterize the genes identified. Cloning of the gene defective in gps1 and genetic complementation revealed that GPS1 encodes CYP705A22, a cytochrome P450 monooxygenase (P450). CYP705A5, a closely related family member, was identified as expressed specifically in roots in response to gravistimulation, and a mutation affecting its expression resulted in a delayed gravity response, increased flavonol levels, and decreased basipetal auxin transport. Molecular modeling coupled with in silico substrate docking and diphenylboric acid 2-aminoethyl ester (DBPA) staining indicated that these P450s are involved in biosynthesis of flavonoids potentially involved in auxin transport. The characterization of two novel P450s (CYP705A22 and CYP705A5) and their role in the gravity response has offered new insights into the regulation of the genetic and physiological controls of plant gravitropism.

Obesity during pregnancy affects sex steroid concentrations depending on fetal gender.

PubMed

Maliqueo, M; Cruz, G; Espina, C; Contreras, I; García, M; Echiburú, B; Crisosto, N

2017-11-01

It is not clear whether maternal obesity along with fetal gender affect sex steroid metabolism during pregnancy. Therefore, we compared sex steroid concentrations and placental expression of steroidogenic enzymes between non-obese and obese pregnant women with non-pathological pregnancies, and investigated the influence of fetal gender on these parameters. In 35 normal weight (body mass index (BMI) 20-24.9 kg m - 2 ) (controls) and 36 obese women (BMI 30-36 kg m - 2 ) (obese), a fasting blood sample was obtained at first and at third trimester of gestation to measure progesterone, dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, testosterone and estradiol by liquid chromatography-tandem mass spectrometry and estrone by radioimmunoassay. In a subset of women, placental mRNA and protein expression of steroidogenic enzymes was measured by quantitative PCR and western blot, respectively. The comparisons were primarily made between controls and obese, and then separately according to fetal gender. At first and third trimesters of gestation serum progesterone was lower whereas testosterone was higher in obese women (P<0.05, respectively). Upon analyzing according to fetal gender, lower progesterone levels were present in obese pregnant women with male fetuses at first trimester and with female fetuses at third trimester (P<0.05, respectively). Testosterone was higher in obese women with male fetuses compared to control women with male fetuses (P<0.05). The placental protein expression of P450scc was higher in obese women compared to controls (P<0.05). P450 aromatase was higher in obese women with female fetuses (P=0.009), whereas in obese women with male fetuses P450 aromatase was lower compared to control women (P=0.026). Obesity in non-pathological pregnancies alters the maternal serum progesterone and testosterone concentrations depending on fetal gender. These changes can be attributed to gender-related placental adaptations, as the expression of P450 aromatase is different in placentas from females compared to males.

Gravity persistent signal 1 reveals a novel cytochrome P450 involved in gravitropic signal transduction

NASA Astrophysics Data System (ADS)

Wyatt, Sarah

Understanding gene expression that occurs during gravitopism is important for studying the processes that link the perception of gravity to the growth response. Arabidopsis plants with a mutation in the GRAVITY PERSISTENT SIGNAL (GPS)1 locus show a "no response" phenotype during gravistimulation experiments. Basepital auxin transport in gps1 mutant was unaffected by the mutation, but auxin was not laterally redistributed after gravistimulation. GPS1 encodes CYP705A22, a cytochrome P450 protein (P450) of unknown function. The wild type CYP705A22 gene was transformed into the gps1 mutant background and successfully rescued the mutant phenotype. Data mining of microarray data collected from gravistimulated root tips of Arabidopsis indicated that although CYP705A22 was not expressed in roots, a family member CYP705A5 was up-regulated within 3 minutes after gravistimulation. Expression profiling of CYP705A5, using real-time quantitative PCR, showed that CYP705A5 was up-regulated nearly five fold within minutes of gravity stimulation. And reporter gene fusions that link the CYP705A5 gene to the green fluorescent protein showed that CYP705A5 was expressed in the root zones of elongation and maturation. Computer modeling of the catalytic domain of CYP705A22 and CYP705A5 and in silico substrate docking simulations generated a list of 130 compounds that are potential substrates of the P450s. Many of the compounds are phenylpropanoid derivatives. Heterologous expression of CYP705A5 in baculovirus and Type 1 binding studies indicate the substrate of the P450 may be quercitin or myricetin. A mutation affecting CYP705A5 expression resulted in a delayed gravity response in roots. The mutant phenotype could be chemically complemented, and DPBA staining in the CYP705A5 mutant indicated a 1.5 fold accumulation of quercetin in mutant roots as compared to WT. These data, taken together, may indicate that we have identified a flavonoid pathway that regulates auxin distribution and thus is involved in gravitropic signal transduction. (Partially support by NSF: 0618506 to SEW)

Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides.

PubMed

Yang, Yuan-Xue; Yu, Na; Zhang, Jian-Hua; Zhang, Yi-Xi; Liu, Ze-Wen

2018-06-01

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over-expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD 85 ), and in N. lugens among three IMI doses (LD 15 , LD 50 and LD 85 ). When IMI and NMI at the LD 85 dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up-regulation of nine genes and down-regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD 85 dose, 10 genes had very similar patterns, such as up-regulation in seven genes, down-regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up-regulation in four genes at all doses and dose-dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up-regulated by the LD 50 dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose-dependent. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Reduction of aromatic and heterocyclic aromatic N-hydroxylamines by human cytochrome P450 2S1.

PubMed

Wang, Kai; Guengerich, F Peter

2013-06-17

Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals, and there is also strong evidence of some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740-1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs.

Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

PubMed Central

Wang, Kai; Guengerich, F. Peter

2013-01-01

Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals and there is also strong evidence for some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anti-cancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions (Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740–1751). In the present study, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs. PMID:23682735

Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

PubMed

Porter, Joanne L; Sabatini, Selina; Manning, Jack; Tavanti, Michele; Galman, James L; Turner, Nicholas J; Flitsch, Sabine L

2018-06-01

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

PubMed Central

Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F.; Potter, Michael F.; Palli, Subba R.

2012-01-01

Background NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. Methodology/Principal Findings The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. Conclusions/Significance These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs. PMID:22347424

RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

PubMed

Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

2012-01-01

NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

Dual-Color Fluorescence Imaging to Monitor CYP3A4 and CYP3A7 Expression in Human Hepatic Carcinoma HepG2 and HepaRG Cells

PubMed Central

Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps. PMID:25101946

Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

PubMed

Tsuji, Saori; Kawamura, Fumihiko; Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

A Novel Multifunctional C-23 Oxidase, CYP714E19, is Involved in Asiaticoside Biosynthesis.

PubMed

Kim, Ok Tae; Um, Yurry; Jin, Mei Lan; Kim, Jang Uk; Hegebarth, Daniela; Busta, Lucas; Racovita, Radu C; Jetter, Reinhard

2018-06-01

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer β-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.

PubMed

Ferrer-Sevillano, Francisco; Fernández-Cañón, José M

2007-03-01

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities▿

PubMed Central

Ferrer-Sevillano, Francisco; Fernández-Cañón, José M.

2007-01-01

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). PMID:17189487

Effect of mosquito mats (pyrethroid-based) vapor inhalation on rat brain cytochrome P450s.

PubMed

Vences-Mejía, Araceli; Gómez-Garduño, Josefina; Caballero-Ortega, Heriberto; Dorado-González, Víctor; Nosti-Palacios, Rosario; Labra-Ruíz, Norma; Espinosa-Aguirre, J Javier

2012-01-01

The effect of transfluthrin (TF) or D-allethrin (DA) pyrethroid (PYR) vapors, often contained as main ingredients in two commercially available mosquito repellent mats, on cytochrome P450 (CYP) enzymes of rat brain and liver was assessed. Immunodetection of CYP2E1 and CYP3A2 proteins revealed their induction in cerebrum and cerebellum, but not in liver microsomes of rats exposed by inhalation to TF or DA. This overexpression of proteins correlated with an increase of their catalytic activities. The specifically increased expression of CYP isoenzymes, due to PYR exposure in the rat brain, could perturb the normal metabolism of endogenous and xenobiotic compounds and leads to increased risks of neurotoxicity by bioactivation, lipid peroxidation and DNA damage.

Effects of cisplatin and letrozole on surgically induced endometriosis and comparison of the two medications in a rat model.

PubMed

Li, Zhanfei; Liu, Huibing; He, Zhengxing; Zhang, Guorui; Lang, Jinghe

2016-10-10

This study was to investigate the effects of cisplatin (CDDP) and letrozole on surgically induced endometriosis and comparison of the two drugs in a rat model. Endometriosis was surgically induced by autologous transplantation of endometrial pieces. Thirty model rats were divided into three groups, randomly. Group 1 (n=10) served as control and received no medication. Group 2 (n=10) received 0.2mg/kg/day of oral letrozole. Group 3 (n=10) received 35mg/m(2) CDDP via peritoneal perfusion every four days. All the rats were treated for 24days. The growth and histologic score of the implants were evaluated. The proliferation- and angiogenesis-associated proteins were assessed using immunohistochemistry and western blotting. The serum sex hormones were assayed using ELISA. After the medication, the growth and histologic score of the implants were significantly lower in the 2 and 3 groups than in the control group. The protein expressions of vascular endothelial growth factor (VEGF), aromatase P450 (P450arom), transforming growth factor-beta (TGF-β), and matrix metalloproteinase (MMP)-2, were significantly lower in groups 2 and 3 than in the control group. Further, the P450arom level was lower in the letrozole group than in the CDDP group. The TGF-β and MMP-2 levels were lower in the CDDP group than in the letrozole group. Serum T level was significantly higher in the letrozole group, and serum E2 level was lower in the letrozole group. In conclusion, cisplatin and letrozole caused similar regression of the implants in the endometriosis model rats. But their effects on the proliferation- and angiogenesis-associated protein expressions and the serum sex hormone levels were different. Cisplatin and letrozole might cause the effects in the endometriotic foci through different mechanism. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-wide identification of 31 cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and analysis of their benzo[α]pyrene-induced expression patterns.

PubMed

Han, Jeonghoon; Kim, Duck-Hyun; Kim, Hui-Su; Kim, Hee-Jin; Declerck, Steven A J; Hagiwara, Atsushi; Lee, Jae-Seong

2018-03-01

While marine invertebrate cytochrome P450 (CYP) genes and their roles in detoxification mechanisms have been studied, little information is available regarding freshwater rotifer CYPs and their functions. Here, we used genomic sequences and RNA-seq databases to identify 31 CYP genes in the freshwater rotifer Brachionus calyciflorus. The 31 Bc-CYP genes with a few tandem duplications were clustered into CYP 2, 3, 4, mitochondrial, and 46 clans with two marine rotifers Brachionus plicatilis and Brachionus koreanus. To understand the molecular responses of these 31 Bc-CYP genes, we also examined their expression patterns in response to benzo[α]pyrene (B[α]P). Three Bc-CYP genes (Bc-CYP3044B3, Bc-CYP3049B4, Bc-CYP3049B6) were significantly upregulated (P<0.05) in response to B[α]P, suggesting that these CYP genes can be involved in detoxification in response to B[α]P exposure. These genes might be useful as biomarkers of B[α]P exposure in B. calyciflorus. Overall, our findings expand the repertoire of known CYPs and shed light on their potential roles in xenobiotic detoxification in rotifers. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterisation of imidacloprid resistance in the bird cherry-oat aphid, Rhopalosiphum padi, a serious pest on wheat crops.

PubMed

Wang, Kang; Zhang, Meng; Huang, Yanna; Yang, Zhuolin; Su, Sha; Chen, Maohua

2018-06-01

Rhopalosiphum padi is a destructive insect pest of wheat worldwide. Studies have shown that R. padi has developed resistance to different insecticides, including imidacloprid. We studied the mechanisms conferring resistance to imidacloprid at the biochemical and molecular levels. An R. padi imidacloprid-resistant (IM-R) strain and a susceptible (SS) strain were established. Fitness analysis using life-tables showed that the IM-R strain had obvious disadvantages in several parameters, indicating reduced fitness. Profiles of cross-resistance of IM-R and SS to seven insecticides were detected. Both synergistic and enzyme activity data suggested that P450 plays a role in resistance. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes CYP6CY3-1 and CYP6CY3-2 were significantly increased in the IM-R strain. No target-site mutation within the nicotinic acetylcholine receptor (nAChR) subunits was detected in the IM-R strain. Interestingly, the expression levels of the nAChR α1, α2, α3, α7-2, and β1 subunit genes were significantly decreased, suggesting that down-regulation of these subunits may be involved in resistance. Multiple mechanisms confer imidacloprid resistance in R. padi. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

PubMed

Chen, Xi'en; Zhang, Yalin

2015-03-10

NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis.

PubMed

Harada, Hisashi; Shindo, Kazutoshi; Iki, Kanoko; Teraoka, Ayuko; Okamoto, Sho; Yu, Fengnian; Hattan, Jun-ichiro; Utsumi, Ryutaro; Misawa, Norihiko

2011-04-01

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses. © Springer-Verlag 2011

In vitro interactions with repeated grapefruit juice administration--to peel or not to peel?

PubMed

Brill, Shlomo; Zimmermann, Christian; Berger, Karin; Drewe, Juergen; Gutmann, Heike

2009-03-01

Interactions of acutely administered grapefruit juice (GFJ) with cytochrome P450 isoform 3A4 (CYP3A4) and P-glycoprotein (Pgp) function are well established. In this study, we investigated in vitro the effect of repeated administration of GFJ and its major constituents (the flavonoid naringin, its aglycone naringenin and the furanocoumarin bergamottin) on mRNA expression of MDR1 and CYP3A4 in LS180 cells. Since the bergamottin content is higher in the peel than in the fruit, we compared GFJ containing peel (GFJP+) with juice without any peel extract (GFJP-). GFJP- (1%) showed no significant effect on MDR1 and CYP3A4 mRNA expression, whereas 1% GFJP+ increased expression of MDR1 3.7-fold (P<0.01) and CYP3A4 2.3-fold (P<0.05). Of the tested constituents, only 10 microM bergamottin and 200 microM naringenin induced MDR1 mRNA levels 2.9- and 4.0-fold, respectively (P<0.01 for both), and CYP3A4 mRNA levels 3.2- and 15.6-fold (P<0.01 for both), respectively. Western blot analysis and rhodamine 123 uptake experiments partly confirmed these findings on the protein and the functional level. In summary, GFJ containing no peel extract may have a lower potential for interactions with CYP3A4 or P-glycoprotein.

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spaggiari, Dany, E-mail: dany.spaggiari@unige.ch

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6,more » 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC{sub 50} values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC{sub 50} values generated 10-fold faster and cheaper compared to individual assays. • P450 2B6 was the most affected by pesticides with IC{sub 50} in the nanomolar range. • Cumulative inhibition of P450 2B6 by mixtures of four low-dosed insecticides.« less

P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

PubMed

Miller, Walter L

2012-10-23

Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts.

PubMed

Martínez, Javier; Moreno, Juan J

2005-09-01

Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

PubMed

Yamamoto, A M; Mura, C; De Lemos-Chiarandini, C; Krishnamoorthy, R; Alvarez, F

1993-06-01

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Characterization of the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 using different in vitro systems.

PubMed

Baranczewski, Pawel; Edlund, Per Olof; Postlind, Hans

2006-03-18

An important step in the drug development process is identification of enzymes responsible for metabolism of drug candidates and determination of enzyme kinetic parameters. These data are used to increase understanding of the pharmacokinetics and possible metabolic-based drug interactions of drug candidates. The aim of the present study was to characterize the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 [1-(3-{2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2(R)-methylpiperazine], a potent and selective 5HT2c-receptor agonist. The enzyme kinetic parameters were determined for formation of three main metabolites of BVT.2938 using human liver microsomes and expressed cytochrome P450 (CYP) isoforms. The major metabolite was formed by hydroxylation of the pyridine ring (CL(int)=27 microl/mgmin), and was catalysed by both CYP2D6*1 and CYP1A1, with K(m) values corresponding to 1.4 and 2.7 microM, respectively. The results from enzyme kinetic studies were confirmed by incubation of BVT.2938 in the presence of the chemical inhibitor of CYP2D6*1, quinidine. Quinidine inhibited the formation of the major metabolite by approximately 90%. Additionally, studies with recombinant expressed CYP isoforms from rat indicated that formation of the major metabolite of BVT.2938 was catalysed by CYP2D2. This result was further confirmed by experiments with liver slices from different rat strains, where the formation of the metabolite correlated with phenotype of CYP2D2 isoform (Sprague-Dawley male, extensive; Dark Agouti male, intermediate; Dark Agouti female, poor metabolizer). The present study showed that the major metabolite of BVT.2938 is formed by hydroxylation of the pyridine ring and catalysed by CYP2D6*1. CYP1A1 is also involved in this reaction and its role in extra-hepatic metabolism of BVT.2938 might be significant.

Unravelling the Molecular Determinants of Bee Sensitivity to Neonicotinoid Insecticides.

PubMed

Manjon, Cristina; Troczka, Bartlomiej J; Zaworra, Marion; Beadle, Katherine; Randall, Emma; Hertlein, Gillian; Singh, Kumar Saurabh; Zimmer, Christoph T; Homem, Rafael A; Lueke, Bettina; Reid, Rebecca; Kor, Laura; Kohler, Maxie; Benting, Jürgen; Williamson, Martin S; Davies, T G Emyr; Field, Linda M; Bass, Chris; Nauen, Ralf

2018-04-02

The impact of neonicotinoid insecticides on the health of bee pollinators is a topic of intensive research and considerable current debate [1]. As insecticides, certain neonicotinoids, i.e., N-nitroguanidine compounds such as imidacloprid and thiamethoxam, are as intrinsically toxic to bees as to the insect pests they target. However, this is not the case for all neonicotinoids, with honeybees orders of magnitude less sensitive to N-cyanoamidine compounds such as thiacloprid [2]. Although previous work has suggested that this is due to rapid metabolism of these compounds [2-5], the specific gene(s) or enzyme(s) involved remain unknown. Here, we show that the sensitivity of the two most economically important bee species to neonicotinoids is determined by cytochrome P450s of the CYP9Q subfamily. Radioligand binding and inhibitor assays showed that variation in honeybee sensitivity to N-nitroguanidine and N-cyanoamidine neonicotinoids does not reside in differences in their affinity for the receptor but rather in divergent metabolism by P450s. Functional expression of the entire CYP3 clade of P450s from honeybees identified a single P450, CYP9Q3, that metabolizes thiacloprid with high efficiency but has little activity against imidacloprid. We demonstrate that bumble bees also exhibit profound differences in their sensitivity to different neonicotinoids, and we identify CYP9Q4 as a functional ortholog of honeybee CYP9Q3 and a key metabolic determinant of neonicotinoid sensitivity in this species. Our results demonstrate that bee pollinators are equipped with biochemical defense systems that define their sensitivity to insecticides and this knowledge can be leveraged to safeguard bee health. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Modulation of steroidogenesis by vitamin D3 in granulosa cells of the mouse model of polycystic ovarian syndrome.

PubMed

Bakhshalizadeh, Shabnam; Amidi, Fardin; Alleyassin, Ashraf; Soleimani, Masoud; Shirazi, Reza; Shabani Nashtaei, Maryam

2017-06-01

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women of reproductive age characterized by polycystic ovarian morphology, anovulation or oligomenorrhea, and hyperandrogenism. It is shown that disruption in the steroidogenesis pathway caused by excess androgen in PCOS is a critical element of abnormal folliculogenesis and failure in dominant follicle selection. Vitamin D plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells. In the present study, we investigated the effects of vitamin D3 on steroidogenic enzyme expression and activities in granulosa cell using a PCOS mouse model. In our study, the PCOS mouse model was developed by the injection of dehydroepiandrosterone (DHEA) for 20 days. The mRNA and protein expression levels of genes involved in steroidogenesis in granulosa cells were compared between polycystic and normal ovaries using real-time PCR and Western blotting assays. Granulosa cells of DHEA-induced PCOS mice were then cultured with and without vitamin D3 and mRNA and protein expression levels of steroidogenic enzymes and serum 17beta-estradiol and progesterone levels were investigated using qRT-PCR, western blot, and radioimmunoassay, respectively. Steroidogenic enzymes including Cyp11a1, StAR, Cyp19a1, and 3β-HSD were upregulated in granulosa cells of PCOS mice when compared to normal mice. Treatment with vitamin D3 decreased mRNA and protein expression levels of steroidogenic enzymes in cultured granulosa cells. Vitamin D3 also decreased aromatase and 3β-HSD activity that leads to decreased 17beta-estradiol and progesterone release. This study suggests that vitamin D3 could modulate the steroidogenesis pathway in granulosa cells of PCOS mice that may lead to improving follicular development and maturation. This is a step towards a possible conceivable treatment for PCOS. AMHR-II: anti-müllerian hormone receptor-II; 3β-HSD: 3β-hydroxysteroid dehydrogenase; Cyp11a1: Cytochrome P450 Family 11 Subfamily A Member 1; Cyp19a1: cytochrome P450 aromatase; DHEA: dehydroepiandrosterone; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; IVF: in vitro fertilization; 25OHD: 25-hydroxy vitamin D; OHSS: ovarian hyperstimulation syndrome; PCOS: polycystic ovarian syndrome; P450scc: P450 side-chain cleavage enzyme; StAR: steroidogenic acute regulatory protein; VDRs: vitamin D receptors.

Disruption of quercetin metabolism by fungicide affects energy production in honey bees (Apis mellifera).

PubMed

Mao, Wenfu; Schuler, Mary A; Berenbaum, May R

2017-03-07

Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera , detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.

Disruption of quercetin metabolism by fungicide affects energy production in honey bees (Apis mellifera)

PubMed Central

Mao, Wenfu; Schuler, Mary A.; Berenbaum, May R.

2017-01-01

Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food. PMID:28193870

Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

PubMed

Rangel, P L; Rodríguez, A; Rojas, S; Sharp, P J; Gutierrez, C G

2009-12-01

The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

A randomized clinical trial of the effects of supplemental calcium and vitamin D3 on markers of their metabolism in normal mucosa of colorectal adenoma patients.

PubMed

Ahearn, Thomas U; McCullough, Marjorie L; Flanders, W Dana; Long, Qi; Sidelnikov, Eduard; Fedirko, Veronika; Daniel, Carrie R; Rutherford, Robin E; Shaukat, Aasma; Bostick, Roberd M

2011-01-15

In cancer cell lines and rodent models, calcium and vitamin D favorably modulate cell proliferation, differentiation, and apoptosis in colonic epithelia. These effects may be modulated by local expression of the calcium receptor (CaR), the vitamin D receptor (VDR), and the P450 cytochromes, CYP27B1 and CYP24A1; however, they have yet to be investigated in humans. To address this gap, we conducted a randomized, double-blinded, placebo-controlled 2×2 factorial clinical trial. Patients with at least one pathology-confirmed colorectal adenoma were treated with 2 g/d elemental calcium and/or 800 IU/d vitamin D3 versus placebo over 6 months (n=92; 23 per group). CaR, VDR, CYP27B1, and CYP24A1 expression and distribution in biopsies of normal appearing rectal mucosa were detected by standardized, automated immunohistochemistry and quantified by image analysis. In the calcium-supplemented group, CaR expression increased 27% (P=0.03) and CYP24A1 expression decreased 21% (P=0.79). In the vitamin D3-supplemented group, CaR expression increased 39% (P=0.01) and CYP27B1 expression increased 159% (P=0.06). In patients supplemented with both calcium and vitamin D3, VDR expression increased 19% (P=0.13) and CaR expression increased 24% (P=0.05). These results provide mechanistic support for further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms, and CaR, VDR, CYP27B1, and CYP24A1 as modifiable, preneoplastic risk biomarkers for colorectal neoplasms. © 2010 AACR.

INDUCTION AND POST-TRANSCRIPTIONAL SUPPRESSION OF HEPATIC CYTOCHROME P450 1A1 BY 3,3',4,4'-TETRACHLOROBIPHENYL. (R827102)

EPA Science Inventory

Abstract

3,3',4,4'-Tetrachlorobiphenyl (TCB) can induce and inhibit cytochrome P450 1A1 (CYP1A1) in vertebrates. TCB may also suppress CYP1A1 protein levels, but the mechanism is unknown. This study examined transcriptional and translational aspects of hepatic ...

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

PubMed

Müller, Gabrielle do Amaral E Silva; Lüchmann, Karim Hahn; Razzera, Guilherme; Toledo-Silva, Guilherme; Bebianno, Maria João; Marques, Maria Risoleta Freire; Bainy, Afonso Celso Dias

2018-06-06

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, β-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Mechanistic Study of the Stereoselective Hydroxylation of [2-2 H1 ,3-2 H1 ]Butanes Catalyzed by Cytochrome P450 BM3 Variants.

PubMed

Yang, Chung-Ling; Lin, Cheng-Hung; Luo, Wen-I; Lee, Tsu-Lin; Ramu, Ravirala; Ng, Kok Yaoh; Tsai, Yi-Fang; Wei, Guor-Tzo; Yu, Steve S-F

2017-02-21

Engineered bacterial cytochrome P450s are noted for their ability in the oxidation of inert small alkanes. Cytochrome P450 BM3 L188P A328F (BM3 PF) and A74E L188P A328F (BM3 EPF) variants are able to efficiently oxidize n-butane to 2-butanol. Esterification of the 2-butanol derived from this reaction mediated by the aforementioned two mutants gives diastereomeric excesses (de) of -56±1 and -52±1 %, respectively, with the preference for the oxidation occurring at the C-H S bond. When tailored (2R,3R)- and (2S,3S)-[2- 2 H 1 ,3- 2 H 1 ]butane probes are employed as substrates for both variants, the obtained de values from (2R,3R)-[2- 2 H 1 ,3- 2 H 1 ]butane are -93 and -92 % for BM3 PF and EPF, respectively; whereas the obtained de values from (2S,3S)-[2- 2 H 1 ,3- 2 H 1 ]butane are 52 and 56 % in the BM3 PF and EPF systems, respectively. The kinetic isotope effects (KIEs) for the oxidation of (2R,3R)-[2- 2 H 1 ,3- 2 H 1 ]butane are 7.3 and 7.8 in BM3 PF and EPF, respectively; whereas KIEs for (2S,3S)-[2- 2 H 1 ,3- 2 H 1 ]butanes are 18 and 25 in BM3 PF and EPF, respectively. The discrepancy in KIEs obtained from the two substrates supports the two-state reactivity (TSR) that is proposed for alkane oxidation in cytochrome P450 systems. Moreover, for the first time, experimental evidence for tunneling in the oxidation mediated by P450 is given through the oxidation of the C-H R bond in (2S,3S)-[2- 2 H 1 ,3- 2 H 1 ]butane. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

PubMed Central

Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

1994-01-01

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Heterologous expression of Helicoverpa armigera cytochrome P450 CYP6B7 in Pichia pastoris and interactions of CYP6B7 with insecticides.

PubMed

Zhao, Chunqing; Song, Genmiao; Duan, Hongxia; Tang, Tao; Wang, Chen; Qiu, Lihong

2017-09-01

Previous studies indicated that constitutive over-expression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P. pastoris was confirmed by SDS-PAGE and western blot. Microsomes containing the expressed HaCYP6B7 showed activities against model substrate p-nitroanisole and 7-ethoxycoumarin, with p-nitroanisole O-demethylation (PNOD) and 7-ethoxycoumarin O-deethylation (ECOD) activities of 15.66- and 4.75-fold of the control, respectively. Moreover, it showed degradation activities against the insecticides bifenthrin, fenvalerate and chlorpyrifos, with clearance activities of 6.88-, 1.49- and 2.27-fold of the control, respectively. The interactions of HaCYP6B7 with insecticides were further confirmed by molecular docking in silico with binding scores of 5.450, 5.295 and 2.197 between putative HaCYP6B7 protein and bifenthrin, fenvalerate and chlorpyrifos, respectively. The results of present study provided more direct and important evidence on the role of HaCYP6B7 conferring pyrethroid resistance in H. armigera. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice.

PubMed

Jourová, L; Anzenbacher, P; Lišková, B; Matušková, Z; Hermanová, P; Hudcovic, T; Kozáková, H; Hrnčířová, L; Anzenbacherová, E

2017-11-01

Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host's health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum NIZO2877 ; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.

Re-engineering Cytochrome P450 2B11dH for Enhanced Metabolism of Several Substrates Including the Anti-cancer Prodrugs Cyclophosphamide and Ifosfamide

PubMed Central

Sun, Ling; Chen, Chong S.; Waxman, David J.; Liu, Hong; Halpert, James R.; Kumar, Santosh

2007-01-01

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced kcat/Km for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in Km (0.72 vs. 18 μM). I209A also demonstrated enhanced selectivity for testosterone 16β-hydroxylation over 16α-hydroxylation. In contrast, V183L showed a 4-fold increased kcat for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased kcat/Km for testosterone 16α-hydroxylation. V183L also displayed a 1.7-fold higher kcat/Km than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a ~4-fold decrease in Km. Introduction of the V183L mutation into full-length P450 2B11 did not enhance the kcat/Km. Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs. PMID:17254539

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes.

PubMed

Hedges, Laura; Brown, Susan; MacLeod, A Kenneth; Vardy, Audrey; Doyle, Edward; Song, Gina; Moreau, Marjory; Yoon, Miyoung; Osimitz, Thomas G; Lake, Brian G

2018-06-04

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL int ) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CL int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL int values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Triterpene Functional Genomics in Licorice for Identification of CYP72A154 Involved in the Biosynthesis of Glycyrrhizin[C][W][OA

PubMed Central

Seki, Hikaru; Sawai, Satoru; Ohyama, Kiyoshi; Mizutani, Masaharu; Ohnishi, Toshiyuki; Sudo, Hiroshi; Fukushima, Ery Odette; Akashi, Tomoyoshi; Aoki, Toshio; Saito, Kazuki; Muranaka, Toshiya

2011-01-01

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin. PMID:22128119

Vitamin D receptor activation induces P-glycoprotein and increases brain efflux of quinidine: an intracerebral microdialysis study in conscious rats.

PubMed

Durk, Matthew R; Fan, Jianghong; Sun, Huadong; Yang, Yingbo; Pang, Henrianna; Pang, K Sandy; de Lannoy, Inés A M

2015-03-01

Since the vitamin D receptor (VDR) was found to up-regulate cerebral P-glycoprotein expression in vitro and in mice, we extend our findings to rats by assessing the effect of rat Vdr activation on brain efflux of quinidine, a P-gp substrate that is eliminated primarily by cytochrome P450 3a. We treated rats with vehicle or the active VDR ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (4.8 or 6.4 nmol/kg i.p. every 2nd day × 4) and examined P-gp expression and cerebral quinidine disposition via microdialysis in control and treatment studies conducted longitudinally in the same rat. The 6.4 nmol/kg 1,25(OH)2D3 dose increased cerebral P-gp expression 1.75-fold whereas hepatic Cyp3a remained unchanged. Although there was no change in systemic clearance elicited by 1,25(OH)2D3, brain extracellular fluid quinidine concentrations were lower in treated rats. We noted that insertion of indwelling catheters increased plasma protein binding of quinidine and serial sampling decreased the blood:plasma concentration ratio, factors that alter distribution ratios in microdialysis studies. After appropriate correction, KECF/P,uu and KECF/B,uu, or ratios of quinidine unbound concentrations in brain extracellular fluid to plasma or blood at steady-state, were more than halved. We demonstrate that VDR activation increases cerebral P-gp expression and delimits brain penetration of P-gp substrates.

Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

USGS Publications Warehouse

Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

1994-01-01

Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

Transcriptomic analysis reveals the flooding tolerant mechanism in flooding tolerant line and abscisic acid treated soybean.

PubMed

Yin, Xiaojian; Hiraga, Susumu; Hajika, Makita; Nishimura, Minoru; Komatsu, Setsuko

2017-03-01

Soybean is highly sensitive to flooding stress and exhibits markedly reduced plant growth and grain yield under flooding conditions. To explore the mechanisms underlying initial flooding tolerance in soybean, RNA sequencing-based transcriptomic analysis was performed using a flooding-tolerant line and ABA-treated soybean. A total of 31 genes included 12 genes that exhibited similar temporal patterns were commonly changed in these plant groups in response to flooding and they were mainly involved in RNA regulation and protein metabolism. The mRNA expression of matrix metalloproteinase, glucose-6-phosphate isomerase, ATPase family AAA domain-containing protein 1, and cytochrome P450 77A1 was up-regulated in wild-type soybean under flooding conditions; however, no changes were detected in the flooding-tolerant line or ABA-treated soybean. The mRNA expression of cytochrome P450 77A1 was specifically up-regulated in root tips by flooding stress, but returned to the level found in control plants following treatment with the P450 inhibitor uniconazole. The survival ratio and root fresh weight of plants were markedly improved by 3-h uniconazole treatment under flooding stress. Taken together, these results suggest that cytochrome P450 77A1 is suppressed by uniconazole treatment and that this inhibition may enhance soybean tolerance to flooding stress.

Metabolism of proposed nerve agent pretreatment, pyridostigmine bromide. Final report, December 1995-December 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leo, K.U.

A reverse phase High Pressure Liquid Chromatography (HPLC) method was developed to separate pyridostigmine bromide from four potential metabolites. Using male and female microsomes from both rat and human, our data suggest that pyridostigmine bromide is not metabolized by the human live microsomes or DNA expressed human CYP-450s via direct observation of no metabolites being formed for incubations up to 90 minutes. Indirect evidence that pyridostigmine metabolism is not via the major human hepatic CYP-450s involved in drug metabolism, 1A2, 2C9, 2E1, 2D6, and 3A4, was observed by failure to inhibit these isozymes while co-incubated with substrates specific for thosemore » isozymes at concentrations of 2-3 times Km. The following CYP-450 substrates were co-incubated with pyridostigmine: phenacetin, tolbutamide, chlorzoxazone, bufuralol, and testosterone. Using unlabelled and 14C-pyridostigmine, metabolite formation was not observed in both male and female rat and human subcellular fractions, specifically cytosol and S9, or under conditions favoring human FMO activity (pH 8.3). These findings indicate the metabolism of pyridostigmine bromide is unlikely to be under any component of sexual dimorphism.« less

Rational engineering of the fungal P450 monooxygenase CYP5136A3 to improve its oxidizing activity toward polycyclic aromatic hydrocarbons.

PubMed

Syed, Khajamohiddin; Porollo, Aleksey; Miller, David; Yadav, Jagjit S

2013-09-01

A promising polycyclic aromatic hydrocarbon-oxidizing P450 CYP5136A3 from Phanerochaete chrysosporium was rationally engineered to enhance its catalytic activity. The residues W129 and L324 found to be critical in substrate recognition were transformed by single (L324F) and double (W129L/L324G, W129L/L324F, W129A/L324G, W129F/L324G and W129F/L324F) mutations, and the engineered enzyme forms were expressed in Pichia pastoris. L324F and W129F/L324F mutations enhanced the oxidation activity toward pyrene and phenanthrene. L324F also altered the regio-selectivity favoring C position 4 over 9 for hydroxylation of phenanthrene. This is the first instance of engineering a eukaryotic P450 for enhanced oxidation of these fused-ring hydrocarbons.

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

USGS Publications Warehouse

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

1993-01-01

Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

USGS Publications Warehouse

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

1993-01-01

Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

Inhibitory effects of kale ingestion on metabolism by cytochrome P450 enzymes in rats.

PubMed

Yamasaki, Izumi; Yamada, Masayoshi; Uotsu, Nobuo; Teramoto, Sachiyuki; Takayanagi, Risa; Yamada, Yasuhiko

2012-01-01

Kale (Brassica oleracea L. var acephala DC) is a leafy green vegetable belonging to the cabbage family (Brassicaceae) that contains a large amount of health-promoting phytochemicals. There are any reports about the effects of kale ingestion on the chemoprevention function and mechanism, but the interactions between kale and drugs have not been researched. We investigated the effects of kale intake on cytochrome P450 (CYP) metabolism by using cocktail probe drugs, including midazolam (for CYP3A4), caffeine (for CYP1A2), dextromethorphan (for CYP2D6), tolbutamide (for CYP2C9), omeprazole (for CYP2C19), and chlorzoxazone (for CYP2E1). Cocktail drugs were administered into rats treated with kale and cabbage (2000 mg/kg) for a week. The results showed that kale intake induced a significant increase in plasma levels and the AUC of midazolam, caffeine, and dextromethorphan. In addition, the plasma concentration and AUC of omeprazole tended to increase. Additionally, no almost differences in the mRNA expression levels of CYP enzymes in the liver were observed. In conclusion, kale ingestion was considered to have an inhibitory effect on the activities of CYP3A4, 1A2, 2D6, and 2C19 for a reason competitive inhibition than inhibitory changes in the mRNA expressions.

BDE47 induces rat CYP3A1 by targeting the transcriptional regulation of miR-23b

NASA Astrophysics Data System (ADS)

Sun, Zhenzhen; Zhang, Zhan; Ji, Minghui; Yang, Hongbao; Cromie, Meghan; Gu, Jun; Wang, Chao; Yang, Lu; Yu, Yongquan; Gao, Weimin; Wang, Shou-Lin

2016-08-01

Cytochrome P450 3A (CYP3A) is the most abundant CYP450 enzyme in the liver and is involved in the metabolism of over 50% of xenobiotics. Our previous studies revealed that 2,2‧,4,4‧-tetrabromodiphenyl ether (BDE47) could induce rat CYP3A1 expression, but the molecular basis remains unclear. Using in silico analysis, we identified a potential miR-23b recognition element (MRE23b) in the 3‧-UTR region of CYP3A1 mRNA, which was verified by the luciferase assay. The miR-23b mimic and inhibitor significantly down- and up-regulated the expression of CYP3A1, respectively. Additionally, BDE47 significantly down-regulated the expression of miR-23b in rats and in hepatic H4IIE cells. Induction or blockage of CYP3A1 by a miR-23b inhibitor or mimic could correspondingly alter BDE47-induced expression of CYP3A1 and cytotoxicity in H4IIE cells. Furthermore, LV-anti-miR-23b significantly decreased endogenous levels of miR-23b and increased the expression and activity of CYP3A1 in rat liver. LV-anti-miR-23b also significantly increased the hydroxylated metabolites of BDE47 (3-OH-BDE47, 4-OH-BDE42, and 4‧-OH-BDE49) in rat serum. In conclusion, we first found that BDE47 induced rat CYP3A1 expression by targeting the transcriptional regulation of miR-23b. This study helps provide a better understanding of CYP3A regulation and offers novel clues for the role of miRNAs in the metabolism and distribution of environmental pollutants.

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

PubMed

Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

1995-04-04

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Comparison of the biotransformation of the 14C-labelled insecticide carbaryl by non-transformed and human CYP1A1-, CYP1A2-, and CYP3A4-transgenic cell cultures of Nicotiana tabacum.

PubMed

Schmidt, Burkhard; Faymonville, Tanja; Gembé, Eva; Joussen, Nicole; Schuphan, Ingolf

2006-08-01

Transgenic tobacco-cell-suspension cultures expressing separately the human cytochrome P450 monooxygenases CYP1A1, CYP1A2, and CYP3A4 were utilized to study the biotransformation of the 14C-labelled insecticide carbaryl (=naphthalen-1-yl methylcarbamate). The resulting data were compared to similar data from the corresponding non-transformed (NT) tobacco-cell culture and commercially available membrane preparations (Bactosomes) of genetically modified bacteria separately containing the same human P450s. A rapid conversion rate of carbaryl was observed with the CYP1A1 and CYP1A2 cells, where only 49.7 and 0.2% of applied carbaryl (1 mg/l), respectively, remained after 24 h, as compared to 77.7% in the non-transformed culture. Unexpectedly, the corresponding results obtained from the CYP3A4 cultures were not definite. With 25 mg/l of carbaryl and 96 h of incubation, it was proven that the insecticide is also substrate of CYP3A4. This finding was supported by GC/EI-MS analysis of the primary metabolite pattern produced by the isozyme. This consisted of naphthalene-1-ol, N-(hydroxymethyl)carbaryl, 4-hydroxycarbaryl, and 5-hydroxycarbaryl, whereas the main product in non-transformed cells was N-(hydroxymethyl)carbaryl. Data obtained from the CYP1A1, CYP1A2, or CYP3A4 Bactosomes agreed with those of the P450-transgenic tobacco cells. Problems with GC/EI-MS analysis of carbaryl and its metabolites are discussed.

Toxic effects of Tripterygium wilfordii Hook F on the reproductive system of adolescent male rats.

PubMed

Jing, Xiaoping; Cheng, Weiwei; Guo, Sheng; Zou, Ya; Zhang, Ting; He, Li

2017-11-01

Tripterygium wilfordii Hook F. (TWHF) is a compound extracted from Lei Gong Teng (Thunder God Vine) that has been used to treat a variety of immune-related diseases in clinical practice, particularly in pediatrics. Nevertheless, clinical data indicated that glycosides from Tripterygium wilfordii Hook F (GTW) are toxic to the male reproductive system, but the mechanism is unknown. Here, the administration of a high dose of GTW for 4 weeks and a low dose for 12 weeks can reduce the body weights and testes weights in adolescent male rats. This effect is accompanied by a significantly reduction in the serum testosterone levels. Notably, short-term use of high-dose GTW or long-term use of low-dose GTW leads to testicular damage in adolescent male rats. Furthermore, the expression of the steroidogenic acute regulatory protein (StAR), P450 side chain cleavage enzyme (P450scc), cytochrome P450 17-hydroxylase (P450c17), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase (17β-HSD) mRNAs and proteins in the testes was down-regulated by a short-term treatment with high-dose GTW and a long-term treatment with low-dose GTW. Therefore, GTW exhibit male reproductive toxicity in a concentration-and time-dependent manner by inhibiting the expression of the key enzymes and total cholesterol level involved in testosterone synthesis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

N-Hydroxylation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity.

PubMed Central

Gill, H J; Tingle, M D; Park, B K

1995-01-01

1. The adverse reactions associated with the administration of dapsone are believed to be caused by metabolism to its hydroxylamine. Previous reports suggest that CYP3A4 is responsible for this biotransformation [1]. 2. Data presented in this paper illustrate the involvement of more than one cytochrome P450 enzyme in dapsone hydroxylamine formation using human liver microsomes. Eadie-Hofstee plots demonstrated bi-phasic kinetics in several livers. No correlation could be established between hydroxylamine formation and CYP3A concentrations in six human livers (r = -0.47; P = 0.34). 3. Studies with low molecular weight inhibitors illustrate the importance of CYP2C9 and CYP3A in dapsone N-hydroxylation. 4. Differential sensitivity of dapsone N-hydroxylation to selective CYP inhibitors indicated that the contribution of individual CYP enzymes varies between livers. Selective inhibition ranged from 6.8 to 44.1% by 5 microM ketoconazole, and from 24.0 to 68.4% by 100 microM sulphaphenazole. The extent of inhibition, by either ketoconazole or sulphaphenazole was dependent on the CYP3A content of the liver. 5. The levels of expression of these cytochrome P450 enzymes may be an important determinant of individual susceptibility to the toxic effects of dapsone, and may influence the ability of an enzyme inhibitor to block dapsone toxicity in vivo. Because of the inability to produce complete inhibition, selective CYP inhibitors are unlikely to offer any clinical advantage over cimetidine in decreasing dapsone hydroxylamine formation in vivo. PMID:8703658

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

PubMed

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

PubMed

Wang, Zhican; Lin, Yvonne S; Dickmann, Leslie J; Poulton, Emma-Jane; Eaton, David L; Lampe, Johanna W; Shen, Danny D; Davis, Connie L; Shuhart, Margaret C; Thummel, Kenneth E

2013-05-01

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia. Copyright © 2013 American Society for Bone and Mineral Research.

[Participation of nitric oxide and arachidonic acid metabolites via cytochrome - P450 in the regulation of arterial blood pressure].

PubMed

Sánchez-Mendoza, M Alicia; Martínez-Ayala, Sonia O; Hernández-Hernández, José A; Zúñiga-Sosa, Leonor; Pastelín-Hernández, Gustavo; Escalante-Acosta, Bruno A

2003-01-01

Nitric oxide and cytochrome P450 arachidonic acid metabolites participate in blood pressure regulation. The synthesis of these autacoids leads to arterial hypertension. However, it is not known whether there is an interaction between them. Therefore, we studied the modulatory effect of nitric oxide and cytochrome P450-arachidonic acid metabolites, their interaction on blood pressure, and the renal content of cytochrome P450. Male Wistar rats were divided: 1) control, 2) L-NAME (100 mg/kg/d p.o.), 3) L-NAME + SnCl2 (10 mg/kg/d i.p.), and 4) L-NAME + dexamethasone (1 mg/kg/d s.c.). We measured blood pressure and collected urine and blood for nitric oxide measurement. NO2 was quantified by HPLC. Blood pressure was: control, 97 +/- 7 mmHg; L-NAME, 151 +/- 4.6 mmHg; L-NAME + SnCl2, 133 +/- 3 mmHg, and L-NAME + dexamethasone 152 +/- 4.5 mmHg. Urine nitrite concentration was: 1) 1.832 +/- 0.32, 2) 1.031 +/- 0.23, 3) 1.616 +/- 0.33, and 4) 1.244 +/- 0.33 mumol/mL, while the concentration in blood was: 1) 0.293 +/- 0.06, 2) 0.150 +/- 0.05, 3) 0.373 +/- 0.13, and 4) 0.373 +/- 0.07 mumol/mL. L-NAME + SnCl2 decreased cytochrome P450 renal content, and L-NAME + dexamethasone showed a similar response. In conclusion, both, nitric oxide and CYP-arachidonic acid metabolites play a role in the regulation of blood pressure. Nitric oxide also partially regulates renal cytochrome P450 content.

Benzo[a]pyrene Reduces Testosterone Production in Rat Leydig Cells via a Direct Disturbance of Testicular Steroidogenic Machinery

PubMed Central

Chung, Jin-Yong; Lee, Seung Gee; Park, Ji-Eun; Yoon, Yong-Dal; Yoo, Ki Soo; Yoo, Young Hyun

2011-01-01

Background: Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is a ubiquitous environmental pollutant that is currently suspected of being an endocrine disruptor. The testis is an important target for PAHs, yet insufficient attention has been paid to their effects on steroidogenesis in Leydig cells. Objective: We hypothesized that long-term exposure to low concentrations of B[a]P might disrupt testosterone production in Leydig cells via an alteration of steroidogenic proteins. Results: Oral exposure to B[a]P reduced serum and intratesticular fluid testosterone levels in rats. However, we did not observe serious testicular atrophy or azoospermia, although spermatogonial apoptosis was significantly increased. Compared with control cells, Leydig cells primed with B[a]P in vivo produced less testosterone in response to human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine monophosphate in vitro. Of note, the reduction of testosterone levels was accompanied by decreased expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD), as well as increased levels of cytochrome P450 side chain cleavage (P450scc), in Leydig cells. The up-regulation of P450scc expression after exposure to B[a]P appears to be associated with a compensatory mechanism for producing the maximum amount of pregnenolone with the minimum amount of transported cholesterol by StAR; the down-regulation of 3β-HSD may occur because B[a]P can negatively target 3β-HSD, which is required for testosterone production. Conclusions: B[a]P exposure can decrease epididymal sperm quality, possibly by disturbing testosterone levels, and StAR may be a major steroidogenic protein that is targeted by B[a]P or other PAHs. PMID:21737371

Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

PubMed Central

Sibbesen, O; Koch, B; Halkier, B A; Møller, B L

1994-01-01

The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C-hydroxylation of p-hydroxyphenylacetonitrile and from cinnamic acid 4-hydroxylase. Images PMID:7937883

Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii.

PubMed

Jia, Longgang; Dong, Jianzhang; Wang, Ruijie; Mao, Shuhong; Lu, Fuping; Singh, Suren; Wang, Zhengxiang; Liu, Xiaoguang

2017-08-01

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

PubMed

Li, Ming; de Graaf, Inge A M; Siissalo, Sanna; de Jager, Marina H; van Dam, Annie; Groothuis, Geny M M

2016-05-01

P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria

PubMed Central

Djouaka, Rousseau F; Bakare, Adekunle A; Coulibaly, Ousmane N; Akogbeto, Martin C; Ranson, Hilary; Hemingway, Janet; Strode, Clare

2008-01-01

Background Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes. Results All mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron) and an urban area (Gbedjromede), low levels of resistance in mosquito samples from an oil contaminated site (Ojoo) and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84) but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold) and Ojoo (7.4-fold) populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations. Conclusion Multiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential resistance mechanisms were also identified that warrant further investigation. Metabolic genes were over expressed irrespective of the presence of kdr, the latter resistance mechanism being absent in one resistant population. The discovery that mosquitoes collected from different types of breeding sites display differing profiles of metabolic genes at the adult stage may reflect the influence of a range of xenobiotics on selecting for resistance in mosquitoes. PMID:19014539

Effects of salinity acclimation on the expression and activity of Phase I enzymes (CYP450 and FMOs) in coho salmon (Oncorhynchus kisutch)

PubMed Central

Lavado, Ramon; Aparicio-Fabre, Rosaura; Schlenk, Daniel

2013-01-01

Phase I biotransformation enzymes are critically important in the disposition of xenobiotics within biota and are regulated by multiple environmental cues, particularly in anadromous fish species. Given the importance of these enzyme systems in xenobiotic/endogenous chemical bioactivation and detoxification, the current study was designed to better characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch) and the effects of salinity acclimation on those enzymes. Livers, gills and olfactory tissues were collected from coho salmon (Oncorhynchus kisutch) after they had undergone acclimation from freshwater to various salinity regimes of seawater (8, 16 and 32 g/L). Using immunoblot techniques coupled with testosterone hydroxylase catalytic activities, 4 orthologs of cytochrome P450 (CYP1A, CYP2K1, CYP2M1 and CYP3A27) were measured in each tissue. Also the expression of 2 transcripts of flavin-containing monooxygenases (FMO A and B) and associated activities were measured. With the exception of CYP1A, which was down-regulated in liver, protein expression of the other 3 enzymes was induced at higher salinity, with the greatest increase observed in CYP2M1 from olfactory tissues. In liver and gills, 6 - and 16 -hydroxylation of testosterone was also significantly increased after hypersaline acclimation. Similarly, FMO A was up-regulated in all 3 tissues in a salinity-dependent pattern, whereas FMO B mRNA was down-regulated. FMO-catalyzed benzydamine N-oxygenase and methyl p-tolyl sulfoxidation were significantly induced in liver and gills by hypersalinity, but was either unchanged or not detected in olfactory tissues. These data demonstrate thatenvironmental conditions may significantly alter the toxicity of environmental chemicals in salmon during freshwater/saltwater acclimation. PMID:23925894

Differences in hepatic microsomal cytochrome P-450 isoenzyme induction by pyrazole, chronic ethanol, 3-methylcholanthrene, and phenobarbital in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

PubMed

Lucas, D; Ménez, J F; Berthou, F; Cauvin, J M; Deitrich, R A

1992-10-01

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ismail, Hanafy M.; O'Neill, Paul M.; Hong, David

2014-01-18

Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the targetmore » tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.« less

Juvenile hormone and colony conditions differentially influence cytochrome P450 gene expression in the termite Reticulitermes flavipes.

PubMed

Zhou, X; Song, C; Grzymala, T L; Oi, F M; Scharf, M E

2006-12-01

In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression.

INDUCTION AND SUPPRESSION OF CYTOCHROME P450 1A BY 3,3', 4,4', 5-PENTACHLOROBIPHENYL AND ITS RELATIONSHIP TO OXIDATIVE STRESS IN THE MARINE FISH SCUP (STENOTOMUS CHRYSOPS). (R827102)

EPA Science Inventory

Abstract

The planar polychlorinated biphenyl (PCB) 3,3',4,4'-tetrachlorobiphenyl (TCB) causes dose-dependent induction and post-transcriptional suppression of hepatic cytochrome P450 1A (CYP1A) in the marine teleost scup (Stenotomus chrysops). That suppr...

Induction of cytochrome P450 1A by cow milk-based formula: a comparative study between human milk and formula

PubMed Central

Xu, Haibo; Rajesan, Ratheishan; Harper, Patricia; Kim, Richard B; Lonnerdal, Bo; Yang, Mingdong; Uematsu, Satoko; Hutson, Janine; Watson-MacDonell, Jo; Ito, Shinya

2005-01-01

During the treatment of neonatal apnea, formula-fed infants, compared to breastfed infants, show nearly three-fold increase in clearance of caffeine, a substrate of cytochrome P450 1A (CYP1A) and in part CYP3A4. However, human milk is known to contain higher concentrations of environmental pollutants than infant formula, which are potent CYP1A inducers. To gain insight into the mechanism underlying this apparent contradiction, we characterized CYP1A and CYP3A4 induction by human milk and cow milk-based infant formula. The mRNA and protein expression of CYP1A1/1A2 were significantly induced by cow milk-based formula, but not by human milk, in HepG2 cells. Luciferase reporter assay demonstrated that cow milk-based formula but not human milk activated aryl hydrocarbon receptor (AhR) significantly. The cotreatment of 3,4-dimethoxyflavone, an AhR antagonist, abolished the formula-induced CYP1A expression. In addition, AhR activation by dibenzo[a,h]anthracene, a potent AhR agonist, was significantly suppressed by infant formula and even more by human milk. In contrast, CYP3A4 mRNA expression was only mildly induced by formula and human milk. Consistently, neither formula nor human milk substantially activated pregnane X receptor (PXR). Effects of whey and soy protein-based formulas on the AhR–CYP1A and the PXR–CYP3A4 pathways were similar to those of cow milk-based formula. In conclusion, infant formula, but not human milk, enhances in vitro CYP1A expression via an AhR-mediated pathway, providing a potential mechanistic basis for the increased caffeine elimination in formula-fed infants. PMID:15997229

Induction of cytochrome P450 1A by cow milk-based formula: a comparative study between human milk and formula.

PubMed

Xu, Haibo; Rajesan, Ratheishan; Harper, Patricia; Kim, Richard B; Lonnerdal, Bo; Yang, Mingdong; Uematsu, Satoko; Hutson, Janine; Watson-MacDonell, Jo; Ito, Shinya

2005-09-01

During the treatment of neonatal apnea, formula-fed infants, compared to breastfed infants, show nearly three-fold increase in clearance of caffeine, a substrate of cytochrome P450 1A (CYP1A) and in part CYP3A4. However, human milk is known to contain higher concentrations of environmental pollutants than infant formula, which are potent CYP1A inducers. To gain insight into the mechanism underlying this apparent contradiction, we characterized CYP1A and CYP3A4 induction by human milk and cow milk-based infant formula. The mRNA and protein expression of CYP1A1/1A2 were significantly induced by cow milk-based formula, but not by human milk, in HepG2 cells. Luciferase reporter assay demonstrated that cow milk-based formula but not human milk activated aryl hydrocarbon receptor (AhR) significantly. The cotreatment of 3,4-dimethoxyflavone, an AhR antagonist, abolished the formula-induced CYP1A expression. In addition, AhR activation by dibenzo[a,h]anthracene, a potent AhR agonist, was significantly suppressed by infant formula and even more by human milk. In contrast, CYP3A4 mRNA expression was only mildly induced by formula and human milk. Consistently, neither formula nor human milk substantially activated pregnane X receptor (PXR). Effects of whey and soy protein-based formulas on the AhR-CYP1A and the PXR-CYP3A4 pathways were similar to those of cow milk-based formula. In conclusion, infant formula, but not human milk, enhances in vitro CYP1A expression via an AhR-mediated pathway, providing a potential mechanistic basis for the increased caffeine elimination in formula-fed infants.

Relationships among Ergot Alkaloids, Cytochrome P450 Activity, and Beef Steer Growth

NASA Astrophysics Data System (ADS)

Rosenkrans, Charles; Ezell, Nicholas

2015-03-01

Determining a grazing animal’s susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 µM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 d of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 d. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

Gene expression profiles of Drosophila melanogaster exposed to an insecticidal extract of Piper nigrum.

PubMed

Jensen, Helen R; Scott, Ian M; Sims, Steve; Trudeau, Vance L; Arnason, John Thor

2006-02-22

Black pepper, Piper nigrum L. (Piperaceae), has insecticidal properties and could potentially be utilized as an alternative to synthetic insecticides. Piperine extracted from P. nigrum has a biphasic effect upon cytochrome P450 monooxygenase activity with an initial suppression followed by induction. In this study, an ethyl acetate extract of P. nigrum seeds was tested for insecticidal activity toward adult Musca domestica and Drosophila melanogaster. The effect of this same P. nigrum extract upon differential gene expression in D. melanogaster was investigated using cDNA microarray analysis of 7380 genes. Treatment of D. melanogaster with P. nigrum extract led to a greater than 2-fold upregulation of transcription of the cytochrome P450 phase I metabolism genes Cyp 6a8, Cyp 9b2, and Cyp 12d1 as well as the glutathione-S-transferase phase II metabolism gene Gst-S1. These data suggests a complex effect of P. nigrum upon toxin metabolism.

Anti-mullerian hormone is expressed by endometriosis tissues and induces cell cycle arrest and apoptosis in endometriosis cells

PubMed Central

2014-01-01

Background The anti-mullerian hormone (AMH) is a member of the transforming growth factor β (TGF-β) superfamily, which is responsible of the regression of the mullerian duct. AMH is expressed in the normal endometrium, where, acting in a paracrine fashion, negatively regulates cellular viability. Our objective was to evaluate the in vitro effects of the treatment with AMH of endometriosic cells. Methods AMH expression in human endometriosis glands was evaluated by immunohistochemistry. RT-PCR has been used to quantify the expression levels of AMH and AMH RII isoforms, as well as of cytochrome P450 in both endometriosis epithelial and stromal cells Effects of AMH and AMH-cleaved treatment in endometriosis cells were evaluated by flow-cytometry analysis. Finally, it has been evaluated the effect of plasmin-digested AMH on cytochrome P450 activity. Results AMH and AMH RII isoforms, as well as cytochrome P450, were expressed in both endometriosis epithelial and stromal cells. Treatment of endometriosis stromal and epithelial cell growth with AMH was able to induce a decrease in the percentage of cells in S phase and increase percentage of cells in G1 and G2 phase; coherently, decreased cell viability and increased percentage of cells death fraction was observed. The plasmin-digested AMH was able to suppress most of the cytochrome P450 activity, causing an increase of pre-G1 phase and of apoptosis induction treating with plasmin-digested AMH in both cell lines, most marked in the epithelial cells. Conclusions The data produced suggest a possible use of AMH as therapeutic agents in endometriosis. PMID:24886254

PAH Particles Perturb Prenatal Processes and Phenotypes: Protection from Deficits in Object Discrimination Afforded by Dampening of Brain Oxidoreductase Following In Utero Exposure to Inhaled Benzo(a)pyrene

PubMed Central

Chadalapaka, Gayathri; Ramesh, Aramandla; Khoshbouei, Habibeh; Maguire, Mark; Safe, Stephen; Rhoades, Raina E.; Clark, Ryan; Jules, George; McCallister, Monique; Aschner, Michael; Hood, Darryl B.

2012-01-01

The wild-type (WT) Cprlox/lox (cytochrome P450 oxidoreductase, Cpr) mouse is an ideal model to assess the contribution of P450 enzymes to the metabolic activation and disposition of environmental xenobiotics. In the present study, we examined the effect of in utero exposure to benzo(a)pyrene [B(a)P] aerosol on Sp4 and N-methyl-D-aspartate (NMDA)–dependent systems as well as a resulting behavioral phenotype (object discrimination) in Cpr offspring. Results from in utero exposure of WT Cprlox/lox mice were compared with in utero exposed brain-Cpr-null offspring mice. Null mice were used as they do not express brain cytochrome P4501B1–associated NADPH oxidoreductase (CYP1B1-associated NADPH oxidoreductase), thus reducing their capacity to produce neural B(a)P metabolites. Subsequent to in utero (E14–E17) exposure to B(a)P (100 μg/m3), Cprlox/lox offspring exhibited: (1) elevated B(a)P metabolite and F2-isoprostane neocortical tissue burdens, (2) elevated concentrations of cortical glutamate, (3) premature developmental expression of Sp4, (4) decreased subunit ratios of NR2B:NR2A, and (5) deficits in a novelty discrimination phenotype monitored to in utero exposed brain-Cpr-null offspring. Collectively, these findings suggest that in situ generation of metabolites by CYP1B1-associated NADPH oxidoreductase promotes negative effects on NMDA-mediated signaling processes during the period when synapses are first forming as well as effects on a subsequent behavioral phenotype. PMID:21987461

A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation

PubMed Central

Meyer, Mark B.; Benkusky, Nancy A.; Kaufmann, Martin; Lee, Seong Min; Onal, Melda; Jones, Glenville; Pike, J. Wesley

2017-01-01

The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1. We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease. PMID:28808057

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

PubMed

Bass, C; Carvalho, R A; Oliphant, L; Puinean, A M; Field, L M; Nauen, R; Williamson, M S; Moores, G; Gorman, K

2011-12-01

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

PubMed Central

Koopmann, Edda; Hahlbrock, Klaus

1997-01-01

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. PMID:9405720

Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs

PubMed Central

Wang, D; Guo, Y; Wrighton, SA; Cooke, GE; Sadee, W

2011-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes ~50% of all clinically used drugs. Although CYP3A4 expression varies widely between individuals, the contribution of genetic factors remains uncertain. In this study, we measured allelic CYP3A4 heteronuclear RNA (hnRNA) and mRNA expression in 76 human liver samples heterozygous for at least one of eight marker SNPs and found marked allelic expression imbalance (1.6–6.3-fold) in 10/76 liver samples (13%). This was fully accounted for by an intron 6 SNP (rs35599367, C>T), which also affected mRNA expression in cell culture on minigene transfections. CYP3A4 mRNA level and enzyme activity in livers with CC genotype were 1.7- and 2.5-fold, respectively, greater than in CT and TT carriers. In 235 patients taking stable doses of atorvastatin, simvastatin, or lovastatin for lipid control, carriers of the T allele required significantly lower statin doses (0.2–0.6-fold, P=0.019) than non-T carriers for optimal lipid control. These results indicate that intron 6 SNP rs35599367 markedly affects expression of CYP3A4 and could serve as a biomarker for predicting response to CYP3A4-metabolized drugs. PMID:20386561

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation.

PubMed

Castro, Jazmin; Araya, Germán; Inostroza, Pamela; Hidalgo, Paulina; González-Ramos, Reinaldo; Sovino, Hugo; Boric, M Angélica; Fuentes, Ariel; Johnson, M Cecilia

2015-10-09

Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

Optical Isomers of Atorvastatin, Rosuvastatin and Fluvastatin Enantiospecifically Activate Pregnane X Receptor PXR and Induce CYP2A6, CYP2B6 and CYP3A4 in Human Hepatocytes

PubMed Central

Korhonova, Martina; Doricakova, Aneta; Dvorak, Zdenek

2015-01-01

Atorvastatin, fluvastatin and rosuvastatin are drugs used for treatment of hypercholesterolemia. They cause numerous drug-drug interactions by inhibiting and inducing drug-metabolizing cytochromes P450. These three statins exist in four optical forms, but they are currently used as enantiopure drugs, i.e., only one single enantiomer. There are numerous evidences that efficacy, adverse effects and toxicity of drugs may be enantiospecific. Therefore, we investigated the effects of optical isomers of atorvastatin, fluvastatin and rosuvastatin on the expression of drug-metabolizing P450s in primary human hepatocytes, using western blots and RT-PCR for measurement of proteins and mRNAs, respectively. The activity of P450 transcriptional regulators, including pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR), was assessed by gene reporter assays and EMSA. Transcriptional activity of AhR was not influenced by any statin tested. Basal transcriptional activity of GR was not affected by tested statins, but dexamethasone-inducible activity of GR was dose-dependently and enantioselectively inhibited by fluvastatin. Basal and ligand-inducible transcriptional activity of PXR was dose-dependently influenced by all tested statins, and the potency and efficacy between individual optical isomers varied depending on statin and optical isomer. The expression of CYP1A1 and CYP1A2 in human hepatocytes was not influenced by tested statins. All statins induced CYP2A6, CYP2B6 and CYP3A4, and the effects on CYP2C9 were rather modulatory. The effects varied between statins and enantiomers and induction potency decreased in order: atorvastatin (RR>RS = SR>SS) > fluvastatin (SR>RS = SS>RR) >> rosuvastatin (only RS active). The data presented here might be of toxicological and clinical importance. PMID:26366873

Responsiveness of cerebral and hepatic cytochrome P450s in rat offspring prenatally exposed to lindane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johri, Ashu; Yadav, Sanjay; Dhawan, Alok

2008-08-15

ABSTRACT: Prenatal exposure to low doses of lindane has been shown to affect the ontogeny of xenobiotic metabolizing cytochrome P450s (CYPs), involved in the metabolism and neurobehavioral toxicity of lindane. Attempts were made in the present study to investigate the responsiveness of CYPs in offspring prenatally exposed to lindane (0.25 mg/kg b. wt.; 1/350th of LD{sub 50}; p. o. to mother) when challenged with 3-methylcholanthrene (MC) or phenobarbital (PB), inducers of CYP1A and 2B families or a sub-convulsant dose of lindane (30 mg/kg b. wt., p. o.) later in life. Prenatal exposure to lindane was found to produce an increasemore » in the mRNA and protein expression of CYP1A1, 1A2, 2B1, 2B2 isoforms in brain and liver of the offspring at postnatal day 50. The increased expression of the CYPs in the offspring suggests the sensitivity of the CYPs during postnatal development, possibly, to low levels of lindane, which may partition into mother's milk. A higher increase in expression of CYP1A and 2B isoenzymes and their catalytic activity was observed in animals pretreated prenatally with lindane and challenged with MC (30 mg/kg, i. p. x 5 days) or PB (80 mg/kg, i. p. x 5 days) when young at age (approx. 7 weeks) compared to animals exposed to MC or PB alone. Further, challenge of the control and prenatally exposed offspring with a single sub-convulsant dose of lindane resulted in an earlier onset and increased incidence of convulsions in the offspring prenatally exposed to lindane have demonstrated sensitivity of the CYPs in the prenatally exposed offspring. Our data assume significance as the subtle changes in the expression profiles of hepatic and cerebral CYPs in rat offspring during postnatal development could modify the adult response to a later exposure to xenobiotics.« less

Effects of aging and rifampicin pretreatment on the pharmacokinetics of human cytochrome P450 probes caffeine, warfarin, omeprazole, metoprolol and midazolam in common marmosets genotyped for cytochrome P450 2C19.

PubMed

Toda, Akiko; Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Kusama, Takashi; Shimizu, Makiko; Uno, Yasuhiro; Mogi, Masayuki; Sasaki, Erika; Yamazaki, Hiroshi

2018-07-01

1. The pharmacokinetics were investigated for human cytochrome P450 probes after single intravenous and oral administrations of 0.20 and 1.0 mg/kg, respectively, of caffeine, warfarin, omeprazole, metoprolol and midazolam to aged (10-14 years old, n = 4) or rifampicin-treated/young (3 years old, n = 3) male common marmosets all genotyped as heterozygous for a cytochrome P450 2C19 variant. 2. Slopes of the plasma concentration-time curves after intravenous administration of warfarin and midazolam were slightly, but significantly (two-way analysis of variance), decreased in aged marmosets compared with young marmosets. The mean hepatic clearances determined by in silico fitting for individual pharmacokinetic models of warfarin and midazolam in the aged group were, respectively, 23% and 56% smaller than those for the young group. 3. Significantly enhanced plasma clearances of caffeine, warfarin, omeprazole and midazolam were evident in young marmosets pretreated with rifampicin (25 mg/kg daily for 4 days). Two- to three-fold increases in hepatic intrinsic clearance values were observed in the individual pharmacokinetic models. 4. The in vivo dispositions of multiple simultaneously administered drugs in old, young and P450-enzyme-induced marmosets were elucidated. The results suggest that common marmosets could be experimental models for aged, induced or polymorphic P450 enzymes in P450-dependent drug metabolism studies.

Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

PubMed Central

Chun, Young-Jin; Kim, Donghak

2016-01-01

Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

Gene and protein expression and cellular localisation of cytochrome P450 enzymes of the 1A, 2A, 2C, 2D and 2E subfamilies in equine intestine and liver.

PubMed

Tydén, Eva; Tjälve, Hans; Larsson, Pia

2014-10-08

Among the cytochrome P450 enzymes (CYP), families 1-3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation. CYP1A and CYP2C were the CYPs with the highest gene expression in the intestine and also showed considerable gene expression in the liver. CYP2E and CYP2A showed the highest gene expression in the liver. CYP2E showed moderate intestinal gene expression, whereas that of CYP2A was very low or undetectable. For CYP2D, rather low gene expression levels were found in both intestine and the liver. In the intestine, CYP gene expression levels, except for CYP2E, exhibited patterns resembling those of the proteins, indicating that intestinal protein expression of these CYPs is regulated at the transcriptional level. For CYP2E, the results showed that the intestinal gene expression did not correlate to any visible protein expression, indicating that intestinal protein expression of this CYP is regulated at the post-transcriptional level. Immunostaining of intestine tissue samples showed preferential CYP staining in enterocytes at the tips of intestinal villi in the small intestine. In the liver, all CYPs showed preferential localisation in the centrilobular hepatocytes. Overall, different gene expression profiles were displayed by the CYPs examined in equine intestine and liver. The CYPs present in the intestine may act in concert with those in the liver to affect the oral bioavailability and therapeutic efficiency of substrate drugs. In addition, they may play a role in first-pass metabolism of feed constituents and of herbal supplements used in equine practice.

Examination of Urinary Beta-Naphthol as a Biomarker Indicative of Jet Fuel Exposures

DTIC Science & Technology

2015-04-01

NPQ) by cytochrome P450 has been shown to alter with age, diminishing at a rate of ~ 3% per year.22 Subject age effects on cytochrome P450 enzymes ...of Ageing on cytochrome P450 enzymes : Consequences for drug biotransformation in the elderly. Current Med Chem. (2007) 14:745-757. 24. Van Winkle...naphthalene 1,2-oxide by the cytochrome P450 monooxygenase system (Fig. 1). This reaction occurs primarily in the liver, although oxidation can also

Novel cytochrome P450 genes, CYP6EB1 and CYP6EC1, are over-expressed in acrinathrin-resistant Frankliniella occidentalis (Thysanoptera: Thripidae).

PubMed

Cifuentes, D; Chynoweth, R; Guillén, J; De la Rúa, P; Bielza, P

2012-06-01

Control of Frankliniella occidentalis (Pergande) is a serious problem for agriculture all over the world because of the limited range of insecticides that are available. Insecticide resistance in F. occidentalis has been reported for all major insecticide groups. Our previous studies showed that cytochrome P450-mediated detoxification is a major mechanism responsible for insecticide resistance in this pest. Degenerate polymerase chain reaction was used to identify P450 genes that might be involved in acrinathrin resistance, in a laboratory population of F. occidentalis. Associated sequences were classified as belonging to the CYP4 and CYP6 families. Real-time quantitative polymerase chain reaction analyses revealed that two genes, CYP6EB1 and CYP6EC1, were over-expressed in adults and L2 larvae of the resistant population, when compared with the susceptible population, suggesting their possible involvement in resistance to acrinathrin.

Age-Related Memory Impairment Associated With Decreased Endogenous Estradiol in the Hippocampus of Female Rats.

PubMed

Chamniansawat, Siriporn; Sawatdiyaphanon, Chattraporn

It is widely known that not only the gonadal estradiol (E2) but also hippocampal E2 plays an essential role in memory process. However, the role of hippocampal E2-enhanced memory mechanism during aging is largely unknown. The aim of the present study was to investigate the effect of age on E2 concentration, the expression level of its receptors, and key steroidogenic enzymes in hippocampus. We also investigated the effect of microglia activation on E2 synthesis in hippocampal neurons. The results showed that serum E2 was higher in 19-month-old (aged) rats, which exhibited spatial memory decline in the Morris water maze (MWM) test when compared to the younger rats. Hence, serum E2 may not be associated with the reduced spatial memory performance in aging. In contrast, the level of E2 and the expressions of its receptors were significantly decreased in hippocampus of aged female rat compared to younger females. Furthermore, the expressions of key hippocampal steroidogenic enzymes, steroidogenic acute regulatory protein (StAR), and cytochrome P450 (P450) also significantly decreased with age, which resulted in lower hippocampal E2 levels. In addition, we found that the microglia of aged brain highly expressed interleukin 6 (IL-6), which directly inhibited E2 synthesis in hippocampal neurons via suppression of P450 synthesis. Taken together, we summarized that the microglia-derived IL-6 inhibited hippocampal E2 synthesis in aged rats which, in turn, contributed to the deficit of spatial memory performance.

Identification of SNPs involved in regulating a novel alternative transcript of P450 CYP6ER1 in the brown planthopper.

PubMed

Liang, Zhi-Kun; Pang, Rui; Dong, Yi; Sun, Zhong-Xiang; Ling, Yan; Zhang, Wen-Qing

2017-04-29

Cytochrome P450-mediated metabolic resistance is one of the major mechanisms involved in insecticide resistance. Although the up-regulation of cytochrome P450 plays a vital role in insecticide metabolism, the molecular basis for the transcriptional regulation of cytochrome P450 remains largely unknown. The P450 gene CYP6ER1, has been reported to confer imidacloprid resistance to the brown planthopper, Nilaparvata lugens. Here, we identified a novel alternative transcript of CYP6ER1 (transcript A2) that had different expression patterns between resistant and susceptible populations, and was more stable after insecticide induction. The promoter of this transcript was sequenced and multiple single nucleotide polymorphisms (SNPs) were detected in individuals from susceptible and resistant field-collected populations. Resistant alleles of four SNPs were found to significantly enhance the promoter activity of the CYP6ER1 transcript A2. Electrophoretic mobility shift assays (EMSAs) revealed that these SNPs might regulate the binding of transcription factors to the promoter. Our findings provide novel evidence regarding the transcriptional regulation of a metabolic resistance-related gene and may be useful to understand the resistance mechanism of N. lugens in the field. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Mechanisms of pyrethroid resistance in the dengue mosquito vector, Aedes aegypti: target site insensitivity, penetration, and metabolism.

PubMed

Kasai, Shinji; Komagata, Osamu; Itokawa, Kentaro; Shono, Toshio; Ng, Lee Ching; Kobayashi, Mutsuo; Tomita, Takashi

2014-06-01

Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4'-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains.

Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism

PubMed Central

Kasai, Shinji; Komagata, Osamu; Itokawa, Kentaro; Shono, Toshio; Ng, Lee Ching; Kobayashi, Mutsuo; Tomita, Takashi

2014-01-01

Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4′-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains. PMID:24945250

miRNA-122 Protects Mice and Human Hepatocytes from Acetaminophen Toxicity by Regulating Cytochrome P450 Family 1 Subfamily A Member 2 and Family 2 Subfamily E Member 1 Expression.

PubMed

Chowdhary, Vivek; Teng, Kun-Yu; Thakral, Sharda; Zhang, Bo; Lin, Cho-Hao; Wani, Nissar; Bruschweiler-Li, Lei; Zhang, Xiaoli; James, Laura; Yang, Dakai; Junge, Norman; Brüschweiler, Rafael; Lee, William M; Ghoshal, Kalpana

2017-12-01

Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122 -/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.

PubMed

Reidy, G F; Mehta, I; Murray, M

1989-05-01

The anti-parkinsonian agent orphenadrine has been shown to form an in vitro metabolic intermediate (MI) complex in hepatic microsomes isolated from phenobarbital (PB)-treated rats. The present study was undertaken to assess the cytochrome P-450 isozyme specificity of inhibition and MI complexation. Spectral studies with untreated and PB-induced rat hepatic microsomes confirmed earlier reports on the selectivity of P-450 complexation by orphenadrine; MI complex formation was only observed with PB-induced microsomes. Inhibition studies with the P-450 substrates androst-4-ene-3,17-dione (androstenedione) and 7-pentoxyresorufin revealed selective inhibition of P-450 PB-B/D-associated monooxygenase activity. Thus, in microsomes from untreated male rats, orphenadrine failed to significantly inhibit (less than 50% inhibition up to a concentration of 300 microM) any of the major pathways of P-450-associated androstenedione metabolism. Preincubation of these microsomal fractions with orphenadrine and NADPH was not associated with increased inhibition of androstenedione metabolism. However, in PB-induced microsomes, P-450 PB-B/D-specific androstenedione 16 beta-hydroxylase activity was significantly and selectively inhibited (IC50 = 90 microM). Preincubation of orphenadrine with NADPH-supplemented PB-induced microsomes for 2, 4, or 8 min before androstenedione addition resulted in increased inhibition toward 16 beta-hydroxylase activity, lowering the observed IC50 to 6.6, 0.47, and 0.06 microM), respectively. Preincubation did not affect the selectivity of inhibition. In the absence of preincubation, orphenadrine appeared to be a potent mixed (competitive/noncompetitive)-type inhibitor of P-450 PB-B/D-associated pentoxyresorufin O-depentylation (Ki = 3.8 microM). Preincubation of orphenadrine with NADPH-supplemented microsomal fractions for 4 min resulted in a 30-fold lowering of the apparent inhibitor constant (Ki = 0.13 microM) and a change in the apparent inhibition kinetics to noncompetitive. Treatment of rats with orphenadrine (75 mg/kg/day intraperitoneally for 3 days) was associated with a 2-fold induction of total hepatic P-450, a 5- and 2.4-fold induction of androstenedione 16 beta- and 6 beta-hydroxylase activity, respectively, and formation of an orphenadrine-P-450 MI complex. Western blots of orphenadrine-induced microsomes revealed a 20-fold increase in P-450 PB-B/D-immunoreactive protein.(ABSTRACT TRUNCATED AT 400 WORDS)

CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for Metabolism of Albendazole and Fenbendazole in Human Liver Microsomes and Recombinant P450 Assay Systems

PubMed Central

Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk

2013-01-01

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo. PMID:23959307

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

PubMed

Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

2013-11-01

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

PubMed

Budzinski, Jason W; Trudeau, Vance L; Drouin, Cathy E; Panahi, Mitra; Arnason, J Thor; Foster, Brian C

2007-09-01

In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions.

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink.

PubMed

Berepiki, Adokiye; Hitchcock, Andrew; Moore, C Mark; Bibby, Thomas S

2016-12-16

Increasing the efficiency of the conversion of light energy to products by photosynthesis represents a grand challenge in biotechnology. Photosynthesis is limited by the carbon-fixing enzyme Rubisco resulting in much of the absorbed energy being wasted as heat or fluorescence or lost as excess reductant via alternative electron dissipation pathways. To harness this wasted reductant, we engineered the model cyanobacterium Synechococcus PCC 7002 to express the mammalian cytochrome P450 CYP1A1 to serve as an artificial electron sink for excess electrons derived from light-catalyzed water-splitting. This improved photosynthetic efficiency by increasing the maximum rate of photosynthetic electron flow by 31.3%. A simple fluorescent assay for CYP1A1 activity demonstrated that the P450 was functional in the absence of its native reductase, that activity was light-dependent and scaled with irradiance. We show for the first time in live cells that photosynthetic reductant can be redirected to power a heterologous cytochrome P450. Furthermore, Synechococcus PCC 7002 expressing CYP1A1 degraded the herbicide atrazine, which is a widespread environmental pollutant.

Evidence for complexation of P-450 IIC6 by an orphenadrine metabolite.

PubMed

Reidy, G F; Murray, M

1990-01-30

Removal of the orphenadrine metabolite from its complex with rat liver P-450 IIB1 is associated with a discrepancy in the reactivation of IIB1 activity. Two possible explanations are that either (1) NADPH-P-450-reductase is inaccessible to the restored IIB1, or (2) complexation of other P-450s may occur. Exogenous P-450 reductase increased all pathways of steroid hydroxylation (1.9 to 3.6-fold) but did not enhance reactivation of IIB1-dependent steroid 16 beta-hydroxylation. Instead, P-450 IIC6-dependent progesterone 21-hydroxylase activity was increased after dissociation to 122% of control. IIC6 activity was also inhibited in vitro in microsomes from phenobarbital-induced rats (ki = 151 microM). Thus, orphenadrine appears to complex P-450 IIC6 as well as IIB1 in rat liver.

Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi

PubMed Central

Syed, Khajamohiddin; Shale, Karabo; Pagadala, Nataraj Sekhar; Tuszynski, Jack

2014-01-01

Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes. PMID:24466198

Effect of triptolide on progesterone production from cultured rat granulosa cells.

PubMed

Zhang, J; Jiang, Z; Mu, X; Wen, J; Su, Y; Zhang, L

2012-06-01

Triptolide(CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), is known to have multiple pharmacological activities. However, studies have also shown that triptolide is highly disrupt to the reproductive system by disrupting normal steroid hormone signaling. In the present study, we investigated the effect of triptolide (5, 10, or 20 nM for 24 h) on progesterone production by rat granulosa cells. Triptolide inhibited both basal and human chorionic gonadotropin (HCG)- and 8-bromo-cAMP-stimulated progesterone production as revealed by RIA assay. Furthermore, the HCG-evoked increase in cellular cAMP content was also inhibited by triptolide, indicating that disruption of the cAMP/PKA signaling pathway may mediate the deleterious effects of triptolide on progesterone regulation. In addition, triptolide inhibited 25-OH-cholesterol-stimulated progesterone production, suggesting that activity of the P450 side chain cleavage (P450scc) enzyme was also be inhibited by triptolide. Western blot and quantitative real-time PCR (qRT-PCR) assays further revealed that triptolide decreased mRNA and protein expression of P450scc and the steroidogenic regulatory (StAR) protein in granulosa cells. In contrast, cell viability tests using 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) indicated that triptolide did not cause measurable cell death at doses that suppressed steroidogenesis. The reproductive toxicity of triptolide may be caused by disruption of cAMP/PKA-mediated expression of a number of progesterone synthesis enzymes or regulatory proteins, leading to reduced progesterone synthesis and reproductive dysfunction. © Georg Thieme Verlag KG Stuttgart · New York.

Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism

PubMed Central

van Herwaarden, Antonius E.; Wagenaar, Els; van der Kruijssen, Cornelia M.M.; van Waterschoot, Robert A.B.; Smit, Johan W.; Song, Ji-Ying; van der Valk, Martin A.; van Tellingen, Olaf; van der Hoorn, José W.A.; Rosing, Hilde; Beijnen, Jos H.; Schinkel, Alfred H.

2007-01-01

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a–/–) mice lacking all functional Cyp3a genes. Cyp3a–/– mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a–/– mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems. PMID:17975676

NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

PubMed Central

Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

2013-01-01

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE{sub 2} induced pain model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun

Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day,more » p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE{sub 2} was monitored. While OME treatment by itself exhibited variable effects on PGE{sub 2} induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.« less

Modulation of Hepatic and Renal Metabolism and Toxicity of Trichloroethylene and Perchloroethylene by Alterations in Status of Cytochrome P450 and Glutathione

PubMed Central

Lash, Lawrence H.; Putt, David A.; Huang, Paul; Hueni, Sarah E.; Parker, Jean C.

2007-01-01

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver. PMID:17433522

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

PubMed

Dettwiler, Ramona; Schmitz, Andrea L; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

2014-01-01

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

Heterologous Expression of Equine CYP3A94 and Investigation of a Tunable System to Regulate Co-Expressed NADPH P450 Oxidoreductase Levels

PubMed Central

Dettwiler, Ramona; Schmitz, Andrea L.; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

2014-01-01

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of “Shield-1” prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR. PMID:25415624

FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

EPA Science Inventory

This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

PubMed Central

Pflugmacher, Stephan; Sandermann, Heinrich

1998-01-01

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants. PMID:9576781

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jan, Yi-Hua; Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu; Baker, Angela A.

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling,more » a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.« less

Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity.

PubMed

Brignac-Huber, Lauren M; Park, Ji Won; Reed, James R; Backes, Wayne L

2016-12-01

Cytochrome P450s (P450s) comprise a superfamily of proteins that catalyze numerous monooxygenase reactions in animals, plants, and bacteria. In eukaryotic organisms, these proteins not only carry out reactions necessary for the metabolism of endogenous compounds, but they are also important in the oxidation of exogenous drugs and other foreign compounds. Eukaryotic P450 system proteins generally reside in membranes, primarily the endoplasmic reticulum or the mitochondrial membrane. These membranes provide a scaffold for the P450 system proteins that facilitate interactions with their redox partners as well as other P450s. This review focuses on the ability of specific lipid components to influence P450 activities, as well as the role of the membrane in P450 function. These studies have shown that P450s and NADPH-cytochrome P450 reductase appear to selectively associate with specific phospholipids and that these lipid-protein interactions influence P450 activities. Finally, because of the heterogeneous nature of the endoplasmic reticulum as well as other biologic membranes, the phospholipids are not arranged randomly but associate to generate lipid microdomains. Together, these characteristics can affect P450 function by 1) altering the conformation of the proteins, 2) influencing the P450 interactions with their redox partners, and 3) affecting the localization of the proteins into specific membrane microdomains. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana.

PubMed

Pedrini, Nicolás; Zhang, Shizhu; Juárez, M Patricia; Keyhani, Nemat O

2010-08-01

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C(33) as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.

Editor's Highlight: Congener-Specific Disposition of Chiral Polychlorinated Biphenyls in Lactating Mice and Their Offspring: Implications for PCB Developmental Neurotoxicity.

PubMed

Kania-Korwel, Izabela; Lukasiewicz, Tracy; Barnhart, Christopher D; Stamou, Marianna; Chung, Haeun; Kelly, Kevin M; Bandiera, Stelvio; Lein, Pamela J; Lehmler, Hans-Joachim

2017-07-01

Chiral polychlorinated biphenyl (PCB) congeners have been implicated by laboratory and epidemiological studies in PCB developmental neurotoxicity. These congeners are metabolized by cytochrome P450 (P450) enzymes to potentially neurotoxic hydroxylated metabolites (OH-PCBs). The present study explores the enantioselective disposition and toxicity of 2 environmentally relevant, neurotoxic PCB congeners and their OH-PCB metabolites in lactating mice and their offspring following dietary exposure of the dam. Female C57BL/6N mice (8-weeks old) were fed daily, beginning 2 weeks prior to conception and continuing throughout gestation and lactation, with 3.1 µmol/kg bw/d of racemic 2,2',3,5',6-pentachlorobiphenyl (PCB 95) or 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) in peanut butter; controls received vehicle (peanut oil) in peanut butter. PCB 95 levels were higher than PCB 136 levels in both dams and pups, consistent with the more rapid metabolism of PCB 136 compared with PCB 95. In pups and dams, both congeners were enriched for the enantiomer eluting second on enantioselective gas chromatography columns. OH-PCB profiles in lactating mice and their offspring were complex and varied according to congener, tissue and age. Developmental exposure to PCB 95 versus PCB 136 differentially affected the expression of P450 enzymes as well as neural plasticity (arc and ppp1r9b) and thyroid hormone-responsive genes (nrgn and mbp). The results suggest that the enantioselective metabolism of PCBs to OH-PCBs may influence neurotoxic outcomes following developmental exposures, a hypothesis that warrants further investigation. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Complementary DNA cloning and functional characterization of cytochrome P450 3A138 in common carp (Cyprinus carpio L.).

PubMed

Ma, Junguo; Bu, Yanzhen; Li, Yao; Niu, Daichun; Li, Xiaoyu

2014-06-01

The full-length sequence of a cytochrome P450 3A 138 (CYP3A138) cDNA in common carp was cloned and sequenced. The transcriptional and microsome enzyme activities of CYP3A138 in the fish liver after rifampicin exposure were also determined in this study. The results showed that the full-length CYP3A138 cDNA is 1912 base pairs (bp) long and contains an open reading frame of 1551 bp encoding a protein of 517 amino acids. Sequence analysis revealed that CYP3A138 is highly conserved in fish. Furthermore, the results of quantitative real-time PCR revealed that CYP3A138 in common carp is constitutively expressed in all tissues, but mainly in the liver and intestine. Additionally, rifampicin exposure promoted both the expression of CYP3A138 at the transcriptional level and the activity of the protein, suggesting that CYP3A138 is a member of the CYP3A subfamily. © 2014 Wiley Periodicals, Inc.

Hepatic microsomal metabolism of montelukast, a potent leukotriene D4 receptor antagonist, in humans.

PubMed

Chiba, M; Xu, X; Nishime, J A; Balani, S K; Lin, J H

1997-09-01

Montelukast (L-706,631, MK-0476, SINGULAIR), a potent and selective leukotriene D4 (CysLT1) receptor antagonist, is currently under development for the treatment of asthma. In vitro studies were conducted using human liver microsomes to evaluate: 1) the difference in the metabolic kinetics of montelukast between adult and pediatric subjects; 2) the relative contribution of flavin-containing monooxygenase and cytochrome P450 (P450) to the sulfoxidation; and 3) the P450 isoforms responsible for montelukast oxidation. No statistically significant difference was observed in the in vitro kinetics for acyl glucuronidation and oxidative metabolism between the two age groups. Results from studies on heat inactivation of flavin-containing monooxygenase and immunochemical inhibition by an anti-rat NADPH P450 reductase antibody on montelukast oxidation indicated that all oxidative metabolism of montelukast-including diastereomeric sulfoxidations, as well as 21- and methyl-hydroxylations-are catalyzed exclusively by P450. Five in vitro approaches have been used to identify the P450 isoforms responsible for the human liver microsomal oxidation of montelukast. The experimental results consistently indicated that CYP3A4 catalyzes sulfoxidation and 21-hydroxylation, whereas CYP2C9 selectively mediates methyl-hydroxylation.

[Effect of bemethyl on cytochrome P-450-dependent monoxygenases in the human liver and lymphocytes].

PubMed

Sorokina, E A; Sibiriak, S V; Sergeeva, S A

2002-01-01

Effects of the actoprotector bemithyl (50 mg/kg, p.o.) upon a single or five-fold administration on the cytochrome P-450 and b5 content and the isoform-specific and nonspecific monooxygenase activity [aminopyrine-N-demethylase, aniline-p-hydroxylase, 4-nitroanisole-o-demethylase,2,5-diphenyloxazole-p-hydroxylase, 7-ethoxyresorufin-o-deethylase (EROD), benzyloxyresorufin-o-debenzylase (BROD)] in rat liver were evaluated. In addition, the influence of bemithyl (0.(1)-100 microM) on the development of EROD and BROD activity was studied on the mitogen-stimulated human lymphocytes in vitro. Administered in rats, bemithyl exhibited the properties of a cytochrome P-450 inductor of the mixed type, which was manifested by an increase in the total cytochrome P-450 content in liver microsomes and in the monooxygenase activity related to both Ah-receptor-dependent and -independent isoforms (except for the aniline-p-hydroxylase activity). The induction of the monooxygenase activity realized by Ah-receptor-dependent isoforms (4-nitroanisole-o-demethylase, 2,5-diphenyloxazole-p-hydroxylase, and EROD activity) was more pronounced, reaching maximum upon a single drug administration. Acting upon the human lymphocytes in vitro, high concentrations of bemithyl increased expression of the EROD activity, while low drug concentrations stimulated the BROD activity.

Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation.

PubMed Central

Roberts, E S; Vaz, A D; Coon, M J

1991-01-01

As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts. PMID:1924356

Anti-inflammatory effect and modulation of cytochrome P450 activities by Artemisia annua tea infusions in human intestinal Caco-2 cells.

PubMed

Melillo de Magalhães, Pedro; Dupont, Isabelle; Hendrickx, Aurélie; Joly, Aurélie; Raas, Thomas; Dessy, Stéphanie; Sergent, Thérèse; Schneider, Yves-Jacques

2012-09-15

In an attempt to understand the beneficial health effects of Artemisia annua other than its anti-malaria properties, extracts from different cultivars prepared as tea infusions were investigated using Caco-2 cells on the intestinal inflammation and cytochrome P450 (CYP) activities. The characterisation of their phenolic compound (PC) profile revealed rosmarinic and chlorogenic acids as the main PCs. The extracts, assayed on Caco-2 cells at a plausible intestinal concentration, significantly decreased the secretion of pro-inflammatory cytokines, IL-8 and IL-6. This effect could be attributable at least to their content in rosmarinic acid, detected as a potent anti-inflammatory compound. The extracts also inhibited the activity of CYP3A4, whose expression was induced by 1,25-dihydroxyvitamin D(3), and of CYP1A1, induced by benzo(a)pyrene. Our results highlight the advantage of drinking A. annua infusions for their potent anti-inflammatory effect, linked to PC content, which could synergise their antimalarial activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nine co-localized cytochrome P450 genes of the CYP2N, CYP2AD, and CYP2P gene families in the mangrove killifish Kryptolebias marmoratus genome: Identification and expression in response to B[α]P, BPA, OP, and NP.

PubMed

Puthumana, Jayesh; Kim, Bo-Mi; Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Jung, Jee-Hyun; Kim, Il-Chan; Hwang, Un-Ki; Lee, Jae-Seong

2017-06-01

The CYP2 genes are the largest and most diverse cytochrome P450 (CYP) subfamily in vertebrates. We have identified nine co-localized CYP2 genes (∼55kb) in a new cluster in the genome of the highly resilient ecotoxicological fish model Kryptolebias marmoratus. Molecular characterization, temporal and tissue-specific expression pattern, and response to xenobiotics of these genes were examined. The CYP2 gene clusters were characterized and designated CYP2N22-23, CYP2AD12, and CYP2P16-20. Gene synteny analysis confirmed that the cluster in K. marmoratus is similar to that found in other teleost fishes, including zebrafish. A gene duplication event with diverged catalytic function was observed in CYP2AD12. Moreover, a high level of divergence in expression was observed among the co-localized genes. Phylogeny of the cluster suggested an orthologous relationship with similar genes in zebrafish and Japanese medaka. Gene expression analysis showed that CYP2P19 and CYP2N20 were consecutively expressed throughout embryonic development, whereas CYP2P18 was expressed in all adult tissues, suggesting that members of each CYP2 gene family have different physiological roles even though they are located in the same cluster. Among endocrine-disrupting chemicals (EDCs), benzo[α]pyrene (B[α]P) induced expression of CYP2N23, bisphenol A (BPA) induced CYP2P18 and CYP2P19, and 4-octylphenol (OP) induced CYP2AD12, but there was no significant response to 4-nonylphenol (NP), implying differential catalytic roles of the enzyme. In this paper, we identify and characterize a CYP2 gene cluster in the mangrove killifish K. marmoratus with differing catalytic roles toward EDCs. Our findings provide insights on the roles of nine co-localized CYP2 genes and their catalytic functions for better understanding of chemical-biological interactions in fish. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional characterization of four naturally occurring variants of human pregnane X receptor (PXR): one variant causes dramatic loss of both DNA binding activity and the transactivation of the CYP3A4 promoter/enhancer region.

PubMed

Koyano, Satoru; Kurose, Kouichi; Saito, Yoshiro; Ozawa, Shogo; Hasegawa, Ryuichi; Komamura, Kazuo; Ueno, Kazuyuki; Kamakura, Shiro; Kitakaze, Masafumi; Nakajima, Toshiharu; Matsumoto, Kenji; Akasawa, Akira; Saito, Hirohisa; Sawada, Jun-Ichi

2004-01-01

Metabolism of administered drugs is determined by expression and activity of many drug-metabolizing enzymes, such as the cytochrome P450 (P450s) family members. Pregnane X receptor (PXR) is a master transcriptional regulator of many drug/xenobiotic-metabolizing enzymes, including P450s and drug transporters. In this study, we describe the functional analysis of four naturally occurring human PXR (hPXR) variants (R98C, R148Q, R381W, and I403V) that we have recently identified. By a reporter gene assay using the CYP3A4 promoter/enhancer reporter in COS-7 or HepG2 cells, it was found that the R98C variant failed to transactivate the CYP3A4 reporter. The R381W and I403V variants also showed varying degrees of reduction in transactivation, depending on the dose of PXR activators, rifampicin, clotrimazole, and paclitaxel. The transcriptional activities of the R148Q variant were not significantly different from that of the wild-type hPXR. The electrophoretic mobility shift assay revealed that only the R98C variant lacked DNA binding. Furthermore, the cellular localization of the hPXR proteins was analyzed. All four variants as well as the wild-type hPXR localized exclusively to the nucleus, regardless of the presence or absence of rifampicin. These data suggest that the R98C, R381W, and I403V hPXR variants, especially R98C, may influence the expression of drug-metabolizing enzymes and transporters, which are transactivated by PXR.

Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

PubMed

Tomankova, Veronika; Liskova, Barbora; Skalova, Lenka; Bartikova, Hana; Bousova, Iva; Jourova, Lenka; Anzenbacher, Pavel; Ulrichova, Jitka; Anzenbacherova, Eva

2015-07-15

Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane). Copyright © 2015 Elsevier Inc. All rights reserved.

NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from Withania somnifera (L.) Dunal

PubMed Central

Rana, Satiander; Lattoo, Surrinder K.; Dhar, Niha; Razdan, Sumeer; Bhat, Wajid Waheed; Dhar, Rekha S.; Vishwakarma, Ram

2013-01-01

Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from W. somnifera. PMID:23437311

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

PubMed Central

Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; Ajikumar, Parayil Kumaran

2016-01-01

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities. PMID:26951651

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.

PubMed

Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; De Mey, Marjan; Ajikumar, Parayil Kumaran

2016-03-22

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Hao, E-mail: hao.hu1@uqconnect.edu.au; Yu, Ting, E-mail: t.yu2@uq.edu.au; Arpiainen, Satu, E-mail: Satu.Juhila@orion.fi

2015-11-15

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3 kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5′-Luc constructs – down to − 160 bp from the TSS – showed p53 responsivenessmore » in p53 overexpressed C3A cells. However, a further deletion from − 160 to − 74 bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene – a well-known p53 activator – increased the expression of the p53 responsive positive control and the CYP2A6-5′-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5′-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6. - Highlights: • CYP2A6 is an immediate target gene of p53. • Six putative p53REs located on 3 kb proximate CYP2A6 promoter region. • The region − 160 bp from TSS is highly homologous with the p53 consensus sequence. • P53 specifically bind to the p53RE on the − 160 bp region. • HNF4α may interact with p53 in regulating CYP2A6 expression.« less

E. coli infection modulates the pharmacokinetics of oral enrofloxacin by targeting P-glycoprotein in small intestine and CYP450 3A in liver and kidney of broilers.

PubMed

Guo, Mengjie; Sun, Yong; Zhang, Yu; Bughio, Shamsuddin; Dai, Xiaohua; Ren, Weilong; Wang, Liping

2014-01-01

P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of abcb1 mRNA and localization of P-gp protein in the liver, kidney, duodenum, jejunum and ileum in healthy and E. coli infected broilers by real time RT-PCR and immunohistochemistry. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P<0.05), but not significantly in the liver and duodenum (P>0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (P<0.05) compared with healthy birds. Furthermore, the infection reduced absorption of orally administered enrofloxacin, significantly decreased Cmax (0.34 vs 0.98 µg mL(-1), P = 0.000) and AUC0-12h (4.37 vs 8.88 µg mL(-1) h, P = 0.042) of enrofloxacin, but increased Tmax (8.32 vs 3.28 h, P = 0.040), T1/2a(2.66 vs 1.64 h(-1), P = 0.050) and V/F (26.7 vs 5.2 L, P = 0.040). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers.

Exogenous steroid substrate modifies the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estradiol production of human luteinized granulosa cells in vitro.

PubMed

Morán, F M; Lohstroh, P; VandeVoort, C A; Chen, J; Overstreet, J W; Conley, A J; Lasley, B L

2003-01-01

The in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid metabolism in human luteinized granulosa cells (hLGC) have been summarized as a decreased estradiol (E(2)) production without altering either E(2) metabolism or cytochrome P450 aromatase activity. In the present study, hLGC were used to analyze the fate of different substrates for cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450(c17)) in the presence or absence of TCDD. Human LGCs were plated directly on plastic culture dishes in medium supplemented with 2 IU/ml of hCG. TCDD (10 nM) or its solvent was added directly to the cells at the time of medium change, every 48 h for 8 days. The objective of the experiment was to test the hypothesis that exogenous steroid, substrate for P450(c17), would reduce the TCDD effects on E(2) synthesis. With dehydroepiandrosterone (DHEA) (a P450(c17) product), a dose-related increase in E(2) production was observed and the effect of TCDD on lowering E(2) production disappeared. In contrast, with increasing doses, up to 10 micro M, of pregnenolone (P(5)), no change in E(2) production was observed. However, 17alpha-hydroxypregnenolone (17P(5)) at 10 micro M produced a modest but significant increase in the E(2) production. Treatments with P(5) and 17P(5) did not alter the effect of TCDD on E(2) production. Radiolabeled substrate utilization by hLGC suggests that the principal metabolic pathway for Delta5 substrates is the conversion to a Delta4 product probably by a very active 3beta-hydroxysteroid dehydrogenase. We conclude that estrogen production by hLGC is limited at the level of lyase activity. Thus, these data suggest that the most likely target for the TCDD-induced inhibition of estrogen synthesis by hLGC is the 17,20-lyase activity of the P450(c17) enzyme complex.

A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

PubMed

Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

1989-02-28

1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug-Flow Bioreactors for the Production of Drug Metabolites

PubMed Central

Wollenberg, Lance A.; Kabulski, Jarod L.; Powell, Matthew J.; Chen, Jifeng; Flora, Darcy R.; Tracy, Timothy S.; Gannett, Peter M.

2013-01-01

Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly (methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug-flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: 1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process. PMID:24166101

Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1.

PubMed

Ding, Zhiping; Wen, Yucong; Yang, Baojun; Zhang, Yixi; Liu, Shuhua; Liu, Zewen; Han, Zhaojun

2013-11-01

Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present. Copyright © 2013 Elsevier Ltd. All rights reserved.

Spinosad resistance in female Musca domestica L. from a field-derived population.

PubMed

Markussen, Mette D K; Kristensen, Michael

2012-01-01

Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC(50), whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression. Copyright © 2011 Society of Chemical Industry.

21-Aminosteroids prevent the down-regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits

PubMed Central

Galal, Ahmed; du Souich, Patrick

1999-01-01

This study was conducted to assess whether a 21-aminosteroid, U74389G, could prevent the down-regulation of hepatic cytochrome P450 (P450) induced by acute moderate hypoxia or an inflammatory reaction.The rabbits of two groups (n=6 per group) were subjected to acute moderate hypoxia (PaO2≈35 mmHg), one pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 48 h). The rabbits of two other groups received 5 ml of turpentine s.c., one of them being pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 72 h). The kinetics of theophylline (2.5 mg kg−1) were assessed to evaluate the activity of the P450. Once the rabbits were sacrificed, the P450 content and the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were estimated in the liver.Compared with control rabbits, hypoxia and inflammation increased theophylline plasma concentrations, as a result of a decrease in theophylline systemic clearance (P<0.05). Both experimental conditions reduced hepatic content of P450 by 40–50% (P<0.05) and increased the amount of hepatic TBARS by around 50% (P<0.05). Pre-treatment with U74389G prevented the hypoxia- and inflammation-induced decrease in theophylline systemic clearance, the down-regulation of hepatic P450, and the increase in liver TBARS.It is concluded that in the rabbit, U74389G prevents hepatic P450 depression produced by acute moderate hypoxia and a turpentine-induced inflammatory reaction, possibly by eliciting a radical quenching antioxidant activity. PMID:10510447

Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

PubMed

Staack, Roland F; Theobald, Denis S; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

2004-04-01

p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.

Are Atlantic cod in Store Lungegårdsvann, a seawater recipient in Bergen, affected by environmental contaminants? A qRT-PCR survey.

PubMed

Olsvik, Pål A; Lie, Kai K; Goksøyr, Anders; Midtun, Torbjørn; Frantzen, Sylvia; Maage, Amund

2009-01-01

The aim of this study was to examine the transcriptional levels of selected genes in liver and head kidney of Atlantic cod Gadus morhua sampled in Store Lungegårdsvann, a seawater recipient situated in the middle of the city of Bergen, Norway, for effects of contaminants released from municipal sewage effluents and former dump sites. Five males and six females were caught with fish traps in Store Lungegårdsvann in 2006. Cod from a location near Jondal in the Hardanger Fjord were used as controls (five males and four females). The following 12 genes were picked as potential markers of contaminant exposure: cytochrome P-450 1A (CYP1A), cytochrome P-450 2C33-like (CYP2C33-like), cytochrome P-450 3C (CYP3C), glutathione S-transcriptase pi (GST) (detoxification and biotransformation), Mn superoxide dismutase (Mn SOD), glutathione reductase (GR), heat-shock protein 70 (HSP70) (oxidative stress), vitellogenin A (VtgA), vitellogenin B (VtgB), zona pellucida 2 (ZP2) (effects of estrogen disruptors), B-cell lymphoma 2 (Bcl-2), and cyclin-dependent kinase inhibitor 1A (CDKN1A) (radiation). The results showed that two males caught in Store Lungegårdsvann possessed high transcriptional levels of VtgA, VtgB, and ZP2 mRNA in the liver. In addition, CYP1A was 4.9-fold higher expressed in males from Store Lungegårdsvann compared to males from the reference population. CYP2C33-like mRNA expression was significantly higher (1.8-fold) in females from Store Lungegårdsvann than in females from the reference population. CYP1A was significantly lower (4.7-fold) expressed in head kidney of females from Store Lungegårdsvann than in females from Hardanger Fjord. In a follow-up examination with sexually mature cod sampled in Store Lungegårdsvann in 2007, the livers were shown to contain high levels of polychlorinated biphenyls (PCB) and dioxin-like PCB. In conclusion, fish inhabiting Store Lungegårdsvann are exposed not only to endocrine disruptors but also to other contaminants that affect the transcription of phase I biotransformation genes.

Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450.

PubMed

Iwasaki, M; Juvonen, R; Lindberg, R; Negishi, M

1991-02-25

The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.

Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate.

PubMed

Venkatakrishnan, K; von Moltke, L L; Greenblatt, D J

2001-04-01

The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase.

PubMed

Henderson, Colin J; Otto, Diana M E; Carrie, Dianne; Magnuson, Mark A; McLaren, Aileen W; Rosewell, Ian; Wolf, C Roland

2003-04-11

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

Detection and diversity of fungal nitric oxide reductase genes ( p450nor) in agricultural soils

DOE PAGES

Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.; ...

2016-03-11

Members of the Fungi convert nitrate (NO 3 -) and nitrite (NO 2 -) to gaseous nitrous oxide (N 2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N 2O from added NO 3 - or NO 2 - in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups,more » of which 151 produced N 2O from NO 2 -. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N 2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N 2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N 2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2O formation.« less

Detection and diversity of fungal nitric oxide reductase genes ( p450nor) in agricultural soils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.

Members of the Fungi convert nitrate (NO 3 -) and nitrite (NO 2 -) to gaseous nitrous oxide (N 2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N 2O from added NO 3 - or NO 2 - in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups,more » of which 151 produced N 2O from NO 2 -. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N 2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N 2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N 2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2O formation.« less

Chemopreventive effects of korean red ginseng extract on rat hepatocarcinogenesis.

PubMed

Kim, Hyemee; Hong, Mi-Kyung; Choi, Haymie; Moon, Hyun-Seuk; Lee, Hae-Jeung

2015-01-01

The objective of this study was to determine a chemopreventive activity of Korean red ginseng extract (KRG) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. After acclimatization for a week, Sprague-Dawley rats were randomized into five groups (n = 15) and fed either KRG (0.5, 1 or 2%) or control diets for 10 weeks. After two weeks of starting of experimental diets, the rats were initiated hepatocarcinogenesis by injection of DEN and were then subjected to two-thirds partial hepatectomy at five-week for developing the medium-term bioassay system. Both 0.5 and 1% KRG diets suppressed the area (55 and 60%; p= 0.0251 and 0.0144) and number (39 and 59%; p= 0.0433 and 0.0012) of glutathione S-transferase placental form (GST-P) positive foci when compared to the DEN-control group. The production of thiobarbituric acid reactive substances (TBARS) was significantly reduced in 0.5 and 1% KRG-treated rats. The supplementation of 1% KRG diet significantly elevated the levels of total glutathione (tGSH) and glutathione-related enzymes including cytosolic glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. It was also observed in cDNA microarray that the gene expressions (Cyp2c6, Cyp2e1, Cyp3a9, and Mgst1) involved in the xenobiotics metabolism via cytochrome P450 signaling pathway were down-regulated in the 1% KRG diet-treated group when compared to the DEN-control. The chemopreventive effects of KRG could be affected by 1) the decrease of lipid peroxidation, 2) the increase of tGSH content and GSH-dependent enzyme activities, and 3) the decrease of the gene expression profile involved in cytochrome P450 signaling pathway. These results suggest that KRG may prove to be a therapeutic agent against hepatocarcinogenesis.

Chemopreventive Effects of Korean Red Ginseng Extract on Rat Hepatocarcinogenesis

PubMed Central

Kim, Hyemee; Hong, Mi-Kyung; Choi, Haymie; Moon, Hyun-Seuk; Lee, Hae-Jeung

2015-01-01

The objective of this study was to determine a chemopreventive activity of Korean red ginseng extract (KRG) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. After acclimatization for a week, Sprague-Dawley rats were randomized into five groups (n = 15) and fed either KRG (0.5, 1 or 2%) or control diets for 10 weeks. After two weeks of starting of experimental diets, the rats were initiated hepatocarcinogenesis by injection of DEN and were then subjected to two-thirds partial hepatectomy at five-week for developing the medium-term bioassay system. Both 0.5 and 1% KRG diets suppressed the area (55 and 60%; p= 0.0251 and 0.0144) and number (39 and 59%; p= 0.0433 and 0.0012) of glutathione S-transferase placental form (GST-P) positive foci when compared to the DEN-control group. The production of thiobarbituric acid reactive substances (TBARS) was significantly reduced in 0.5 and 1% KRG-treated rats. The supplementation of 1% KRG diet significantly elevated the levels of total glutathione (tGSH) and glutathione-related enzymes including cytosolic glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. It was also observed in cDNA microarray that the gene expressions (Cyp2c6, Cyp2e1, Cyp3a9, and Mgst1) involved in the xenobiotics metabolism via cytochrome P450 signaling pathway were down-regulated in the 1% KRG diet-treated group when compared to the DEN-control. The chemopreventive effects of KRG could be affected by 1) the decrease of lipid peroxidation, 2) the increase of tGSH content and GSH-dependent enzyme activities, and 3) the decrease of the gene expression profile involved in cytochrome P450 signaling pathway. These results suggest that KRG may prove to be a therapeutic agent against hepatocarcinogenesis. PMID:25553083

[Steroid and xenobiotic receptor (SXR), multidrug resistance gene (MDR1) and GSTs, SULTs and CYP polymorphism expression in invasive bladder cancer, analysis of their expression and correlation with other prognostic factors].

PubMed

Rioja Zuazu, J; Bandrés Elizalde, E; Rosell Costa, D; Rincón Mayans, A; Zudaire Bergera, J; Gil Sanz, M J; Rioja Sanz, L A; García Foncillas, J; Berián Polo, J M

2007-01-01

Steroid and Xenobiotic Receptor (SXR) has demonstrated its activation by numerous drugs, including cytochrome P450 potent inducers like rifampicina or cotrimazol. The role of SXR is well known, and lies regulating in a positive manner cytochrome P450 3A4 (CYP3A4) transcription and the multidrug resistance gene (MDR1), it's considered a key in the xenobiotic detoxification mechanism, being involved in all phases of the detoxification process. Enzymes involved in Policyclic Aromatic hidrocarbures (PAH) metabolism and degradation are polymorphic in humans, including glutation S-transferases (GSTs), N-acetiltransferases (NATs), sulfotransferases (SULTs)1A1 and cytochrome p450 (CYP)1B1. The objectives we've planned are: 1. Analyze the expression of the transcription factor SXR and MDR1 in bladder by means of RT-PCR real time, both in normal bladder and in tumoral bladder. 2. Analyze the relation between clinical and pathological factors with the expression of SXR and MDR1. 3. Analyze the expression of the polymorphims CYP1B1, GSTM1 GSTT1 and SULT1A1 and their correlation with different clinic-pathological and molecular factors. In a prospective way the size of the sample was estimated. In 67 patients from two institutions (Hospital Universitario Miguel Servet (49 HUMS) and Clinica Universitaria de Navarra (18 CUN)), diagnosed of invasive bladder cancer and treated by means of radical cystectomy, were determined the expression of both SXR and MDR1 by means of real time PCR, as well as the polymorphisms CYP1B1, GSTM1 GSTT1 y SULT1A1 by means of RFLP (Restriction fragment length polymorphism). Correlations with other prognostic factors by contingency tables were performed. Average follow up was 23.7 months with a median of 28.26 months. Of the 67 patients studied, 31 patients (46.3) presented disease progression, in form of local recurrence or in distant metastasis or both. With a average time to progression of 12.4 months and a median of 10 months, with a range of 1.1 month to 31.9 month. 36 patients (53.7%) did not have any evidence of disease progression during follow up. The Steroid and Xenobiotic Receptor as well as the Multidrug Resistance Gene (MDR1) are expressed in both normal bladder (0.94DeltaCt y 0.94DeltaCt) and tumoral bladder in the cystectomy specimen (1.09 DeltaCt y 0.45 DeltaCt). We've analyzed their expression in a quantitative manner and in a qualitative manner. The expression of SXR correlates with the presence of ca. in situ (p=0.024), vasculo-lymphatic invasion (p=0.05) mean while MDR1 correlates with presence of vasculo-lymphatic invasion (p=0.05) Both factors are correlate between each others (p=0.011). Polymorphisms: CYP1B1, GSTM1, GSTT1 and SULT1A1, are expressed in these patients but their expression doesn't correlates with any prognostic factor Both SXR and MDR1 are expressed in normal bladder as well as in tumoral bladder. And their expression correlates with different prognostic factors with influence in the survival described in the literature.

Imidacloprid is degraded by CYP353D1v2, a cytochrome P450 overexpressed in a resistant strain of Laodelphax striatellus.

PubMed

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Wu, Min; Zhang, Haomiao; Pu, Jian; Jiang, Ling; Han, Zhaojun

2017-07-01

Cytochrome P450s are associated with the metabolising of a wide range of compounds, including insecticides. CYP353D1v2 has been found to be overexpressed in an imidacloprid-resistant strain of Laodelphax striatellus. Thus, this study was conducted to express CYP353D1v2 in Sf9 cells as a recombinant protein, to assess its ability to metabolise imidacloprid. Western blot and carbon monoxide difference spectrum analysis indicated that the intact CYP353D1v2 protein had been successfully expressed in Sf9 insect cells. Catalytic activity tests with four traditional P450-activity-probing substrates found that the expressed CYP353D1v2 preferentially metabolised p-nitroanisole, ethoxycoumarin and ethoxyresorufin with specific activities of 32.70, 0.317 and 1.22 pmol min -1 pmol -1 protein respectively, but no activity to luciferin-H EGE. The enzyme activity for degrading imidacloprid was tested by measuring substrate depletion and formation of the metabolite. Kinetic parameters for imidacloprid were K m 5.99 ± 0.95 µm and k cat 0.03 ± 0.0004 min -1 . The chromatogram analysis showed clearly the NADPH-dependent depletion of imidacloprid and the formation of an unknown metabolite. The UPLC-MS mass spectrum demonstrated that the metabolite was an oxidative product of imidacloprid, 5-hydroxy-imidacloprid. These results suggest that CYP353D1v2 in L. striatellus is capable of degrading imidacloprid, and that enzyme activity can be evaluated well only by some traditional probing substrates. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne benzo(a)pyrene exposure: implications for biomarker determination.

PubMed

Lee, Jin Wuk; Kim, Yong Hwa; Yoon, Seokjoo; Lee, Sung Kyu

2014-09-01

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t-test, p < 0.01) after 4 days of exposure in 100 μg/L BaP. These results indicate that the only use of mRNA expression level of CYP system as a biomarker make us underestimate prolonged toxicity (4-14 days) of BaP and the only use of protein expression level of CYP system make us underestimate acute toxicity (1-2 days) of BaP. Therefore, we suggests that a combinational use of the mRNA expression level and protein expression level of CYP system, hepatosomatic index is a useful biomarker in risk assessment of waterborne BaP exposure. In addition, DNA adduct formation was a useful biomarker in risk assessment of waterborne BaP exposure at 4 days. CYP1A was a more sensitive biomarker than CYP reductase for BaP exposure when considering both the mRNA and protein level. Furthermore, our results show that Z. platypus is a useful species for assessing the risk of waterborne BaP exposure. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

R-warfarin clearances from plasma associated with polymorphic cytochrome P450 2C19 and simulated by individual physiologically based pharmacokinetic models for 11 cynomolgus monkeys.

PubMed

Utoh, Masahiro; Kusama, Takashi; Miura, Tomonori; Mitsui, Marina; Kawano, Mirai; Hirano, Takahiro; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

2018-02-01

1. Cynomolgus monkey cytochrome P450 2C19 (formerly known as P450 2C75), homologous to human P450 2C19, has been identified as R-warfarin 7-hydroxylase. In this study, simulations of R-warfarin clearance in individual cynomolgus monkeys genotyped for P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling. 2. Pharmacokinetic parameters and absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearances for individual PBPK models were estimated for eleven cynomolgus monkeys. 3. One-way ANOVA revealed significant effects of the genotype (p < 0.01) on the observed elimination half-lives and areas under the curves of R-warfarin among the homozygous mutant, heterozygous mutant, and wild-type groups. R-Warfarin clearances in individual cynomolgus monkeys genotyped for P450 2C19 were simulated by simplified PBPK modeling. The modeled hepatic intrinsic clearances were significantly associated with the P450 2C19 genotypes. The liver microsomal elimination rates of R-warfarin for individual animals after in vivo administration showed significant reductions associated with the genotype (p < 0.01). 4. This study provides important information to help simulate clearances of R-warfarin and related medicines associated with polymorphic P450 2C19 in individual cynomolgus monkeys, thereby facilitating calculation of the fraction of hepatic clearance.

Influence of amino acid residues near the active site of cytochrome P450 from Bacillus megaterium on the selectivity of n-octane oxidation to octanol regioisomers

NASA Astrophysics Data System (ADS)

Miyaji, Akimitsu; Baba, Toshihide

2017-09-01

A mutant of cytochrome P450 from Bacillus megaterium (CYP450BM-3) was prepared by replacing two alanine residues around active site of the enzyme, alanine 328 and alanine 82, with leucine and tryptophan, respectively. The CYP450BM-3 mutant produced 2-octanol selectively from n-octane under atmospheric temperature and pressure; its selectivity was 74%. Furthermore, the mutant produced 1-octanol, which is not produced by wild-type enzyme.

Effects of cytochrome P450 3A modulators ketoconazole and carbamazepine on quetiapine pharmacokinetics

PubMed Central

Grimm, Scott W; Richtand, Neil M; Winter, Helen R; Stams, Karen R; Reele, Stots B

2006-01-01

Aims To explore the potential for drug interactions on quetiapine pharmacokinetics using in vitro and in vivo assessments. Methods The CYP enzymes responsible for quetiapine metabolite formation were assessed using recombinant expressed CYPs and CYP-selective inhibitors. P-glycoprotein (Pgp) transport was tested in MDCK cells expressing the human MDR1 gene. The effects of CYP3A4 inhibition were evaluated clinically in 12 healthy volunteers that received 25 mg quetiapine before and after 4 days of treatment with ketoconazole 200 mg daily. To assess CYP3A4 induction in vivo, 18 patients with psychiatric disorders were titrated to steady-state quetiapine levels (300 mg twice daily), then titrated to 600 mg daily carbamazepine for 2 weeks. Results CYP3A4 was found to be responsible for formation of quetiapine sulfoxide and N- and O-desalkylquetiapine and not a Pgp substrate. In the clinical studies, ketoconazole increased mean quetiapine plasma Cmax by 3.35-fold, from 45 to 150 ng ml−1 (mean Cmax ratio 90% CI 2.51, 4.47) and decreased its clearance (Cl/F) by 84%, from 138 to 22 l h−1 (mean ratio 90% CI 0.13, 0.20). Carbamazepine decreased quetiapine plasma Cmax by 80%, from 1042 to 205 ng ml−1 (mean Cmax ratio 90% CI 0.14, 0.28) and increased its clearance 7.5-fold, from 65 to 483 l h−1 (mean ratio 90% CI 6.04, 9.28). Conclusions Cytochrome P450 3A4 is a primary enzyme responsible for the metabolic clearance of quetiapine. Quetiapine pharmacokinetics were affected by concomitant administration of ketoconazole and carbamazepine, and therefore other drugs and ingested natural products that strongly modulate the activity or expression of CYP3A4 would be predicted to change exposure to quetiapine. PMID:16390352

Differential regulation by heat stress of novel cytochrome P450 genes from the dinoflagellate symbionts of reef-building corals.

PubMed

Rosic, Nedeljka N; Pernice, Mathieu; Dunn, Simon; Dove, Sophie; Hoegh-Guldberg, Ove

2010-05-01

Exposure to heat stress has been recognized as one of the major factors leading to the breakdown of the coral-alga symbiosis and coral bleaching. Here, we describe the presence of three new cytochrome P450 (CYP) genes from the reef-building coral endosymbiont Symbiodinium (type C3) and changes in their expression during exposure to severe and moderate heat stress conditions. Sequence analysis of the CYP C-terminal region and two conserved domains, the "PERF" and "heme-binding" domains, confirmed the separate identities of the CYP genes analyzed. In order to explore the effects of different heat stress scenarios, samples of the scleractinian coral Acropora millepora were exposed to elevated temperatures incrementally over an 18-h period (rapid thermal stress) and over a 120-h period (gradual thermal stress). After 18 h of gradual heating and incubation at 26 degrees C, the Symbiodinium CYP mRNA pool was approximately 30% larger, while a further 6 degrees C increase to a temperature above the average sea temperature (29 degrees C after 72 h) resulted in a 2- to 4-fold increase in CYP expression. Both rapid heat stress and gradual heat stress at 32 degrees C resulted in 50% to 90% decreases in CYP gene transcript abundance. Consequently, the initial upregulation of expression of CYP genes at moderately elevated temperatures (26 degrees C and 29 degrees C) was followed by a decrease in expression under the greater thermal stress conditions at 32 degrees C. These findings indicate that in the coral-alga symbiosis under heat stress conditions there is production of chemical stressors and/or transcriptional factors that regulate the expression of genes, such as the genes encoding cytochrome P450 monooxygenases, that are involved in the first line of an organism's chemical defense.

N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

PubMed Central

Jennings, Gareth K.; Ritchie, Caroline M.; Shock, Lisa S.; Lyons, Charles E.

2016-01-01

Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH <7.4, the spectra are definitively type I, whereas at pH ≥7.4 the spectra have split Soret bands, including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development. PMID:27126611

Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells.

PubMed

Tran, Christine D H; Timmins, Peter; Conway, Barbara R; Irwin, William J

2002-01-01

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 microM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 microM 1alpha,25-dihydroxyvitamin D(3) to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213 +/- 54.35%) compared to that of parent cells (100 +/- 6.05%). Furthermore, they retained significantly less [(3)H]vincristine sulphate (p < 0.05), a P-gp substrate, after efflux (272.89 +/- 11.86 fmol/mg protein) than the parent cells (381.39 +/- 61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1alpha,25-dihydroxyvitamin D(3) treatment was quantified to be 76.2 +/- 7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0 +/- 20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0 +/- 20.0 to 1057.0 +/- 46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. Copyright 2002 Wiley-Liss, Inc.

Protopine and allocryptopine increase mRNA levels of cytochromes P450 1A in human hepatocytes and HepG2 cells independently of AhR.

PubMed

Vrba, Jiri; Vrublova, Eva; Modriansky, Martin; Ulrichova, Jitka

2011-06-10

The isoquinoline alkaloids protopine and allocryptopine are present in phytopreparations from medicinal plants, such as Fumaria officinalis. Since nothing is known about effects of the alkaloids on the expression of xenobiotic-metabolizing enzymes, we examined whether protopine or allocryptopine affect the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells. In HepG2 cells, protopine and allocryptopine significantly increased CYP1A1 mRNA levels after 24h exposure at concentrations from 25 and 10 μM, respectively, as shown by real-time PCR. Both protopine and allocryptopine also dose-dependently increased CYP1A1 and CYP1A2 mRNA levels in human hepatocytes. However, the effects of the tested alkaloids on both cell models were much lower than the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. Using gene reporter assays performed in transiently transfected HepG2 cells, we demonstrated that the induction of CYP1A1 expression by either protopine or allocryptopine was associated with mild or negligible activation of the aryl hydrocarbon receptor. In contrast to TCDD, CYP1A mRNA levels induced by protopine or allocryptopine in both HepG2 cells and human hepatocytes did not result in elevated CYP1A protein or activity levels as shown by western blotting and EROD assays, respectively. We conclude that the use of products containing protopine and/or allocryptopine may be considered safe in terms of possible induction of CYP1A enzymes. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxidemore » back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO{sub 3} and FMO{sub 5} was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO{sub 1} showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO{sub 1}-catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation.« less

Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

EPA Science Inventory

EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

Cytochrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4beta-hydroxycholesterol.

PubMed

Kanebratt, K P; Diczfalusy, U; Bäckström, T; Sparve, E; Bredberg, E; Böttiger, Y; Andersson, T B; Bertilsson, L

2008-11-01

The Karolinska cocktail, comprising caffeine, losartan, omeprazole, and quinine, was given before and after administration of rifampicin (20, 100, or 500 mg daily) to measure induction of cytochrome P450 (P450) enzymes. Rifampicin was given for 14 days to eight healthy subjects (all of whom possessed at least one wild-type CYP2C9 and one wild-type CYP2C19 gene) in each dose group. 4beta-hydroxycholesterol was assessed as an endogenous marker of CYP3A4 induction. A fourfold induction of CYP3A4 was seen at the highest dose by both quinine:3'-hydroxyquinine and 4beta-hydroxycholesterol measurements (P < 0.001). CYP3A4 was also induced at the two lower doses of rifampicin when measured by these two markers (P < 0.01 or P < 0.001). CYP1A2, CYP2C9, and CYP2C19 were induced after 500 mg rifampicin daily (1.2-fold, P < 0.05; 1.4-fold, P < 0.05; and 4.2-fold, P < 0.01, respectively). In conclusion, we have shown that the Karolinska cocktail and 4beta-hydroxycholesterol can be used for an initial screening of the induction properties of a drug candidate.

Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

PubMed

Itoh, S; Yanagimoto, T; Tagawa, S; Hashimoto, H; Kitamura, R; Nakajima, Y; Okochi, T; Fujimoto, S; Uchino, J; Kamataki, T

1992-03-24

P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

Pathway-Targeted Pharmacogenomics of CYP1A2 in Human Liver

PubMed Central

Klein, Kathrin; Winter, Stefan; Turpeinen, Miia; Schwab, Matthias; Zanger, Ulrich M.

2010-01-01

The human drug metabolizing cytochrome P450 (CYP) 1A2, is one of the major P450 isoforms contributing by about 5–20% to the hepatic P450 pool and catalyzing oxidative biotransformation of up to 10% of clinically relevant drugs including clozapine and caffeine. CYP1A2 activity is interindividually highly variable and although twin studies have suggested a high heritability, underlying genetic factors are still unknown. Here we adopted a pathway-oriented approach using a large human liver bank (n = 150) to elucidate whether variants in candidate genes of constitutive, ligand-inducible, and pathophysiological inhibitory regulatory pathways may explain different hepatic CYP1A2 phenotypes. Samples were phenotyped for phenacetin O-deethylase activity, and the expression of CYP1A2 protein and mRNA was determined. CYP1A2 expression and function was increased in smokers and decreased in patients with inflammation and cholestasis. Of 169 SNPs in 17 candidate genes including the CYP1A locus, 136 non-redundant SNPs with minor allele frequency >5% were analyzed by univariate and multivariate methods. A total of 13 strong significant associations were identified, of which 10 SNPs in the ARNT, AhRR, HNF1α, IL1β, SRC-1, and VDR genes showed consistent changes for at least two phenotypes by univariate analysis. Multivariate linear modeling indicated that the polymorphisms and non-genetic factors together explained 42, 38, and 33% of CYP1A2 variation at activity, protein and mRNA levels, respectively. In conclusion, we identified novel trans-associations between regulatory genes and hepatic CYP1A2 function and expression, but additional genetic factors must be assumed to explain the full extent of CYP1A2 heritability. PMID:21918647

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

PubMed

Arlt, Volker M; Poirier, Miriam C; Sykes, Sarah E; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C Roland; Henderson, Colin J; Phillips, David H

2012-09-03

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

PubMed Central

Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D.; da Silva, Carlos Antonio Trindade; Morisseau, Christophe; Hammock, Bruce D.

2015-01-01

Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE2 was monitored. While OME treatment by itself exhibited variable effects on PGE2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. PMID:26522832

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition.

PubMed

Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio

2006-07-15

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

PubMed

Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

2017-05-01

Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Evidence for tangeretin O-demethylation by rat and human liver microsomes.

PubMed

Canivenc-Lavier, M C; Brunold, C; Siess, M H; Suschetet, M

1993-03-01

1. Tangeretin, a polymethoxylated flavone, was studied as a substrate for cytochrome P450-catalysed demethylation reactions by rat and human liver microsomes. Evidence has been presented for the production of formaldehyde in the presence of tangeretin and NAD(P)H. Kinetic studies showed a Km value for tangeretin of about 18 microM in both species. 2. The reaction was inhibited by CO, piperonyl butoxide, 7,8-benzoflavone, propyl gallate, aminobenzothiazole and metyrapone. 3. Rats pretreated with classical cytochrome P450 inducers (Aroclor 1254, 3-methylcholanthrene, phenobarbital, dexamethasone and ciprofibrate) or with flavonoids (flavone, flavanone, quercetin and tangeretin) resulted in increased microsomal demethylation of tangeretin after 3-methylcholanthrene and flavone only. Tangeretin did not enhance its own metabolism. 4. Tangeretin interacted with the oxidized form of cytochrome P450 to produce a reverse type I spectrum. 5. Results indicate that tangeretin is metabolized in liver microsomes by an O-demethylation reaction involving cytochrome P450.

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense[C][W

PubMed Central

Irmisch, Sandra; Clavijo McCormick, Andrea; Boeckler, G. Andreas; Schmidt, Axel; Reichelt, Michael; Schneider, Bernd; Block, Katja; Schnitzler, Jörg-Peter; Gershenzon, Jonathan; Unsicker, Sybille B.; Köllner, Tobias G.

2013-01-01

Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus × canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom. PMID:24220631

Liver Zonation Index of Drug Transporter and Metabolizing Enzyme Protein Expressions in Mouse Liver Acinus.

PubMed

Tachikawa, Masanori; Sumiyoshiya, Yuna; Saigusa, Daisuke; Sasaki, Kazunari; Watanabe, Michitoshi; Uchida, Yasuo; Terasaki, Tetsuya

2018-05-01

The purpose of the present study was to clarify the molecular basis of zonated drug distributions in mouse liver based on the protein expression levels of transporters and metabolizing enzymes in periportal (PP) and pericentral (PC) vein regions of mouse hepatic lobules. The distributions of sulforhodamine 101 (SR-101), a substrate of organic anion transporting polypeptides (Oatps), and ribavirin, a substrate of equilibrative nucleoside transporter 1 (Ent1), were elucidated in frozen liver sections of mice, to which each compound had been intravenously administered. Regions strongly positive for SR-101 (SR-101 + ) and regions weakly positive or negative for SR-101 (SR-101 - ) were separated by laser microdissection. The zonated distribution of protein expression was quantified in terms of the liver zonation index. Quantitative targeted absolute proteomics revealed the selective expression of glutamine synthetase in the SR-101 + region, indicating predominant distribution of SR-101 in hepatocytes of the PC vein region. The protein levels of Oatp1a1, Oatp1b2, organic cation transporter 1 (Oct1), and cytochrome P450 (P450) 2e1 were greater in the PC vein regions, whereas the level of organic anion transporter 2 (Oat2) was greater in the PP vein regions. Mouse Oatp1a1 mediated SR-101 transport. On the other hand, there were no statistically significant differences in expression of Ent1, Na + -taurocholate cotransporting polypeptide, several canalicular transporters, P450 enzymes, and UDP-glucuronosyltransferases between the PP and PC vein regions. This is consistent with the almost uniform distribution of ribavirin in the liver. In conclusion, sinusoidal membrane transporters such as Oatp1a1, Oatp1b2, Oct1, and Oat2 appear to be determinants of the zonated distribution of drugs in the liver. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause.

PubMed

Yang, Ping; Tanaka, Hiromasa; Kuwano, Eiichi; Suzuki, Koichi

2008-03-01

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

PubMed Central

Lammers, P J; McLaughlin, S; Papin, S; Trujillo-Provencio, C; Ryncarz, A J

1990-01-01

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria. Images PMID:2123860

Infrasound-induced changes on sexual behavior in male rats and some underlying mechanisms.

PubMed

Zhuang, Zhiqiang; Pei, Zhaohui; Chen, Jingzao

2007-01-01

To investigate some bioeffects of infrasound on copulation as well as underlying mechanisms, we inspected the changes of sexual behavior, serum testosterone concentration and mRNA expression levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in testes of rats exposed to infrasound of 8Hz at 90 or 130dB for 1, 7, 14 and 21 days (2h/day), respectively. Rats exposed to 90dB exhibited significant decrement in sexual behavior, serum testosterone levels and mRNA expression levels of StAR and P450scc at the time point of 1 day but not at the rest time points, and no significantly change of SF-1 mRNA expression was observed over the period of 21 days in spite of mild fluctuation. Rats exposed to 130dB exhibited significant decrement in all aspects above, which became more profound with prolonged exposure. Our conclusion is that adverse bioeffects of infrasound on reproduction depend on some exposure parameters, the mechanism of which could involve in the decreased expression of some key enzymes or regulator for testosterone biosynthesis. Copyright © 2006. Published by Elsevier B.V.

Nicotine Component of Cigarette Smoke Extract (CSE) Decreases the Cytotoxicity of CSE in BEAS-2B Cells Stably Expressing Human Cytochrome P450 2A13.

PubMed

Ji, Minghui; Zhang, Yudong; Li, Na; Wang, Chao; Xia, Rong; Zhang, Zhan; Wang, Shou-Lin

2017-10-13

Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC 50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.

Role of P450 monooxygenases in the degradation of the endocrine-disrupting chemical nonylphenol by the white rot fungus Phanerochaete chrysosporium.

PubMed

Subramanian, Venkataramanan; Yadav, Jagjit S

2009-09-01

The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (approximately 75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant.

Role of P450 Monooxygenases in the Degradation of the Endocrine-Disrupting Chemical Nonylphenol by the White Rot Fungus Phanerochaete chrysosporium▿

PubMed Central

Subramanian, Venkataramanan; Yadav, Jagjit S.

2009-01-01

The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (∼75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant. PMID:19542331

High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions

PubMed Central

Li, Guannan; Huang, Ke; Nikolic, Dejan

2015-01-01

Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry–based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography–tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. PMID:26285764

Enhanced Biotransformation of Fluoranthene by Intertidally Derived Cunninghamella elegans under Biofilm-Based and Niche-Mimicking Conditions

PubMed Central

Mitra, Sayani; Pramanik, Arnab; Banerjee, Srijoni; Haldar, Saubhik; Gachhui, Ratan

2013-01-01

The aims of the investigation were to ascertain if surface attachment of Cunninghamella elegans and niche intertidal conditions provided in a bioreactor influenced biotransformation of fluoranthene by C. elegans. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disc allowed comparison of fluoranthene biotransformation between CCFs with a hydrophobic surface (PMMA-CCF) and a hydrophilic glass surface (GS-CCF) and a 500-ml Erlenmeyer flask (EF). Fluoranthene biotransformation was higher by 22-fold, biofilm growth was higher by 3-fold, and cytochrome P450 gene expression was higher by 2.1-fold when C. elegans was cultivated with 2% inoculum as biofilm culture in PMMA-CCF compared to planktonic culture in EF. Biotransformation was enhanced by 7-fold with 10% inoculum. The temporal pattern of biofilm progression based on three-channel fluorescence detection by confocal laser scanning microscopy demonstrated well-developed, stable biofilm with greater colocalization of fluoranthene within extracellular polymeric substances and filaments of the biofilm grown on PMMA in contrast to a glass surface. A bioreactor with discs rotating at 2 revolutions per day affording 6-hourly emersion and immersion mimicked the niche intertidal habitat of C. elegans and supported biofilm formation and transformation of fluoranthene. The amount of transformed metabolite was 3.5-fold, biofilm growth was 3-fold, and cytochrome P450 gene expression was 1.9-fold higher in the process mimicking the intertidal conditions than in a submerged process without disc rotation. In the CCF and reactor, where biofilm formation was comparatively greater, higher concentration of exopolysaccharides allowed increased mobilization of fluoranthene within the biofilm with consequential higher gene expression leading to enhanced volumetric productivity. PMID:24038685

Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450.

PubMed

Zong, Cai; Garner, C Edwin; Huang, Chinyen; Zhang, Xiao; Zhang, Lingyi; Chang, Jie; Toyokuni, Shinya; Ito, Hidenori; Kato, Masashi; Sakurai, Toshihiro; Ichihara, Sahoko; Ichihara, Gaku

2016-09-06

Neurotoxicity of 1-bromopropane (1-BP) has been reported in both human cases and animal studies. To date, neurotoxicity of 1-BP has been induced in rats but not in mice due to the lethal hepatotoxicity of 1-BP. Oxidization by cytochromes P450 and conjugation with glutathione (GSH) are two critical metabolism pathways of 1-BP and play important roles in toxicity of 1-BP. The aim of the present study was to establish a murine model of 1-BP neurotoxicity, by reducing the hepatotoxicity of 1-BP with 1-aminobenzotriazole (1-ABT); a commonly used nonspecific P450s inhibitor. The results showed that subcutaneous or intraperitoneal injection of 1-ABT at 50mg/kg body weight BID (100mg/kg BW/day) for 3days, inhibited about 92-96% of hepatic microsomal CYP2E1 activity, but only inhibited about 62-64% of CYP2E1 activity in brain microsomes. Mice treated with 1-ABT survived even after exposure to 1200ppm 1-BP for 4 weeks and histopathological studies showed that treatment with 1-ABT protected mice from 1-BP-induced hepatic necrosis, hepatocyte degeneration, and hemorrhage. After 4-week exposure to 1-BP, the brain weight of 1-ABT(+)/1200ppm 1-BP group was decreased significantly. In 1-ABT-treated groups, expression of hippocampal Ran protein and cerebral cortical GRP78 was dose-dependently increased by exposure to 1-BP. We conclude that the control of hepatic P450 activity allows the observation of effects of 1-BP on the murine brain at a higher concentration by reduction of hepatotoxicity. The study suggests that further experiments with liver-specific control of P450 activity using gene technology might provide better murine models for 1-bromopropane-induced neurotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

PubMed

Liu, Nannan; Li, Ming; Gong, Youhui; Liu, Feng; Li, Ting

2015-05-01

P450s are known to be critical for the detoxification and/or activation of xenobiotics such as drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In insects, P450s play an important role in detoxifying exogenous compounds such as insecticides and plant toxins and their overexpression can result in increased levels of P450 proteins and P450 activities. This has been associated with enhanced metabolic detoxification of insecticides and has been implicated in the development of insecticide resistance in insects. Multiple P450 genes have been found to be co-overexpressed in individual insect species via several constitutive overexpression and induction mechanisms, which in turn are co-responsible for high levels of insecticide resistance. Many studies have also demonstrated that the transcriptional overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of regulatory genes/factors and resistance genes. This chapter reviews our current understanding of how the molecular mechanisms of P450 interaction/gene regulation govern the development of insecticide resistance in insects and our progress along the road to a comprehensive characterization of P450 detoxification-mediated insecticide resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

PubMed

Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane

2007-03-01

Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

PubMed

Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

2015-10-01

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. Copyright © 2015 Elsevier Inc. All rights reserved.

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: functional characterisation and signatures of selection.

PubMed

Wilding, Craig S; Smith, Ian; Lynd, Amy; Yawson, Alexander Egyir; Weetman, David; Paine, Mark J I; Donnelly, Martin J

2012-09-01

Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Culex quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in Escherichia coli we have now demonstrated time- and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10× expression compared to the equivalent region (lacking CuRE1) from the susceptible strain. Close correspondence with the gene expression fold-change implicates the upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1 bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic. When applied to C. quinquefasciatus larvae from Ghana we have demonstrated a significant association with permethrin resistance in multiple field sites (mean Odds Ratio = 3.86) suggesting this marker has relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs contribute regulatory motifs involved in gene expression may be necessary. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detection and Diversity of Fungal Nitric Oxide Reductase Genes (p450nor) in Agricultural Soils

PubMed Central

Welsh, Allana; Orellana, Luis H.; Konstantinidis, Konstantinos T.; Chee-Sanford, Joanne C.; Sanford, Robert A.; Schadt, Christopher W.

2016-01-01

ABSTRACT Members of the Fungi convert nitrate (NO3−) and nitrite (NO2−) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3− or NO2− in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N2O from NO2−. Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N2O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from NO2−, whereas nirK (encoding the NO-forming NO2− reductase) was amplified in only 13 to 74% of the N2O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. IMPORTANCE A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N2O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N2O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N2O emissions. PMID:26969694

SLC6A19 is a novel putative gene, induced by dioxins via AhR in human hepatoma HepG2 cells.

PubMed

Tian, Wenjing; Fu, Hualing; Xu, Tuan; Xu, Sherry Li; Guo, Zhiling; Tian, Jijing; Tao, Wuqun; Xie, Heidi Qunhui; Zhao, Bin

2018-06-01

The aryl hydrocarbon receptor (AhR) plays an important role in mediating dioxins toxicity. Currently, genes of P450 families are major research interests in studies on AhR-mediated gene alterations caused by dioxins. Genes related to other metabolic pathways or processes may be also responsive to dioxin exposures. Amino acid transporter B0AT1 (encoded by SLC6A19) plays a decisive role in neutral amino acid transport which is present in kidney, intestine and liver. However, effects of dioxins on its expression are still unknown. In the present study, we focused on the effects of dioxin and dioxin-like compounds on SLC6A19 expression in HepG2 cells. We identified SLC6A19 as a novel putative target gene of AhR activation in HepG2 cells. 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) increased the expression of SLC6A19 in time- and concentration-dependent manners. Using AhR antagonist CH223191 and/or siRNA assays, we demonstrated that certain AhR agonists upregulated SLC6A19 expression via AhR, including TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (1,2,3,7,8-PeCDD), 2,3,4,7,8- pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and PCB126. In addition, the expression of B0AT1 was also significantly induced by TCDD in HepG2 cells. Our study suggested that dioxins might affect the transcription and translation of SLC6A19 in HepG2 cells, which might be a novel putative gene to assess dioxins' toxicity in amino acid transport and metabolism in liver. Copyright © 2018 Elsevier Ltd. All rights reserved.

Conformational change of cytochrome P450 1A2 induced by phospholipids and detergents.

PubMed

Yun, C H; Song, M; Kim, H

1997-08-08

Recently, it was reported that the activity of rabbit P450 1A2 is markedly increased at elevated salt concentration (Yun, C-H., Song, M., Ahn, T., and Kim, H. (1996) J. Biol. Chem. 271, 31312-31316). The activity increase of P450 1A2 coincides with the raised alpha-helix content and decreased beta-sheet content. The presence of phospholipid magnified this effect. Here, possible structural change of rabbit P450 1A2 accompanying the phospholipid-induced increase in its enzyme activity was investigated by circular dichroism, fluorescence spectroscopy, and absorption spectroscopy. Studies with the reconstituted system supported by cumene hydroperoxide or NADPH showed that the P450 1A2 activities were found to be dependent on the head group and hydrocarbon chain length of phospholipid. Phosphatidylcholines having short hydrocarbon chains with a carbon number of 8-12 were very efficient for reconstitution of the P450-catalyzed reactions supported by both cumene hydroperoxide and NADPH. It was found that the phospholipid increased the alpha-helix content and lowered the beta-sheet content of P450. Intrinsic fluorescence intensity is also increased in the presence of phospholipid. The low spin iron configuration of P450 1A2 shifted toward the high spin configuration by most of the phospholipids in the endoplasmic reticulum. Some synthetic phospholipids having short hydrocarbon chains with a carbon number of 10-12 caused a shift in the spin equilibrium of P450 1A2 toward low spin. The effect of detergents on the activity and conformation of P450 1A2 was also studied. It was found that the addition of detergents to P450 1A2 solution increased the enzyme activity of P450 1A2. Detergents also increased the alpha-helix content and lowered the beta-sheet content of P450 1A2. Intrinsic fluorescence emissions also increased with the presence of detergents. Octyl glucoside and deoxycholate caused a shift toward high spin. On the other hand, cholate caused a shift toward low spin. It was found that the activity increase of rabbit P450 1A2 coincides with the conformational change including raised alpha-helix content. It is proposed that the interaction with the phospholipid molecules surrounding P450 1A2 in the endoplasmic reticulum is important for a functional conformation of P450 1A2 in a monooxygenase system including NADPH-P450 reductase.

Deltamethrin is metabolized by CYP6FU1, a cytochrome P450 associated with pyrethroid resistance, in Laodelphax striatellus.

PubMed

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Peng, Yingchuan; Zhang, Haomiao; Jiang, Ling; Wu, Min; Han, Zhaojun

2018-06-01

Cytochrome P450s (CYPs) are known to play a major role in metabolizing a wide range compounds. CYP6FU1 has been found to be over-expressed in a deltamethrin-resistant strain of Laodelphax striatellus. This study was conducted to express CYP6FU1 in Sf9 cells as a recombinant protein, to confirm its ability to degrade deltamethrin, chlorpyrifos, imidacloprid and traditional P450 probing substrates. Carbon monoxide difference spectrum analysis indicated that the intact CYP6FU1 protein was expressed in insect Sf9 cells. Catalytic activity tests with four traditional P450 probing substrates revealed that the expressed CYP6FU1 preferentially metabolized p-nitroanisole and ethoxyresorufin, but not ethoxycoumarin and luciferin-HEGE. The enzyme kinetic parameters were tested using p-nitroanisole. The michaelis constant (K m ) and catalytic constant (K cat ) values were 17.51 ± 4.29 µm and 0.218 ± 0.001 pmol min -1 mg -1 protein, respectively. Furthermore, CYP6FU1 activity for degradation of insecticides was tested by measuring substrate depletion and metabolite formation. The chromatogram analysis showed obvious nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of deltamethrin, and formation of the unknown metabolite. Mass spectra and the molecular docking model showed that the metabolite was 4-hydroxy-deltamethrin. However, the recombinant CYP6FU1 could not metabolize imidacloprid and chlorpyrifos. These results confirmed that the over-expressed CYP6FU1 contributes to deltamethrin resistance in L. striatellus, and p-nitroanisole might be a potential diagnostic probe for deltamethrin metabolic resistance detection and monitoring. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, C.-C.; Lii, C.-K.; Liu, K.-L.

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation bymore » n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 {mu}M arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.« less

Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper.

PubMed

Zimmer, Christoph T; Garrood, William T; Singh, Kumar Saurabh; Randall, Emma; Lueke, Bettina; Gutbrod, Oliver; Matthiesen, Svend; Kohler, Maxie; Nauen, Ralf; Davies, T G Emyr; Bass, Chris

2018-01-22

Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3-5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6-8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

PubMed Central

2011-01-01

Abstract Background Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. Results One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues. Conclusions A collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins. PMID:22047182

Identification of the rat liver cytochrome P450 enzymes involved in the metabolism of the calcium channel blocker dipfluzine hydrochloride.

PubMed

Guo, Wei; Shi, Xiaowei; Wang, Wei; Zhang, Weili; Li, Junxia

2014-11-01

This study aimed to identify the specific cytochrome P450 (CYP450) enzymes involved in the metabolism of dipfluzine hydrochloride using the combination of a chemical inhibition study, a correlation analysis and a panel of recombinant rat CYP450 enzymes. The incubation of Dip with rat liver microsomes yielded four metabolites, which were identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS). The results from the assays involving eight selective inhibitors indicated that CYP3A and CYP2A1 contributed most to the metabolism of Dip, followed by CYP2C11, CYP2E1 and CYP1A2; however, CYP2B1, CYP2C6 and CYP2D1 did not contribute to the formation of the metabolites. The results of the correlation analysis and the assays involving the recombinant CYP450 enzymes further confirmed the above results and concluded that CYP3A2 contributed more than CYP3A1. The results will be valuable in understanding drug-drug interactions when Dip is coadministered with other drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

PubMed Central

Chen, Song; Li, Xianchun

2007-01-01

Background Transposons, i.e. transposable elements (TEs), are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), MITEs (miniature inverted-repeat transposable elements), one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1) implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1) involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes. PMID:17381843

Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug-Drug Interactions.

PubMed

Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

2016-02-24

Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug-drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug-drug interactions.

Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug–Drug Interactions

PubMed Central

Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

2016-01-01

Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug–drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug–drug interactions. PMID:26927160

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2*

PubMed Central

Pallan, Pradeep S.; Nagy, Leslie D.; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M.; Azumaya, Caleigh M.; Wawrzak, Zdzislaw; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

2015-01-01

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116–119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation.

PubMed

Meyer, Mark B; Benkusky, Nancy A; Kaufmann, Martin; Lee, Seong Min; Onal, Melda; Jones, Glenville; Pike, J Wesley

2017-10-20

The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D 3 to its hormonal form, 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1 , are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH) 2 D 3 -mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH) 2 D 3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH) 2 D 3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytochrome P450 induction in mallard duck (MD), black-crowned night heron (BCNH) and Fisher-344 rat

USGS Publications Warehouse

Melancon, M.J.; Rattner, B.A.; Stegeman, John J.

1991-01-01

P450 induction was studied in adult and pipping MDs, pipping BCNHs, and rats. Adult MDs and rats received i.p. injection of corn oil, 3-methylcholanthrene (MC) in corn oil (20 mg/kg), saline or phenobarbital (PB) in saline (80 mg/kg) for 3 days. MD and BCNH embryos received MC and PB by injection into the aircell approximately 2 days before pipping and were sacrificed at pipping. Hepatic microsomes were assayed for protein, arylhydrocarbon hydroxylase (AHH), benzphetamine-N-demethylase (BEND), ethoxy-resorufin-O-dealkylase (EROD), pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD), ethoxycoumarin-O-dealkylase (ECOD), and by SDS-PAGE with western blot using a polyclonal anti-P4S0IIB antibody and a monoclonal anti-P450IA antibody (MAb 1-12-3). Although species and age caused substantial differences in responses, all treated groups showed an increase in one or more monooxygenase assays. All animals treated with MC showed a strong induction of a protein recognized by anti-P450IA, and all those treated with PB showed strong induction of a band recognized by anti-P450IIB.

Atrazine-xenobiotic nuclear receptor interactions induce cardiac inflammation and endoplasmic reticulum stress in quail (Coturnix coturnix coturnix).

PubMed

Li, Xue-Nan; Zuo, Yu-Zhu; Qin, Lei; Liu, Wei; Li, Yan-Hua; Li, Jin-Long

2018-05-09

Atrazine (ATR) is one of the most extensively used herbicide that eventually leaches into groundwater and surface water from agricultural areas. Exposure to ATR does harm to the health of human and animals, especially the heart. However, ATR exposure caused cardiotoxicity in bird remains unclear. To evaluate ATR-exerted potential cardiotoxicity in heart, quail were exposed with 0, 50, 250, and 500 mg/kg BW/day ATR by gavage treatment for 45 days. Cardiac histopathological alternation was observed in ATR-induced quail. ATR exposure increased the Cytochrome P450s and Cytochrome b5 contents, Cytochrome P450 (CYP) enzyme system (APND, ERND, AH, and NCR) activities and the expression of CYP isoforms (CYP1B1, CYP2C18, CYP2D6, CYP3A4, CYP3A7, and CYP4B1) in quail heart. The expression of nuclear xenobiotic receptors (NXRs) was also influenced in the heart by ATR exposure. ATR exposure significantly caused the up-regulation of pro-inflammatory cytokines (TNF-α, IL-6, NF-κB, and IL-8), down-regulation of anti-inflammatory cytokines (IL-10) expression levels and increased NO content and iNOS activity. The present research provides new insights into the mechanism that ATR-induced cardiotoxicity through up-regulating the expression levels of GRP78 and XBP-1s, triggering ER stress, activating the expression of IRE1α/TRAF2/NF-κB signaling pathway related factors (IRE1α, TRAF2, IKK, and NF-κB) and inducing an inflammatory response in quail hearts. In conclusion, ATR exposure could induce cardiac inflammatory injury via activating NXRs responses, disrupting CYP homeostasis and CYP isoforms transcription, altering NO metabolism and triggering ER stress and inflammatory response by activating IRE1α/TRAF2/NF-κB signaling pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

Contributions of Human Enzymes in Carcinogen Metabolism

PubMed Central

Rendic, Slobodan; Guengerich, F. Peter

2012-01-01

Considerable support exists for roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are procarcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s—1A1, 1A2, 1B1, 2A6, 2E1, and 3A4—accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, inter-individual variations, and risk assessment. PMID:22531028

Protection by Nigella sativa against carbon tetrachloride-induced downregulation of hepatic cytochrome P450 isozymes in rats.

PubMed

Ibrahim, Zein S; Ishizuka, Mayumi; Soliman, Mohamed; ElBohi, Khlood; Sobhy, Wageh; Muzandu, Kaampwe; Elkattawy, Azza M; Sakamoto, Kentaro Q; Fujita, Shoichi

2008-11-01

Nigella sativa (family Ranunculaceae) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflammatory IL-10.

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size.

PubMed

Ma, Meng; Wang, Qian; Li, Zhanjie; Cheng, Huihui; Li, Zhaojie; Liu, Xiangli; Song, Weining; Appels, Rudi; Zhao, Huixian

2015-07-01

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over-expression induced production of more cells in the seed coat, leading to an 11-48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over-expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over-expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Mechanism-based inactivation of benzo(a)pyrene hydroxylase by aryl acetylenes and aryl olefins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gan, L.S.; Lu, J.Y.L.; Alworth, W.L.

A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxgenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, 3-ethynylperylene, 2-ethynylfluorene, methyl 1-pyrenyl acetylene, cis- and trans-1-(2-bromovinyl)pyrene, and 1-allylpyrene serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene hydroxylase, while 1-vinylpyrene and phenyl 1-pyrenyl acetylene do not cause a detectable suicide inhibition of benzo(a)pyrene hydroxylase. The mechanism-based loss of benzo(a)pyrene hydroxylase caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes (suicide destruction). The suicide inhibition by these aryl acetylenesmore » therefore does not involve covalent binding to the heme moiety of the monooxygenase. Nevertheless, in the presence of NADPH, /sup 3/H-labeled 1-ethynylpyrene becomes covalently attached to the cytochrome P-450 protein; the measured stoichiometry of binding is one 1-ethynylpyrene per P-450 heme unit. The authors conclude that the inhibition of benzo(a)pyrene hydroxylase produced by 1-ethynylpyrene may be related to the mechanism of suicide inhibition of P-450 activity by chloramphenicol rather than the mechanism of suicide destruction of P-450 previously described for acetylene and propyne.« less

Interactive effects of nutrition, reproductive state and pollution on molecular stress responses of mussels, Mytilus galloprovincialis Lamarck, 1819.

PubMed

González-Fernández, Carmen; Albentosa, Marina; Sokolova, Inna

2017-10-01

Marine bivalves including mussels Mytilus galloprovincialis are commonly used as sentinels for pollution monitoring and ecosystem health assessment in the coastal zones. Use of biomarkers to assess the pollution effects assumes that the effects of pollutants on the biomarkers exceed the natural background variability; yet this assumption has rarely been tested. We exposed mussels at different reproductive stages and nutritive states to two concentrations of a polycyclic aromatic hydrocarbon (fluoranthene, 3 and 60 μg L -1 ) for three weeks. Expression levels of the molecular biomarkers related to the detoxification and general stress response [cytochrome P450 oxidase (CYP450), glutathione S-transferases (GST-α; GST-S1; GST-S2), the multixenobiotic resistance protein P-glycoprotein (PgP), metallothioneins (MT10 and MT20), heat shock proteins (HSP22, HSP70-2; HSP70-3; HSP70-4), as well as mRNA expression of two reproduction-related genes, vitellogenin (Vitel) and vitelline coat lysin M7 (VCLM7)] were measured. The mussels' nutrition and reproductive state affected the baseline mRNA levels of molecular biomarkers and modulated the transcriptional responses of biomarker genes to the pollutant exposure. Thus, mussel physiological state could act as a confounding factor in the evaluation of the response of pollution through molecular biomarkers. The biomarker baseline levels must be determined across a range of physiological states to enable the use of biomarkers in monitoring programs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

PubMed Central

Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

2013-01-01

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

PubMed

Solari, J J F; Pozzi, A G; Ceballos, N R

2002-12-01

In Bufo arenarum, the biosynthesis of testosterone and 5alpha-dihydrotestosterone takes place through a complete 5-ene pathway, 5-androsten-3beta,17beta-diol being the immediate precursor of testosterone. Besides androgens, testes are able to synthesise 5alpha-pregnan-3,20-dione and several 3alpha and 20alpha reduced derivatives. During the breeding season, steroid biosynthesis turns from androgen to C21-steroid production. As a consequence, the cytochrome P450 17-hydroxylase, C17,20-lyase (CypP450(c17)) could be a key enzyme in that metabolic shift. The present study demonstrates that in testes of B. arenarum, CypP450(c17) co-localises with glucose-6-phosphatase in the microsomal fraction. CypP450(c17) possesses more affinity for pregnenolone than for progesterone in both non-reproductive (Km = 43.76 +/- 4.63 nM and 2,170 +/- 630 nM, respectively) and reproductive (Km = 37.46 +/- 4.19 nM and 3,060 +/- 190 nM, respectively) seasons. These results could explain the predominance of the 5-ene pathway for testosterone biosynthesis. Toad CypP450(c17) activity is higher in the non-reproductive period than the reproductive period, suggesting that this enzyme is an important factor in toad steroidogenic changes. Animals in reproductive conditions showed a significant reduction in circulating androgens. This is in agreement with the decrease in Vmax of cytochrome P450 17-hydroxylase activity, enhancing the physiological relevance of these in vitro results.