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1

Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.  

PubMed

Estrogens are the major steroids produced by equine gonads. To identify the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immunoblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromatase (P450arom) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for P450arom and the granulosa cells of large, viable more follicles showed a high degree of immunoreactivity. In the corpus luteum, all the luteinized cells showed immunoreactivity. No immunoreactivity was detected in other cells of small and large viable follicles. Immunolocalization of P450arom in the equine testicular Leydig cells and in ovarian granulosa and luteinized cells indicates that these cells have the ability to metabolize androgens to estrogens and possibly to catechol estrogens. PMID:7769228

Almadhidi, J; Seralini, G E; Fresnel, J; Silberzahn, P; Gaillard, J L

1995-06-01

2

Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization  

SciTech Connect

A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B couples with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-{alpha}-phosphatidylcholine with (1{beta}-{sup 3}H,4-{sup 14}C)androstenedione as substrate. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The very high K{sub m} value for 16{alpha}-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at {minus}80C.

Yoshida, Nobutaka; Osawa, Yoshio (Medical Foundation of Buffalo Research Institute, NY (USA))

1991-03-26

3

Molecular characterization and expression of equine testicular cytochrome P450 aromatase  

Microsoft Academic Search

We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least

Gilles-Eric Seralini; Alexey Tomilin; Celine Nativelle-Serpentini; Pascal Sourdaine; Safa Moslemi

2003-01-01

4

Aromatase Cytochrome P450 and Extragonadal Estrogen Play a Role in Ischemic Neuroprotection  

Microsoft Academic Search

Female animals are protected from many forms of neurological injury and degeneration relative to their male counterparts, in part attributable to their native estrogens. We hypothesized that estradiol aromatized from precursor androgens via the cytochrome P450 aromatase contributes to ischemic neuroprotection in the female. Female homozygous aromatase knock-out (ArKO) mice and randomly cycling, wild-type (WT) female littermates were treated with

Louise D. McCullough; Kathleen Blizzard; Evan R. Simpson; Orhan K. Oz; Patricia D. Hurn

2003-01-01

5

Molecular characterization and expression of equine testicular cytochrome P450 aromatase.  

PubMed

We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androstenedione as substrate in a dose-dependent manner. The isolation of testicular aromatase by a new immunoaffinity method demonstrated that the protein could exist either glycosylated or not with a 2 kDa difference. All these results taken together allow new structural studies to progress in the understanding of this cytochrome P450. PMID:12591609

Seralini, Gilles Eric; Tomilin, Alexey; Auvray, Pierrïck; Nativelle-Serpentini, Celine; Sourdaine, Pascal; Moslemi, Safa

2003-02-20

6

Stereochemistry of estrogen biosynthesis by a reconstituted aromatase cytochrome P-450 preparation from human placenta  

SciTech Connect

According to the literature, the multistep reaction mechanism of estrogen biosynthesis proceeds with stereospecific loss of the equatorial 1 beta-, and axial 2 beta-protons. These results were deduced from experiments carried out, either with crude microsomes, or at best with impure enzyme extracts. However, when (1,2- /sup 3/H)4-androstene-3,17-dione of known absolute /sup 3/H-label distribution was incubated with a reconstituted enzyme system, consisting of homogeneous NADPH-cytochrome P-450 reductase and highly purified aromatase, we obtained results that can only be logically explained by a trans- and antiparallel elimination reaction of both the axially oriented C-2 beta-, and C-1-alpha protons. We further demonstrate that the reconstituted enzyme has an aromatase activity optimum at pH 7.2, and an apparent Km of 0.66 microM for NADPH and of 0.24 microM for 4-androstene-3,17-dione. Also, the enzyme requires 3 nmoles of NADPH for each nmole of estrogen that is formed.

Muto, N.; Tan, L.

1986-04-29

7

Expression of cytochrome P450 aromatase transcripts in buffalo (Bubalus bubalis)-ejaculated spermatozoa and its relationship with sperm motility.  

PubMed

The cytochrome P450 aromatase (aromP450) deficient mice are infertile due to an impairment of spermatogenesis associated with a decrease in sperm motility and inability to fertilize oocytes. The sperm analysis showed decreased sperm motility in humans, having Cyp19 gene mutations. Further, in human, it was hypothesized that aromatase could be used as marker of sperm quality, particularly in the acquisition of its motility. However, there is no information regarding the expression of aromP450 in spermatozoa of farm animals including cattle and buffalo. In the present study, the expression of aromP450 in ejaculated buffalo spermatozoa and its relationship with sperm motility of ejaculated spermatozoa was studied by RT-PCR using total RNA isolated from buffalo-ejaculated spermatozoa. The results showed that conventional RT-PCR could not amplify aromatase transcript, while a nested PCR detected the presence of P450arom mRNA in buffalo-ejaculated spermatozoa. RT reaction followed by nested PCR was performed to compare the expression of aromatase transcripts in buffalo-ejaculated spermatozoa of two category semen graded on the basis of mass motility and motile and non-motile spermatozoa separated by swim-up. A higher (P<0.01) expression of aromP450 transcript was found in spermatozoa obtained from the good quality semen (higher mass motility) to that in spermatozoa of poor quality semen (low mass motility). Similarly, higher (P<0.01) expression of aromP450 mRNA was observed in the motile spermatozoa as compared to non-motile spermatozoa separated from good quality semen by swim-up. It is concluded that the present study demonstrates a positive relation between aromatase transcript and mass motility of buffalo-ejaculated spermatozoa, which could be a putative marker for the quality of semen in farm animals, particularly the acquisition of sperm motility. PMID:17851018

Tiwari, Ashutosh; Singh, Dheer; Kumar, O Suneel; Sharma, M K

2008-04-01

8

Immunoexpression of aromatase cytochrome P450 and 17?-hydroxysteroid dehydrogenase in women's ovaries after menopause  

PubMed Central

Background Menopause results in a lack of regular menstrual cycles, leading to the reduction of estrogen production. On the other hand, ovarian androgen synthesis is still present at reduced levels and requires expression of several steroidogenic enzymes. Methods This study was performed on 104 postmenopausal women hospitalized due to uterine leiomyomas, endometriosis, and/or a prolapsed uterus. Patients were divided into three groups depending on the time from menopause. Group A patients experienced menopause 1–5 years before enrollment in the study (42 women). Group B included women who had their last menstruation 5–10 years before the study (40 women). Group C consisted of 22 women who were more than 10 years past menopause. Hysterectomy or removal of the uterine corpus with adnexa was performed during laparotomy. We evaluated the expression of aromatase cytochrome P450 (CYP 19) and 17?-hydroxysteroid dehydrogenase (17? HSD) by employing immunohistochemistry. Results Activity of 17?-HSD and CYP19 was demonstrated in the cytoplasm of stromal cells of postmenopausal ovaries, epithelium cells coating the ovaries, vascular endothelial cells, and epithelial inclusion cysts. However, overall expression of both 17?-HSD and CYP 19 decreased with time after menopause. Conclusion Demonstration of the activity of the key enzymes of ovarian steroidogenesis, CYP 19 and 17?-HSD, confirms steroidogenic activity in the ovaries of postmenopausal women. Nevertheless, ovarian steroidogenic activity decreases with time, and its significant decrease occurs 10 years after menopause. PMID:24855493

2014-01-01

9

Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.  

PubMed

Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen. PMID:9009238

Almadhidi, J; Moslemi, S; Drosdowsky, M A; Séralini, G E

1996-09-01

10

Kinetic Analysis of the Three-step Steroid Aromatase Reaction of Human Cytochrome P450 19A1*  

PubMed Central

Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (kcat of 0.06 s?1 for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone. PMID:20385561

Sohl, Christal D.; Guengerich, F. Peter

2010-01-01

11

Cytochromes P450  

PubMed Central

There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

Werck-Reichhart, Daniele; Bak, S?ren; Paquette, Suzanne

2002-01-01

12

Cytochromes P450  

PubMed Central

There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

Bak, S?ren; Beisson, Fred; Bishop, Gerard; Hamberger, Bjorn; Hofer, Rene; Paquette, Suzanne; Werck-Reichhart, Daniele

2011-01-01

13

Estrogen synthetase (aromatase) reconstitution using cytochrome P-450 and NADPH-cytochrome C reductase from human term placental microsomes  

SciTech Connect

The physiologically important cytochrome P-450 enzyme system responsible for the aromatization of androgen to form estrogen has not been systematically studied in reconstitution systems. This study utilized homogeneous NADPH-cytochrome c reductase and partially purified cytochrome P-450. Activity was stimulated several-fold by optimal concentrations of phosphatidylcholine, and demonstrated hyperbolic kinetics with increasing amounts of NADPH-cytochrome c reductase or cytochrome P-450. Aromatization was linear for 30-60 min, then under certain conditions terminated prematurely with up to 80% of the substrate remaining. This termination did not appear to be due to limiting cofactor, production of an inhibitor during aromatization, or lipid peroxidation. An identical effect was observed with placental microsomes when the activity to substrate ratio was decreased to the value in the reconstitution system. Examination of the steroid product and intermediates by 2-D tlc using /sup 3/H-androstenedione (A) as substrate gave 19-OH (2%) and 19-oxoA(3%) and estrone (50%). Radioactivity in areas corresponding to testosterone and estradiol was observed only in trace amounts (< 0.2%). Two as yet unidentified radioactive compounds with R/sub f/'s close to 19-OH and 19-oxo A (not corresponding to 2-OH estrone or 19-nor A) were formed in small amounts (1%).

Lobo, J.O.; Bellino, F.L.

1986-05-01

14

Ketamine attenuates cytochrome p450 aromatase gene expression and estradiol-17? levels in zebrafish early life stages.  

PubMed

Ketamine, a dissociative anesthetic, is a noncompetitive antagonist of N-methyl-D-aspartate-type glutamate receptors. In rodents and non-human primates as well as in zebrafish embryos, ketamine has been shown to be neurotoxic. In cyclic female rats, ketamine has been shown to decrease serum estradiol-17? (E2) levels. E2 plays critical roles in neurodevelopment and neuroprotection. Cytochrome p450 (CYP) aromatase catalyzes E2 synthesis from androgens. Although ketamine down-regulates a number of CYP enzymes in rodents, its effect on the CYP aromatase (CYP19) is not known. Zebrafish have been used as a model system for examining mechanisms underlying drug effects. Here, using wild-type (WT) zebrafish (Danio rerio) embryos, we demonstrate that ketamine significantly reduced E2 levels compared with the control. However, the testosterone level was elevated in ketamine-treated embryos. These results are concordant with data from mammalian studies. Ketamine also attenuated the expression of the ovary form of CYP aromatase (cyp19a1a) at the transcriptional level but not the brain form of aromatase, cyp19a1b. Exogenous E2 potently induced the expression of cyp19a1b and vtg 1, both validated biomarkers of estrogenicity and endocrine disruption, but not cyp19a1a expression. Attenuation of activated ERK/MAPK levels, reportedly responsible for reduced human cyp19 transcription, was also observed in ketamine-treated embryos. These results suggest that reduced E2 levels in ketamine-treated embryos may have resulted from the suppression of cyp19a1a transcription. PMID:23696345

Trickler, William J; Guo, Xiaoqing; Cuevas, Elvis; Ali, Syed F; Paule, Merle G; Kanungo, Jyotshna

2014-05-01

15

Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro  

Microsoft Academic Search

Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water

Jamal Almadhidi; Safa Moslemi; Michel A. Drosdowsky; Gilles-Eric Séralini

1996-01-01

16

Immunolocalization of androgen receptor, aromatase cytochrome P450, estrogen receptor alpha and estrogen receptor beta proteins during the breeding season in scent glands of muskrats (Ondatra zibethicus).  

PubMed

Aromatase cytochrome P450 (P450arom) is an enzyme that catalyzes the conversion of androgen to estrogen. Expression of P450arom in extra-gonadal sites and locally-synthesized estrogen play an important role in physiological conditions. The purpose of this study was to investigate the cellular immunolocalization of androgen receptor (AR), P450arom, estrogen receptor alpha (ERa) and estrogen receptor beta (ER?) in muskrat scent glands during the breeding season. Histological observation and immunohistochemistry of AR, P450arom, ERa and ER? were performed in the muskrat scent glands. In addition, total proteins were extracted from scent glandular tissues in the breeding season and were used for Western blotting analysis for AR, P450arom, ER? and ER?. Histologically, glandular cells, interstitial cells, epithelial cells of the excretory duct and the excretory tubules were identified in the muskrat scent glands during the breeding season. AR was only observed in glandular cells of scent glands; P450arom was expressed in glandular cells and epithelial cells of the excretory duct; ER? was found in glandular cells, interstitial cells and epithelial cells of the excretory duct, whereas ER? was present in glandular cells and epithelial cells of the excretory duct. Also, the positive signals of AR, P450arom, ER? and ER? by Western blotting were all observed in scent glandular tissues. These results suggested that the scent gland is the target organ of androgens and estrogens, and that estrogens may play an important autocrine or paracrine role in glandular function of the muskrats. PMID:21967219

Lu, Lu; Zhang, Haolin; Lv, Na; Ma, Xiaoting; Tian, Long; Hu, Xiao; Liu, Shuqiang; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

2011-10-01

17

Cloning of two mRNA variants of brain aromatase cytochrome P450 in rainbow trout ( Oncorhynchus mykiss Walbaum)  

Microsoft Academic Search

This work describes the molecular cloning of the cDNA encoding the rainbow trout (Oncorhynchus mykiss Walbaum) brain cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5?- and 3?-rapid amplification of cDNA ends (RACE) analyses. The results obtained demonstrate that, as in other teleost fishes, the trout genome contains, besides the gene previously identified in the

Luisa Dalla Valle; Annalisa Ramina; Silvia Vianello; Paola Belvedere; Lorenzo Colombo

2002-01-01

18

Mechanism of the Third Oxidative Step in the Conversion of Androgens to Estrogens by Cytochrome P450 19A1 Steroid Aromatase.  

PubMed

Aromatase is the cytochrome P450 enzyme that cleaves the C10-C19 carbon-carbon bond of androgens to form estrogens, in a three-step process. Compound I (FeO(3+)) and ferric peroxide (FeO2(-)) have both been proposed in the literature as the active iron species in the third step, yielding an estrogen and formic acid. Incubation of purified aromatase with its 19-deutero-19-oxo androgen substrate was performed in the presence of (18)O2, and the products were derivatized using a novel diazo reagent. Analysis of the products by high-resolution mass spectrometry showed a lack of (18)O incorporation in the product formic acid, supporting only the Compound I pathway. Furthermore, a new androgen 19-carboxylic acid product was identified. The rates of nonenzymatic hydration of the 19-oxo androgen and dehydration of the 19,19-gem-diol were shown to be catalytically competent. Thus, the evidence supports Compound I and not ferric peroxide as the active iron species in the third step of the steroid aromatase reaction. PMID:25252141

Yoshimoto, Francis K; Guengerich, F Peter

2014-10-22

19

Mechanism of the Third Oxidative Step in the Conversion of Androgens to Estrogens by Cytochrome P450 19A1 Steroid Aromatase  

PubMed Central

Aromatase is the cytochrome P450 enzyme that cleaves the C10–C19 carbon–carbon bond of androgens to form estrogens, in a three-step process. Compound I (FeO3+) and ferric peroxide (FeO2–) have both been proposed in the literature as the active iron species in the third step, yielding an estrogen and formic acid. Incubation of purified aromatase with its 19-deutero-19-oxo androgen substrate was performed in the presence of 18O2, and the products were derivatized using a novel diazo reagent. Analysis of the products by high-resolution mass spectrometry showed a lack of 18O incorporation in the product formic acid, supporting only the Compound I pathway. Furthermore, a new androgen 19-carboxylic acid product was identified. The rates of nonenzymatic hydration of the 19-oxo androgen and dehydration of the 19,19-gem-diol were shown to be catalytically competent. Thus, the evidence supports Compound I and not ferric peroxide as the active iron species in the third step of the steroid aromatase reaction. PMID:25252141

2014-01-01

20

[Pharmacokinetics of cytochrome P-450].  

PubMed

Pharmacogenetics of cytochromes P-450. Cytochromes P-450 are a large family of enzymes found in all living species whose function is the activation of molecular oxygen which, in turn, will oxidize an organic substrate. They are divided in two groups: one including the constitutive enzymes that intervene in vital processes such as cholesterol synthesis, cholesterol transfer into steroid and sex hormones, prostaglandin synthesis, etc.; the other group including the inducible enzymes, responsible of the metabolism of exogenous substances. Their concentration increases in the presence of specific substrates, like herbicides, cigarette smoke, hydrocarbons, insecticides, etc.. Of the latter group, the genetic polymorphism of two families is described. Family I is involved in the metabolism of polycyclic aromatic hydrocarbons: an allele codifying for a low activity cytoplasmic receptor (autosomic recessive inheritance) and a high affinity one (recessive inheritance) are present. The transformations carried out by the cytochromes P-450 give origin to intermediate reactive products, epoxides, that bonding to nucleoproteins or nucleic acids, can have either toxic or carcinogenic action. Therefore, the subjects with high affinity genes have an increased risk of cancer. This phenomenon, relating to pulmonary cancer, has been demonstrated in cigarette smokers. Family II is the group of greatest pharmacogenetic and clinical interest, since it is responsible of the polymorphism of the response to different drugs, such as halothane, (malignant hyperthermia), ethanol (alcohol intolerance), nitrosamine, (cancer), debrisoquine (hypotension), spartein (excessive uterine contractions). An increased or reduced ability to metabolize specific substances is the consequence: the pharmacological effects can therefore vary very much in the two classes of carriers of different alleles. Possible future applications of these polymorphisms in clinical practice are discussed. PMID:8233656

Rasore-Quartino, A; Frenquellucci, G

1993-01-01

21

Cytochrome P450-activated prodrugs  

PubMed Central

A prodrug is a compound that has negligible, or lower, activity against a specified pharmacological target than one of its major metabolites. Prodrugs can be used to improve drug delivery or pharmacokinetics, to decrease toxicity, or to target the drug to specific cells or tissues. Ester and phosphate hydrolysis are widely used in prodrug design because of their simplicity, but such approaches are relatively ineffective for targeting drugs to specific sites. The activation of prodrugs by the cytochrome P450 system provides a highly versatile approach to prodrug design that is particularly adaptable for targeting drug activation to the liver, to tumors or to hypoxic tissues. PMID:23360144

Ortiz de Montellano, Paul R

2013-01-01

22

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

PubMed

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 degrees C, 100% females), mixed-sex producing (24 and 25 degrees C, 73.3 and 26.7% females, respectively), and masculinizing (29 degrees 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. PMID:17726668

Karube, Makiko; Fernandino, Juan Ignacio; Strobl-Mazzulla, Pablo; Strüssmann, Carlos Augusto; Yoshizaki, Goro; Somoza, Gustavo Manuel; Patiño, Reynaldo

2007-11-01

23

Luminogenic cytochrome P450 assays.  

PubMed

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery. PMID:16859410

Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

2006-08-01

24

Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus  

Microsoft Academic Search

To understand the effect of endocrine-disrupting chemicals (EDCs) on cytochrome P450 aromatase (rm-cyp19) gene expression between gender types in the hermaphroditic fish Rivulus marmoratus, we cloned two distinct rm-cyp19 genes using RT-PCR with degenerative primers, obtained full-length cDNAs using 5?- and 3?-RACE-PCR methods, and completely sequenced them. The brain aromatase (rm-cyp19b) cDNA consisted of 2,124bp including the open reading frame

Young-Mi Lee; Jung Soo Seo; Il-Chan Kim; Yong-Dal Yoon; Jae-Seong Lee

2006-01-01

25

Cytochromes P450: Roles in Diseases*  

PubMed Central

The cytochrome P450 superfamily consists of a large number of heme-containing monooxygenases. Many human P450s metabolize drugs used to treat human diseases. Others are necessary for synthesis of endogenous compounds essential for human physiology. In some instances, alterations in specific P450s affect the biological processes that they mediate and lead to a disease. In this minireview, we describe medically significant human P450s (from families 2, 4, 7, 11, 17, 19, 21, 24, 27, 46, and 51) and the diseases associated with these P450s. PMID:23632021

Pikuleva, Irina A.; Waterman, Michael R.

2013-01-01

26

Unusual Cytochrome P450 Enzymes and Reactions*  

PubMed Central

Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

Guengerich, F. Peter; Munro, Andrew W.

2013-01-01

27

A world of cytochrome P450s  

PubMed Central

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

Nelson, David R.

2013-01-01

28

Cooperative properties of cytochromes P450  

PubMed Central

Cytochromes P450 form a large and important class of heme monooxygenases with a broad spectrum of substrates and corresponding functions, from steroid hormone biosynthesis to the metabolism of xenobiotics. Despite decades of study, the molecular mechanisms responsible for the complex non-Michaelis behavior observed with many members of this super-family during metabolism, often termed ‘cooperativity,’ remain to be fully elucidated. Although there is evidence that oligomerization may play an important role in defining the observed cooperativity, some monomeric cytochromes P450, particularly those involved in xenobiotic metabolism, also display this behavior due to their ability to simultaneously bind several substrate molecules. As a result, formation of distinct enzyme-substrate complexes with different stoichiometry and functional properties can give rise to homotropic and heterotropic cooperative behavior. This review aims to summarize the current understanding of cooperativity in cytochromes P450, with a focus on the nature of cooperative effects in monomeric enzymes. PMID:19555717

Denisov, Ilia G.; Frank, Daniel J.; Sligar, Stephen G.

2009-01-01

29

Intronic polymorphisms of cytochromes P450.  

PubMed

The cytochrome P450 enzymes active in drug metabolism are highly polymorphic. Most allelic variants have been described for enzymes encoded by the cytochrome P450 family 2 (CYP2) gene family, which has 252 different alleles. The intronic polymorphisms in the cytochrome P450 genes account for only a small number of the important variant alleles; however, the most important ones are CYP2D6*4 and CYP2D6*41 , which cause abolished and reduced CYP2D6 activity, respectively, and CYP3A5* 3 and CYP3A5*5 , common in Caucasian populations, which cause almost null activity. Their discoveries have been based on phenotypic alterations within individuals in a population, and their identification has, in several cases, been difficult and taken a long time. In light of the next-generation sequencing projects, it is anticipated that further alleles with intronic mutations will be identified that can explain the hitherto unidentified genetic basis of inter-individual differences in cytochrome P450-mediated drug and steroid metabolism. PMID:20846929

Ingelman-Sundberg, Magnus; Sim, Sarah C

2010-08-01

30

Cytochromes P450 as versatile biocatalysts  

Microsoft Academic Search

Cytochromes P450 are ubiquitously distributed enzymes, which were discovered about 50 years ago and which possess high complexity and display a broad field of activity. They are hemoproteins encoded by a superfamily of genes converting a broad variety of substrates and catalysing a variety of interesting chemical reactions. This enzyme family is involved in the biotransformation of drugs, the bioconversion

Rita Bernhardt

2006-01-01

31

THE HUMAN INTESTINAL CYTOCHROME P450 “PIE”  

PubMed Central

Cytochromes P450 (P450s) 3A, 2C, and 1A2 constitute the major “pieces” of the human liver P450 “pie” and account, on average, for 40, 25, and 18%, respectively, of total immunoquantified P450s (J Pharmacol Exp Ther 270:414–423, 1994). The P450 profile in the human small intestine has not been fully characterized. Therefore, microsomes prepared from mucosal scrapings from the duodenal/jejunal portion of 31 human donor small intestines were analyzed by Western blot using selective P450 antibodies. P450s 3A4, 2C9, 2C19, and 2J2 were detected in all individuals and ranged from 8.8 to 150, 2.9 to 27, <0.6 to 3.9, and <0.2 to 3.1 pmol/mg, respectively. CYP2D6 was detected in 29 individuals and ranged from <0.2 to 1.4 pmol/mg. CYP3A5 was detected readily in 11 individuals, with a range (average) of 4.9 to 25 (16) pmol/mg that represented from 3 to 50% of total CYP3A (CYP3A4 + CYP3A5) content. CYP1A1 was detected readily in three individuals, with a range (average) of 3.6 to 7.7 (5.6) pmol/mg. P450s 1A2, 2A6, 2B6, 2C8, and 2E1 were not or only faintly detected. As anticipated, average CYP3A content (50 pmol/mg) was the highest. Excluding CYP1A1, the remaining enzymes had the following rank order: 2C9 > 2C19 > 2J2 > 2D6 (8.4, 1.1, 0.9, and 0.5 pmol/mg, respectively). Analysis of a pooled preparation of the 31 donor specimens substantiated these results. In summary, as in the liver, large interindividual variation exists in the expression levels of individual P450s. On average, CYP3A and CYP2C9 represents the major pieces of the intestinal P450 pie, accounting for 80 and 15%, respectively, of total immunoquantified P450s. PMID:16467132

Paine, Mary F.; Hart, Heather L.; Ludington, Shana S.; Haining, Robert L.; Rettie, Allan E.; Zeldin, Darryl C.

2007-01-01

32

Novel extrahepatic cytochrome P450s  

SciTech Connect

The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis.

Karlgren, Maria [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden)]. E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Ingelman-Sundberg, Magnus [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden)

2005-09-01

33

Flower colour and cytochromes P450  

PubMed Central

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

Tanaka, Yoshikazu; Brugliera, Filippa

2013-01-01

34

Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals  

SciTech Connect

The roles of different forms of cytochrome P-450 in activation and deactivation of toxic chemicals, such as benzopyrene synthesis and breakdown of steroid hormones, and other functions, indicate the significance of these enzymes. Monooxygenase systems have been studied in species from several phyla of aquatic organisms. However, cytochrome P-450, the dominant catalyst in xenobiotic monoxygenase activity, is best studied in fish. Forms of cytochrome P-450 have been purified from several teleost species, including scup (Stenotomus chrysops), rainbow trout (Salmo gairdneri), and cod (Gadus morhua). Cytochrome P-450E from scup, cytochrome P-450 LM/sub 4b/ from trout, and cytochrome P-450c from cod have properties similar to each other and appear to be homologous hydrocarbons or BNF-inducible isozymes. Cytochrome P-450E from scup is immunochemically related to the major BNF-inducible isozyme (cytochrome P-450c or BNF-B) in rats, indicating homology between the fish and mammalian BNF-inducible isozymes. Several other cytochrome P-450 forms with interesting or unusual properties have been purified from aquatic species. Mammalian homologs are not yet known for these isozymes. Further studies of cytochrome P-450 forms in aquatic species should establish additional homologies and the regulation of these forms by chemical and biological variables, possibly providing fundamental insights into the function and evolution of these proteins.

Stegeman, J.J.; Kloepper-Sams, P.J.

1987-04-01

35

Cytochrome P450-Dependent Lipid Metabolism in Preovulatory Follicles  

E-print Network

Cytochrome P450-Dependent Lipid Metabolism in Preovulatory Follicles J. W. NEWMAN, J. E. STOK, J. D follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites spectrometry. Ara- chidonate oxidation and epoxyeicosatrienoic acid hydrolysis to dihydroxyeicosatrienoic acids

Hammock, Bruce D.

36

African variation at Cytochrome P450 genes  

PubMed Central

The genomics revolution has provided a plethora of data from many previously uncharacterized populations. The increase in the amount of genetic data has improved our understanding of why individuals and populations differ in their susceptibility to multiple diseases. It has also enabled researchers to identify how genomic variation, including at the Cytochrome P450 (CYP450) super-family, affects the safety and efficacy of therapeutic drugs. CYP450 metabolize ?90% of clinically administered drugs. Variability in CYP450 expression is known to affect the safety and efficacy of therapeutic drugs, including many used in the treatment and control of infectious diseases. There are inter-ethnic differences in the frequencies of clinically relevant CYP450 variants which affect CYP450 expression. Comparative studies of African populations have identified population structuring at CYP450 genes. This is associated with intra-African differences in the success of drug therapies used in the treatment of infectious diseases. Therapeutic drugs dominate control strategies for infectious diseases and are widely administered through mass drug administration campaigns. However, resistance to chemotherapy is spreading across endemic regions. The most common response has been to increase chemotherapeutic dosages, and administer combination therapies. However, there are few pharmacovigilance data examining how these changes influence adverse drug reactions. This review provides an overview of current knowledge of intra-Africa CYP450 variation, and the known associations with sub-optimal clinical outcomes in the treatment of infectious diseases. In addition, the potential for evolutionary approaches in the study of CYP450 variation is discussed to examine their potential in preventative medicine and intervention strategies within Africa. PMID:24481193

Bains, Ripudaman K.

2013-01-01

37

Cytochrome P450 pharmacogenetics in African populations.  

PubMed

The Cytochrome P450 (CYP450) family of enzymes is involved in the oxidative metabolism of many therapeutic drugs, carcinogens and various endogenous substrates. These enzymes are highly polymorphic at an inter-individual and inter-ethnic level. Polymorphisms or genetic variations account for up to 30% of inter-individual differences seen in a variety of drug responses. The frequencies of the different metabolizer categories (slow, intermediate, extensive and ultra-rapid), the distribution of genetic variants, genotype-phenotype correlations and the clinical importance of the CYP450 enzymes have been extensively documented in Caucasian and Oriental populations. Limited data exists for African populations, despite the fact that this knowledge is critically important for these populations who experience a heavy burden of communicable and non-communicable diseases. In addition, the costs incurred through adverse drug reactions and non-responsiveness to therapy could be reduced through the wide-scale application of pharmacogenetics. This review provides an overview and investigation of CYP450 genotypic and phenotypic reports published from 1980 to present in African populations. Our findings confirm the high degree of variability that is expected when comparing individuals of African origin to other ethnic groups and also highlight the distribution of clinically relevant CYP450 alleles amongst the various African populations. The notable discordance in genotypic and phenotypic data amongst African populations exemplifies the need for in-depth and well-orchestrated molecular and pharmacological investigations of these populations in the future, for which whole genome sequencing and association studies will be critical. PMID:23590174

Alessandrini, Marco; Asfaha, Sahle; Dodgen, Tyren Mark; Warnich, Louise; Pepper, Michael Sean

2013-05-01

38

Regulation of cytochrome P450 activity by physicochemical methods  

NASA Astrophysics Data System (ADS)

Physicochemical factors influencing the catalytic activities of hemoproteins (of cytochrome P450, in particular) that find use in biosensors and bioreactors are considered. The authors' data on the preparation of semisynthetic hemoproteins based on cytochrome P450 2B4 and NADPH-dependent cytochrome P450 reductase are presented. The use of alternative electron sources (electrochemical reduction and photoreduction) for the redox cycle of hemoproteins is discussed. The effects of temperature, pressure, chemical modification of proteins and organic solvents on the efficiency of hemoprotein-catalysed enzymic reactions are analysed. The bibliography includes 86 references.

Shumyantseva, Viktoriya V.; Bulko, Tat'yana V.; Archakov, Aleksandr I.

1999-10-01

39

Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana)  

PubMed Central

The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48 h exposure to 1 and 100 ?g/L of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5 ng/L of the estrogenic chemical, 17?-ethynylestradiol (EE2), and 673 ng/L of the thyroid hormone, 3,5, 3?-triiodothyronine (T3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48 h exposure to EE2 but not T3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development. PMID:21371610

Gunderson, Mark P.; Veldhoen, Nik; Skirrow, Rachel C.; Macnab, Magnus K.; Ding, Wei; van Aggelen, Graham; Helbing, Caren C.

2011-01-01

40

Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)  

ClinicalTrials.gov

Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

2014-09-24

41

TERATOGEN METABOLISM: THALIDOMIDE ACTIVATION IS MEDIATED BY CYTOCHROME P-450  

EPA Science Inventory

A metabolite of thalidomide generated by hepatic microsomes inhibited the attachment of tumor cells to concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by microsomal cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a...

42

Homotropic cooperativity of monomeric cytochrome P450 3A4  

Microsoft Academic Search

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in

Bradley J. Baas; Ilia G. Denisov; Stephen G. Sligar

2010-01-01

43

Cytochromes P450 Catalyze the Reduction of ?,?-Unsaturated Aldehydes  

PubMed Central

The metabolism of ?,?-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 ?-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 ?,?-unsaturated aldehydes in liver. PMID:21766881

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

44

Cytochromes P450 catalyze the reduction of ?,?-unsaturated aldehydes.  

PubMed

The metabolism of ?,?-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, O(2), 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 and 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 a 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 ?-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 the reduction of ?,?-unsaturated aldehydes in the liver. PMID:21766881

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

2011-08-15

45

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

PubMed Central

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

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

2013-01-01

46

DNA shuffling of cytochromes P450 for indigoid pigment production.  

PubMed

DNA family shuffling is a powerful method of directed evolution applied for the generation of novel enzymes with the aim of improving their existing features or creating completely new enzyme properties. This method of evolution in vitro requires parental sequences containing a high level of sequence similarity, such as is found in family members of cytochrome P450 enzymes. Cytochromes P450 (P450s or CYPs) are capable of catalyzing a variety of chemical reactions and generating a wide range of products including dye production (e.g., pigments indigo and indirubin). Application of the method of DNA family shuffling described here has enabled us to create novel P450 enzymes and to further extend the capacity of P450 to oxidize indole, leading to pigment formation. PMID:23475680

Rosic, Nedeljka N

2013-01-01

47

Characterization of Drosophila melanogaster cytochrome P450 genes  

PubMed Central

Cytochrome P450s form a large and diverse family of heme-containing proteins capable of carrying out many different enzymatic reactions. In both mammals and plants, some P450s are known to carry out reactions essential for processes such as hormone synthesis, while other P450s are involved in the detoxification of environmental compounds. In general, functions of insect P450s are less well understood. We characterized Drosophila melanogaster P450 expression patterns in embryos and 2 stages of third instar larvae. We identified numerous P450s expressed in the fat body, Malpighian (renal) tubules, and in distinct regions of the midgut, consistent with hypothesized roles in detoxification processes, and other P450s expressed in organs such as the gonads, corpora allata, oenocytes, hindgut, and brain. Combining expression pattern data with an RNA interference lethality screen of individual P450s, we identify candidate P450s essential for developmental processes and distinguish them from P450s with potential functions in detoxification. PMID:19289821

Chung, Henry; Sztal, Tamar; Pasricha, Shivani; Sridhar, Mohan; Batterham, Philip; Daborn, Phillip J.

2009-01-01

48

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s.  

PubMed

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the 'cytochrome P450 genesis locus', where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution. PMID:23297357

Nelson, David R; Goldstone, Jared V; Stegeman, John J

2013-02-19

49

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s  

PubMed Central

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution. PMID:23297357

Nelson, David R.; Goldstone, Jared V.; Stegeman, John J.

2013-01-01

50

Cytochrome P450 monooxygenases and insecticide resistance in insects.  

PubMed Central

Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the sequencing of a cytochrome P450 candidate for resistance in resistant and susceptible flies. Several mutations leading to amino-acid substitutions have been detected in the P450 gene CYP6A2 of a resistant strain. The location of these mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule. This has been verified by heterologous expression of wild-type and mutated cDNA in Escherichia coli. When other resistance mechanisms are considered, relatively few genetic mutations are involved in insecticide resistance, and this has led to an optimistic view of the management of resistance. Our observations compel us to survey in more detail the genetic diversity of cytochrome P450 genes and alleles involved in resistance. PMID:10021770

Berge, J B; Feyereisen, R; Amichot, M

1998-01-01

51

Cytochrome P450 monooxygenases: perspectives for synthetic application.  

PubMed

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs. PMID:16759725

Urlacher, Vlada B; Eiben, Sabine

2006-07-01

52

Homotropic cooperativity of monomeric cytochrome P450 3A4  

SciTech Connect

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G. (UIUC)

2010-11-16

53

Domain-domain interaction in cytochrome P450BM3  

Microsoft Academic Search

The influence of ionic strength on the interactions between individually expressed functional domains of cytochrome P450BM-3 and the domains in the holoenzyme has been analyzed by spectrophotometric and fluorometric techniques. High ionic strength facilitated electron transfer from NADPH to the FMN moiety of the reductase domain (BMR) of P450BM-3 and did not affect the first electron transfer from FMN to

I. Sevrioukova; J. A. Peterson

1996-01-01

54

Cytochrome P450: taming a wild type enzyme  

PubMed Central

Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable'--they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability. PMID:21411308

Jung, Sang Taek; Lauchli, Ryan; Arnold, Frances H

2011-01-01

55

Bioelectronic delivery of electrons to cytochrome P450 enzymes.  

PubMed

Cytochrome P450s (cyt P450s) are the major oxidative enzymes in human oxidative metabolism of drugs and xenobiotic chemicals. In nature, the iron heme cyt P450s utilize oxygen and electrons delivered from NADPH by a reductase enzyme to oxidize substrates stereo- and regioselectively. Significant research has been directed toward achieving these events electrochemically. This Feature Article discusses the direct electrochemistry of cyt P450s in thin films and the utilization of such films for electrochemically driven biocatalysis. Maintaining and confirming structural integrity and catalytic activity of cyt P450s in films is an essential feature of these efforts. We highlight here our efforts to elucidate the influence of iron heme spin state and secondary structure of human cyt P450s on voltammetric and biocatalytic properties, using methodologies to quantitatively describe the dynamics of these processes in thin films. We also describe the first cyt P450/reductase films that accurately mimic the natural biocatalytic pathway and show how they can be used with voltammetry to elucidate key mechanistic features. Such bioelectronic cyt P450 systems have high value for future drug development, toxicity screening, fundamental investigations, and chemical synthesis systems. PMID:21591685

Krishnan, Sadagopan; Schenkman, John B; Rusling, James F

2011-07-01

56

The oxidative inactivation of cytochrome P450 in monooxygenase reactions.  

PubMed

Possible mechanisms of cytochrome P450 self-inactivation during catalytic turnover have been considered. Two ways of hemoprotein inactivation are so far known. The first, studied extensively by many authors, is the formation of active substrate intermediates, capable of modifying heme and apoenzyme. The second way, revealed quite recently and resulting from uncoupled cytochrome P450-catalyzed monooxygenase reactions, is yet to be clarified. Briefly, it involves formation of hydrogen peroxide in the hemoprotein active center, which interacts with the enzyme associated Fe2+, thereby generating hydroxyl radicals that bleach the heme and modify the apoenzyme. This mechanism operates with substrates and cytochrome P450 forms with partially coupled monooxygenase reactions, thus causing the formation of hydrogen peroxide as a byproduct. PMID:8299999

Karuzina, I I; Archakov, A I

1994-01-01

57

STRUCTURAL DIFFERENCES BETWEEN SOLUBLE AND MEMBRANE BOUND CYTOCHROMES P450  

PubMed Central

The superfamily of cytochromes P450 forms a large class of heme monooxygenases with more than 13000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochromes P450 from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochromes P450 reveal the presence of two distinct structural classes of cytochromes P450. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which results in an increase of the volume of the substrate binding pocket and an opening of a the potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochromes P450, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochromes P450 can be rationalized through the presence of several amino acids inserts in the latter class which are involved in direct interactions with the membrane, namely the F’ - and G’ – helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B-C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B-C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochromes P450 with important functional implications induced by the lipid bilayer. PMID:22244217

Denisov, I. G.; Shih, A.Y.; Sligar, S.G.

2014-01-01

58

The cytochrome P450 (CYP) gene superfamily in Daphnia pulex  

PubMed Central

Background Cytochrome P450s (CYPs) in animals fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. Results Data mining and manual curation of the Daphnia pulex genome identified 75 functional CYP genes, and three CYP pseudogenes. These CYPs belong to 4 clans, 13 families, and 19 subfamilies. The CYP 2, 3, 4, and mitochondrial clans are the same four clans found in other sequenced protostome genomes. Comparison of the CYPs from D. pulex to the CYPs from insects, vertebrates and sea anemone (Nematostella vectensis) show that the CYP2 clan, and to a lesser degree, the CYP4 clan has expanded in Daphnia pulex, whereas the CYP3 clan has expanded in insects. However, the expansion of the Daphnia CYP2 clan is not as great as the expansion observed in deuterostomes and the nematode C. elegans. Mapping of CYP tandem repeat regions demonstrated the unusual expansion of the CYP370 family of the CYP2 clan. The CYP370s are similar to the CYP15s and CYP303s that occur as solo genes in insects, but the CYP370s constitute ~20% of all the CYP genes in Daphnia pulex. Lastly, our phylogenetic comparisons provide new insights into the potential origins of otherwise mysterious CYPs such as CYP46 and CYP19 (aromatase). Conclusion Overall, the cladoceran, D. pulex has a wide range of CYPs with the same clans as insects and nematodes, but with distinct changes in the size and composition of each clan. PMID:19383150

Baldwin, William S; Marko, Peter B; Nelson, David R

2009-01-01

59

Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor).  

PubMed

Denitrifying bacteria and fungi efficiently detoxify the toxic metabolite nitric oxide (NO) through reduction to nitrous oxide (N2O) using nitric oxide reductase (NOR) enzymes. In fungi, for example Fusarium oxysporum, NO is reduced by a Cytochrome P450 NOR (P450nor). This enzyme contains a heme b center coordinated to a proximal cysteinate ligand in the active site. In the proposed mechanism of P450nor, the ferric heme binds NO first to form a ferric heme-nitrosyl complex, which is subsequently reduced by NAD(P)H to generate a ferrous HNO species as the next key intermediate. Recently, key progress has been made in our understanding of the electronic structures and fundamental reactivity of these important intermediates, using suitable model complexes. In this review, model complexes of ferric heme-nitrosyls with varied axial anionic ligands (such as N-donors, O-donors, and S-donors) are discussed first. Then, the generation and reactivity of ferrous heme-HNO complexes is summarized and related back to the mechanism of P450nor. PMID:24658055

McQuarters, Ashley B; Wirgau, Nathaniel E; Lehnert, Nicolai

2014-04-01

60

Biochemistry 1995,34, 10113-101 19 10113 Dynamic Interactions of Rabbit Liver Cytochromes P450IA2 and P450IIB4 with  

E-print Network

drug oxidation. Rotational diffusion of cytochrome P450 was measured by observing the decayBiochemistry 1995,34, 10113-101 19 10113 Dynamic Interactions of Rabbit Liver Cytochromes P450IA2 and P450IIB4 with Cytochrome b5 and NADPH-Cytochrome P450 Reductase in Proteoliposomest Makoto Yamada

Kawato, Suguru

61

Unusual properties of the cytochrome P450 superfamily  

PubMed Central

During the early years of cytochrome P450 research, a picture of conserved properties arose from studies of mammalian forms of these monooxygenases. They included the protohaem prosthetic group, the cysteine residue that coordinates to the haem iron and the reduced CO difference spectrum. Alternatively, the most variable feature of P450s was the enzymatic activities, which led to the conclusion that there are a large number of these enzymes, most of which have yet to be discovered. More recently, studies of these enzymes in other eukaryotes and in prokaryotes have led to the discovery of unexpected P450 properties. Many are variations of the original properties, whereas others are difficult to explain because of their unique nature relative to the rest of the known members of the superfamily. These novel properties expand our appreciation of the broad view of P450 structure and function, and generate curiosity concerning the evolution of P450s. In some cases, structural properties, previously not found in P450s, can lead to enzymatic activities impacting the biological function of organisms containing these enzymes; whereas, in other cases, the biological reason for the variations are not easily understood. Herein, we present particularly interesting examples in detail rather than cataloguing them all. PMID:23297356

Lamb, David C.; Waterman, Michael R.

2013-01-01

62

Increase in a specific cytochrome P-450 isoenzyme in the liver of congenitally jaundiced Gunn rats.  

PubMed Central

Congenitally jaundiced (jj) Gunn rats had a greater hepatic microsomal content of a cytochrome P-450 isoenzyme, P-450c, than did the non-jaundiced (Jj) rats. No differences in content of P-450b, P-450d and pregnenolone-16 alpha-carbonitrile-induced (PCN) P-450 were found between jj and Jj rats. This is the first demonstration of a constitutive increase in a specific cytochrome P-450 isoenzyme in association with a genetic defect. Images Fig. 4. PMID:3593244

Kapitulnik, J; Hardwick, J P; Ostrow, J D; Webster, C C; Park, S S; Gelboin, H V

1987-01-01

63

Does differing metabolism by cytochrome P450 have clinical importance?  

Microsoft Academic Search

The cytochrome P450 (CYP) is a group of enzymes that oxidatively modify drugs to a more water-soluble form for renal excretion.\\u000a Nearly 50% of all clinically used medications and endogenous steroids are metabolized by the CYP enzyme 3A4, which explains\\u000a why many of the important potential drug interactions involved this enzyme. Despite an excellent safety record, CYP 3A4 statins\\u000a (lovastatin,

Michael H. Davidson

2000-01-01

64

Modulation by phytochemicals of cytochrome P450-linked enzyme activity  

Microsoft Academic Search

Compounds derived from plant sources with putative anticancer properties were studied for their effects on alkoxyresorufin O-dealkylase activity, a measure of cytochrome P450 activity. The phytochemicals investigated included benzyl isothiocyanate, caffeic acid, chlorogenic acid, diosmin, ferulic acid, indole-3-carbinol, phenethyl isothiocyanate and resveratrol. Each phytochemical at concentrations of 0.25 and 0.5 ?M was incubated with 0.2 mg hamster liver microsomal protein

Robert W. Teel; Huong Huynh

1998-01-01

65

KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES  

EPA Science Inventory

Kinetics of Bromodichloromethane Metabolism by Cytochrome P450 Isoenzymes in Human Liver Microsomes Guangyu Zhao and John W. Allis ABSTRACT The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

66

Sex Differences in Cytochrome P-450 and Mixed-Function Oxygenase Activity in Gonadally Mature Trout.  

National Technical Information Service (NTIS)

Levels of microsomal cytochrome P-450 and aminopyrine demethylase activity in liver and of cytochrome P-450 in kidney of gonadally mature rainbow and brook trout were markedly greater in males than in females. Similar differences appeared in hepatic micro...

J. J. Stegeman, M. Chevion

1980-01-01

67

Microfluidic-based measurements of cytochrome P450 enzyme activity of primary mammalian hepatocytes  

E-print Network

cytochrome P450 (CYP450) is a family of variant enzymes that catalyses the oxidative metabolism of a wideMicrofluidic-based measurements of cytochrome P450 enzyme activity of primary mammalian hepatocytes

68

Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3 Edgardo T. Farinas,  

E-print Network

Catalyzing a wide range of oxidative reactions under mild conditions in aqueous solutions, cytochrome P450Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3 Edgardo T. Farinas, Miguel Alcalde conversion of cytochrome P450 BM-3, a medium-chain (C12 ­C18) fatty acid monooxygenase, into a highly

Arnold, Frances H.

69

Aromatase (P450arom) and 11?-hydroxylase (P45011?) genes are differentially expressed during the sex change process of the protogynous rice field eel, monopterus albus  

Microsoft Academic Search

Steroids are known to play a crucial role in gonadal sex differentiation in many non-mammalian vertebrates, but also in the\\u000a gonadal sex change of hermaphroditic teleosts. We investigated the expression of two genes encoding key steroidogenic enzymes,\\u000a i.e., cytochrome P450 aromatase (P450arom) and cytochrome P45011?-hydroxylase (P45011?), during the sex change of the protogynous\\u000a rice field eel, Monopterus albus. Using RT-PCR

Ji-Fang Liu; Yann Guiguen; Shao-Jun Liu

2009-01-01

70

Cytochrome P450-derived eicosanoids: the neglected pathway in cancer.  

PubMed

Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ?-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

Panigrahy, Dipak; Kaipainen, Arja; Greene, Emily R; Huang, Sui

2010-12-01

71

Regulation of cytochrome P450 (CYP) genes by nuclear receptors.  

PubMed Central

Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

Honkakoski, P; Negishi, M

2000-01-01

72

Human cytochromes P450 in health and disease.  

PubMed

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases-confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

Nebert, Daniel W; Wikvall, Kjell; Miller, Walter L

2013-02-19

73

Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus.  

PubMed Central

Streptomyces griseolus ATCC 11796 contains two inducible, herbicide-metabolizing cytochromes P-450 previously designated P-450SU1 and P-450SU2 (P-450CVA1 and P-450CVB1, respectively, using nomenclature of Nebert et al. [D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6:1-11, 1987]). Using antibodies directed against cytochrome P-450SU1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450SU1. Similar information about the cytochrome P-450SU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2. The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-450CAM from Pseudomonas putida. Both cytochromes P-450SU1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-450CAM, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group. Images PMID:2345149

Omer, C A; Lenstra, R; Litle, P J; Dean, C; Tepperman, J M; Leto, K J; Romesser, J A; O'Keefe, D P

1990-01-01

74

Unusual regioselectivity and active site topology of human cytochrome P450 2J2  

E-print Network

1 Unusual regioselectivity and active site topology of human cytochrome P450 2J2 . : This work.mansuy@univ-paris5.fr #12;2 ABBREVIATIONS COSY: correlation spectroscopy; CYP or P450: cytochrome P450; EDTA magnetic resonance; UV: ultraviolet. #12;3 ABSTRACT The oxidation of six derivatives of terfenadone

Boyer, Edmond

75

Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium.  

PubMed Central

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals. PMID:9173903

Longo, V; Amato, G; Santucci, A; Gervasi, P G

1997-01-01

76

Resveratrol Is a Selective Human Cytochrome P450 1A1 Inhibitor  

Microsoft Academic Search

Resveratrol (trans-3,4?,5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase

Young Jin Chun; Mie Young Kim; F. Peter Guengerich

1999-01-01

77

Cytochrome P450-mediated metabolism of xanthotoxin by Papilio multicaudatus.  

PubMed

Within the genus Papilio, the P. glaucus group contains the most polyphagous Papilio species within the Papilionidae. The majority of Papilio species are associated with hostplants in the families Rutaceae and Apiaceae, and characterizing most are secondary metabolites called furanocoumarins. Recent phylogenetic studies suggest that furanocoumarin metabolism is an ancestral trait, with the glaucus group derived from ancestors associated with furanocoumarin-containing Rutaceae. In this study, we examined this relationship by conducting a gravimetric analysis of growth that used various concentrations of the furanocoumarin xanthotoxin. Papilio multicaudatus, the putative ancestor of the glaucus group, includes at least one furanocoumarin-containing rutaceous species among its hostplants; this species can consume leaf tissue containing up to 0.3% xanthotoxin with no detectable effect on relative growth rate, relative consumption rate, or efficiency of conversion of ingested food. As is the case for other Papilio species, xanthotoxin metabolism is mediated by cytochrome P450 monooxygenases (P450s). Ingestion of xanthotoxin by ultimate instar P. multicaudatus increases activity up to 30-fold in a dose-dependent fashion. Midguts of induced larvae can also effectively metabolize six other furanocoumarins, including both linear (bergapten, isopimpinellin, imperatorin) and angular (angelicin, sphondin) forms. A metabolite of xanthotoxin in the frass from xanthotoxin-treated larvae, identified as 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid by MS-MS and NMR analyses, is identical to one from the frass of P. polyxenes. The occurrence of this metabolite in two swallowtails and the presence of a second metabolite of xanthotoxin, 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol in the frass of both P. polyxenes and Depressaria pastinacella are consistent with the suggestion that lepidopterans share as the first step of xanthotoxin metabolism the P450-mediated epoxidation of the furan ring 2'-3' double bond. PMID:16572296

Mao, Wenfu; Berhow, Mark A; Zangerl, Arthur R; McGovern, Jennifer; Berenbaum, May R

2006-03-01

78

Cholesterol ester oxidation by mycobacterial cytochrome p450.  

PubMed

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58-67 and 100-109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth. PMID:25210044

Frank, Daniel J; Madrona, Yarrow; Ortiz de Montellano, Paul R

2014-10-31

79

Nanodiscs in the studies of membrane-bound cytochrome P450 enzymes  

PubMed Central

SUMMARY Cytochromes P450 from Eukaryotes and their native redox partners cytochrome P450 reductases both belong to the class of monotopic membrane proteins containing one transmembrane anchor. Incorporation into the lipid bilayer significantly affects their equilibrium and kinetic properties and plays an important role in their interactions. We describe here the detailed protocols developed in our group for the functional self-assembly of mammalian cytochromes P450 and cytochrome P450 reductases into Nanodiscs with controlled lipid composition. The resulting preparations are fully functional, homogeneous in size, composition and oligomerization state of the heme enzyme, and show an improved stability with respect to P420 formation. We provide a brief overview of applications of Nanodisc technology to the biophysical and biochemical mechanistic studies of cytochromes P450 involved in steroidogenesis, and of the most abundant xenobiotic metabolizing human cytochrome P450 CYP3A4. PMID:23475672

Luthra, A.; Gregory, M.; Grinkova, Y.V.; Denisov, I.G.; Sligar, S.G.

2014-01-01

80

Active site dynamics of toluene hydroxylation by cytochrome P-450  

SciTech Connect

Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH{sub 2}D and PhCHD{sub 2} suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggsting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH{sub 3} vs PhCD{sub 3} indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH{sub 3} and PhCD{sub 3}, {sup D}V for benzyl alcohol formation is only 1.92, whereas {sup D}V for total product formation is 0.67 (i.e., inverse). From competitive incubations of PhCH{sub 3}/PhCD{sub 3} mixtures {sup D}(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, {sup D}(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate.

Hanzlik, R.P.; Kahhiing John Ling (Univ. of Kansas, Lawrence (United States))

1990-06-22

81

Modulation of cytochrome P450 isozymes in human liver, by ethanol and drug intake.  

PubMed

Cytochromes P450 (P450) are a family of isozymes which play an important role in xenobiotic metabolism. The concentration of three P450 isozymes, namely P450-IIE1(A1c),-IIIA(NF) and -IIC8-10(MP) has been measured in human liver biopsies of patients with different alcohol and drug intake status. All these three P450s were expressed in all subjects. Ethanol intake increased P450-IIE1(A1c) content with no effect on the content of the two other P450s. Drug intake (barbiturates) increased both P450-IIIA(NF) and -IIC8-10(MP) content without any effect on P450-IIE1(A1c). This paper brought, at protein level, further evidence of the importance of environmental conditions on P450 isozyme pattern, and therefore, on drug metabolizing capacity of human liver. PMID:2515975

Perrot, N; Nalpas, B; Yang, C S; Beaune, P H

1989-12-01

82

Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis.  

PubMed Central

Extensive screening of aromatase cDNA was carried out in cDNA libraries from various human tissues. The DNA sequences of all the isolated cDNA clones were identical in the region encoded by exons 2-10 of the aromatase gene. However, tissue-specific sequences, which were classified into four groups, were observed in the 5' portions of the clones corresponding to the region encoded by exon 1. All of them were also found in clones isolated from a human genomic library and mapped between exons 1 and 2 of the human aromatase gene reported previously, suggesting the presence of multiple exons 1 and promoters in the gene. Reverse transcription-PCR analyses of aromatase mRNAs in various tissues revealed that aromatase transcripts are tissue-specifically spliced by alternative use of multiple exons 1, although minor forms of the transcripts were also present in each tissue. Aromatase mRNA is spliced from 10 exons in most tissues, but from 9 exons in the prostate and from 10 or 11 exons in the placenta. This suggests that tissue-specific regulation of the aromatase gene in various tissues may be explained by alternative use of multiple exons 1 flanked with tissue-specific promoters. The alternative use of multiple exons 1 for liver transcripts was found to change developmentally. Furthermore, switch from an adipose-specific exon 1 to another type of exon 1 was observed in aromatase transcripts of adipose tissues of three of five breast cancer patients. Images Fig. 3 Fig. 4 PMID:8248245

Harada, N; Utsumi, T; Takagi, Y

1993-01-01

83

Bacterial cytochrome P450 system catabolizing the Fusarium toxin deoxynivalenol.  

PubMed

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (k(cat)/K(m)) of 6.4 mM(-1) s(-1). The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat. PMID:23275503

Ito, Michihiro; Sato, Ikuo; Ishizaka, Masumi; Yoshida, Shin-ichiro; Koitabashi, Motoo; Yoshida, Shigenobu; Tsushima, Seiya

2013-03-01

84

Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol  

PubMed Central

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM?1 s?1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat. PMID:23275503

Ito, Michihiro; Sato, Ikuo; Ishizaka, Masumi; Yoshida, Shin-ichiro; Koitabashi, Motoo; Yoshida, Shigenobu

2013-01-01

85

Regulation of cytochrome P450 gene transcription by phenobarbital.  

PubMed

The ability of phenobarbital to induce levels of drug metabolism in mammals has been known for over 40 years. However, the molecular mechanisms underlying increased expression of the genes of the key enzyme in drug metabolism, cytochrome P450, have not been elucidated, primarily because in vitro model systems in which the induction could be studied were not available. Transfected primary cultured hepatocytes, transfection of liver in situ, and transgenic mice now provide suitable models for phenobarbital induction. In this review, progress toward understanding the mechanism of phenobarbital induction of gene expression is discussed with an emphasis on the mammalian genes, CYP2B1, CYP2B2, and Cyp2b10, which are most highly inducible by phenobarbital. Barbiturate induction of P450s in Bacillus megaterium, which is the system best understood, and its relevance to mammalian mechanisms of induction are also discussed. In B. megaterium, the binding of a repressor to several motifs is reversed by direct effects of barbiturates and by induction of positively acting factors. One of the repressor binding sites, the barbie box, is present in many mammalian phenobarbital-inducible genes, including the promimal promoter regions of CYP2B1, CYP2B2, and Cyp2B10. In the mammalian P450 genes, evidence has been proposed for phenobarbital-regulated elements both in the proximal promoter region and in a distal enhancer region. The role of the proximal region is controversial. A positively acting element that overlaps the barbie box sequence and a negative element have been proposed to mediate induction of CYP2B1/2, based primarily on protein binding and cell-free transcription assays. In contrast, other investigators have not found differences in phenobarbital-dependent protein binding in the proximal promoter region nor mediation of phenobarbital induction by this region. A distal gene fragment, at about -2000 kb in CYP2B1, CYP2B2, and Cyp2b10, has been shown to be a phenobarbital-responsive enhancer independent of proximal promoter elements. This fragment contains several binding sites for proteins and several functional elements, including an NF-1 site, and, therefore, has been designated as a phenobarbital-responsive unit. Possible models are presented in which phenobarbital treatment induces altered chromatin structure, which allows the binding of positively acting factors, or activates factors already bound, to the distal enhancer and the proximal promoter. PMID:9752718

Kemper, B

1998-01-01

86

Differential effects of B and T-lymphocyte mitogens on cytochrome P450 in mice  

Microsoft Academic Search

Lipopolysaccharide (LPS) is widely used as a B-lymphocyte mitogen and is known to depress expression of the cytochrome P450 (P450). However, there have been no studies regarding to the effects of the other mitogens on the expression of P450. This study investigated the effects of mitogens on the constitutive and inducible expression of mouse hepatic P450. Following treatment with B-lymphocyte

Hye Gwang Jeong

1999-01-01

87

Gender differences in the regulation of P450 aromatase expression and activity in human adipose tissue  

Microsoft Academic Search

OBJECTIVE: To investigate the hormonal regulation of P450 aromatase activity (responsible for the conversion of C19 androgens to C18 oestrogens) in human adipose tissue from men and pre- and post-menopausal women.SUBJECTS: Subcutaneous abdominal adipose tissue was obtained from 19 subjects: six pre-menopausal females (mean age 41.8±(s.e.m.) 2.5; mean weight 76.01±5.6 kg), eight post-menopausal females (mean age 59.9±2.0; mean weight 63.5±2.6

PG McTernan; A Anwar; MC Eggo; AH Barnett; PM Stewart; S Kumar

2000-01-01

88

Profiling Cytochrome P450 Expression in Ovarian Cancer: Identification of Prognostic Markers  

Microsoft Academic Search

Purpose: The cytochromes P450 are a multigene family of enzymes with a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs and biologically active endogenous compounds. The purpose of this study was to define the cytochrome P450 profile of ovarian cancer and identify novel therapeutic targets and establish the prognostic signif- icance of expression

Diane Downie; Morag C. E. McFadyen; Patrick H. Rooney; Margaret E. Cruickshank; David E. Parkin; Iain D. Miller; Colin Telfer; William T. Melvin; Graeme I. Murray

89

Evaluation of Norcarane as a Probe for Radicals in Cytochrome P450-and Soluble Methane Monooxy-  

E-print Network

Evaluation of Norcarane as a Probe for Radicals in Cytochrome P450- and Soluble Methane Monooxy related to this paper, we recently discovered that norcarane is oxidized to 2-norcarene and 3-norcarene in de- saturase reactions catalyzed by cytochrome P450 and diiron- containing enzymes, including some

Leigh, William J.

90

Characterization of cytochrome P450-mediated drug metabolism in cats.  

PubMed

In this study we examined activities of cytochrome P450 (CYP)1A, 2C, 2D and 3A using hepatic microsomes from five male and five female cats. CYP1A, 2C, 2D and 3A activities were referred by ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation (TBH), bufuralol 1'-hydroxylation (BLH) and midazolam 1'- and 4-hydroxylation respectively. The anti-rat CYP1A2 and CYP3A2 serum significantly inhibited EROD and midazolam 1'- and 4-hydroxylation, suggesting that EROD and midazolam 1'- and 4-hydroxylation were catalysed by CYP1A and 3A in cats respectively. Quinidine inhibited BLH in cats microsomes at quite low concentrations, suggesting that BLH was catalysed by CYP2D in cats. Tolbutamide hydroxylation activities were negligible in hepatic microsomes from both male and female cats, suggesting CYP2C activities of cats are extremely low. This suggests that CYP2C substrates should be carefully administered to cats. Although there is no sexual difference in CYP1A activities, there are differences in CYP2D and 3A activities of cats. CYP2D activities were higher (3-fold), but CYP3A activities were lower (one-fifth) in female cats. These results might suggest that CYP2D and 3A substrates should be prescribed for male and female cats using different dosage regimen. PMID:17803734

Shah, S S; Sanda, S; Regmi, N L; Sasaki, K; Shimoda, M

2007-10-01

91

First principles calculation of the activity of cytochrome P450  

NASA Astrophysics Data System (ADS)

The cytochrome P450 superfamily of enzymes is of enormous interest in the biological sciences due to the wide range of endogenous and xenobiotic compounds which it metabolises, including many drugs. We describe the use of first principles quantum mechanical modeling techniques, based on density functional theory, to determine the outcome of interactions between an enzyme and a number of compounds. Specifically, we calculate the spin state of an Fe3+ ion present in a haem moiety at the active site of these enzymes. The spin state of this ion indicates if the catalytic reaction will proceed. The computational results obtained compare favorably with experimental data. Only the principle components of the active site of the enzyme are included in the computational models, demonstrating that only a small fragment of the protein needs to be included in the models in order to accurately reproduce this aspect of the enzymes' function. These results open the way for further investigation of this superfamily of enzymes using the methods detailed in this paper.

Segall, M. D.; Payne, M. C.; Ellis, S. W.; Tucker, G. T.; Boyes, R. N.

1998-04-01

92

A specific cytochrome P450 hydroxylase in herboxidiene biosynthesis.  

PubMed

The anti-cholesterol natural product herboxidiene is synthesized by a noniterative modular polyketide synthase (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG) in Streptomyces chromofuscus A7847. In this work, the putative monooxygenase HerG was expressed in Escherichia coli and the purified enzyme was subjected to biochemical studies. It was identified as a cytochrome P450 enzyme responsible for the stereospecific hydroxylation at C-18. This enzyme is highly substrate-specific as it efficiently hydroxylates 18-deoxy-25-demethyl-herboxidiene, but showed no activity towards 18-deoxy-herboxidiene. The kcat/Km value for the HerG-catalyzed hydroxylation of 18-deoxy-25-demethyl-herboxidiene was determined to be 1669.70±47.36M(-1)s(-1). In vitro co-reaction of HerG with the methyltransferase HerF and analysis of the product formation in S. chromofuscus A7847 revealed that the biosynthetic intermediate 18-deoxy-25-demethyl-herboxidiene is successively hydroxylated at C-18 by HerG and methylated at 17-OH to yield the final product herboxidiene. The minor metabolite 18-deoxy-hereboxidiene is a byproduct of the biosynthetic pathway. PMID:25139567

Yu, Dayu; Xu, Fuchao; Shao, Lei; Zhan, Jixun

2014-09-15

93

Nanoscale-engineered cytochrome p450 system with a branch structure.  

PubMed

Most of the bacterial cytochrome P450 s require two kinds of electron transfer proteins, ferredoxin and ferredoxin reductase, and thus P450 s do not show catalytic activity by themselves. A microbial transglutaminase-mediated site-specific cross-linking enables the formation of fusion P450 protein with a branched structure, which is generated from a genetic fusion protein of P450-ferredoxin reductase and ferredoxin, an interactive nanoscale protein structure. This fusion P450 system is self-sufficient due to intramolecular electron transfer, which means the system does not require additional electron-transferring proteins. Because some components of bacterial cytochrome P450 system are interchangeable, this self-sufficient system can be applied to non-natural combination of P450 and electron transfer proteins from different species of bacteria. PMID:21553178

Hirakawa, Hidehiko; Nagamune, Teruyuki

2011-01-01

94

Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency  

SciTech Connect

Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland); Mallet, Delphine [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France)] [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Hofer, Gaby [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland)] [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland); Samara-Boustani, Dinane [Hopital Necker-Enfants malades, Paris (France)] [Hopital Necker-Enfants malades, Paris (France); Leger, Juliane [Hopital Robert Debre, Paris (France)] [Hopital Robert Debre, Paris (France); Polak, Michel [Hopital Necker-Enfants malades, Paris (France)] [Hopital Necker-Enfants malades, Paris (France); Morel, Yves [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France)] [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland)

2011-09-09

95

Plant cytochrome P450s from moss to poplar  

Microsoft Academic Search

This review represents the first attempt to define the origins of the major P450-containing pathways in plants. Comparative genomics with five complete P450 gene sets from Chlamydomonas reinhardtii with 39 sequences, Physcomitrella patens (moss) with 71 sequences, rice with 356 sequences, Arabidopsis with 246 sequences and Populus with 312 sequences is used to estimate how old each gene family is

David R. Nelson

2006-01-01

96

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency.  

PubMed

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17?-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase. PMID:21843508

Flück, Christa E; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V

2011-09-01

97

Inhibition of cholesterol 7 alpha-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC.  

PubMed Central

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification. Images Fig. 4. Fig. 5. PMID:2510717

Eldredge, E R; Jackson, B; Suckling, K E; Wolf, C R

1989-01-01

98

Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies.  

PubMed

A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b. PMID:3935002

Rothwell, C E; Khazaeli, M B; Bernstein, I A

1985-08-15

99

P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis  

PubMed Central

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost. PMID:18930820

Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, Rene

2008-01-01

100

Laboratory Evolution of Cytochrome P450 BM-3 Monooxygenase for  

E-print Network

, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles, have a significant market value. One serious drawback, however, is that the Km values of P450 BM-3

Arnold, Frances H.

101

Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2.  

PubMed

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a Ni(2+)-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies. PMID:24795797

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

2014-03-01

102

Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2  

PubMed Central

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a Ni2+-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies. PMID:24795797

Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee

2014-01-01

103

Ginsenoside Metabolites, Rather Than Naturally Occurring Ginsenosides, Lead to Inhibition of Human Cytochrome P450 Enzymes  

Microsoft Academic Search

There is still an argument about ginseng-prescription drug interactions. To evaluate the influence on cytochrome P450 (P450) activities of ginseng in the present study, the influence on P450 activities of naturally occurring ginsenosides and their degrada- tion products in human gut lumen was assayed by using human liver microsomes and cDNA-expressed CYP3A4. The results showed that the naturally occurring ginsenosides

Yong Liu; Jiang-Wei Zhang; Wei Li; Hong Ma; Jie Sun; Mai-Cun Deng; Ling Yang

2006-01-01

104

[The role of cytochrome p450 in metabolism of S-ethynylthiophosphates].  

PubMed

We studied interaction between S-ethynyl ethers of phosphorus acids with cytochrome P-450 from rat liver and housefly abdomen. High thionic effect, i.e., considerable selectivity for the studied compounds in homoiotherms and arthropods, proved to the related to the triple bond in these compounds. Apparently, cytochrome P-450 participates in S-ethynylthiophosphates metabolism and breaks the P-S bond. This gives rise to "self-destroying" metabolites, namely, alkylthioketenes, which decelerate deactivation reactions through destruction of the corresponding isoform of cytochrome P-450 in the microsomal fraction in both homoiotherms and insects. However, the activation reaction goes much faster in insects than in homoiotherms. PMID:10881425

Vikhreva, L A; Pudova, T A; Godovikov, N N; Kabachnik, M I; Salganik, R Ia; Nedel'kina, S V

2000-01-01

105

Interaction of methadone with substrates of human hepatic cytochrome P450 3A4.  

PubMed

Methadone, a synthetic drug, is one of the most widely used drugs for opiate dependency treatment. This drug has been demonstrated to be extensively metabolized by cytochrome P450 3A4 in human liver microsomes. Thus, the aim of this in vitro study was to determine if methadone is an inhibitor of other P450s characterized by their specific catalytic activities. Enzymatic activities specific to P450 2E1, P450 1A, P450 2B and P450 2C were not inhibited by methadone. Conversely, nifedipine oxidation, mediated by cytochrome P450 3A4, was potently inhibited by methadone by a mixed-type inhibition mechanism with a Ki of 100 microM. Fluvoxamine, a new antidepressant, was shown to be a potent mixed-type inhibitor of methadone N-demethylation with a Ki of 7 microM. Finally, methadone appears to be a mixed-type inhibitor and not a suicide inhibitor of cytochrome P450 3A family. Accordingly, caution should be advised in the clinical use of methadone when other drugs are administered that are able to induce or inhibit P450 3A4, such as rifampicin or nifedipine, diazepam and fluvoxamine. PMID:9020195

Iribarne, C; Dréano, Y; Bardou, L G; Ménez, J F; Berthou, F

1997-02-14

106

Cytochrome P450IA mRNA expression in feral Hudson River tomcod.  

PubMed

We 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, we 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 by 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. PMID:1855491

Kreamer, G L; Squibb, K; Gioeli, D; Garte, S J; Wirgin, I

1991-06-01

107

Cytochrome P450IA mRNA expression in feral Hudson River tomcod  

SciTech Connect

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 by 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.

Kreamer, G.L.; Squibb, K.; Gioeli, D.; Garte, S.J.; Wirgin, I. (New York University Medical Center, Institute of Environmental Medicine, Tuxedo (USA))

1991-06-01

108

Interleukin-6 and cytochrome-P450, reason for concern?  

PubMed

Interleukin 6 (IL-6) plays a central role in the immunopathogenesis of rheumatoid arthritis (RA) and tocilizumab [TCZ] (an anti-IL-6 receptor antibody) has been shown to be effective in the treatment of the condition. As up-regulation of IL-6 reduces the activity of cytochrome P450 (CYP) enzymes, blockade of this cytokine may enhance CYP function. This may lead to reduced bioavailability of CYP-metabolized drugs. Due to the increasing use of TCZ, we undertook a systematic literature review to explore such interactions. Our search was conducted in MEDLINE, EMBASE, Web of Science, FDA and EMEA websites for in vitro and in vivo studies, clinical trials and reviews mentioning TCZ and CYP on the basis of the title and abstract. Appropriate articles were further screened based on full-text review to select only those reporting IL-6, TCZ and their potential interaction with CYP-metabolized drugs. Two in vitro studies showed that TCZ-reversed IL-6 induced reduction of CYP isozymes. CYP3A4 mRNA expression was most reduced by IL-6 followed by CYP2C9 and CYP2C19. This change was prevented with TCZ. Three clinical studies investigated the interaction showing simvastatin (CYP3A4 substrate) bioavailability reduced by TCZ and omeprazole bioavailability was decreased by TCZ-induced CYP2C19 activity. The bioavailability of dextromethorphan (CYP2D6 and CYP3A4 substrates) was shown to be unaffected by TCZ treatment. The observed increase in CYP isozyme activity by TCZ is of clinical relevance as the bioavailability of the CYP isozyme substrates were decreased in vivo. As CYP3A4 is the isozyme responsible for the largest proportion of drug metabolism, it is probable that the bioavailability of other drugs may be reduced by TCZ. Thus, clinicians should exercise caution when co-prescribing TCZ and CYP-metabolized drugs. More studies are required to investigate this interaction further. PMID:22451032

Kim, Sooha; Östör, Andrew J K; Nisar, Muhammad K

2012-09-01

109

Protein-Surfactant Film Voltammetry of Wild-Type and Mutant Cytochrome P450 BM3  

E-print Network

their biological importance (e.g., xenobiotic elimination),2 but also from their ability to perform valuable differ strikingly in their reactions with dioxygen. The cytochromes P450 (P450s) catalyze challenging oxidation reactions under physiological conditions.1 Interest in these proteins stems not only from

Arnold, Frances H.

110

Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450  

E-print Network

/en/index.html). The major causes are the high susceptibil- ity of immunocompromised individuals strategies and drugs to combat this human disease. The complete sequence of the M. tuberculosis genome re cytochrome P450 (P450) enzymes are encoded in the genome, presumably linked to the extensive lipid metabolism

111

CYTOCHROME P450-DEPENDENT METABOLISM OF TRICHLOROETHYLENE IN THE RAT KIDNEY  

EPA Science Inventory

The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite con...

112

Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.  

PubMed Central

A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species. Images Fig. 1. Fig. 6. PMID:1445245

Puccini, P; Menicagli, S; Longo, V; Santucci, A; Gervasi, P G

1992-01-01

113

Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.  

PubMed Central

In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative. Images PMID:3186722

Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

1988-01-01

114

Electron Paramagnetic Resonance Studies on Hepatic Microsomal Cytochrome P-450 from a Marine Teleost Fish.  

National Technical Information Service (NTIS)

Hepatic microsomal cytochrome P-450 from fish (Stenotomus versicolor), untreated or treated with 3-methylcholanthrene, 5,6-benzo-flavone, or tricaine methanesulfonate, exhibited an absorption maximum at 450 nm when reduced and ligated to CO. Microsomes fr...

M. Chevion, J. J. Stegeman, J. Peisach, W. E. Blumberg

1977-01-01

115

Steroid transformation by Rhodococcus strains and bacterial cytochrome P450 enzymes.  

E-print Network

??The thesis describes activities of four bacterial steroid-induced cytochrome P450 enzymes in terms of their potential biotechnological use for steroid transformations by engineered Rhodococcus strains… (more)

Du Plessis-Rosloniec, Kamila Zofia

2011-01-01

116

Approaches to Deorphanization of Human and Microbial Cytochrome P450 Enzymes  

PubMed Central

One of the general problems in biology today is that we are characterizing genomic sequences much faster than identifying the functions of the gene products, and the same problem exists with cytochromes P450 (P450). One-fourth of the human P450s are not well-characterized and therefore considered “orphans.” A number of approaches to deorphanization are discussed generally. Several liquid chromatography-mass spectrometry approaches have been applied to some of the human and Streptomyces coelicolor P450s. One current limitation is that too many fatty acid oxidations have been identified and we are probably missing more relevant substrates, possibly due to limits of sensitivity. PMID:20493973

Guengerich, F. Peter; Tang, Zhongmei; Cheng, Qian; Salamanca-Pinzón, S. Giovanna

2010-01-01

117

Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment  

Microsoft Academic Search

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein–protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in

Bradley J. Baas; Ilia G. Denisov; Stephen G. Sligar

2004-01-01

118

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450  

Microsoft Academic Search

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

Barnett A. Rattner; Mark J. Melancon; Thomas W. Custer; Roger L. Hothem; Kirke A. King; Leonard J. LeCaptain; James W. Spann; Bruce R. Woodin; John J. Stegeman

1993-01-01

119

Enhanced bacterial expression of several mammalian cytochrome P450s by codon optimization and chaperone coexpression  

PubMed Central

To elucidate the effect of codon optimization and chaperone coexpression on the heterologous expression of mammalian cytochrome P450s in Escherichia coli, the expression of P450s 2B1, 2S1, 2U1, 2W1, and 27C1 were investigated. With codon optimization for N-terminus or the entire gene, the expression levels of P450 27C1, 2U1 and 2W1 increased 22-fold, 3.6-fold and 2.1-fold respectively, while those for P450s 2B1 and 2S1 remained unchanged. With coexpression of E. coli molecular chaperones GroEL/ES, the expression level increased up to 14-fold for P450 27C1, and 3- to 5-fold for P450s 2B1, 2S1, and 2W1. Simultaneous application of these two techniques resulted in synergetic effects. PMID:19557307

Qiao, Jing; Zhang, Zhi-Gang; Guengerich, F. Peter; Liu, Yan; Pei, Xiao-Qiong

2014-01-01

120

Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail.  

PubMed

Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at 0.5 or 1.0% fresh weight. Although xanthotoxin exerts much of its toxic effects when photoactivated by ultraviolet light, induction of P450 activity did not differ in the presence or absence of ultraviolet light. Despite a 4.7-fold induction of xanthotoxin-metabolizing P450 activity, total P450 content measured in the same microsomal samples did not increase significantly. These data indicate that multiple forms of P450 exist in the black swallowtail midgut and that they are differentially induced by xanthotoxin. PMID:24272422

Cohen, M B; Berenbaum, M R; Schuler, M A

1989-09-01

121

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450  

SciTech Connect

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 or phenobarbital. Compared to controls, 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases 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[prime]-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP1B) were significantly associated with total PCB burdens.

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W. (U.S. Fish and Wildlife Service, Laurel, MD (United States). Patuxent Wildlife Research Center); Woodin, B.R.; Stegeman, J.J. (Woods Hole Oceanographic Inst., MA (United States))

1993-09-01

122

Involvement of Cytochrome P450 in Pentachlorophenol Transformation in a White Rot Fungus Phanerochaete chrysosporium  

PubMed Central

The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min?1 (mg protein)?1, which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research. PMID:23029295

Ning, Daliang; Wang, Hui

2012-01-01

123

Potent inhibition of human cytochrome P450 1 enzymes by dimethoxyphenylvinyl thiophene.  

PubMed

Cytochrome P450 (P450) 1 enzymes such as P450 1A1, 1A2, and 1B1 are known to be involved in the oxidative metabolism of various procarcinogens and are regarded as important target enzymes for cancer chemoprevention. Previously, several hydroxystilbene compounds were reported to inhibit P450 1 enzymes and were rated as candidate chemopreventive agents. In this study, we investigated the inhibitory effect of 2-[2-(3,5-dimethoxyphenyl)vinyl]-thiophene (DMPVT), produced from the chemical modification of oxyresveratrol, on the activities of P450 1 enzymes. The inhibitory potential by DMPVT on the P450 1 enzyme activity was evaluated with the Escherichia coli membranes of the recombinant human cytochrome P450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P450 reductase. DMPVT significantly inhibited ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 61, 11, and 2 nM for 1A1, 1A2, and 1B1, respectively. The EROD activity in DMBA-treated rat lung microsomes was also significantly inhibited by DMPVT in a dose-dependent manner. The modes of inhibition by DMPVT were non-competitive for all three P450 enzymes. The inhibition of P450 1B1-mediated EROD activity by DMPVT did not show the irreversible mechanism-based effect. The loss of EROD activity in P450 1B1 with DMPVT incubation was not blocked by treatment with the trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Taken together, the results suggested DMPVT to be a strong noncompetitive inhibitor of human P450 1 enzymes that should be considered as a good candidate for a cancer chemopreventive agent in humans. PMID:15022723

Lee, Sang-Kwang; Kim, Yongmo; Kim, Mie Young; Kim, Sanghee; Chun, Young-Jin

2004-02-01

124

Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver.  

PubMed

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s. PMID:20538623

Yang, Xia; Zhang, Bin; Molony, Cliona; Chudin, Eugene; Hao, Ke; Zhu, Jun; Gaedigk, Andrea; Suver, Christine; Zhong, Hua; Leeder, J Steven; Guengerich, F Peter; Strom, Stephen C; Schuetz, Erin; Rushmore, Thomas H; Ulrich, Roger G; Slatter, J Greg; Schadt, Eric E; Kasarskis, Andrew; Lum, Pek Yee

2010-08-01

125

The catalytic pathways of soluble methane monooxygenase (sMMO) and cytochrome P450CAM, iron-containing enzymes,  

E-print Network

236 The catalytic pathways of soluble methane monooxygenase (sMMO) and cytochrome P450CAM, iron. All rights reserved. Abbreviations cP450 cytochrome P450 DFT density functional theory MMOH methane, as in a biochemical context. A highly reactive oxidant is required, yet this species must perform only the requisite C

Gherman, Benjamin F.

126

HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity  

ERIC Educational Resources Information Center

Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

Furge, Laura Lowe; Fletke, Kyle J.

2007-01-01

127

An artificial electron donor supported catalytic cycle of Pseudomonas putida cytochrome P450{sub cam}  

SciTech Connect

Putidaredoxin (PdX), the physiological effector of cytochrome P450{sub cam} (P450{sub cam}), serves to gate electron transfer into oxy-P450{sub cam} during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450{sub cam} turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450{sub cam}. We demonstrate that the catalytic cycle of wild-type P450{sub cam} can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450{sub cam}, we observed an intermediate with spectral features characteristic of compound I. The rate constant for the formation of compound I in the presence of ferrocyanide supported reaction cycle was found to be comparable to the ones observed for H{sub 2}O{sub 2} supported compound I formation in wild-type P450{sub cam}, but was much lower than those observed for classical peroxidases. The results presented in this paper form the first kinetic analysis of this intermediate for an artificial electron-driven P450{sub cam} catalytic pathway in solution.

Prasad, Swati [Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005 (India)]. E-mail: swati@scripps.edu; Murugan, Rajamanickam [Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005 (India); Mitra, Samaresh [Indian Institute of Chemical Biology, Kolkata 700 032 (India)

2005-09-23

128

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450  

USGS Publications Warehouse

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.

Rattner, B. A.; Melancon, M. J.; Custer, T. W.; Hothem, R. L.; King, K. A.; LeCaptain, L. J.; Spann, J. W.; Woodin, B. R.; Stegeman, J. J.

1993-01-01

129

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450  

USGS Publications Warehouse

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.

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, B.R.; Stegeman, J.J.

1993-01-01

130

Role of cytochrome P-450 in reperfusion injury of the rabbit lung.  

PubMed Central

Reactive oxygen species are a major cause of damage occurring in ischemic tissue after reperfusion. During reperfusion transitional metals such as iron are required for reactive oxygen species to mediate their major toxic effects. Xanthine oxidase is an important source of reactive oxygen species during ischemia-reperfusion injury, but not in all organs or species. Because cytochrome P-450 enzymes are an important pulmonary source of superoxide anion (O2-.) generation under basal conditions and during hyperoxia, and provide iron catalysts necessary for hydroxyl radical (.OH) formation and propagation of lipid peroxidation, we postulated that cytochrome P-450 might have a potential role in mediating ischemia-reperfusion injury. In this report, we explored the role of cytochrome P-450 enzymes in a rabbit model of reperfusion lung injury. The P-450 inhibitors 8-methoxypsoralen, piperonyl butoxide, and cimetidine markedly decreased lung edema from transvascular fluid flux. Cimetidine prevented the reperfusion-related increase in lung microvascular permeability, as measured by movement of 125I-albumin from the vascular space into lung water and alveolar fluid. P-450 inhibitors also prevented the increase in lung tissue levels of thiobarbituric acid reactive products in the model. P-450 inhibitors did not block enhanced O2-. generation by ischemic reperfused lungs, measured by in vivo reduction of succinylated ferricytochrome c in lung perfusate, but did prevent the increase in non-protein-bound low molecular weight chelates of iron after reperfusion. Thus, cytochrome P-450 enzymes are not likely a major source of enhanced O2-. generation, but serve as an important source of iron in mediating oxidant injury to the rabbit lung during reperfusion. These results suggest an important role of cytochrome P-450 in reperfusion injury to the lung and suggest potential new therapies for the disorder. PMID:2173718

Bysani, G K; Kennedy, T P; Ky, N; Rao, N V; Blaze, C A; Hoidal, J R

1990-01-01

131

Inhibition of Cytochrome P450 Enzymes by the E- and Z-Isomers of Norendoxifen  

PubMed Central

Aromatase catalyzes the conversion of testosterone to estradiol and is the main source of endogenous estrogen in postmenopausal women. Aromatase inhibitors (AIs) are used to treat postmenopausal women with hormone receptor–positive breast cancer. Norendoxifen [4-(1-(4-(2-aminoethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenol], an active metabolite of the selective estrogen receptor modulator tamoxifen, has been shown to be a potent competitive AI, with an IC50 of 90 nM. To obtain data relevant to the clinical use of norendoxifen, the primary objective of this study was to investigate norendoxifen’s inhibitory capability on enzymes related to drug-drug interactions. We determined the inhibitory ability of norendoxifen against important drug-metabolizing cytochrome P450 enzymes, including CYP1A2, CYP2A6, CYP3A4, CYP3A5, and CYP2C19, to establish the potency of norendoxifen as a potential cause of drug-drug interactions. A second objective was to determine the effects of E- and Z-norendoxifen on the inhibition of these enzymes to further characterize the isomers’ selectivity. The inhibitory abilities of E-, mixed, and Z-norendoxifen against recombinant aromatase (CYP19), CYP1A2, CYP3A4, CYP3A5, and CYP2C19 were tested using microsomal incubations. Mixed norendoxifen inhibited these enzymes with Ki values of 70 ± 9, 76 ± 3, 375 ± 6, 829 ± 62, and 0.56 ± 0.02 nM, respectively. E-Norendoxifen had a 9.3-fold-higher inhibitory ability than Z-norendoxifen against CYP19, while E- and Z-norendoxifen had similar potencies against CYP1A2, CYP3A4, CYP3A5, and CYP2C19. These results suggest that norendoxifen is able to act as a potent AI, and that its E-isomer is 9.3-fold more potent than the Z-isomer. PMID:23824607

Flockhart, Peter J.; Lu, Deshun; Lv, Wei; Lu, Wenjie Jessie; Han, Xu; Cushman, Mark; Flockhart, David A.

2013-01-01

132

Regulation of intestinal cytochrome P450 expression by hepatic cytochrome P450: possible involvement of fibroblast growth factor 15 and impact on systemic drug exposure.  

PubMed

Tissue-specific deletion of the gene for NADPH-cytochrome P450 (P450) reductase (CPR), the essential electron donor to all microsomal P450 enzymes, in either liver or intestine, leads to upregulation of many P450 genes in the tissue with the Cpr deletion. Here, by studying the liver-specific Cpr-null (LCN) mouse, we examined whether an interorgan regulatory pathway exists, such that a loss of hepatic CPR would cause compensatory changes in intestinal P450 expression and capacity for first-pass metabolism of oral drugs. We show for the first time that intestinal expression of CYP2B, 2C, and 3A proteins was increased in LCN mice by 2- to 3-fold compared with wild-type (WT) mice, accompanied by significant increases in small intestinal microsomal lovastatin-hydroxylase activity and systemic clearance of oral lovastatin (at 5 mg/kg). Additional studies showed that the hepatic Cpr deletion, which caused large decreases in bile acid (BA) levels in the liver, intestine, plasma, and intestinal content, led to drastic decreases in the mRNA levels of intestinal fibroblast growth factor 15 (FGF15), a target gene of the BA receptor farnesoid X receptor. Furthermore, treatment of mice with FGF19 (the human counterpart of mouse FGF15) abolished the difference between WT and LCN mice in small intestinal (SI) CYP3A levels at 6 hours after the treatment. Our findings reveal a previously unrecognized direct role of intestinal FGF15/19 in the regulation of SI P450 expression and may have profound implications for the prediction of drug exposure in patients with compromised hepatic P450 function. PMID:24184963

Zhu, Yi; Ding, Xinxin; Fang, Cheng; Zhang, Qing-Yu

2014-01-01

133

A rapid screening for cytochrome P450 catalysis on new chemical entities: cytochrome P450 BM3 and 1,2,5-oxadiazole derivatives.  

PubMed

This work presents the validation of a rapid screening procedure for the catalysis of cytochrome P450 on new chemical entities. The assay is tested on the prototypical, catalytically self-sufficient and soluble cytochrome P450 BM3 from Bacillus megaterium that shares a high degree of homology with mammalian counterparts. The so-called alkali assay developed in our laboratory is validated here also by product formation and molecular modeling on a number of derivatives sharing the molecular scaffold of the 1,2,5-oxadiazole ring, a class of molecules very different from the long-chain fatty acids known to be oxidized by cytochrome P450 BM3. The alkali assay reveals the ability of this cytochrome to oxidize NADPH in the presence of nine out of thirteen 1,2,5-oxadiazole derivatives tested. The enzyme shows high affinity and coupling efficiencies when incubated with four 1,2,5-oxadiazole derivatives. The presence of oxidation products deriving from catalysis was also confirmed by high-performance liquid chromatography (HPLC). Molecular docking suggests that a key factor for the 1,2,5-oxadiazole derivatives to enter the active site and induce catalysis is the presence of the -SO(2) moiety bridging the 1,2,5-oxadiazole and phenyl rings. These data indicate that the alkali assay is able to quickly and cheaply detect the recognition of new substrates by cytochrome P450. The assay is not intended to substitute HPLC-mass spectrometry analysis, but it is a preliminary screening that allows elimination of obvious nonsubstrates from the start. PMID:22983164

Tsotsou, Georgia E; Di Nardo, Giovanna; Sadeghi, Sheila J; Fruttero, Roberta; Lazzarato, Loretta; Bertinaria, Massimo; Gilardi, Gianfranco

2013-02-01

134

Cytochrome P450IA mRNA expression in feral hudson river tomcod  

Microsoft Academic Search

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

G. L. Kreamer; K. Squibb; D. Gioeli; S. J. Garte; I. Wirgin

1991-01-01

135

Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future  

Microsoft Academic Search

The field of cytochrome P450 pharmacogenetics has progressed rapidly during the past 25 years. All the major human drug-metabolizing P450 enzymes have been identified and cloned, and the major gene variants that cause inter-individual variability in drug response and are related to adverse drug reactions have been identified. This information now provides the basis for the use of predictive pharmacogenetics

Magnus Ingelman-Sundberg

2004-01-01

136

Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail  

Microsoft Academic Search

Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at

Michael B. Cohen; May R. Berenbaum; Mary A. Schuler

1989-01-01

137

Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions  

Microsoft Academic Search

Much of the interest in the cytochrome P450 (P450) enzymes has been because of oxidation of chemicals to reactive products. The epoxides (oxiranes) have been a major topic of interest with olefins and aryl compounds. Epoxides vary considerably in their reactivity, with t1\\/2 varying from 1s to several hours. The stability and reactivity influences not only the overall damage to

F Peter Guengerich

2003-01-01

138

Involvement of Singlet Oxygen in Cytochrome P450Dependent Substrate Oxidations  

Microsoft Academic Search

Cytochrome P450 (P450)-dependent p-hydroxylation of aniline and o-deethylation of 7-ethoxycoumarin were examined in rat liver microsomes in the presence of radical scavengers. The addition of ?-carotene, a quencher of singlet oxygen species (1O2), suppressed the aniline hydroxylation, while the addition of sodium azide (NaN3) (1O2 quencher) enhanced the reaction. No other reactive oxygen scavengers or chelating agents such as superoxide

Mayuko Osada; Yuhko Ogura; Hiroyuki Yasui; Hiromu Sakurai

1999-01-01

139

Induction and inactivation of a cytochrome P450 confering herbicide resistance in wheat seedlings  

Microsoft Academic Search

Summary  Cytochrome P450-dependent enzymes from wheat catalyze the oxidation of endogenous compounds (lauric and oleic acids) and of\\u000a several herbicides (diclofop, chlortoluron, bentazon). Treatment of wheat seedlings with the safener, naphthalic anhydride\\u000a and with phenobarbital increases dramatically several P450-dependent enzyme activities including diclofop and lauric acid\\u000a hydroxylation. The parallel induction of lauric acid (?-1)-hydroxylase and diclofop hydroxylase activities suggests that both

N. Forthoffer; C. Helvig; N. Dillon; I. Benveniste; A. Zimmerlin; F. Tardif; J.-P. Salaün

2001-01-01

140

Bell pepper fruit fatty acid hydroperoxide lyase is a cytochrome P450 (CYP74B)  

Microsoft Academic Search

Fatty acid hydroperoxide lyases cleave a C?C bond adjacent to a hydroperoxide group in lipoxygenase derived lipid hydroperoxides to form short-chain aldehydes and oxo-acids. Previously, we showed that fatty acid hydroperoxide lyase from bell pepper fruits is a heme protein whose spectrophotometric properties greatly resemble a cytochrome P450. In order to ascertain the relationship of it to the P450 gene

Kenji Matsui; Mizuyoshi Shibutani; Toshiharu Hase; Tadahiko Kajiwara

1996-01-01

141

Natural variation in the expression of cytochrome P-450 and dimethylnitrosamine demethylase in Drosophila  

SciTech Connect

Electrophoresis of Drosophila microsomes resolves two major heme-containing protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b. 22 references, 2 figures, 1 table.

Waters, L.C.; Simms, S.I.; Nix, C.E.

1984-09-28

142

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver  

Microsoft Academic Search

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally\\u000a with BPA daily for 4 days, at doses of 10, 20, and 40 mg\\/kg. Among the P450-dependent monooxygenase activities, testosterone\\u000a 2?-hydroxylase (T2AH) and testosterone 6?-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40

Nobumitsu Hanioka; Hideto Jinno; Tetsuji Nishimura; Masanori Ando

1998-01-01

143

Human cytochrome P450 oxidation of 5-hydroxythalidomide and pomalidomide, an amino analogue of thalidomide.  

PubMed

The sedative and antiemetic drug thalidomide [?-(N-phthalimido)glutarimide] was withdrawn in the early 1960s because of its potent teratogenic effects but was approved for the treatment of lesions associated with leprosy in 1998 and multiple myeloma in 2006. The mechanism of teratogenicity of thalidomide still remains unclear, but it is well-established that metabolism of thalidomide is important for both teratogenicity and cancer treatment outcome. Thalidomide is oxidized by various cytochrome P450 (P450) enzymes, the major one being P450 2C19, to 5-hydroxy-, 5'-hydroxy-, and dihydroxythalidomide. We previously reported that P450 3A4 oxidizes thalidomide to the 5-hydroxy and dihydroxy metabolites, with the second oxidation step involving a reactive intermediate, possibly an arene oxide, that can be trapped by glutathione (GSH) to GSH adducts. We now show that the dihydroxythalidomide metabolite can be further oxidized to a quinone intermediate. Human P450s 2J2, 2C18, and 4A11 were also found to oxidize 5-hydroxythalidomide to dihydroxy products. Unlike P450s 2C19 and 3A4, neither P450 2J2, 2C18, nor 4A11 oxidized thalidomide itself. A recently approved amino analogue of thalidomide, pomalidomide (CC-4047, Actimid), was also oxidized by human liver microsomes and P450s 2C19, 3A4, and 2J2 to the corresponding phthalimide ring-hydroxylated product. PMID:24350712

Chowdhury, Goutam; Shibata, Norio; Yamazaki, Hiroshi; Guengerich, F Peter

2014-01-21

144

Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions  

PubMed Central

Pyrethroid insecticides are used to control 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 nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in 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 unique tools for disease control. PMID:24248381

Ismail, Hanafy M.; O'Neill, Paul M.; Hong, David W.; Finn, Robert D.; Henderson, Colin J.; Wright, Aaron T.; Cravatt, Benjamin F.; Hemingway, Janet; Paine, Mark J. I.

2013-01-01

145

The NADPH-cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis.  

PubMed

The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN, and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA(3), GA(4), and GA(7) production, but low levels of non-hydroxylated C(20)-GAs (GA(15) and GA(24)) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism. PMID:15037621

Malonek, Stefan; Rojas, Maria C; Hedden, Peter; Gaskin, Paul; Hopkins, Paul; Tudzynski, Bettina

2004-06-11

146

Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench 1  

PubMed Central

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration. ImagesFigure 3Figure 4 PMID:16668136

Halkier, Barbara Ann; M?ller, Birger Lindberg

1991-01-01

147

Isolation of cytochrome P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR strategy  

Microsoft Academic Search

Cytochrome P-450 monooxygenases are membrane-bound enzymes involved in a wide range of biosynthetic pathways in plants. An efficient PCR strategy for isolating cytochrome P-450 cDNA clones from plant cDNA libraries is described. A set of degenerate primers for PCR amplification was designed to recognize nucleotide sequences specifying the highly conserved haembinding region of cytochrome P-450 proteins. Using this primer set

Annemarie H. Meijer; Erik Souer; Robert Verpoorte; J. Harry C. Hoge

1993-01-01

148

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection  

Microsoft Academic Search

BACKGROUND: Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B

Davis W Cheng; Hong Lin; Yuri Takahashi; M Andrew Walker; Edwin L Civerolo; Drake C Stenger

2010-01-01

149

Comparative induction of cytochrome P450IVA1 and peroxisome proliferation by ciprofibrate in the rat and marmoset  

Microsoft Academic Search

Chronic ciprofibrate administration resulted in distinct differences in hepatic responses between the two species examined. In the rat, hepatomegaly was observed with the coordinate induction of carnitine acetyltransferase, peroxisomal ?-oxidation and cytochrome P450IVA1 activities. The latter induction of cytochrome P450IVA1-dependent fatty acid hydroxylase activity was specific to this cytochrome P450 sub family, as ciprofibrate pretreatment resulted in an inhibition of

Janet M. Makowska; Frank W. Bonner; G. Gordon Gibson

1991-01-01

150

The Cytochrome P450 Superfamily Complement (CYPome) in the Annelid Capitella teleta.  

PubMed

The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ?65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 ?-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism. PMID:25390889

Dejong, Chris A; Wilson, Joanna Y

2014-01-01

151

The Cytochrome P450 Superfamily Complement (CYPome) in the Annelid Capitella teleta  

PubMed Central

The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ?65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 ?-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism. PMID:25390889

Dejong, Chris A.; Wilson, Joanna Y.

2014-01-01

152

Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor  

PubMed Central

The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed ‘bld’ (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules. PMID:23357279

Tian, Zhenghua; Cheng, Qian; Yoshimoto, Francis K.; Lei, Li; Lamb, David C.; Guengerich, F. Peter

2013-01-01

153

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.  

PubMed Central

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals. PMID:7644480

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

1995-01-01

154

Effects of 2-acetylaminofluorene, dietary fats and antioxidants on nuclear envelope cytochrome P-450  

SciTech Connect

The authors reported a marked loss of cytochrome P-450 in hepatic nuclear envelope (NE) but not in microsomes of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks. This may reflect loss of NE capacity to detoxify AAF metabolites generated by microsomal P-450. They are now investigating if dietary effects such as progressive decrease in the incidence of AAF-induced tumors in rats fed high polyunsaturated fat diet (HPUF) vs. high saturated fat diet (HSF) vs. low fat diet (LF), and the anticarcinogenic activity of butylated hydroxytoluene (BHT; 0.3% w/w) correlate with preservation of NE P-450. Rats fed AAF HSF (25.6% w/w corn oil) showed marked loss of NE P-450 after 3 weeks; BHT protected against this loss. Rats fed AAF in HSF (25.6% w/w; 18 parts beef tallow + 2 parts corn oil), on the other hand, experienced a marked drop in NE P-450 after 9 weeks; BHT protected against this loss. Comparison of NE P-450 levels in control rats fed HPUF or HSF for 3 weeks with those of rats fed a semipurified diet with 10% fat or Purina chow (ca. 5% fat), support the prediction of an inverse correlation between the levels of dietary fat and the NE P-450 content. Studies on AAF and BHT effects using LF (2% w/w corn oil) are in progress.

Carubelli, R.; Graham, S.A.; Griffin, M.J.; McCay, P.B.

1986-05-01

155

Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides.  

PubMed

Escherichia coli BL21 as production strain for the production of cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. in high yields was developed. The expression was first optimized with a series of flask experiments testing several key parameters for their influence on the expression level and enzyme activity. The optimal process parameters found in the flask experiments were verified in a cultivation process in a 5-L bioreactor. Glycerol proved to be superior over glucose as carbon source. Low dissolved oxygen (DO) concentration (<10%) during expression was found to be critical for active P450s production, resulting in expression level of 400 nM for P450SMO. Intact cells were used to establish an efficient bioconversion system for the production of sulfoxidation product. With p-chlorothioanisole as a representative substrate, the desired product (S-sulfoxide) was afforded with 99% ee and highest production of 130 mg/L within 12 h. PMID:20424864

Zhang, Jian-Dong; Li, Ai-Tao; Xu, Jian-He

2010-11-01

156

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1  

PubMed Central

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

Pflugmacher, Stephan; Sandermann, Heinrich

1998-01-01

157

Cytochrome P450-mediated metabolic engineering: current progress and future challenges.  

PubMed

Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. PMID:24709279

Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Björn; Werck-Reichhart, Danièle

2014-06-01

158

CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY  

EPA Science Inventory

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

159

CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 GENE FAMILY  

EPA Science Inventory

The P450ALK gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. tructural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures a...

160

Effects of avermectin on microsomal cytochrome P450 enzymes in the liver and kidneys of pigeons.  

PubMed

Residues of avermectin (AVM) drugs have toxic effects on non-target organisms. Analyses of cytochrome P450 enzymes are among the most frequently employed indicators in pharmacology and toxicology studies. In this study, the responses of cytochrome P450 enzymes and pathological changes in the liver and kidney tissues of King pigeons (Columba livia) following subchronic exposure to avermectin for 30, 60 and 90d were investigated. Dose- and time-dependent decreases in the activities of P450 enzymes (i.e., aminopyrine-N-demethylase, erythromycin N-demethylase, aniline 4-hydroxylase and NADPH-cytochrome C reductase) and down-regulation of the P450 and b5 contents were observed. The microscopic structures were clearly altered, the severity of these alterations increased with the concentration of AVM and the exposure time. These results imply that AVM can inhibit the P450 enzyme systems in the liver and kidney tissues of pigeons. This research provides insight into the safe use of AVM and a comprehensive evaluation of the toxicological effects of AVM in birds. PMID:25194326

Zhu, Wen-Jun; Zhang, Zi-Wei; Wang, Xian-Song; Xu, Shi-Wen; Li, Ming; Li, Shu

2014-09-01

161

Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11.  

PubMed

Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ?-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ?-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ?-hydroxylation ?2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ?-oxidation. PMID:25203493

Kim, Donghak; Cha, Gun-Su; Nagy, Leslie D; Yun, Chul-Ho; Guengerich, F Peter

2014-10-01

162

Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.  

PubMed

Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function. PMID:25236845

Park, Ji Won; Reed, James R; Brignac-Huber, Lauren M; Backes, Wayne L

2014-12-01

163

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

PubMed

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 ?M for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 ?M 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. PMID:22819650

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

2012-12-01

164

Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101).  

PubMed

Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics. For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns). Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site. The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam. PMID:15236583

Meilleur, Flora; Contzen, Jörg; Myles, Dean A A; Jung, Christiane

2004-07-13

165

Orphans in the Human Cytochrome P450 Superfamily: Approaches to Discovering Functions and Relevance in Pharmacology  

PubMed Central

As a result of technical advances in recombinant DNA technology and nucleotide sequencing, entire genome sequences have become available in the past decade and offer potential in understanding diseases. However, a central problem in the biochemical sciences is that the functions of only a fraction of the genes/proteins are known, and this is also an issue in pharmacology. This review is focused on issues related to the functions of cytochrome P450 (P450) enzymes. P450 functions can be categorized in several groups: 1) Some P450s have critical roles in the metabolism of endogenous substrates (e.g., sterols and fat-soluble vitamins). 2) Some P450s are not generally critical to normal physiology but function in relatively nonselective protection from the many xenobiotic chemicals to which mammals (including humans) are exposed in their diets [as well as more anthropomorphic chemicals (e.g., drugs, pesticides)]. 3) Some P450s have not been extensively studied and are termed “orphans” here. With regard to elucidation of any physiological functions of the orphan P450s, the major subject of this review, it is clear that simple trial-and-error approaches with individual substrate candidates will not be very productive in addressing questions about function. A series of liquid chromatography/mass spectrometry/informatics approaches are discussed, along with some successes with both human and bacterial P450s. Current information on what are still considered “orphan” P450s is presented. The potential for application of some of these approaches to other enzyme systems is also discussed. PMID:21737533

Cheng, Qian

2011-01-01

166

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.  

PubMed Central

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function. PMID:7731980

Bestervelt, L L; Vaz, A D; Coon, M J

1995-01-01

167

A Host-Inducible Cytochrome P-450 from a Host-Specific Caterpillar: Molecular Cloning and Evolution  

Microsoft Academic Search

Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant

Michael B. Cohen; Mary A. Schuler; May R. Berenbaum

1992-01-01

168

Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4  

Microsoft Academic Search

Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the

Pham Thi Thanh Ha; Inge Sluyts; Sigrid Van Dyck; Jie Zhang; Ron A. H. J. Gilissen; Jos Hoogmartens; Ann Van Schepdael

2006-01-01

169

PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER  

EPA Science Inventory

Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

170

Cytochrome P450 enzyme functionalized-quantum dots as photocatalysts for drug metabolism.  

PubMed

On the basis of the photo-induced electron transfer (PET) from CdTe quantum dots (QDs) to cytochrome P450 2C9 (CYP2C9), a light-controlled drug metabolism system was successfully designed by using CYP2C9 functionalized-CdTe QDs as photocatalysts. PMID:24821498

Xu, Xuan; Qian, Jing; Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

2014-07-21

171

ANTHONY Y. H. LU COMMEMORATIVE ISSUE PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL REGULATION OF CYTOCHROME P450  

Microsoft Academic Search

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Fran- cisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay be-

EDWARD T. MORGAN; MARION B. SEWER; HEINRICH IBER; FRANK J. GONZALEZ; YING-HUE LEE; ROBERT H. TUKEY; STEVE OKINO; TIEN VU; YUE-HWA CHEN; JASPREET S. SIDHU; CURTIS J. OMIECINSKI

172

Metabolic Activity of Cytochrome P450 Isoforms in Hepatocytes Cryopreserved with Wheat Protein Extract  

E-print Network

Metabolic Activity of Cytochrome P450 Isoforms in Hepatocytes Cryopreserved with Wheat Protein adequate safety testing for drug toxicity before new drugs can be adminis- tered to patients. Hepatocytes- preservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques

Sarhan, Fathey

173

Regio-and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3  

E-print Network

Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3 Matthew W alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee

Arnold, Frances H.

174

TRANSFECTION OF CYTOCHROME P450 CDNAS INTO MAMMALIAN CELLS USED IN MUTATION AND TRANSFORMATION ASSAYS  

EPA Science Inventory

The present work demonstrates that cDNAs coding for cytochrome P450 enzymes can be tranfected into mammalian cells and expressed, n the present studies, two different cell systems were used for transfection: 0T1/2 cells which can be used to study initiation and promotion (Diamond...

175

METABOLISM OF MYCLOBUTANIL AND TRIADIMEFON BY HUMAN AND RAT CYTOCHROME P450 ENZYMES AND LIVER MICROSOMES.  

EPA Science Inventory

Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil wa...

176

INDUCTION OF CYTOCHROME P450 ISOFORMS IN RAT LIVER BY TWO CONAZOLES, TRIADIMEFON AND MYCLOBUTANIL  

EPA Science Inventory

1. 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 by gavage for 1...

177

Transcription of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta: Activating  

E-print Network

Transcription of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta: Activating initiates when cholesterol is converted in the mitochondria to the first steroid, pregnenolone. This reaction is cata- lyzed by a specialized enzyme complex that includes the cholesterol side-chain cleavage

Lebendiker, Mario

178

Pharmacogenetics affects dosing, efficacy, and toxicity of cytochrome P450–metabolized drugs  

Microsoft Academic Search

Drug-metabolizing enzyme activity is one of many factors affecting patient response to medications. The objective of this review is to highlight the potential for genetic variability in cytochrome P450 enzyme activity that can lead to interperson differences in response to drugs. Awareness and application of this knowledge will improve drug use in clinical practice and provide the physician with further

Janyce F Rogers; Anne N Nafziger; Joseph S Bertino

2002-01-01

179

Differential induction of cytochrome P450 transcripts in Papilio polyxenes by linear and angular furanocoumarins  

Microsoft Academic Search

Cytochrome P450 monooxygenases play critical roles in the detoxification of linear and angular furanocoumarins present in the host plants of Papilio polyxenes (black swallowtail) larvae. To determine the spectrum of linear and angular furanocoumarins that induce isozymes responsible for these detoxifications, the metabolic activities of individual P. polyxenes larvae were monitored in response to individual furanocoumarins. Two linear furanocoumarins, xanthotoxin

Chien-Fu Hung; Hataichanoke Prapaipong; May R. Berenbaum; Mary A. Schuler

1995-01-01

180

Mechanisms of Inhibitory and Regulatory Effects of Methylenedioxyphenyl Compounds on Cytochrome P450Dependent Drug Oxidation  

Microsoft Academic Search

Cytochrome P450 (CYP) enzymes catalyse the oxidative conversion of drugs and other lipophilic compounds to hydrophilic metabolites. Thus, CYPs play a dominant role in the elimination of drugs from the body. Inhibitory interactions occur when drugs compete for oxidation by specific CYPs whereas certain drugs increase the capacity for oxidative biotransformation by inducing the synthesis of new CYPs. Methylenedioxyphenyl (MDP)

Michael Murray

2000-01-01

181

Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation  

E-print Network

Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation Edgardo T. Farinas of contempor- ary catalysis is the controlled oxidation of hydrocar- bons.[1] Processes for controlled, stereo- and regioselective oxidation of hydrocarbon feed stocks to more valuable and useful products such as alco- hols

Arnold, Frances H.

182

Regioselective and stereoselective metabolism of ibuprofen by human cytochrome P450 2C  

Microsoft Academic Search

The cytochrome P450s responsible for the regio- and stereoselectivity in the 2- and 3-hydroxylation of the chiral non-steroidal antiinflammatory drug ibuprofen were characterized in human liver microsomes. The rates of formation of both the 2- and 3-hydroxy metabolites exhibited monophasic (N = 2; N is the number of microsomal preparations) and biphasic (N = 2) substrate concentration dependence for both

Mitchell A. Hamman; Gary A. Thompson; Stephen D. Hall

1997-01-01

183

EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2  

EPA Science Inventory

EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2. T M Ross1, B P Anderson1, G Zhao2, R A Pegram1 and J W Allis1. 1U.S. EPA, ORD, NHEERL, Research Triangle Park, NC; 2University of North Carolina, Chapel Hill, NC. Sponsor: H Barton Bromodichlorometh...

184

Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.  

ERIC Educational Resources Information Center

Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)

Groves, John T.

1985-01-01

185

QUANTITATIVE EVALUATION OF BROMODICHLOROMETHANE METABOLISM BY RECOMBINANT RAT AND HUMAN CYTOCHROME P450S  

EPA Science Inventory

ABSTRACT We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. BDCM is a drinking water disinfectant byproduct that has been implicated in liver, kidn...

186

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...

187

Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase  

PubMed Central

Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this. PMID:23911089

Huang, Wei-Cheng; Ellis, Jacqueline; Moody, Peter C.E.; Raven, Emma L.; Roberts, Gordon C.K.

2013-01-01

188

Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases  

SciTech Connect

The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical oxidants of various P450s directly using a chemical approach to generate the radical in situ. This resulted in the first report of direct methane to methanol conversion by a heme porphyrin catalyst using the soluble P450 from Mycobacterium sp, CYP153A6.

Arnold, Frances H.

2012-02-27

189

Clay-bridged electron transfer between cytochrome p450(cam) and electrode.  

PubMed

We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam). The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution. UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution. However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential. PMID:10679275

Lei, C; Wollenberger, U; Jung, C; Scheller, F W

2000-02-24

190

Cyclopropylamine inactivation of cytochromes P450: role of metabolic intermediate complexes.  

PubMed

The inactivation of cytochrome P450 enzymes by cyclopropylamines has been attributed to a mechanism involving initial one-electron oxidation at nitrogen followed by scission of the cyclopropane ring leading to covalent modification of the enzyme. Herein, we report that in liver microsomes N-cyclopropylbenzylamine (1) and related compounds inactivate P450 to a large extent via formation of metabolic intermediate complexes (MICs) in which a nitroso metabolite coordinates tightly to the heme iron, thereby preventing turnover. MIC formation from 1 does not occur in reconstituted P450 systems with CYP2B1/2, 2C11 or 2E1, or in microsomes exposed to gentle heating to inactivate the flavin-containing monooxygenase (FMO). In contrast, N-hydroxy-N-cyclopropylbenzylamine (3) and N-benzylhydroxylamine (4) generate MICs much faster than 1 in both reconstituted and microsomal systems. MIC formation from nitrone 5 (PhCH = N(O)cPr) is somewhat faster than from 1, but very much faster than the hydrolysis of 5 to a primary hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5' (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to alpha-nitrosotoluene as the precursor to oxime 2 and the major MIC from 1. PMID:15797239

Cerny, Matthew A; Hanzlik, Robert P

2005-04-15

191

Conformational Plasticity and Structure/Function Relationships in Cytochromes P450  

PubMed Central

Abstract The cytochrome P450s are a superfamily of enzymes that are found in all kingdoms of living organisms, and typically catalyze the oxidative addition of atomic oxygen to an unactivated C-C or C-H bond. Over 8000 nonredundant sequences of putative and confirmed P450 enzymes have been identified, but three-dimensional structures have been determined for only a small fraction of these. While all P450 enzymes for which structures have been determined share a common global fold, the flexibility and modularity of structure around the active site account for the ability of P450 enzymes to accommodate a vast number of structurally dissimilar substrates and support a wide range of selective oxidations. In this review, known P450 structures are compared, and some structural criteria for prediction of substrate selectivity and reaction type are suggested. The importance of dynamic processes such as redox-dependent and effector-induced conformational changes in determining catalytic competence and regio- and stereoselectivity is discussed, and noncrystallographic methods for characterizing P450 structures and dynamics, in particular, mass spectrometry and nuclear magnetic resonance spectroscopy are reviewed. Antioxid. Redox Signal. 13, 1273–1296. PMID:20446763

Kazanis, Sophia; Dang, Marina

2010-01-01

192

Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea.  

PubMed Central

Cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of plant secondary metabolites including phenylpropanoids and phytoalexins. To investigate the regulation of these P450s in the phenylpropanoid biosynthetic pathway of pea (Pisum sativum), partial cDNAs representing four distinct P450s expressed in pea seedlings were cloned using a reverse transcription-polymerase chain reaction strategy. One of the corresponding full-length cDNA clones, designated CYP73A9, encodes pea trans-cinnamic acid 4-hydroxylase, which catalyzes the second core reaction in the phenylpropanoid pathway. As expected from its central role in the production of lignin precursors and defense compounds, northern analysis of poly(A)+ mRNA demonstrates that transcripts encoding CYP73A9 are induced appreciably within 3 h after wounding. A second cDNA clone, designated CYP82, encodes a novel P450, whose transcripts are also induced in response to wounding at approximately the same time as CYP73A9 transcripts. Despite the multitude of environmental stimuli known to induce expression of phenylpropanoid pathway enzymes, genomic DNA Southern analysis indicates that each of these P450s is encoded by a low copy number (possibly a single copy) gene family. PMID:8819874

Frank, M R; Deyneka, J M; Schuler, M A

1996-01-01

193

Genome-wide identification and expression analyses of cytochrome P450 genes in mulberry (Morus notabilis).  

PubMed

Cytochrome P450s play critical roles in the biosynthesis of physiologically important compounds in plants. These compounds often act as defense toxins to prevent herbivory. In the present study, a total of 174 P450 genes of mulberry (Morus notabilis C.K.Schn) were identified based on bioinformatics analyses. These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner. These genes were compared to the P450 genes in Arabidopsis thaliana. Families CYP80, CYP92, CYP728, CYP733, CYP736, and CYP749 were found to exist in mulberry, and they may play important roles in the biosynthesis of mulberry secondary metabolites. Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids, other secondary metabolites, xenobiotics, amino acids, cofactors, vitamins, terpenoids, and polyketides. These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes. PMID:24304637

Ma, Bi; Luo, Yiwei; Jia, Ling; Qi, Xiwu; Zeng, Qiwei; Xiang, Zhonghuai; He, Ningjia

2014-09-01

194

Structural and Thermodynamic Basis of (+)-?-Pinene Binding to Human Cytochrome P450 2B6  

PubMed Central

Despite recent advances in atomic level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-?-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-?-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-?-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-?-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility. PMID:23786449

Wilderman, P. Ross; Shah, Manish B.; Jang, Hyun-Hee; Stout, C. David; Halpert, James R.

2013-01-01

195

Crystal Structure of Human Cytochrome P450 2D6 with Prinomastat Bound*  

PubMed Central

Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 Å resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for ? sheet 1, helices A, F, F?, G?, G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F? that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6. PMID:22308038

Wang, An; Savas, Uzen; Hsu, Mei-Hui; Stout, C. David; Johnson, Eric F.

2012-01-01

196

Possibility of application of cytochrome P450 to bioremediation of dioxins.  

PubMed

Dioxins, including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans, and coplanar polychlorinated biphenyls, are known to be metabolized by enzymes such as cytochrome (CYP) P450, angular dioxygenase, lignin peroxidase, and dehalogenase. It is noted that all of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase each have a distinct electron transport chain. Among these enzyme systems, we have focused on cytochrome P450-dependent metabolism of dioxins from the viewpoint of practical use for bioremediation. Mammalian and fungal cytochromes P450 showed remarkable activity toward low-chlorinated PCDDs. In particular, mammalian cytochromes P450 belonging to the CYP1 family showed high activity. Rat CYP1A1 showed high activity toward 2,3,7-trichloro-dibenzo-p-dioxin but no detectable activity for 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD). On the basis of these results, we assumed that enlarging the space of the substrate-binding pocket of rat CYP1A1 might generate TCDD-metabolizing enzyme. Large-sized amino acids located at putative substrate-recognition sites and F-G loop were substituted for alanine by site-directed mutagenesis. Finally, we successfully generated 2,3,7,8-TCDD-metabolizing enzyme by site-directed mutagenesis of rat CYP1A1. We hope that recombinant microorganisms harboring genetically engineered cytochrome P450 will be used for bioremediation of soil contaminated with PCDDs, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls in the future. PMID:23586993

Sakaki, Toshiyuki; Yamamoto, Keiko; Ikushiro, Shinichi

2013-01-01

197

Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse  

SciTech Connect

Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by {sup 32}P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

Stiborova, Marie [Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2 (Czech Republic)], E-mail: stiborov@natur.cuni.cz; Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Henderson, Colin J.; Wolf, C. Roland [Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Dundee DD1 9SY (United Kingdom); Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri [Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2 (Czech Republic); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Frei, Eva [Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

2008-02-01

198

Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions  

SciTech Connect

The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11{beta}-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O{sup 18} studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17{alpha}-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17{alpha}-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11{beta}-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction.

Estabrook, Ronald W. [Virginia Lazenby O'Hara Professor of Biochemistry, Ida and Cecil Green Chair in the Biomedical Sciences, Department of Biochemistry, Room Y7.206B, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9038 (United States)]. E-mail: Ronald.estabrook@utsouthwestern.edu

2005-12-09

199

Sensitization of Human Breast Cancer Cells to Cyclophosphamide and Ifosfamide by Transfer of a Liver Cytochrome P450 Gene1  

Microsoft Academic Search

The cancer chemotherapeutic agent Cyclophosphamide (CPA) and its isomer ifosfamide (IFA) are alkylating agent prodrugs that require me tabolism by liver cytochrome P450 (P450) enzymes for antitumor activity. The therapeutic effectiveness of these oxazaphosphorines is limited by the hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. Transfer of a liver cytochrome P450 gene,

Ling Chen; David J. Waxman; Dongshu Chen; Donald W. Kufe

1996-01-01

200

The human cytochrome P450 2A family: Comparisons and identification of amino acids essential for substrate recognition  

E-print Network

The goal of this research was to identify the differential structure-activity relationships between cytochromes P450 (CYP) 2A13 and 2A6 and their substrates. Cytochromes P450 2A13 and 2A6 are very closely related, having ...

DeVore, Natasha M.

2008-01-01

201

THE DIFFERENTIAL HEPATOTOXICITY AND CYTOCHROME P450 RESPONSE OF F344 RATS TO THE THREE ISOMERS OF DICHLOROBENZENE  

EPA Science Inventory

The acute hepatotoxicity and response of hepatic cytochrome P450 to treatment with the three isomers of dichlorobenzene (DCB) have been investigated. The objectives were to estimate toxic thresholds and to further e1ucidate the role of cytochrome P450 in the metabolism and toxici...

202

DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE  

EPA Science Inventory

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

203

DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONANZOLE  

EPA Science Inventory

Strains of Saccharomyces cerevisiae deleted in the NADPH-Cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14a-demethylase. esistance is restored through complementation by the plasmid-born...

204

Characterization of the cytochrome P450 involved in side-chain oxidation of cyclophosphamide in humans  

Microsoft Academic Search

Objective:\\u000a \\u000a \\u000a Cyclophosphamide (CP) is an antineoplastic prodrug which requires bioactivation (4-hydroxylation) by the cytochrome P450 (CYP)\\u000a enzymes in human liver. In parallel, P450-mediated side-chain oxidation (N-dealkylation) leads to the formation of the non-alkylating\\u000a dechloroethylcyclophosphamide (DCl-CP) and chloroacetaldehyde, the latter being a potential neurotoxic agent. The enzyme responsible\\u000a for side-chain oxidation has not been identified yet. We therefore used an in

F. Bohnenstengel; U. Hofmann; M. Eichelbaum; H. K. Kroemer

1996-01-01

205

Molecular cloning and expression analysis of a cytochrome P450 gene in tomato  

Microsoft Academic Search

A full-length cytochrome P450 cDNA, CYP71A2, was cloned from tomato (Lycopersicon esculentum Mill.) by RT-PCR and RACE. CYP71A2 (GenBank accession no. GQ370622) encoded a single polypeptide of 495 amino acid residues and shared 46–68% of identity with CYP71A1 which associated with\\u000a avocado fruit ripening. The polypeptide, which held the conserved domains in all P450s, was classified as CYP71. CYP71A2-GFP\\u000a fusion

Zhengguo Li; Yanwei Hao; Yingwu Yang; Wei Deng

2010-01-01

206

Circular dichroism of partially purified cytochrome P-450 from rabbit liver microsomes.  

PubMed

The heme related circular dichroic bands of solubilized cytochrome P-450 from rabbit liver and some of its complexes with type I- and type II-substrayes and heme iron ligands were measured in the Soret region. All P-450 dersorption. The positions of the dichroic bands together with their ellipticities vary with ligand substitution and the oxidative state of the iron. The results are compared with CD-date of other hemoproteins and discussed with respect to stereochemical properties of the heme pocket. PMID:1015157

Jänig, G R; Winkler, W; Ruckpaul, K; Rein, H

1976-01-01

207

Mechanistic aspects of CYP74 allene oxide synthases and related cytochrome P450 enzymes  

PubMed Central

The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a “different look” at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As new enzyme reactions and new P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture. PMID:19747698

Brash, Alan R.

2009-01-01

208

Pharmacogenetic biomarkers as tools for improved drug therapy; emphasis on the cytochrome P450 system.  

PubMed

Important interindividual differences in drug pharmacokinetics cause absence of drug response or adverse drug reactions in significant fractions of the populations. The identification of the major enzymes participating, and the elucidation of the genetic basis for this variation in particular among cytochromes P450, provide tools for a personalized medicine treatment, which can make drug therapy much more effective at a lower cost. Much of the pioneering work linking drug metabolizing phenotype to genetic polymorphism among the P450 enzymes has been carried out at Karolinska Institutet. In this review we give a background and description of this work as well as the important implications for future medicine. PMID:20494117

Ingelman-Sundberg, Magnus; Sim, Sarah C

2010-05-21

209

Ab Initio Electronic Structure Calculations of Cytochrome P450 -- Ligand Interactions  

NASA Astrophysics Data System (ADS)

The Cytochrome P450 superfamily of enzymes are of great interest in pharmacology as they participate in an enormous range of physiological processes including drug deactivation and xenobiotic detoxification. We apply ab initio electronic structure calculations to model the interactions of the haem molecule at the P450 active site with substrate and inhibitor ligands. These calculations, based on density function theory, were performed with the CETEP code which uses a plane wave basis set and pseudopotentials to perform efficient LDA, GGA and spin dependent calculations. A change in the spin state of the haem iron atom is observed on binding of a substrate molecule, consistent with the accepted reaction mechanism.

Segall, M. D.; Payne, M. C.; Ellis, S. W.; Tucker, G. T.

1997-03-01

210

Protection against chemical-induced lung injury by inhibition of pulmonary cytochrome P-450  

SciTech Connect

Protection afforded by trialkyl phosphorothionates against the lung injury caused by trialkyl phosphorothiolates probably results from the inhibition by the P{double bond}S moiety of the thionates, of one or more pulmonary cytochrome P-450 isozymes. The aromatic hydrocarbons p-xylene and pseudocumene also protect against this injury and inhibit some P-450 isozymes, but by a different mechanism. OOS-Trimethylphosphorothionate and p-xylene were compared as protective agents against the effect of OOS-trimethylphosphorothiolate and two other lung toxins ipomeanol and 1-nitronaphthalene that are known to be activated by cytochrome P-450. The effects of these protective compounds, in vivo, on pulmonary cytochrome P-450 activity were also determined. Both compounds inhibited pentoxyresorufin O-deethylase activity, but not ethoxyresorufin O-deethylase. The phosphorothionate was most effective against lung injury caused by the phosphorothiolates and 1-nitronaphthalene, whereas p-xylene was much more effective against ipomeanol. {beta}-Naphthoflavone, which induces pulmonary ethoxyresorufin O-deethylase activity, did not protect against phosphorothiolate or 1-nitronaphthalene injury, and it was only marginally effective in decreasing the toxicity or ipomeanol.

Verschoyle, R.D.; Dinsdale, D. (Medical Research Council Laboratories, Carshalton Surrey (England))

1990-04-01

211

Potential effects of environmental contaminants on P450 aromatase activity and DNA damage in swallows from the Rio Grande and Somerville, Texas  

Microsoft Academic Search

Cliff swallows (Petrochelidon pyrrhonota) and cave swallows (P. fulva) were sampled during the breeding season at several locations in the Rio Grande, Texas, to evaluate the potential effects\\u000a of environmental contaminants on P450 aromatase activity in brain and gonads and DNA damage in blood cells. The tritiated\\u000a water-release aromatase assay was used to measure aromatase activity and flow cytometry was

M. A. Sitzlar; M. A. Mora; J. G. W. Fleming; F. W. Bazer; J. W. Bickham; C. W. Matson

2009-01-01

212

Studies on covalent binding of (-)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to cytochromes P-450 LM2 and LM4 and NADPH-cytochrome P-450 reductase.  

PubMed

1. Metabolism of 14C-labelled benzo[a]pyrene (-)trans-7,8-dihydrodiol to protein- and DNA-binding products in a reconstituted enzyme system proceeds 5 to 10 times faster with rabbit cytochrome P-450 LM4 than with LM2. 2. Either cytochrome converts the substrate to ethyl acetate- and water-soluble metabolites, identified by h.p.l.c. Water-soluble metabolites comprise 78% of the total products with cytochrome P-450 LM2, but only 50% of those formed by LM4. The relative proportion of the two types of metabolites is differentially affected by certain modifiers such as 7,8-benzoflavone. 3. Half of the radioactivity in the aqueous phase of reaction mixtures containing cytochrome P-450 LM4 represents (-)trans-7,8-diol metabolites in complex primarily with NADPH and phosphate. The remaining water-soluble products are bound covalently to proteins in the reconstituted system. 4. Polyacrylamide gel electrophoresis, autoradiography, and measurement of the radioactivity in individual bands indicate that a larger fraction of metabolites is bound to cytochrome P-450 LM4 than to NADPH-cytochrome P-450 reductase, and only marginal binding to cytochrome P-450 LM2 is seen. Metabolite binding to added DNA is likewise substantially greater in magnitude when cytochrome P-450 LM4, as opposed to LM2, catalyses (-)trans-7,8-diol oxygenation. Thus, the degree of metabolite binding to monoxygenase proteins and to DNA correlates well with the catalytic activity of cytochrome P-450 LM4 and LM2 towards (-)trans-7,8-diol. 5. DNA causes a dramatic enhancement in the activity of cytochrome P-450 LM4 with (-)trans-7,8-diol, indicating that the cytochrome and/or the reductase may be functionally impaired by metabolites of this substrate. Such an effect may alter the balance between detoxication and activation of the carcinogenic benzo[a]pyrene. PMID:2515665

Deutsch, J; Vatsis, K P; Leutz, J C; Coon, M J; Gelboin, H V

1989-12-01

213

Human cytochrome P-450 PB-1: A multigene family involved in mephenytoin and steroid oxidations that maps to chromosome 10  

PubMed Central

The cytochrome P-450 monooxygenase system possesses catalytic activity toward many exogenous compounds (e.g., drugs, insecticides, and polycyclic aromatic hydrocarbons) and endogenous compounds (e.g., steroids, fatty acids, and prostaglandins). Multiple forms of cytochrome P-450 with different substrate specificities have been isolated. In the present paper we report the isolation and sequence of a cDNA clone for the human hepatic cytochrome P-450 responsible for mephenytoin (an anticonvulsant) oxidation. The mephenytoin cytochrome P-450 is analogous to the rat cytochrome P-450 form termed PB-1 (family P450C2C). We also report that human PB-1 is encoded by one of a small family of related genes all of which map to human chromosome 10q24.1-10q24.3. The endogenous role of this enzyme appears to be in steroid oxidations. This cytochrome P-450 family does not correspond to any of the hepatic cytochrome P-450 gene families previously mapped in humans. ImagesFigure 1Figure 4Figure 5Figure 6 PMID:2827463

Meehan, R. R.; Gosden, J. R.; Rout, D.; Hastie, N. D.; Friedberg, T.; Adesnik, M.; Buckland, R.; van Heyningen, V.; Fletcher, J.; Spurr, N. K.; Sweeney, J.; Wolf, C. R.

1988-01-01

214

Purification and immunochemical detections of ?-naphthoflavone- and phenobarbital-induced avian cytochrome P450 enzymes  

USGS Publications Warehouse

Livers from mallards (Anas platyrhynchos) were treated with either -naphthoflavone (50 mg/kg) or phenobarbital (70 mg/kg). Purification of induced hepatic cytochrome P450 was accomplished using both DEAE and hydroxyapatite columns, as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. Polyclonal antibodies to these proteins were then produced in young male New Zealand White rabbits. ?-naphthoflavone (?NF)- and phenobarbital(PB)-treated red-winged blackbird, screech owl, European starling and lesser scaup liver microsomes were analyzed in western blots for species cross-reactivity. Although all four of these avian species exhibited cross-reactivity with antibodies to ?NF-induced mallard P450, all but the lesser scaup revealed a protein of higher molecular weight than that of the ?NF-induced mallard. In addition, only the lesser scaup exhibited cross-reactivity with the anti-PB-induced mallard P450 antibodies.

Brown, R.L.; Levi, P.E.; Hodgson, E.; Melancon, M.J.

1996-01-01

215

Pulmonary oxygen toxicity in rats treated with cytochrome P-450 inducers  

SciTech Connect

Pulmonary oxygen toxicity is assumed to result from damage caused by superoxide (O/sub 2//sup -/) hydrogen peroxide (H/sub 2/O/sub 2/) and/or hydroxyl radical (OH) produced by the partial reduction of molecular oxygen (O/sub 2/). The microsomal cytochrome P-450 (P-450) monooxygenase system is known to produce O/sub 2//sup -/ and H/sub 2/O/sub 2/. They have studied the influence of monooxygenase induction using phenobarbital (PB) and ..beta..-naphthoflavone (..beta..-NF) on O/sub 2/ toxicity in the rat. PB- or ..beta..-NF induce hepatic P-450 but only ..beta..-NF induces pulmonary P-450. Pulmonary microsomes produced O/sub 2//sup -/ and H/sub 2/O/sub 2/ at rates (expressed per mg microsomal protein) which did not vary as a function of pretreatment. Rats were exposed to 100% O/sub 2/ for up to 3 days. After 3 days of O/sub 2/, lung weights were about 50% above controls regardless of pretreatment. The microsomal monooxygenase enzymes (P-450, b/sub 5/ and NADPH P-450 reductase) were quantified in liver and lung. Lung microsomal P-450 was reduced after 3 days of O/sub 2/ exposure regardless of pretreatment. The protective enzymes (catalase, superoxide dismutase (SOD) and glutathione (GSH) peroxidase) and non-protein sulfhydryl groups (NPSH) were also quantified in lung and liver samples. Lung NPSH and GSH peroxidase were increased after 3 days of O/sub 2/ exposure regardless of pretreatment while SOD was increased in controls and PB- but not ..beta..-NF-treated rats. Three of 14 ..beta..-NF-treated rats died during O/sub 2/ exposure while no animals in the control or PB-treated groups died.

Ebel, R.E.; Barlow, R.L.; Gregory, E.M.

1987-05-01

216

Significance of neuronal cytochrome P450 activity in opioid-mediated stress-induced analgesia.  

PubMed

Stressful environmental changes can suppress nociceptive transmission, a phenomenon known as "stress-induced analgesia". Depending on the stressor and the subject, opioid or non-opioid mechanisms are activated. Brain ? opioid receptors mediate analgesia evoked either by exogenous agents (e.g. morphine), or by the release of endogenous opioids following stressful procedures. Recent work with morphine and neuronal cytochrome P450 (P450)-deficient mice proposed a signal transduction role for P450 enzymes in µ analgesia. Since µ opioid receptors also mediate some forms of stress-induced analgesia, the present studies assessed the significance of brain P450 activity in opioid-mediated stress-induced analgesia. Two widely-used models of opioid stress-induced analgesia (restraint and warm water swim) were studied in both sexes of wild-type control and P450-deficient (Null) mice. In control mice, both stressors evoked moderate analgesic responses which were blocked by pretreatment with the opioid antagonist naltrexone, confirming the opioid nature of these responses. Consistent with literature, sex differences (control female>control male) were seen in swim-induced, but not restraint-induced, analgesia. Null mice showed differential responses to the two stress paradigms. As compared with control subjects, Null mice showed highly attenuated restraint-induced analgesia, showing a critical role for neuronal P450s in this response. However, warm water swim-induced analgesia was unchanged in Null vs. control mice. Additional control experiments confirmed the absence of morphine analgesia in Null mice. These results are the first to show that some forms of opioid-mediated stress-induced analgesia require brain neuronal P450 activity. PMID:25020125

Hough, Lindsay B; Nalwalk, Julia W; Yang, Weizhu; Ding, Xinxin

2014-08-26

217

In vitro activity of uva-ursi against cytochrome P450 isoenzymes and P-glycoprotein.  

PubMed

Some natural health products (NHPs) affect drug metabolism enzymes and transport proteins, potentially affecting the safety and efficacy of the drug or other NHPs. This study was undertaken to characterize the effect of uva-ursi (Arctostaphylos uva-ursi) on cytochrome P450 isozyme (3A4, 3A5, 3A7, 2C19, and 19)-mediated metabolism and P-glycoprotein (P-gp) transport. Three bulk and 2 capsulated uva-ursi samples were obtained from commercial outlets. The capsules were batched, and herbal samples were ground to a common consistency. Aqueous and methanol extracts were freshly prepared. Cytochrome P450 isozyme-mediated metabolism was determined by using in vitro bioassays. P-gp transport function was determined by using a rhodamine 123 (Rh123) uptake test in human (THP-1) monocytes and human Caco-2 cells. All products were analyzed by HPLC for arbutin, gallic acid, myricitrin, and isoquercetin. A large variation was observed in the biomarkers found between the bulk and capsulated samples. Our data indicate that both the aqueous and methanol extracts of all 5 uva-ursi products showed high cytochrome P450 isozyme inhibition, with the exception of the methanol extracts against cytochromes P3A4 and P19, which had low to moderate activity. The aqueous extracts of uva-ursi showed an inhibitory effect on Rh123 efflux by P-gp at 1 h and an inductive effect at 18 h for both cell lines. Our results show that the uva-ursi herbal products tested here have pharmacological properties, including the potential capacity to affect drug safety and efficacy. Further studies are warranted against a wider range of cytochrome P450 isozymes and to determine whether these effects are clinically significant. PMID:18066112

Chauhan, B; Yu, C; Krantis, A; Scott, I; Arnason, J T; Marles, R J; Foster, B C

2007-11-01

218

Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450  

PubMed Central

Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. We show that CsA (1?–?10??M) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. Using cytochrome c as substrate, we show that CsA (10??M) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. CsA (10??M) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. CsA (10??M) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes. PMID:11861326

Krauskopf, Alexandra; Buetler, Timo M; Nguyen, Nathalie S D; Mace, Katherine; Ruegg, Urs T

2002-01-01

219

Black swallowtail (Papilio polyxenes) alleles encode cytochrome P450s that selectively metabolize linear furanocoumarins.  

PubMed

Cytochrome P450 monooxygenases are involved in metabolism of hostplant allelochemicals by larval Lepidoptera. Biochemical purification of the P450 polypeptide induced in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin, has allowed us to clone cDNAs encoding two allelic variants of the CYP6B1 locus. Expression of these alleles in lepidopteran cell lines using baculovirus expression vectors has demonstrated that both P450 isoforms metabolize substantial amounts of linear furanocoumarins, such as xanthotoxin and bergapten, but not angular furanocoumarins, such as angelicin and sphondin. These linear furanocoumarins are ubiquitous constituents of the hostplants of P. polyxenes. The efficiency of linear furanocoumarin metabolism is strongly affected by the nature of the substituents on the benzene ring; methoxy-derivatives are metabolized more efficiently than are derivatives with smaller (hydroxy-) or larger (8-O isopentenyl) groups. Metabolism of either bergapten or xanthotoxin is inhibited in the presence of the other. In addition, metabolism of linear furanocoumarins is inhibited by the presence of nonmetabolizable angular furanocoumarins, indicating that the active site of CYP6B1 binds angular furanocoumarins. The reactivities described here indicate that P. polyxenes larvae express at least two selective furanocoumarin-metabolic P450s: CYP6B1, which metabolizes a discrete set of linear furanocoumarins, and another P450, as yet unidentified, which metabolizes angular furanocoumarins more efficiently than does CYP6B1. PMID:8179316

Ma, R; Cohen, M B; Berenbaum, M R; Schuler, M A

1994-05-01

220

Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).  

PubMed Central

One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape. PMID:12223771

Bennett, R. N.; Kiddle, G.; Wallsgrove, R. M.

1997-01-01

221

Novel approaches to the use of cytochrome P450 activities in wildlife toxicity studies  

SciTech Connect

Many wildlife toxicity studies, e.g. with avian species, use cytochrome P450 activities as markers for biological activities of environmental contaminants. It has been established that induction of CYP1A1 correlates with Ah-receptor mediated toxicity of dioxin-like compounds in many species. In addition, CYP1A1 plays a significant role in bioactivation of polycyclic aromatics. So far very few studies focused on the natural function of P450 isoenzymes in wildlife species. Besides classical hepatic CYP1A(1) associated activities, like EROD and AHH, several new techniques are available to study the activities of various CYP isoenzymes. Caffeine N-demethylation, testosterone and 17ss-estradiol hydroxylation patterns can provide new insights in the physiological function of P450 isoenzymes and the induction of the basal activities by chemicals. So far little interest was given to processes which occur after the DNA-receptor binding, e.g. changes in steroid hormone metabolism and pathways in environmental toxicology. This in spite of the fact that very subtle changes in steroid hormone levels may have significant physiological implications. This presentation will focus on some P450 activities, besides CYP1A(1), which might be important for development and reproduction. Some experimental approaches, limitations and techniques will be discussed which could lead to elucidation of the possible endocrine function of P450s.

VandenBerg, M. [Utrecht Univ. (Netherlands). Research Inst. of Toxicology; Bosveld, A.T.C.

1995-12-31

222

Pungent ginger components modulates human cytochrome P450 enzymes in vitro  

PubMed Central

Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 ?mol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

2013-01-01

223

Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum  

PubMed Central

The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals. PMID:20877397

Busi, R; Vila-Aiub, M M; Powles, S B

2011-01-01

224

Effects of aprepitant on cytochrome P450 3A4 activity using midazolam as a probe  

Microsoft Academic Search

Background: Aprepitant is a neurokinin1 receptor antagonist that enhances prevention of chemotherapy-induced nausea and vomiting when added to conventional therapy with a corticosteroid and a 5-hydroxytryptamine3 (5-HT3) antagonist. Because aprepitant may be used with a variety of chemotherapeutic agents and ancillary support drugs, which may be substrates of cytochrome P450 (CYP) 3A4, assessment of the potential of this drug to

Anup K. Majumdar; Jacqueline B. McCrea; Deborah L. Panebianco; Michael Hesney; James Dru; Marvin Constanzer; Michael R. Goldberg; Gail Murphy; Keith M. Gottesdiener; Christopher R. Lines; Kevin J. Petty; Robert A. Blum

2003-01-01

225

Effects of non-ionic surfactants on cytochrome P450-mediated metabolism in vitro  

Microsoft Academic Search

The purpose of the study was to investigate the impact of commonly used non-ionic surfactants on cytochrome P450 (CYP) 3A4-mediated metabolism of testosterone and the CYP2C9-mediated metabolism of diclofenac. Polysorbate 80 (PS 80), D-?-tocopheryl polyethylene glycol (1000) succinate (TPGS), sucrose laurate, Cremophor EL (CR EL), and Cremophor RH 40 (Cr RH 40) were incubated with human liver microsomes at different

Anne Christiansen; Thomas Backensfeld; Karsten Denner; Werner Weitschies

2011-01-01

226

Effect of Cytochrome P450 3A4 Inhibition on the Pharmacokinetics of Docetaxel  

Microsoft Academic Search

Objective: In vitro studies indicate that the anticancer drug docetaxel is primarily eliminated by cytochrome P450 (CYP) 3A4–mediated metabolism. Coadministration of drugs that modulate the activity of CYP3A4 is, therefore, likely to have undesirable clinical consequences. We investigated the effects of the potent CYP3A4 inhibitor ketoconazole on the pharmacokinetics of docetaxel in patients with cancer.Methods: Seven patients were treated in

Frederike K. Engels; Albert J. ten Tije; Sharyn D. Baker; Carlton K. K. Lee; Walter J. Loos; Arnold G. Vulto; Jaap Verweij; Alex Sparreboom

2004-01-01

227

From the Cover: Structural basis for ligand promiscuity in cytochrome P450 3A4  

Microsoft Academic Search

Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of 50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4-ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal

Marika Ekroos; Tove Sjögren

2006-01-01

228

Interaction of pefloxacin and enoxacin with the human cytochrome P450 enzyme CYP1A2  

Microsoft Academic Search

Background and Objectives: Pefloxacin is reported to cause clinically relevant inhibition of theophylline metabolism in vivo, but in vitro pefloxacin was only a weak inhibitor of the cytochrome P450 CYP1A2, mediating main theophylline biotransformation. We therefore further characterized the interaction between pefloxacin and CYP1A2.Methods: A randomized 3-period change-over study was conducted in 12 healthy young volunteers on the steady-state interactions

Martina Kinzig-Schippers; Uwe Fuhr; Michael Zaigler; Jörg Dammeyer; Guido Rüsing; Andreas Labedzki; Jürgen Bulitta; Fritz Sörgel

1999-01-01

229

Expression and Induction of Cytochrome P450 Isoenzymes in Human Skin Equivalents  

Microsoft Academic Search

Organotypic skin models are frequently used for a wide range of applications and latterly also for dermatotoxicological studies. To evaluate their practicability for the investigation of xenobiotic metabolism in human skin we compared three types of organotypic skin models, acquired by purchase from different manufacturers, to a self-constructed in-house model with regard to cytochrome P450 (CYP) isoenzyme expression on mRNA

M. M. Neis; A. Wendel; T. Wiederholt; Y. Marquardt; S. Joussen; J. M. Baron; H. F. Merk

2010-01-01

230

Distinct roles of cytochrome P450 reductase in mitomycin c redox cycling and cytotoxicity  

PubMed Central

Mitomycin c (MMC), a quinone-containing anticancer drug, is known to redox cycle and generate reactive oxygen species. A key enzyme mediating MMC redox cycling is cytochrome P450 reductase, a microsomal NADPH-dependent flavoenzyme. In the present studies, CHO cells overexpressing this enzyme (CHO-OR cells) and corresponding control cells (CHO-WT cells) were used to investigate the role of cytochrome P450 reductase in the actions of MMC. In lysates from both cell types, MMC was found to redox cycle and generate H2O2; this activity was greater in CHO-OR cells (Vmax = 1.2 ± 0.1 nmol H2O2/min/mg protein in CHO-WT cells vs. 32.4 ± 3.9 nmol H2O2/min/mg protein in CHO-OR cells). MMC was also more effective in generating superoxide anion and hydroxyl radicals in CHO-OR cells, relative to CHO-WT cells. Despite these differences in MMC redox cycling, MMC-induced cytotoxicity, as measured by growth inhibition, was similar in the two cell types (IC50 = 72 ± 20 nM for CHO-WT and 75 ± 23 nM for CHO-OR cells), as was its ability to induce G2/M and S phase arrest. Additionally, in 9 different tumor cell lines, although a strong correlation was observed between MMC-induced H2O2 generation and cytochrome P450 reductase activity, there was no relationship between redox cycling and cytotoxicity. Hypoxia, which stabilizes MMC radicals generated by redox cycling, also had no effect on the sensitivity of tumor cells to MMC-induced cytotoxicity. These data indicate that NADPH cytochrome P450 reductase-mediated MMC redox cycling is not involved in cytotoxicity of this chemotherapeutic agent. PMID:20501808

Wang, Yun; Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2010-01-01

231

Cytochrome P450-dependent hydroxylation in the biosynthesis of rosmarinic acid in Coleus  

Microsoft Academic Search

Three membrane-bound, cytochrome P450-dependent hydroxylases which are involved in the biosynthesis of rosmarinic acid have been characterized in microsomal preparations from cell cultures of Coleus blumei. Cinnamic acid 4-hydroxylase introduces the 4-hydroxyl group into cinnamic acid and forms 4-coumaric acid. This enzyme from Coleus blumei displayed saturation concentrations of 0.5 mM for both cinnamic acid and NADPH. The apparent Km-values

Maike Petersen

1997-01-01

232

Monitoring Compound Integrity With Cytochrome P450 Assays and qHTS  

Microsoft Academic Search

The authors describe how room temperature storage of a 1120-member compound library prepared in either DMSO or in a hydrated-DMSO\\/water (67\\/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z? factors of 0.71 and 0.62, with 17% and 32% of the library found as active against the

Ryan MacArthur; William Leister; Henrike Veith; Paul Shinn; Noel Southall; Christopher P. Austin; James Inglese; Douglas S. Auld

2009-01-01

233

Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays  

Microsoft Academic Search

DNA damage is a critical factor in carcinogenesis. The Ames assay is a short-term test that screens for DNA-damaging agents. To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro. A combination of iron(III) porphyrin

Keiko Inami; Masataka Mochizuki

2002-01-01

234

Influence of cytochrome P450 polymorphisms on drug therapies: Pharmacogenetic, pharmacoepigenetic and clinical aspects  

Microsoft Academic Search

The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies

Magnus Ingelman-Sundberg; Sarah C. Sim; Alvin Gomez; Cristina Rodriguez-Antona

2007-01-01

235

CYP5122A1, a Novel Cytochrome P450 Is Essential for Survival of Leishmania donovani  

Microsoft Academic Search

BackgroundCytochrome P450s (CYP450s) are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene

Smriti Verma; Ashish Mehta; Chandrima Shaha; Mauricio Martins Rodrigues

2011-01-01

236

An evaluation of the cytochrome P450 induction potential of pantoprazole in primary human hepatocytes  

Microsoft Academic Search

Primary human hepatocytes contain a full complement of human drug-metabolizing enzymes and therefore represent a relevant experimental system for the evaluation of pharmacokinetic drug–drug interaction potential in human. In this study, the cytochrome P450 (CYP) induction potential of pantoprazole (PAN) was evaluated and compared to two other proton pump inhibitors (PPIs), omeprazole (OM) and lansoprazole (LAN). Primary human hepatocytes from

Noriko Masubuchi; Albert P Li; Osamu Okazaki

1998-01-01

237

Cytochrome P450 enzymes in aquatic invertebrates: recent advances and future directions  

Microsoft Academic Search

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. These enzymes metabolize a wide variety of substrates including endogenous molecules (e.g. fatty acids, eicosenoids, steroids) and xenobiotics (e.g.

Mark J Snyder

2000-01-01

238

Cytochromes P450-mediated degradation of fuel oxygenates by environmental isolates.  

PubMed

The degradation of fuel oxygenates [methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME)] by Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005 and Gordonia sp. IFP 2009 (formerly Mycobacterium sp.) isolated from different environments was compared. Strains IFP 2001, IFP 2005 and IFP 2009 grew on ETBE due in part to the activity of a cytochrome P450, CYP249. All of these strains were able to degrade ETBE to tert-butyl alcohol and are harboring the CYP249 cytochrome P450. They were also able to degrade MTBE and TAME, but ETBE was degraded in all cases most efficiently, with degradation rates measured after growth on ETBE of 2.1, 3.5 and 1.6 mmol ETBE g(-1) dry weight h(-1) for strains IFP 2001, IFP 2005 and IFP 2009, respectively. The phylogenetic relationships between the different ethR (encoding the regulator) and ethB (encoding the cytochrome P450) genes were determined and showed high identity between different ethB genes (>99%). Only ETBE was able to induce the expression of ethB in strains IFP 2001 and IFP 2005 as measured by reverse transcriptase quantitative PCR. Our results are a first indication of the possible role played by the ethB gene in the ecology of ETBE degradation. PMID:20337704

Malandain, Cédric; Fayolle-Guichard, Françoise; Vogel, Timothy M

2010-05-01

239

Identification of a functional water channel in cytochrome P450 enzymes  

PubMed Central

Cytochrome P450 enzymes are monooxygenases that contain a functional heme b group linked to a conserved cysteine with a thiolate bond. In the native state, the central iron atom is hexacoordinated with a covalently bound water molecule. The exclusion of solvent molecules from the active site is essential for efficient enzymatic function. Upon substrate binding, water has to be displaced from the active site to prevent electron uncoupling that results in hydrogen peroxide or water. In contrast to typical hemoproteins, the protein surface is not directly accessible from the heme of cytochromes P450. We postulate a two-state model in which a conserved arginine, stabilizing the heme propionate in all known cytochrome P450 crystal structures, changes from the initial, stable side-chain conformation to another rotamer (metastable). In this new state, a functional water channel (aqueduct) is formed from the active site to a water cluster located on the thiolate side of the heme, close to the protein surface. This water cluster communicates with the surface in the closed state and is partly replaced by the flipping arginine side chain in the open state, allowing water molecules to exit to the surface or to reaccess the active site. This two-state model suggests the presence of an exit pathway for water between the active site and the protein surface. PMID:9122160

Oprea, Tudor I.; Hummer, Gerhard; Garcia, Angel E.

1997-01-01

240

Comparison of basal and induced cytochromes P450 in 6 species of waterfowl  

USGS Publications Warehouse

Cytochrome P450-associated monooxygenase activities were measured in control and prototype inducer-treated mallard duck, black duck, wood duck, lesser scaup, Canada goose and mute swan. Ages of the birds ranged from pipping embryos (that were treated approximately 3 days before pipping) to adults. Three or more of the following hepatic microsomal monooxygenases were assayed in each species: Benzyloxyresorufin-O-dealkylase (BROD), Ethoxyresorufin-O-dealkylase (EROD), methoxyresorufin-O-dealkylase (MROD), and pentoxyresorufin-O-dealkylase (PROD). Baseline activities differed between species, but because of differences in ages, sources of the eggs or birds, and diets, these cannot be viewed as absolute differences. The cytochrome P450 inducers utilized were beta-naphthoflavone (BNF), 3-methylcholanthrene (3MC) and phenobarbital (PB). In general, there was little response to PB; only lesser scaup were induced to greater than three times control level and most species were well under this. Responses to BNF and 3MC occurred in each species studied, but differed in which of the monooxygenases was most induced (absolute values and ratios to control values) and in relative induction between species. BROD frequently had an induction ratio EROD. Overall, lesser scaup were the most responsive, canada geese the least responsive, and the other species intermediate in responsiveness to the cytochrome P450 inducers studied.

Melancon, M.J.; Rattner, B.A.; Hoffman, D.J.; Beeman, D.; Day, D.; Custer, T.

1999-01-01

241

Construction and engineering of a thermostable self-sufficient cytochrome P450  

SciTech Connect

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)] [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)] [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

2009-06-19

242

Species differences in ciprofibrate induction of hepatic cytochrome P450 4A1 and peroxisome proliferation.  

PubMed

Six species (CD-1 mouse, Fischer 344 rat, Syrian golden hamster, Duncan-Hartley guinea pig, half-lop rabbit and marmoset monkey) were treated orally with ciprofibrate, a potent oxyisobutyrate hypolipidaemic drug for 14 days. A dose-dependent liver enlargment was observed in the mouse and rat and at the high dose level in the hamster. A marked dose-dependent increase in the 12-hydroxylation of lauric acid was observed in the treated mouse, hamster, rat, and rabbit, associated with a concomitant elevation in the specific content of cytochrome P-450 4A1 apoprotein, determined by an ELISA technique. Similarly, in these responsive species, an increase in mRNA levels coding for cytochrome P450 4A1 was observed. Lauric acid 12-hydroxylation was unchanged in the guinea pig and marmoset after ciprofibrate pretreatment, and cytochrome P-450 4A1 was not detected immunochemically in liver microsomes from these latter species. In the untreated mouse, hamster, rat, and rabbit, the 12-hydroxylation of lauric acid was more extensive than the 11-hydroxylation, whereas in the guinea pig and marmoset the activity ratios were reversed, with 11-hydroxylation predominating. Peroxisomal fatty acid beta-oxidation was markedly induced in the mouse, hamster, rat, and rabbit on treatment at the higher dose level (39-, 3-, 13- and 5-fold, respectively) and was slightly increased in the marmoset (2-fold), yet was unchanged in the guinea pig following treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1282571

Makowska, J M; Gibson, G G; Bonner, F W

1992-01-01

243

Modulation of mitomycin C mutagenicity on Saccharomyces cerevisiae by glutathione, cytochrome P-450, and mitochondria interactions.  

PubMed

It is well established that most anticancer drugs also have mutagenic effects and require metabolic activation before exerting their mutagenic/antiblastic activity. Antitumoral compound effects strongly depend on the biochemical/physiological conditions of the tumoral cells, and especially on the activation of specific drugs metabolizing enzymes and on respiration. We examined the mitomycin C-induced mutagenic effects on the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant p degrees at different contents of glutathione and cytochrome P-450, molecules able to activate/detoxicate xenobiotics. The mutagenic activity of the drug was evaluated as frequency of mitotic gene conversion and reversion in different physiological conditions. The highest frequencies of reversion and especially of gene conversion were observed at the highest cytochrome P-450 contents in the D7 strain with a further increase at high glutathione level. In the respiratory-deficient strain, the highest frequency of convertants was shown at low glutathione level and lack of cytochrome P-450. These results suggest the relevance of mitochondrial functionality for the expression of genotoxic activity of this anticancer drug. PMID:9150759

Rossi, C; Poli, P; Candi, A; Buschini, A

1997-04-24

244

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*  

PubMed Central

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and ?-bulge of cytP450 and residues at the end of helix ?4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. PMID:23709268

Ahuja, Shivani; Jahr, Nicole; Im, Sang-Choul; Vivekanandan, Subramanian; Popovych, Nataliya; Le Clair, Stephanie V.; Huang, Rui; Soong, Ronald; Xu, Jiadi; Yamamoto, Kazutoshi; Nanga, Ravi P.; Bridges, Angela; Waskell, Lucy; Ramamoorthy, Ayyalusamy

2013-01-01

245

Die Bedeutung genetischer Polymorphismen im Enzym Cytochrom P450 2C9 für Pharmakokinetik und Wirkungen der nichtsteroidalen Antiphlogistika Diclofenac und Ibuprofen.  

E-print Network

??Die Bedeutung genetischer Polymorphismen im Enzym Cytochrom P450 2C9 für Pharmakokinetik und Wirkungen von Diclofenac und Ibuprofen Es wird angenommen, dass Cytochrom-P450 2C9 die 4’-Hydroxylierung… (more)

Freytag, Georg Tobias Heinrich

2005-01-01

246

Distinct forms of cytochrome P-450 are responsible for 6 beta-hydroxylation of bile acids and of neutral steroids.  

PubMed Central

Cytochrome P-450-dependent 6 beta-hydroxylation of bile acids in rat liver contributes to the synthesis of the quantitatively important pool of 6-hydroxylated bile acids, as well as to the detoxification of hydrophobic bile acids. The lithocholic acid 6 beta-hydroxylation reaction was investigated and compared with androstenedione 6 beta-hydroxylation. Differential responses of these two activities to inducers and inhibitors of microsomal P-450 enzymes, lack of mutual inhibition by the two substrates and differential inhibition by antibodies raised against several purified hepatic cytochromes P-450 were observed. From these results it was concluded that 6 beta-hydroxylation of lithocholic acid is catalysed by P-450 form(s) different from the subfamily IIIA cytochromes P-450 which are responsible for the bulk of microsomal androstenedione 6 beta-hydroxylation. Similar, but more tentative, results revealed that the 7 alpha-hydroxylation of lithocholic acid and of androstenedione may be also catalysed by distinct P-450 enzymes. The results indicate that cytochromes P-450 hydroxylating bile acids are distinct from analogous enzymes that carry out reactions of the same regio- and stereo-specificity on neutral steroids (steroid hormones). A comparison of pairs of cytochromes P-450 that catalyse the same reaction on closely related steroid molecules will help to define those structural elements in the proteins that determine the recognition of their respective substrates. PMID:2018466

Zimniak, P; Holsztynska, E J; Radominska, A; Iscan, M; Lester, R; Waxman, D J

1991-01-01

247

Time course for induction of cytochrome P450 - Dependent activities by ethylbenzene  

SciTech Connect

The goal of this study was to examine the time course for ethylbenzene-mediated induction of cytochrome P450-dependent activities. Male Holtzman rats were treated with a single i.p. injection of ethylbenzene (EB) suspended in corn oil. In this study, the rats were injected at different times so all animals were killed at the same time P450 levels were transiently elevated at 1 hr after EB treatment, and returned to control levels by 2 hrs. Levels increased again at 10 hrs to a maximum at 24 hrs, returning to control after 48 hrs. Cytochrome b{sub 5} levels reached a minimum at 5 hrs and returned to controls after 10 hrs. In general, P450-dependent activities produced maximal induction after 24 hrs. Most of the P450-dependent activities examined returned to controls by 48 hrs; however, p-nitroanisole demethylation remained elevated after 48 hrs. Toluene metabolism was also induced by EB treatment, with each of the three metabolites exhibiting its own pattern of induction. Benzyl alcohol formation dropped to a minimum at 5-10 hrs, returning to controls by 24 hrs. Production of o-cresol was elevated more than 10 fold at 24 hrs and remained elevated after 48 hrs. Production of p-cresol followed a biphasic patter of induction increasing about 7 fold after 1 hr, and further increasing to a maximum between 10 and 24 hrs. P-cresol levels remained elevated at 48 hrs. Western blotting showed induction of both P450 1A1 and 2B1 at 1 and 2 hr, respectively, reaching a maximum at 24 hrs.

Sequeira, D.J.; Eyer, C.S.; Cawley, G.F.; Backes, W.L. (Louisiana State Univ., New Orleans, LA (United States))

1992-02-26

248

Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films.  

PubMed

Electrochemical methods continue to present an attractive means for achieving in vitro biocatalysis with cytochromes P450; however understanding fully the nature of electrode-bound P450 remains elusive. Herein we report thermodynamic parameters using electrochemical analysis of full-length mammalian microsomal cytochrome P450 2B4 (CYP 2B4) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of CYP 2B4-DDAB films on silica slides reveal an absorption maximum at 418nm, characteristic of low-spin, six-coordinate, water-ligated Fe(III) heme in P450. The Fe(III/II) and Fe(II/I) redox couples (E1/2) of substrate-free CYP 2B4 measured by cyclic voltammetry are -0.23V and -1.02V (vs. SCE, or 14mV and -776mV vs. NHE) at 21°C. The standard heterogeneous rate constant for electron transfer from the electrode to the heme for the Fe(III/II) couple was estimated at 170s(-1). Experiments indicate that the system is capable of catalytic reduction of dioxygen, however substrate oxidation was not observed. From the variation of E1/2 with temperature (18-40°C), we have measured entropy and enthalpy changes that accompany heme reduction, -151Jmol(-1)K(-1) and -46kJmol(-1), respectfully. The corresponding entropy and enthalpy values are less for the six-coordinate low-spin, imidazole-ligated enzyme (-59Jmol(-1)K(-1) and -18kJmol(-1)), consistent with limited conformational changes upon reduction. These thermodynamic parameters are comparable to those measured for bacterial P450 from Bacillus megaterium (CYP BM3), confirming our prior reports that the surfactant environment exerts a strong influence on the redox properties of the heme. PMID:24013063

Hagen, Katharine D; Gillan, James M; Im, Sang-Choul; Landefeld, Sally; Mead, Griffin; Hiley, Megan; Waskell, Lucy A; Hill, Michael G; Udit, Andrew K

2013-12-01

249

Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes  

Microsoft Academic Search

Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive

Yasuhito Kobayashi; Yutaka Motohashi; Yoshifumi Miyazaki; Mitsuyoshi Yatagai; Takehito Takano

1991-01-01

250

Development of a caffeine breath test to measure cytochrome P450-1A activity in birds  

Microsoft Academic Search

Cytochrome P450-1A activity is induced by planar polyhalogenated diaromatic hydrocarbons, and is often used as a biomarker of exposure of wildlife to these compounds. P450-1A activity is usually measured ex vivo in liver tissue. The purpose of this study was to develop a less invasive breath test to measure P450-1A activity in birds. Such an assay would allow measurement of

Lori A. Feyk; John P. Giesy

1996-01-01

251

Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi  

PubMed Central

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

Syed, Khajamohiddin; Shale, Karabo; Pagadala, Nataraj Sekhar; Tuszynski, Jack

2014-01-01

252

Systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi.  

PubMed

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

Syed, Khajamohiddin; Shale, Karabo; Pagadala, Nataraj Sekhar; Tuszynski, Jack

2014-01-01

253

Crystal Structure of H2O2-dependent Cytochrome P450SP? with Its Bound Fatty Acid Substrate  

PubMed Central

Cytochrome P450SP? (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H2O2-dependent P450. P450SP? hydroxylates fatty acids with high ?-regioselectivity. Herein we report the crystal structure of P450SP? with palmitic acid as a substrate at a resolution of 1.65 ?. The structure revealed that the C? of the bound palmitic acid in one of the alternative conformations is 4.5 ? from the heme iron. This conformation explains the highly selective ?-hydroxylation of fatty acid observed in P450SP?. Mutations at the active site and the F–G loop of P450SP? did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450BS? (CYP152A1), which shows ?-regioselectivity. This implies that the high regioselectivity of P450SP? is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450BS?. PMID:21719702

Fujishiro, Takashi; Shoji, Osami; Nagano, Shingo; Sugimoto, Hiroshi; Shiro, Yoshitsugu; Watanabe, Yoshihito

2011-01-01

254

Solubilization and reconstitution of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca  

SciTech Connect

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tests was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2',5'-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase. 24 references, 4 figures, 4 tables.

Desjardins, A.E.; Matthews, D.E.; VanEtten, H.D.

1984-07-01

255

Oxidation of 7-dehydrocholesterol and desmosterol by human cytochrome P450 46A1.  

PubMed

Cytochrome P450 (P450 or CYP) 46A1 is expressed in brain and has been characterized by its ability to oxidize cholesterol to 24S-hydroxycholesterol. In addition, the same enzyme is known to further oxidize 24S-hydroxycholesterol to the 24,25- and 24,27-dihydroxy products, as well as to catalyze side-chain oxidations of 7?-hydroxycholesterol and cholestanol. As precursors in the biosynthesis of cholesterol, 7-dehydrocholesterol has not been found to be a substrate of P450 46A1 and desmosterol has not been previously tested. However, 24-hydroxy-7-dehydrocholesterol was recently identified in brain tissues, which prompted us to reexamine this enzyme and its potential substrates. Here we report that P450 46A1 oxidizes 7-dehydrocholesterol to 24-hydroxy-7-dehydrocholesterol and 25-hydroxy-7-dehydrocholesterol, as confirmed by LC-MS and GC-MS. Overall, the catalytic rates of formation increased in the order of 24-hydroxy-7-dehydrocholesterol < 24-hydroxycholesterol < 25-hydroxy-7-dehydrocholesterol from their respective precursors, with a ratio of 1:2.5:5. In the case of desmosterol, epoxidation to 24S,25-epoxycholesterol and 27-hydroxylation was observed, at roughly equal rates. The formation of these oxysterols in the brain may be of relevance in Smith-Lemli-Opitz syndrome, desmosterolosis, and other relevant diseases, as well as in signal transduction by lipids. PMID:25017465

Goyal, Sandeep; Xiao, Yi; Porter, Ned A; Xu, Libin; Guengerich, F Peter

2014-09-01

256

CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450.  

PubMed

We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of protein-bound water make P450 a unique subject for the studies of high-pressure adaptation. Earlier, we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1). Here, we report purification and initial characterization of its mesophilic ortholog from the shallow-water P. profundum 3TCK (CYP261C2), as well as another piezophilic enzyme, CYP261D1, from deep-sea Moritella sp. PE36. Comparison of the three enzymes revealed a striking peculiarity of the piezophilic enzymes. Both CYP261C1 and CYP261D1 possess an apparent pressure-induced conformational toggle actuated at the pressures commensurate with the physiological pressure of habitation of the host bacteria. Furthermore, in contrast to CYP261C2, the piezophilic CYP261 enzymes may be chromatographically separated into two fractions with different properties, and different thermodynamic parameters of spin equilibrium in particular. According to our concept, the changes in the energy landscape that evolved in pressure-tolerant enzymes must stabilize the less-hydrated, closed conformers, which may be transient in the catalytic mechanisms of nonpiezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle. PMID:23586990

Davydov, Dmitri R; Sineva, Elena V; Davydova, Nadezhda Y; Bartlett, Douglas H; Halpert, James R

2013-01-01

257

Mössbauer and EPR Study of Reaction Intermediates of Cytochrome P450  

NASA Astrophysics Data System (ADS)

We present a complementary Mössbauer and EPR study on reaction intermediates of substrate-free and substrate-bound cytochrome P450cam from Pseudomonas putida prepared by the freeze-quench method from 57Fe-labeled P450cam using peroxy acetic acid as oxidizing agent. When reacting the substrate-free P450cam for 8 ms reaction time the reaction mixture consists of ˜85% of ferric low-spin iron (Fe(III)) with g-factors and hyperfine parameters of the starting material; the remaining ˜15% are identified as ferryl iron (Fe(IV); S Fe=1) by its Mössbauer signature. Parallel to the ferryl iron a tyrosine radical ( S rad=1/2) is formed. The two paramagnetic species are not exchange-coupled; however, they are close enough to significantly influence the (EPR) relaxation behavior of the radical spin. In the case of substrate-bound P450cam only trace amounts of the tyrosine radical are formed within 8 ms (<3%); within the accuracy of Mössbauer spectroscopy (5%) iron(IV) can not be detected. The results point to Tyr-96, which is hydrogen-bonded to the substrate camphor, as the candidate for the observed tyrosine radical.

Schünemann, V.; Trautwein, A. X.; Jung, C.; Terner, J.

2002-06-01

258

Reduced Oxy Intermediate Observed in D251N Cytochrome P450cam David E. Benson, Kenneth S. Suslick,* and Stephen G. Sligar*  

E-print Network

established the oxidation and oxygenation state of the cytochrome D251N P450cam spectral intermediateReduced Oxy Intermediate Observed in D251N Cytochrome P450cam David E. Benson, Kenneth S. SuslickVised Manuscript ReceiVed February 24, 1997X ABSTRACT: Cytochrome P450s are ubiquitous heme proteins responsible

Suslick, Kenneth S.

259

Global Analysis of Protein-Protein Interactions Reveals Multiple Cytochrome P450 2E1–Reductase Complexes  

PubMed Central

Although a single binary functional complex between cytochrome P450 and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain experimentally observed catalytic activity of recombinant P450 2E1 in dependence on the total concentration of added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450:CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450•CPR, the quaternary complex (P450)2• (CPR)2, and the homodimer (P450)2. The second plausible model additionally involves a weakly bound ternary complex (P450)2•CPR. Importantly, only the binary complex P450•CPR seems catalytically active in either of the two most probable mechanisms. PMID:17685587

Jamakhandi, Arvind P.; Kuzmic, Petr; Sanders, Daniel E.; Miller, Grover P.

2008-01-01

260

SEASONAL HEPATIC CYTOCHROME P-450 INDUCTION IN COTTON RATS (SIGMODON HISPIDUS) INHABITING PETROCHEMICAL WASTE SITES. (R826242)  

EPA Science Inventory

Abstract Wildlife species inhabiting contaminated sites are often exposed to complex mixtures of chemicals that have known effects on physiological and biochemical function. We evaluated the induction of major hepatic cytochrome P-450 isoenzymes through O -dealky...

261

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)  

SciTech Connect

Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)] [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

2011-08-05

262

Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme  

DOEpatents

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Marrone, Babetta L. (Los Alamos, NM); Simpson, Daniel J. (Los Alamos, NM); Unkefer, Clifford J. (Los Alamos, NM); Whaley, Thomas W. (Santa Fe, NM)

1992-01-01

263

Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme  

DOEpatents

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Marrone, Babetta L. (Los Alamos, NM); Simpson, Daniel J. (Los Alamos, NM); Unkefer, Clifford J. (Los Alamos, NM); Whaley, Thomas W. (Santa Fe, NM)

1993-01-01

264

Inhibition of cytochrome P450 2E1 expression by 2-(allylthio)Pyrazine, a Potential chemoprotective agent: hepatoprotective Effects  

Microsoft Academic Search

Cytochrome P450 2E1 (P450 2E1) is active in both the detoxification and activation of small organic molecules. The effects of 2-(allylthio)pyrazine (2-AP) on P450 2El-catalytic activity and the expression of rat hepatic P450 2E1 were examined. 2-AP competitively inhibited 4-nitrophenol hydroxylase activity in vitro (Ki, 12 ?M). 2-AP treatment of rats (200 mg\\/kg\\/day, po, 1–3 days old) resulted in 20–30%

Nak Doo Kim; Mi Kyong Kwak; Sang Geon Kim

1997-01-01

265

The sequence and characterization of TRI1, a cytochrome P450 monooxygenase involved in T-2 toxin biosynthesis  

E-print Network

THE Sf QUENCE AND CHARACTERIZATION OF TRI1, A CYTOCHROME P450 MONOOXYGENASE INVOLVED IN T-2 TOXIN BIOSYNTHESIS A Thesis by ISAAC BURTON MEEK Submitted to the Office of Graduate Studies of Texas A8 M University in partial fulfillment... of the requirements for the degree of MASTER OF SCIENCE December 2001 Major Subject: Plant Pathology THE SEQUENCE AND CHARACTERIZATION OF TRI1, A CYTOCHROME P450 MONOOXYGENASE INVOLVED IN T-2 TOXIN BIOSYNTHESIS A Thesis by ISAAC BURTON MEEK Submitted...

Meek, Isaac Burton

2012-06-07

266

Regulation of NADPH-cytochrome P450 reductase expressed during Douglas-fir germination and seedling development  

Microsoft Academic Search

NADH-cytochrome P450 is a key enzyme that transfers electrons from NADPH to the cytochrome P450 family of enzymes. To begin to determine the regulation of CPR gene expression and enzyme activity in Douglas-fir a full-length cDNA was isolated from a seedling ?ZAP cDNA library and the ORF was used to develop a synthetic CPR-peptide-based antiserum. Northern blot analysis indicated CPR

Timothy J. Tranbarger; Benjamin S. Forward; Santosh Misra

2000-01-01

267

Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera  

PubMed Central

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

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

2012-01-01

268

Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.  

PubMed Central

A cytochrome P450-like gene, tentatively named P450CMEF, was amplified by a mixed oligonucleotide-primed amplification of cDNA from C3H mouse embryo fibroblast cells, designated 10T1/2, that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) or benz[a]anthracene (BA). A set of inosine-containing degenerate primers that were targeted to two conserved regions of known cytochrome P450 cDNAs were used. One primer was coded for the well-described and conserved heme-binding region of P450 enzymes, and the second was designed based upon other considerations of homology among P450 molecules. One of the four PCR-amplified cDNA products hybridized to two major RNA bands, 4.2 and 5.3 kb, that were induced by DMBA or BA. The amino acid sequence of the fragment deduced from the base-sequence data indicate that the amplified cDNA has a 50-55% identity with the cytochrome P450 subfamily 1A. The induction of P450CMEF mRNA preceded the induction of aryl hydrocarbon hydroxylase activity after DMBA or BA treatment, suggesting that the product of P450CMEF is involved in the metabolism of these polycyclic aromatic hydrocarbons in 10T1/2 cells. From the partial sequence of the cDNA identified by this procedure, we propose that P450CMEF is a member of the P450 superfamily, possibly in a subfamily of family 1, that is induced in 10T1/2 cells by DMBA and BA. This method should be useful in identifying additional P450 genes and genes in other gene families. Images Fig. 2 Fig. 5 PMID:7505439

Shen, Z; Wells, R L; Liu, J; Elkind, M M

1993-01-01

269

Purification and Characterization of an NADPH-Cytochrome P450 (Cytochrome c) Reductase from Spearmint ( Mentha spicata) Glandular Trichomes  

Microsoft Academic Search

Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2?,5?-adenosine diphosphate agarose. SDS–PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot

Krishan Ponnamperuma; Rodney Croteau

1996-01-01

270

Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera.  

PubMed

A complete cDNA encoding the NADPH-cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis-Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50=1.69 ?M). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90 ?M) was also observed. PMID:24768738

Liu, Dong; Zhou, Xiaojie; Li, Mei; Zhu, Shunyi; Qiu, Xinghui

2014-07-25

271

The role of brain noradrenergic system in the regulation of liver cytochrome P450 expression.  

PubMed

The aim of the present study was to examine the effect of the brain noradrenergic system on the expression of cytochrome P450 in the liver. The experiment was carried out on male Wistar rats. Intracerebroventricular injection of the noradrenergic neurotoxin DSP-4 diminished noradrenaline level in the brain. Simultaneously, significant decreases in the serum concentration of the growth hormone, testosterone and the thyroid hormone thyroxine, as well as an increase in corticosterone level were observed. The concentrations of triiodothyronine and the cytokines interleukine 2 (IL-2) and 6 (IL-6) were not changed by DSP-4. The neurotoxin produced complex changes in the functioning of cytochrome P450. Significant decreases in the activity of liver CYP2C11 (measured as a rate of the 2?- and 16?-hydroxylation of testosterone) and CYP3A (measured as a rate of the 2?- and 6?-hydroxylation of testosterone) were found. In contrast, the activity of CYP1A (measured as a rate of caffeine metabolism) rose, while that of CYP2A (measured as a rate of the 7?-hydroxylation of testosterone), CYP2C6 (measured as a rate of the 7-hydroxylation of warfarin) and CYP2D (the 1'-hydroxylation of bufuralol) remained unchanged. The changes in the activity of CYP1A, CYP2C11 and CYP3A correlated positively with those in CYP protein levels and with the CYP mRNA levels of CYP1A1, CYP2C11 and CYP3A1/2 genes, respectively. The obtained results indicate an important role of the brain noradrenergic system in the neuroendocrine regulation of liver cytochrome P450 expression, which may be of significance in pathological states involving this system, or during pharmacotherapy with drugs affecting noradrenergic transmission. PMID:23924605

Sadakierska-Chudy, Anna; Haduch, Anna; Rysz, Marta; Go?embiowska, Krystyna; Daniel, W?adys?awa A

2013-09-15

272

Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes  

SciTech Connect

Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES) was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study. Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.

Froehlich, Eleonore, E-mail: eleonore.froehlich@klinikum-graz.a [Center for Medical Research, Medical University of Graz (Austria); Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz (Austria); Kueznik, Tatjana [Center for Medical Research, Medical University of Graz (Austria); Samberger, Claudia [Center for Medical Research, Medical University of Graz (Austria); Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz (Austria); Roblegg, Eva [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens-University of Graz (Austria); Wrighton, Christopher [Institute of Medical Technologies and Health Management, Joanneum Research, Auenbruggerplatz 20/3, A-8036 Graz (Austria); Pieber, Thomas R. [Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz (Austria)

2010-02-01

273

Certain tryptophan photoproducts are inhibitors of cytochrome P450-dependent mutagenicity.  

PubMed

Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomyces cerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Salmonella typhimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100 + S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-450IA1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecarpine and dehydrorutaecarpine. PMID:1330548

Rannug, U; Agurell, E; Rannug, A; Cederberg, H

1992-01-01

274

Metabolism of the Tricyclic Antidepressant Amitriptyline by cDNA-Expressed Human Cytochrome P450 Enzymes  

Microsoft Academic Search

The metabolism of amitriptyline was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6 and 2E1. CYP 2C19 was the most important enzyme with regard to the demethylation of amitriptyline, the quantitatively most important metabolic pathway. CYP 1A2, 3A4, 2C9 and CYP 2D6 also participated in the demethylation of amitriptyline. CYP 2D6 was the

Ole V. Olesen; Kristian Linnet

1997-01-01

275

Genetic polymorphism of cytochrome P450 as a biomarker of susceptibility to environmental toxicity.  

PubMed Central

Cytochrome P450 (CYP) enzymes are responsible for the metabolism of numerous xenobiotics and endogenous compounds, including the metabolic activation of most environmental toxic chemicals and carcinogens. Both metabolic and genetic polymorphisms have been identified for human CYP enzymes. The association of CYP genetic polymorphism and human cancer risk, and susceptibility to environmental hazards, have received increasing attention. This article briefly reviews the approaches and methods currently used in CYP genetic polymorphism studies. In addition, the current status and perspectives of using CYP genetic polymorphism as a biomarker of individual susceptibility to cancer and environmental toxicity are discussed. PMID:9255558

Hong, J Y; Yang, C S

1997-01-01

276

Valence bond all the way: from the degenerate H-exchange to cytochrome P450.  

PubMed

This is a personalized Perspective on the development of a valence bond (VB) view of chemical reactivity (J. Am. Chem. Soc., 1981, 103, 3692) as a LEGO process whereby one constructs "reactivity objects", such as barriers, transition states, and reaction intermediates from VB building blocks, and thereby understands and predicts chemical reactivity in a unified manner. In so doing, I have tried to give the reader a panoramic set of applications, from the simple H-exchange reaction all the way to alkane hydroxylation by cytochrome P450 (Prog. Phys. Org. Chem. 1985, 15, 197; Angew. Chem., Int. Ed., 1999, 38, 586). PMID:20574583

Shaik, Sason

2010-08-21

277

Epoxidation of Olefins by Cytochrome P-450 Model Compounds: Mechanism of Oxygen Atom Transfer  

NASA Astrophysics Data System (ADS)

The mechanism of the Mn(III) porphyrincatalyzed epoxidation of olefins by lithium hypochlorite is examined. The active oxidant is thought to be a high-valent manganese--oxo complex. It is shown that a relatively stable intermediate is reversibly formed upon interaction of the olefin and the oxo complex. The decomposition of this intermediate to Mn(III) porphyrin and epoxide is the rate-determining step of the catalytic cycle. Some analogies to the biochemical epoxidation of olefins catalyzed by cytochrome P-450 are discussed.

Collman, James P.; Brauman, John I.; Meunier, Bernard; Raybuck, Scott A.; Kodadek, Thomas

1984-05-01

278

Cytochrome P450 1A1, cigarette smoking, and risk of endometrial cancer (United States)  

Microsoft Academic Search

Cytochrome P450 1A1 (CYP1A1) is involved in the metabolism of estradiol and the activation of polycyclic aromatic hydrocarbons\\u000a found in tobacco products. Polymorphic variation in CYP1A1 activity may modify susceptibility to endometrial cancer through\\u000a the oxidative metabolism of estradiol and the activation of tobacco-smoke constituents. We prospectively evaluated the associations\\u000a between three common CYP1A1 polymorphisms and endometrial cancer risk, as

Monica McGrath; Susan E. Hankinson; Immaculata De Vivo

2007-01-01

279

Cytochrome P450-catalyzed degradation of nicotine: fundamental parameters determining hydroxylation by cytochrome P450 2A6 at the 5'-carbon or the n-methyl carbon.  

PubMed

The oxidation of (2'S)-nicotine in the active site of human cytochrome P450 2A6 has been subjected to a detailed analysis by theoretical quantum mechanical/molecular mechanical (QM/MM) calculations linked with a theoretical and experimental study of the associated isotope effects. The study has focused on seeking an explanation as to why oxidation at the 5'-carbon position (A) is favored over oxidation at the methyl carbon (CMe) position (B). It is deduced that the choice of hydrogen for abstraction is not determined by geometric features of the active site, but by the lower energy barrier associated with 5' oxidation. N-Demethylation leading to N-hydroxymethylnornicotine requires ca. 6.5 kcal/mol more energy to transfer a hydrogen atom than is required for oxidation on the carbon 5'. Neither protonation of the pyrrolidine nitrogen (N1') nor inclusion of a water molecule in the reaction process influences the balance between the two oxidation pathways. In both cases, the hydrogen transfer step is rate limiting. An analysis of the calculated kinetic isotope effects indicates that the presence of a (2)H in either the C5' or the CMepositions has a significant effect on the reaction kinetics. However, the experimental values of around 2.2-2.6 are considerably lower than those predicted by theoretical calculations (9.3 and 6.9 for C5' or the CMe positions, respectively, in the LS state of Cpd I), typical of the masking commonly found for CYP450 reactions. The fact that similar values are found for cotinine formation from both substrates, however, may indicate that the measured value is not that for H-abstraction but, rather, is a combined value for (2)H influence on electronic redistribution between iminium states of the pyrrolidine ring. This is the first time that oxidation at the C5' or the CMe positions has been directly compared and that isotope effects have been obtained for this reaction in a human cytochrome P450 reaction. PMID:22676413

Kwiecie?, Renata A; Le Questel, Jean-Yves; Lebreton, Jacques; Delaforge, Marcel; André, François; Pihan, Emilie; Roussel, Anaïs; Fournial, Anaïs; Paneth, Piotr; Robins, Richard J

2012-07-12

280

CYP6B3: a second furanocoumarin-inducible cytochrome P450 expressed in Papilio polyxenes.  

PubMed

Cytochrome P450 monooxygenases in the larvae of Papilio polyxenes (black swallowtail) (Lepidoptera: Papilionidae) are capable of detoxifying linear and angular furanocoumarins found in their host plants. The CYP6B1 locus, which is transcriptionally induced in these larvae in response to xanthotoxin, encodes a P450 that principally metabolizes linear furanocoumarins such as xanthotoxin and bergapten. We have now cloned CYP6B3 cDNA derived from a second locus that is evolutionarily related to the CYP6B1 locus. Reverse transcription-PCR Southern analyses have demonstrated that CYP6B3 transcripts are expressed in response to a wider range of linear and angular furanocoumarins but at lower abundance than CYP6B1 transcripts. Whereas CYP6B1 transcripts are expressed at a low detectable level in uninduced control larvae and at high levels in xanthotoxin-induced larvae, CYP6B3 transcripts are nearly undetectable in control larvae and are highly induced by xanthotoxin and bergapten (linear furanocoumarins) as well as by angelicin and sphondin (angular furanocoumarins). The fact that these two CYP6B loci are differentially regulated by these four furanocoumarins indicates that P. polyxenes has adapted to the presence of the wide range of furanocoumarins in its host plants by diversifying its P450 isozyme structure and its furanocoumarin-responsive regulatory cascades. PMID:8589841

Hung, C F; Harrison, T L; Berenbaum, M R; Schuler, M A

1995-08-01

281

Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process  

SciTech Connect

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

Miao, Yinglong [ORNL; Baudry, Jerome Y [ORNL

2011-01-01

282

Oxidase uncoupling in heme monoxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs  

PubMed Central

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons : 1 dioxygen : 1 product. However, three alternate unproductive pathways exist where the intermediate iron-oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen-oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4 reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase. PMID:23266608

Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.; Sligar, Stephen G.

2014-01-01

283

Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.  

PubMed

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17?-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site. PMID:23620596

Estrada, D Fernando; Laurence, Jennifer S; Scott, Emily E

2013-06-01

284

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR*  

PubMed Central

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17?-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site. PMID:23620596

Estrada, D. Fernando; Laurence, Jennifer S.; Scott, Emily E.

2013-01-01

285

Interaction of buprenorphine and its metabolite norbuprenorphine with cytochromes p450 in vitro.  

PubMed

Buprenorphine is a thebaine derivative used in the treatment of heroin and other opiate addictions. In this study, the selective probe reactions for each of the major hepatic cytochromes P450 (P450s) were used to evaluate the effect of buprenorphine and its main metabolite norbuprenorphine on the activity of these P450s. The index reactions used were CYP1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2C19 (omeprazole 5-hydrxoylation), CYP2D6 (dextromethorphan O-demethylation), CYP2B6 (7-ethoxy-4-trifluoromethyl-coumarin 7-deethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (omeprazole sulfoxidation). Buprenorphine exhibited potent, competitive inhibition of CYP2D6 (Ki 10 +/- 2 microM and 1.8 +/- 0.2 microM) and CYP3A4 (Ki 40 +/- 1.6 microM and 19 +/- 1.2 microM) in microsomes from human liver and cDNA-expressing lymphoblasts, respectively. Compared with buprenorphine, norbuprenorphine demonstrated a lower inhibitory potency with CYP2D6 (22.4% inhibition at 20 microM norbuprenorphine) and CYP3A4 (13.6% inhibition at 20 microM) in microsomes from human cDNA-expressing lymphoblast cells. Furthermore, buprenorphine was shown to be a substrate of CYP2D6 (Km = 600 microM; Vmax = 0.40 nmol/min/mg protein) and CYP3A4 (Km = 36 microM; Vmax = 0.19 nmol/min/mg protein). The present in vitro study suggests that buprenorphine and its major metabolite norbuprenorphine are inhibitors of CYP2D6 and CYP3A4; however, at therapeutic concentrations they are not predicted to cause potentially clinically important drug interactions with other drugs metabolized by major hepatic P450s. PMID:12756210

Zhang, Wenjiang; Ramamoorthy, Yamini; Tyndale, Rachel F; Sellers, Edward M

2003-06-01

286

Covalent Attachment of an Electroactive Sulfydryl Reagent in the Active Site of Cytochrome P450cam as Revealed by the Crystal  

E-print Network

Covalent Attachment of an Electroactive Sulfydryl Reagent in the Active Site of Cytochrome P450cam to cytochrome P450cam from Pseudomonas putida. The crystal structure of the modified enzyme was determined at 2 a striking positive shift in the heme redox potential of the ferrocene-containing P450cam from -380 m

Fülöp, Vilmos

287

CMER RESEARCH TOPICS -2005 #3: PREPARATION OF MONOCLONAL ANTIBODIES FOR CYTOCHROME P450 1A (CYP1A) AND VITELLOGENIN (Vtg) PROTEINS THAT ARE  

E-print Network

1 of 12 CMER RESEARCH TOPICS - 2005 #3: PREPARATION OF MONOCLONAL ANTIBODIES FOR CYTOCHROME P450 1A and accumulation of Vtg in the serum of responding males and females. The other pathway, containing cytochrome P450 1A (CYP1A), is one member of the extensive P450 oxidative degradation pathway that is often enhanced

Kunkel, Joseph G.

288

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?  

PubMed Central

The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo- and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14?-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated. PMID:23297358

Kelly, Steven L.; Kelly, Diane E.

2013-01-01

289

Cytochrome P450 induction and metabolism of alkoxyresorufins, ethylmorphine and testosterone in cultured hepatocytes from goats, sheep and cattle.  

PubMed

Very little is known of cytochrome P450 (P450) patterns and enzyme characteristics in food-producing animal species. Oxidative metabolism of alkoxyresorufins, ethylmorphine (EtM) and testosterone (TST) was used to monitor the effects of the P450 inducers phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX) and triacetyloleandomycin (TAO) in primary cultured hepatocytes from goats, sheep and cattle. BNF effectively and specifically induced ethoxyresorufin deethylase (> 20-fold), indicating the presence of an inducible P450 1A form, and down-regulated EtM demethylation and most selected TST hydroxylations. In non-induced hepatocyte cultures, TST was metabolized to 6 beta-, 2 beta-, 12 beta-, and 11 alpha-hydroxy-TST (OHT). PB and, to a lesser extent, DEX non-specifically induced all OHT formations, and EtM demethylation. TAO almost completely inhibited OHT formation and EtM demethylation. These results indicate the involvement of principally one P450 form, or a restricted number of related P450 forms, presumably belonging to the P450 3A subfamily. In western blot analysis, cross reactivity was found with rat anti-P450 3A1 and anti-sheep P450 3A. A more specific PB effect was observed for 16 alpha-OHT, which may be formed though a ruminant P450 2B form. None of the inducers influenced pentoxyresorufin depentylase (PROD) or EtM O-deethylation. Metabolite patterns and inducibility of selected activities in ruminant hepatocytes are in accordance with previous findings in goats in vivo. Cytochrome P450 characteristics in ruminants appear to differ from those in rats whereas similarities to the situation in humans appear to exist. PMID:8250964

van 't Klooster, G A; Blaauboer, B J; Noordhoek, J; van Miert, A S

1993-11-17

290

Cytochrome P450 responses and PCB congeners in pipping heron embryos from Virginia, the Great Lakes and San Francisco Bay  

USGS Publications Warehouse

Pipping black-crowned night-heron (Nvcticorax nvcticorax) embryos were collected from undisturbed (Chincoteague National Wildlife Refuge VA; CNWR) and industrialized (Cat Island, Green Bay WI and San Francisco Bay, CA; SFB) locations. Hepatic monooxygenases (AHH, EROD, BROD, ECOD) were induced up to 100-fold, and were correlated (r=0.50 to 0.72) with total PCB burdens (N =61 embryos). A subset of 30 embryos have now been analyzed by GC/MS for 12 AHH-active PCB congeners and by Western blot for cytochromes P450lA and P450llB. At Cat Island, concentrations of 8 congeners were greater (P <0.05) than at CNWR. P450lA and P450llB were detected in 44% and 100% of the Cat Island embryos compared to 8% and 33% of the CNWR + SFB embryos. Cytochrome P450 parameters were correlated with the total PCBs (r =0.44 to 0.67) and with at least 9 PCB congeners (r =0.39 to 0.77). Since P450 responses might be affected by other contaminants, sample extract potency in the H411E rat hepatoma bioassay is being determined to study relationships among dioxin equivalents and cytochrome P450 parameters.

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Tillett, D.E.; Woodin, B.R.; Stegeman, J.J.

1992-01-01

291

Differential hepatotoxicity and cytochrome P450 responses of Fischer-344 rats to the three isomers of dichlorobenzene  

SciTech Connect

The acute hepatotoxicity and response of hepatic cytochrome P450 to treatment with the three isomers of dichlorobenzene (DCB) have been investigated. The objectives were to estimate the onset of toxicity and to further elucidate the role of cytochrome P450 in the metabolism and toxicity of these compounds. In a study design employing one animal per dose level, Fischer-344 rats were gavaged with up to 25 different dosages, then evaluated 24 h later. Hepatic necrosis, serum alanine aminotransferase, and serum aspartate aminotransferase exhibited similar patterns demonstrating that ortho-DCB (o-DCB) was the most toxic in terms of both earliest onset and degree of response at higher dosages. For these three endpoints, meta-DCB (m-DCB) exhibited a lesser toxicity. Para-DCB (p-DCB) did not cause changes in these three endpoints, but hepatic degenerative changes were found. Total hepatic cytochrome P450 responses were also different after treatment with each isomer. The o-DCB produced a dose-dependent decrease in P450 beginning at dosages lower than the onset of necrosis and appeared to be a suicide substrate for P450. The m-DCB treatment increased P450 at dosages below the onset of necrosis and decreased P450 at higher dosages, with the decline preceding the onset of hepatocyte death.

Allis, J.W.; Simmons, J.E.; House, D.E.; Robinson, B.L.; Berman, E.

1992-01-01

292

Evidence for the involvement of several cytochromes P-450 in the first steps of caffeine metabolism by human liver microsomes.  

PubMed

Caffeine biotransformation and four monooxygenase activities involving cytochrome P-450IA2, namely ethoxy- and methoxyresorufin O-dealkylases, phenacetin O-deethylase, and acetanilide 4-hydroxylation were studied in 25 human liver microsomes. All these activities were highly significantly intercorrelated (r greater than 0.72, p less than 0.001) and correlated with the level of immunoreactive P-450IA2 content (r greater than 0.65; p less than 0.001). P-450IA content was measured by immunoblotting with anti-rat P-450 beta-naphthoflavone-B, an antibody that detects only a single band corresponding to P-450IA2. The formation rate of two caffeine metabolites, namely paraxathine and theobromine, was correlated with the four monooxygenase activities measured and P-450IA2-specific content (r greater than 0.75). However, inhibition studies of caffeine metabolism by phenacetin, a specific substrate of P-450IA2, clearly indicated that only the N-3 demethylation of caffeine was supported by this enzyme. These in vitro data demonstrate that P-450IA2 is predominantly responsible for the major metabolic pathway of caffeine and that the formation of other demethylated metabolites is mediated, at least partly, by other P-450 enzymes. PMID:1680620

Berthou, F; Flinois, J P; Ratanasavanh, D; Beaune, P; Riche, C; Guillouzo, A

1991-01-01

293

Cytochrome P450 gene CYP337 and heritability of fitness traits in the Glanville fritillary butterfly.  

PubMed

Fitness-related life history traits often show substantial heritable genetic variation in natural populations, but knowledge of the genetic architecture of these traits is limited. In the Glanville fritillary butterfly, we measured the heritability of key life history traits in a large outdoor population cage during 2 years and generations and combined this experiment with an association study of a set of candidate genes. The genes were selected on the basis of previous genomic and transcriptomic studies and have been linked to the physiology and life history of this or other arthropod species. Heritability was high and significant for two traits, post-diapause larval development time (h(2) = 0.37) and lifetime egg (and larval) production (h(2) = 0.62); the latter is closely related to lifetime reproductive success and therefore fitness. We discovered a strong association between genetic polymorphism in the cytochrome P450 gene CYP337 and lifetime egg production, which accounted for 14% of the additive variance in egg production. This gene belongs to a group of cytochrome P450 genes that have a well-documented role in host plant adaptations in Lepidoptera and other insects and is likely to play an important role in the ecology and microevolution of the Glanville fritillary. This study provides a prime example of a gene associated with heritable fitness variation, measured under semi-natural ecological conditions. PMID:24552294

de Jong, M A; Wong, S C; Lehtonen, R; Hanski, I

2014-04-01

294

The beet R locus encodes a new cytochrome P450 required for red betalain production.  

PubMed

Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species. PMID:22660548

Hatlestad, Gregory J; Sunnadeniya, Rasika M; Akhavan, Neda A; Gonzalez, Antonio; Goldman, Irwin L; McGrath, J Mitchell; Lloyd, Alan M

2012-07-01

295

PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism  

SciTech Connect

As part of the pksX gene cluster of Bacillus subtilis strain 168, pksS has been preliminarily annotated as a cytochrome P450 homolog that hydroxylates the polyketide product of this cluster, which was recently shown to be involved in the biosynthesis of bacillaene and dihydrobacillaene. Here we report that there is a frame-shift error in the reported sequence for pksS, and that we have successfully cloned, overexpressed, and purified the protein encoded by the corrected sequence. By utilizing electronic absorption spectrophotometry, we have observed that the ferrous CO complex of PksS absorbs maximally near 450 nm, which confirms the annotation that this protein is a cytochrome P450. We have also established a cell-free system derived from crude cytosolic B. subtilis protein extracts which provides reductase activity essential to sustaining the putative catalytic cycle of PksS. Using LC-MS analysis we have collected data which suggests that the substrate for PksS is dihydrobacillaene.

Reddick, Jason J. [Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, 435 Science Building, P.O. Box 26170, Greensboro, NC 27402-6170 (United States)]. E-mail: jjreddic@uncg.edu; Antolak, Stephanie A. [Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, 435 Science Building, P.O. Box 26170, Greensboro, NC 27402-6170 (United States); Raner, Gregory M. [Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, 435 Science Building, P.O. Box 26170, Greensboro, NC 27402-6170 (United States)

2007-06-22

296

Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol.  

PubMed

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length I. pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly (Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. PMID:17046597

Sandstrom, Pamela; Welch, William H; Blomquist, Gary J; Tittiger, Claus

2006-11-01

297

Inter-relation of cytochrome P450 and contaminants burdens in sibling heron embryos and nestlings  

USGS Publications Warehouse

Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of arylhydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) weremodestly elevated (cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

Rattner, B.; Melancon, M.; Custer, T.; Hothem, R.

1995-01-01

298

Considerations and recent advances in QSAR models for cytochrome P450-mediated drug metabolism prediction.  

PubMed

Quantitative structure-activity relationships (QSAR) methods are urgently needed for predicting ADME/T (absorption, distribution, metabolism, excretion and toxicity) properties to select lead compounds for optimization at the early stage of drug discovery, and to screen drug candidates for clinical trials. Use of suitable QSAR models ultimately results in lesser time-cost and lower attrition rate during drug discovery and development. In the case of ADME/T parameters, drug metabolism is a key determinant of metabolic stability, drug-drug interactions, and drug toxicity. QSAR models for predicting drug metabolism have undergone significant advances recently. However, most of the models used lack sufficient interpretability and offer poor predictability for novel drugs. In this review, we describe some considerations to be taken into account by QSAR for modeling drug metabolism, such as the accuracy/consistency of the entire data set, representation and diversity of the training and test sets, and variable selection. We also describe some novel statistical techniques (ensemble methods, multivariate adaptive regression splines and graph machines), which are not yet used frequently to develop QSAR models for drug metabolism. Subsequently, rational recommendations for developing predictable and interpretable QSAR models are made. Finally, the recent advances in QSAR models for cytochrome P450-mediated drug metabolism prediction, including in vivo hepatic clearance, in vitro metabolic stability, inhibitors and substrates of cytochrome P450 families, are briefly summarized. PMID:18574695

Li, Haiyan; Sun, Jin; Fan, Xiaowen; Sui, Xiaofan; Zhang, Lan; Wang, Yongjun; He, Zhonggui

2008-11-01

299

Inter-relation of cytochrome P450 and contaminants burdens in sibling heron embryos and nestlings  

SciTech Connect

Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of aryl hydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) were modestly elevated ({<=} three-fold) in nestlings from polluted sites. Concentrations of p,p{prime}DDE, other organochlorine pesticides and total PCBs in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos, At these low pollutant concentrations there was little correlation between monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBS, aryl hydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

Rattner, B.; Melancon, M.; Custer, T.; Hothem, R. [National Biological Service, Laurel, MD (United States). Patuxent Environmental Science Center; [Upper Mississippi Science Center, La Crosse, WI (United States); [California Pacific Science Center, Davis, CA (United States)

1995-12-31

300

Absolute quantification and modulation of cytochrome P450 3A isoforms in cattle liver.  

PubMed

In humans and laboratory animals, knowledge about cytochrome P450 (CYP) regulation and function is detailed and very extensive. However, CYPs have still been incompletely characterized in veterinary species so far. In this study, mRNA levels of three CYP3A enzymes (CYP3A28, CYP3A38 and CYP3A48) were measured in cattle liver by using quantitative real-time RT-PCR (qPCR) assays and an absolute quantification approach. In particular, the possible presence of breed-differences in CYP3A expression was investigated in five different meat cattle breeds (Charolais, CH; Piedmontese, PM; Blonde d'Aquitaine, BA; Marchigiana, MA; Valdostana, VALD) and the potential transcriptional effect of the prototypical inducer phenobarbital (PB) upon the CYP3A isoforms was evaluated. Cytochrome P450 3A38 showed the highest amounts of gene copy numbers, followed by CYP3A48 and CYP3A28. Significant breed-differences in CYP3A gene abundances were found, and PB significantly up-regulated all the CYP3A isoforms. The data provide new information about CYP3A expression in cattle, particularly the heterogeneity in the pattern of expression of distinct hepatic CYP3As (CYP3A38?>?3A48?>?3A28), the significant effect of breed, and their common up-regulation following the exposure to PB, although with different orders of magnitude. PMID:25193407

Zancanella, V; Giantin, M; Dacasto, M

2014-10-01

301

1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity in liver microsomes  

SciTech Connect

The preparation of 1-ethynylpyrene (EP) by incubation of EP with liver microsomes in the presence of NADPH yields fluorescent products briefly. Addition of microsomes restores the original rate. The metabolism of EP is initially more rapid in microsomes from 5,6-benzoflavone- (BF) pretreated rats than in those from phenobarbital (PB) pretreated rats or controls. Ep inhibits the hydroxylation of benzo(a)pyrene (BP) by liver microsomes. Ep more effectively inhibits the oxidation of BP in liver microsomes from BF rats than from PB rats or from controls. The inhibition of BP hydroxylation activity due to EP is dependent upon NADPH and is apparently irreversible. Kinetic analyses show that the inhibition of BP hydroxylation is due to loss of the activity by a process that is first order in EP and that reaches a limiting value at infinite EP concentrations. A self-catalyzed inhibition of the cytochrome P-450 dependent BP hydroxylation may occur in the presence of EP. Incubation with EP under conditions that result in loss of BP hydroxylase activity in microsomes from BF rats and 66% of the activity from PB rats causes the loss of 6 and 12% of the cytochrome P-450, respectively. Thus the loss of P-450 content is an insensitive measure of the effect of this inhibitor upon this cytochrome P-450 dependent enzyme activity. Selectivity of the loss of P-450 due to the incubation of the different microsomal preparations with EP is observed to be different than the selectivity for loss of BP hydroxylase activity. It is proposed that the inhibition of cytochrome P-450 dependent enzymes by alkynes need not involve heme alkylation and a resulting loss of P-450 content. In vivo EP does not cause a significant change in the cytochrome P-450 content in the microsomes isolated, or result in the change in BP hydroxylation.

Gan, L.S.L.; Acebo, A.L.; Alworth, W.L.

1984-08-14

302

Human recombinant cytochrome p450 enzymes display distinct hydrogen peroxide generating activities during substrate independent NADPH oxidase reactions.  

PubMed

Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as "oxidase" activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the "oxidase" activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (?0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5-7), and when CPR was present in excess (CYP/CPR = 0.2-0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

2014-10-01

303

Assessing the Geometric Diversity of Cytochrome P450 Ligand Conformers by Hierarchical Clustering with a Stop Criterion  

E-print Network

applied on a database containing 70 ligands of the cytochrome CYP 3A4, showing that the structural is valid for all small molecules. 1. INTRODUCTION The cytochrome CYP 3A4 is a member of the P450 of roughly 50% of the drugs on the market.1 The CYP 3A4 ligands are structurally diverse compounds

Paris-Sud XI, Université de

304

Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase  

Microsoft Academic Search

One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450’s substrates.

Zhimou Wen; Liping Pan; May R Berenbaum; Mary A Schuler

2003-01-01

305

A Panel of Cytochrome P450 BM3 Variants To Produce Drug Metabolites and Diversify Lead Compounds  

PubMed Central

Here we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant. PMID:19774562

Sawayama, Andrew M.; Chen, Michael M. Y.; Kulanthaivel, Palaniappan; Kuo, Ming-Shang; Hemmerle, Horst; Arnold, Frances H.

2011-01-01

306

CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123  

PubMed Central

Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair and disposal. These less well-appreciated aspects are reviewed herein. PMID:20860521

Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

2011-01-01

307

[Role of antioxidants in electro catalytic activity of cytochrome P450 3A4].  

PubMed

The electrochemical analysis of cytochrome ?450 3?4 catalytic activity has shown that vitamins C, A and ? influence on electron transfer and Fe3+/Fe2+ reduction process of cytochrome ?450 3?4. These data allow to assume possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome ?450 3?4, at carrying out of complex therapy. This class of vitamins shows antioxidant properties that lead to increase of the cathodic current corresponding to heme reduction of this functionally significant haemoprotein. Ascorbic acid of 0.028-0.56 mM concentration stimulates cathodic peak (an electrochemical signal) of cytochrome ?450 3?4. At the presence of diclofenac (Voltaren) - a typical substrate of cytochrome ?450 3?4 - the increase growth of a catalytic current testifying to an electrocatalysis and stimulating action of ascorbic acid is observed. In the presence of vitamins A and ? also is registered dose-dependent (in a range of 10-100 M) increase in a catalytic current of cytochrome ?450 3?4: the maximum increase corresponds to 229 ± 20% for 100 M of vitamin A, and 162±10% for 100 M of vitamin E. Vitamin E in the presence of P450's inhibitor itraconazole doesn't give essential increase in a reductive current, unlike retinol (vitamin A). This effect can manifest substrate properties of tocopherol (vitamin E). The electrochemical approach for the analysis of catalytic activity of cytochrome ?450 3?4 and studies of influence of biologically active compounds on an electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (till 10-15 mol/electrode), to carry out the analysis without participation of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments. PMID:24837311

Shumiantseva, V V; Makhova, A A; Bulko, T V; Shikh, E V; Kukes, V G; Usanov, S A; Archakov, A I

2014-01-01

308

Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes  

PubMed Central

Background Cytochrome P450 proteins (CYPs) play diverse and pivotal roles in fungal metabolism and adaptation to specific ecological niches. Fungal genomes encode extremely variable “CYPomes” ranging from one to more than 300 CYPs. Despite the rapid growth of sequenced fungal and oomycete genomes and the resulting influx of predicted CYPs, the vast majority of CYPs remain functionally uncharacterized. To facilitate the curation and functional and evolutionary studies of CYPs, we previously developed Fungal Cytochrome P450 Database (FCPD), which included CYPs from 70 fungal and oomycete species. Here we present a new version of FCPD (1.2) with more data and an improved classification scheme. Results The new database contains 22,940 CYPs from 213 species divided into 2,579 clusters and 115 clans. By optimizing the clustering pipeline, we were able to uncover 36 novel clans and to assign 153 orphan CYP families to specific clans. To augment their functional annotation, CYP clusters were mapped to David Nelson’s P450 databases, which archive a total of 12,500 manually curated CYPs. Additionally, over 150 clusters were functionally classified based on sequence similarity to experimentally characterized CYPs. Comparative analysis of fungal and oomycete CYPomes revealed cases of both extreme expansion and contraction. The most dramatic expansions in fungi were observed in clans CYP58 and CYP68 (Pezizomycotina), clans CYP5150 and CYP63 (Agaricomycotina), and family CYP509 (Mucoromycotina). Although much of the extraordinary diversity of the pan-fungal CYPome can be attributed to gene duplication and adaptive divergence, our analysis also suggests a few potential horizontal gene transfer events. Updated families and clans can be accessed through the new version of the FCPD database. Conclusions FCPD version 1.2 provides a systematic and searchable catalogue of 9,550 fungal CYP sequences (292 families) encoded by 108 fungal species and 147 CYP sequences (9 families) encoded by five oomycete species. In comparison to the first version, it offers a more comprehensive clan classification, is fully compatible with Nelson’s P450 databases, and has expanded functional categorization. These features will facilitate functional annotation and classification of CYPs encoded by newly sequenced fungal and oomycete genomes. Additionally, the classification system will aid in studying the roles of CYPs in the evolution of fungal adaptation to specific ecological niches. PMID:23033934

2012-01-01

309

Enzymes involved in the metabolism of the carcinogen 2-nitroanisole: evidence for its oxidative detoxication by human cytochromes P450.  

PubMed

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Determining the capability of humans to metabolize 2-NA and understanding which human cytochrome P450 (P450) enzymes are involved in its activation and/or detoxification are important to assess an individual's susceptibility to this environmental carcinogen. We compared the ability of hepatic microsomal samples from different species including human to metabolize 2-NA. Comparison between experimental animals and human P450 enzymes is essential for the extrapolation of animal carcinogenicity data to assess human health risk. Human hepatic microsomes generated a pattern of 2-NA metabolites, reproducing that formed by hepatic microsomes of rats and rabbits. An O-demethylated metabolite of 2-NA (2-nitrophenol) and two ring-oxidized derivatives of this metabolite (2,6-dihydroxynitrobenzene and 2,X-dihydroxynitrobenzene) were produced. No nitroreductive metabolism leading to the formation of o-anisidine was evident with hepatic microsomes of any species. Likewise, no DNA binding of 2-NA metabolite(s) measured with either tritium-labeled 2-NA or the (32)P-postlabeling technique was detectable in microsomes. Therefore, hepatic microsomal P450 enzymes participate in the detoxication reactions of this environmental carcinogen. Using hepatic microsomes of rabbits pretreated with specific P450 inducers, microsomes from Baculovirus transfected insect cells expressing recombinant human P450 enzymes, purified P450 enzymes, and selective P450 inhibitors, we found that human recombinant P450 2E1, 1A1, and 2B6, as well as orthologous rodent P450 enzymes, are the most efficient enzymes metabolizing 2-NA. The role of specific P450 enzymes in the metabolism of 2-NA in human hepatic microsomes was investigated by correlating specific P450-dependent reactions with the levels of 2-NA metabolites formed by the same microsomes and by examining the effects of specific inhibitors of P450 enzymes on 2-NA metabolism. On the basis of these studies, we attribute most of the 2-NA oxidation metabolism in human microsomes to P450 2E1. These results, the first report on the metabolism of 2-NA by human P450 enzymes, clearly demonstrate that P450 2E1 is the major human enzyme oxidizing this carcinogen in human liver. PMID:15144223

Miksanová, Markéta; Sulc, Miroslav; Rýdlová, Helena; Schmeiser, Heinz H; Frei, Eva; Stiborová, Marie

2004-05-01

310

Cytochrome P-450scc a review of the specificity and properties of the cholesterol binding site.  

PubMed

Cytochrome P-450scc is unusual among members of this class of enzymes in showing a high degree of substrate specificity. Features of the cholesterol structure which are particularly important for binding include the 3 beta-hydroxyl, the delta 5-ring configuration, and the side-chain organization in the 20-22 region. Regarding the ring system, binding appears to require planarity and limited size at the 4-5-6 carbons (the A-B ring juncture). In the region of the 3 beta-hydroxyl, a "cleft" in the binding site extends about 4 A beyond the hydroxyl and can accommodate two additional ether-linked carbons. Evidence indicates that an enzyme residue hydrogen-bonds to the oxygen of the 3 beta hydroxyl, providing much of the energy for the initial enzyme-substrate interaction. The cytochrome shows less specificity for the side-chain structure, except in the region of carbons 20-22 where hydroxylation/side-chain cleavage takes place. The binding cleft for the side-chain is limited to approximately the length of the isocaproic group but can accommodate structural variations beyond the 22-position. Evidence indicates that the region near the 20-22 bond is more limited in size, and that an amino acid residue near the heme iron binds strongly and stereospecifically to the 22R-hydroxyl of the cleavage intermediates, 22R-hydroxycholesterol and 20 alpha, 22R-dihydroxycholesterol. The 22R-hydrogen of cholesterol is very close to the heme iron (approximately 3 A), while the 22S-hydrogen is slightly further (about 4 A). The size and bonding properties of the steroid binding/active site suggest a mechanism which accounts for the stereospecificity and sequence of reactions catalyzed by cytochrome P-450scc. PMID:3549273

Lambeth, J D

1986-01-01

311

Cj1411c Encodes for a Cytochrome P450 Involved in Campylobacter jejuni 81-176 Pathogenicity  

PubMed Central

Cytochrome P450s are b-heme-containing enzymes that are able to introduce oxygen atoms into a wide variety of organic substrates. They are extremely widespread in nature having diverse functions at both biochemical and physiological level. The genome of C. jejuni 81-176 encodes a single cytochrome P450 (Cj1411c) that has no close homologues. Cj1411c is unusual in its genomic location within a cluster involved in the biosynthesis of outer surface structures. Here we show that E. coli expressed and affinity-purified C. jejuni cytochrome P450 is lipophilic, containing one equivalent Cys-ligated heme. Immunoblotting confirmed the association of cytochrome P450 with membrane fractions. A Cj1411c deletion mutant had significantly reduced ability to infect human cells and was less able to survive following exposure to human serum when compared to the wild type strain. Phenotypically following staining with Alcian blue, we show that a Cj1411c deletion mutant produces significantly less capsular polysaccharide. This study describes the first known membrane-bound bacterial cytochrome P450 and its involvement in Campylobacter virulence. PMID:24086558

Alvarez, Luis A. J.; Bourke, Billy; Pircalabioru, Gratiela; Georgiev, Atanas Y.; Knaus, Ulla G.; Daff, Simon; Corcionivoschi, Nicolae

2013-01-01

312

HPLC determination of caffeine and paraxanthine in urine: An assay for cytochrome P450 1A2 activity.  

PubMed

Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among individuals and, therefore, so does drug efficacy as well as susceptibility to side effects and toxicity. Thus, assessing P450 activity is of great interest in drug development and clinical pharmacology. This study investigates the phenotyping of a single P450 activity by analyzing urine samples using isocratic reverse-phase HPLC. Specifically, the activity of human P450 1A2, which converts caffeine into paraxanthine, can be investigated by measuring the change in caffeine and paraxanthine concentrations in urine over time following a single dose of caffeine. There is an observable relationship between caffeine intake and paraxanthine formation that varies among individuals. This laboratory exercise provides a means for simple assessment of P450 1A2 metabolic activity using an HPLC method without additional extraction or purification steps and introduces students to the complexities of individualized medicine as well as the basic biochemical techniques of sample preparation and quantitative HPLC. Furthermore, students may design and test their own hypothesis using these methods. PMID:21591074

Furge, Laura Lowe; Fletke, Kyle J

2007-03-01

313

Reconstitution of the Fatty Acid Hydroxylase Activity of Cytochrome P450BM3 Utilizing Its Functional Domains  

Microsoft Academic Search

Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase fromBacillus megaterium,is a multidomain protein containing heme, FAD, and FMN. Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme

Irina Sevrioukova; Gilles Truan; Julian A. Peterson

1997-01-01

314

Regulation of an insect cytochrome P450 monooxygenase gene ( CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades  

Microsoft Academic Search

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site.

Rebecca Petersen Brown; Cynthia M. McDonnell; May R. Berenbaum; Mary A. Schuler

2005-01-01

315

Structure, Genetic Mapping, and Function of the Cytochrome P450 3A37 Gene in the Turkey (Meleagris gallopavo)  

Microsoft Academic Search

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity

S. Rawal; K. M. Mendoza; K. M. Reed

2009-01-01

316

Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1  

PubMed Central

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min?1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 ?M indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the O? of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, ?1/?2 loop and ?4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. PMID:23276288

Zhang, Haoming; Gay, Sean C.; Shah, Manish; Foroozesh, Maryam; Liu, Jiawang; Osawa, Yoichi; Zhang, Qinghai; Stout, C. David; Halpert, James R.; Hollenberg, Paul F.

2013-01-01

317

Selective Filling of Nanowells in Nanowell Arrays Fabricated Using Polystyrene Nanosphere Lithography with Cytochrome P450 Enzymes  

PubMed Central

This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned-array fabrication methods, while ensuring the protein retains the normal biological activity. Nanosphere-lithography was used to fabricate a nanowell array with nanowells that were 100 nm in diameter and a periodicity of 500 nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high throughput screening assays or as nanoelectrode arrays. PMID:22947619

Wollenberg, Lance A.; Jett, John E.; Wu, Yueting; Flora, Darcy R.; Wu, Nianqiang; Tracy, Timothy S.; Gannett, Peter M.

2012-01-01

318

Selective filling of nanowells in nanowell arrays fabricated using polystyrene nanosphere lithography with cytochrome P450 enzymes.  

PubMed

This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned array fabrication methods, while ensuring the protein retains normal biological activity. Nanosphere lithography was used to fabricate a nanowell array with nanowells 100 nm in diameter with a periodicity of 500 nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high-throughput screening assays or as nanoelectrode arrays. PMID:22947619

Wollenberg, Lance A; Jett, John E; Wu, Yueting; Flora, Darcy R; Wu, Nianqiang; Tracy, Timothy S; Gannett, Peter M

2012-09-28

319

Selective filling of nanowells in nanowell arrays fabricated using polystyrene nanosphere lithography with cytochrome P450 enzymes  

NASA Astrophysics Data System (ADS)

This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned array fabrication methods, while ensuring the protein retains normal biological activity. Nanosphere lithography was used to fabricate a nanowell array with nanowells 100 nm in diameter with a periodicity of 500 nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high-throughput screening assays or as nanoelectrode arrays.

Wollenberg, Lance A.; Jett, John E.; Wu, Yueting; Flora, Darcy R.; Wu, Nianqiang; Tracy, Timothy S.; Gannett, Peter M.

2012-09-01

320

Fusion protein bilayer fabrication composed of recombinant azurin/cytochrome P450 by the sortase-mediated ligation method.  

PubMed

Recently, the fabrication of protein bilayer has been required for the development of protein or enzyme complex formation. In the present study, we fabricated a fusion protein bilayer composed of recombinant azurin-cytochrome P450, which was synthesized by a site-specific sortase-mediated ligation method. The Pseudomonas aeruginosa azurin was modified by DNA recombinant technique, for enzymatic ligation and immobilization. The Pseudomonas putida cytochrome P450 was also modified for enzymatic ligation. The recombinant metalloproteins were conjugated via the sortase A. The conjugation was confirmed by SDS-PAGE and UV-vis. Then, the prepared fusion protein was immobilized on Au substrate, by the self-assembly method. The Azu-P450 (recombinant azurin-cytochrome P450) fusion protein layer was confirmed by AFM (Atomic Force Microscopy) and SERS (Surface-enhanced Raman Spectroscopy), to confirm the fusion protein bilayer orientation. Moreover, the electrochemical property of Azu-P450 was observed by cyclic voltammetry (CV). As a result, the Azu-P450 fusion protein bilayer shows good orientation on the Au substrate. Also, the original redox property of this fusion protein bilayer has been well maintained. The proposed fusion protein bilayer can. PMID:24924834

Lee, Taek; Min, Junhong; Hirakawa, Hidehiko; Nagamune, Teruyuki; Choi, Jeong-Woo

2014-08-01

321

Evaluation of inhibition selectivity for human cytochrome P450 2A enzymes.  

PubMed

Cytochrome P450 (P450) enzymes are mixed-function oxidases that catalyze the metabolism of xenobiotics and endogenous biochemicals. Selective inhibitors are needed to accurately distinguish the contributions of individual P450 enzymes in the metabolism of drugs and the activation of procarcinogens in human tissues, but very frequently these enzymes have substantial overlapping selectivity. We evaluated a chemically diverse set of nine previously identified CYP2A6 inhibitors to determine which are able to discriminate between human CYP2A enzymes CYP2A6 and the 94%-identical CYP2A13 enzyme. Inhibitor binding to recombinant purified enzyme was evaluated, and affinities were determined. K(i) values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (k(cat)/K(m) 0.19 and 0.12 ?M?¹ · min?¹, respectively) than hydroxylation of the classic substrate coumarin (0.11 and 0.53 ?M?¹ · min?¹, respectively). Of the nine compounds assayed, only tranylcypromine and (R)-(+)-menthofuran had a greater than 10-fold preference for CYP2A6 inhibition versus CYP2A13 inhibition. Most compounds evaluated [tryptamine, 4-dimethylaminobenzaldehyde, phenethyl isothiocyanate, ?-nicotyrine, (S)-nicotine, and pilocarpine] demonstrated only moderate or no preference for inhibition of one CYP2A enzyme over the other. However, 8-methoxypsoralen has a 6-fold lower K(i) for CYP2A13 than for CYP2A6. This information is useful to inform reinterpretation of previous data with these inhibitors and to guide future studies seeking to determine which human CYP2A enzyme is responsible for the in vivo metabolism of compounds in human tissues expressing both enzymes. PMID:22696418

Stephens, Eva S; Walsh, Agnes A; Scott, Emily E

2012-09-01

322

Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes  

PubMed Central

The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11?-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11?-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11?-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH for several adrenal P450 enzymes in the steroid pathway. Although the reduced pyridine nucleotides are produced intra-luminally, these compounds may also slowly transverse the endoplasmic reticulum membrane by unknown mechanisms. PMID:23665046

Foster, Christy A.; Mick, Gail J.; Wang, Xudong; McCormick, Kenneth

2014-01-01

323

Effects of Cytochrome P450 2C9 Polymorphism on Bosentan Metabolism.  

PubMed

Cytochrome P450 (P450) 2C9 is an important member of the P450 enzyme superfamily, with 58 CYP2C9 allelic variants previously reported. Genetic polymorphisms of CYP2C9 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to assess the catalytic activities of 38 human CYP2C9 alleles, including 24 novel alleles (*36-*60) found in the Han Chinese population, toward bosentan (BOS) in vitro. Insect microsomes expressing the 38 CYP2C9 alleles were incubated with 10-625 ?M bosentan for 30 minutes at 37°C and terminated by cooling to -80°C immediately. BOS and hydroxyl bosentan, the major metabolite of BOS, were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry system. Thirty-eight defective alleles can be classified into three categories according to the relative clearance value compared with wild type: nine alleles exhibited significantly increased intrinsic clearance values (Vmax/Km) compared with the wild type (1.5-fold-?4.9-fold relative clearance); nine alleles exhibited significantly reduced intrinsic clearance values compared with the wild type (0.6-28.9% relative clearance). The remaining 20 alleles exhibited no significant difference (1-fold) in enzyme activity compared with the wild type. These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2C9 alleles when administering BOS in the clinic. This is the first report of all these rare alleles for BOS metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles. PMID:25142737

Chen, Mengchun; Zhang, Youting; Pan, Peipei; Wang, Li; Zhan, Yunyun; Jin, Hui; Xia, Mengmin; Wang, Xianqin; Dai, Dapeng; Cai, Jianping; Hu, Guoxin

2014-11-01

324

Key Residues Controlling Phenacetin Metabolism By Human Cytochrome P450 2A Enzymes  

SciTech Connect

Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K{sub D}) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2{prime}-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K{sub D} values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2{prime}-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structure-based quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu{sup 370}.

DeVore, N.M.; Smith, B.D.; Urban, M.J.; Scott, E.E.

2009-05-14

325

Aromatase gene expression in the stallion  

Microsoft Academic Search

Adult stallion secretes very high estrogen levels in its testicular vein and semen, and the responsible enzyme cytochrome P450 aromatase (P450 arom) is known to be present mainly in Leydig cells. We studied in further details the distribution of equine aromatase in various adult tissues including the brain (hypothalamic area), liver, kidney, small intestine, muscle, bulbourethral gland and testes. The

Emmanuel Lemazurier; Pascal Sourdaine; Céline Nativelle; Bruno Plainfossé; Gilles-Eric Séralini

2001-01-01

326

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana  

PubMed Central

It was previously proposed that regio-specific hydroxylation of an immunosuppressive cyclosporine (CsA) at the 4th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycete Sebekia benihana. This modification is thought to be the reason for the hair growth-promoting side effect without the immunosuppressive activity of CsA. Through S. benihana genome sequencing and in silico analysis, we identified the complete cytochrome P450 complement (CYPome) of S. benihana, including 21 CYPs and their electron transfer partners, consisting of 7 ferredoxins (FDs) and 4 ferredoxin reductases (FDRs). Using Escherichia coli conjugation-based S. benihana CYPome-targeted disruption, all of the identified CYP, FD, and FDR genes in S. benihana were individually inactivated. Among the 32 S. benihana exconjugant mutants tested, only a single S. benihana CYP mutant, ?CYP-sb21, failed to exhibit CsA hydroxylation activity. The hydroxylation was restored by CYP-sb21 gene complementation. Since all S. benihana FD and FDR disruption mutants maintained CsA hydroxylation activity, it can be concluded that CYP-sb21, a new member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific CsA hydroxylation process in S. benihana. Moreover, expression of an extra copy of the CYP-sb21 gene increased CsA hydroxylation in wild-type S. benihana and an NADPH-enriched Streptomyces coelicolor mutant, by 2-fold and 1.5-fold, respectively. These results show for the first time that regio-specific hydroxylation of CsA is carried out by a specific P450 hydroxylase present in S. benihana, and they set the stage for the biotechnological application of regio-specific CsA hydroxylation through heterologous CYP-sb21 expression. PMID:23354713

Lee, Mi-Jin; Kim, Hyun-Bum; Yoon, Yeo Joon; Han, Kyuboem

2013-01-01

327

Ronald Estabrook's early guidance of a postdoctoral fellow concerning the intricacies of steroid metabolism by cytochromes P450.  

PubMed

Ronald Estabrook made his initial impact studying cytochrome P450 by demonstrating the oxidative metabolism function of this unique class of enzymes, which had an unusual spectral peak at 450 nm when reduced and in the presence of carbon monoxide. Utilizing a photochemical action spectrum, he demonstrated that a cytochrome P450 was responsible for steroid 21 hydroxylation catalyzed by microsomes prepared from adrenal cortex tissue. As a young postdoctoral student, I was given the unique opportunity to learn from a true pioneer in this field. Ron had a surprisingly small laboratory at that time that allowed me to closely interact with a great scientist to learn about the important role cytochrome P450 proteins play in a wide variety of different organisms catalyzing oxidative metabolism reactions essential to life and to provide organisms, with the means to defend against xenobiotics. PMID:17786620

Sheets, Joel J

2007-01-01

328

Regio- and stereochemical studies on the alpha-carbon oxidation of (S)-nicotine by cytochrome P-450 model systems.  

PubMed

Results from previous studies indicate that rabbit liver microsomal cytochrome P-450 catalyzes the C-5' two-electron oxidation of (S)-nicotine stereoselectivity with preferential loss of the pro-(E)-hydrogen atom trans to the pyridine ring. We now have examined the regio- and stereochemical features of the oxidation of (S)-nicotine by peroxides in the presence of various hemoproteins and by electrochemical and photochemical methods. None of these systems gave rise to the stereochemical outcomes observed with the cytochrome P-450 mediated reaction. The results of these studies are interpreted as additional evidence for the formation of a highly ordered complex between (S)-nicotine and cytochrome P-450 that directs the regio- and diasterioselective alpha-carbon oxidation of this substrate. PMID:3346880

Peterson, L A; Castagnoli, N

1988-03-01

329

Significance of Cytochrome P450 System Responses and Levels of Bile Fluorescent Aromatic Compounds in Marine Wildlife Following Oil Spills  

SciTech Connect

The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 in marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a linkage to higher order biological effects, e.g. toxicity, lesions, reproductive failure.

Lee, Richard F.; Anderson, Jack W.

2005-07-01

330

Glutathione depletion by aniline analogs in vitro associated with liver microsomal cytochrome P-450.  

PubMed

Enzymic depletion of glutathione (GSH) in vitro by aniline analogs was mostly dependent on the cytochrome P-450 level in liver microsomes. In a case of acetaminophen (AAP), active metabolite of AAP formed through liver microsomal drug metabolizing enzymes consumed GSH. The active metabolite formed binds, at least in part, covalently to liver microsomal proteins. In addition, species differences in the extent of GSH depletion by AAP in vitro was related to the amounts of the active metabolite of AAP bound covalently to liver microsomal protein(s) by experiments using 14C-AAP. Similar depletion of GSH was also seen with other aniline analogs such as aniline itself and p-chloroaniline, but not with acetanilide, in four animal species. These in vitro results obtained here strongly support the well-known findings concerning both GSH depletion and covalent binding in vivo of the active metabolite after AAP treatment. PMID:722999

Aikawa, K; Satoh, T; Kobayashi, K; Kitagawa, H

1978-10-01

331

Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cama, b  

PubMed Central

Residual dipolar couplings were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450cam (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The 1DNH residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site, and largely support previously reported differences between the open and closed states of CYP101A1. PMID:21265500

Asciutto, Eliana K.; Dang, Marina; Pochapsky, Susan Sondej; Madura, Jeffry D.; Pochapsky, Thomas C.

2011-01-01

332

Advances in the electrochemical simulation of oxidation reactions mediated by cytochrome p450.  

PubMed

Combining electrochemistry with mass spectrometry constitutes an increasingly useful approach for simulating reactions catalyzed by cytochrome P450 (CYP450). In this review, we discuss the ability of the electrochemical cell to act as a reliable tool to mimic CYP450. The electrochemical oxidation process and CYP450-catalyzed reactions are compared in terms of mechanistic pathways, chemical structures of reactive intermediate metabolites, and final chemical structures of oxidation products. The oxidation reactions mediated by CYP450 are known to occur by either a single electron transfer (SET) or a hydrogen atom transfer (HAT) mechanism. The similarities between the reactions mediated electrochemically or by CYP450 are discussed in terms of SET and HAT mechanisms. PMID:25285807

Bussy, Ugo; Boujtita, Mohammed

2014-10-20

333

Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis  

PubMed Central

Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(?)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound. PMID:24688524

Ishikawa, Noemia Kazue; Tahara, Satoshi; Namatame, Tomohiro; Farooq, Afgan; Fukushi, Yukiharu

2013-01-01

334

Whole-cell biotransformation with recombinant cytochrome P450 for the selective oxidation of Grundmann's ketone.  

PubMed

25-Hydroxy-Grundmann's ketone is a key building block in the chemical synthesis of vitamin D3 and its derivatives through convergent routes. Generally, the chemical synthesis of this compound involves tedious procedures and results in a mixture of several products. Recently, the selective hydroxylation of Grundmann's ketone at position C25 by cytochrome P450 (CYP) 154E1 from Thermobifida fusca YX was described. In this study a recombinant whole-cell biocatalyst was developed and applied for hydroxylation of Grundmann's ketone. Biotransformation was performed by Escherichia coli cells expressing CYP154E1 along with two redox partner systems, Pdx/PdR and YkuN/FdR. The system comprising CYP154E1/Pdx/PdR showed the highest production of 25-hydroxy-Grundmann's ketone and resulted in 1.1mM (300mgL(-1)) product concentration. PMID:25023538

Hernández-Martín, Alba; von Bühler, Clemens J; Tieves, Florian; Fernández, Susana; Ferrero, Miguel; Urlacher, Vlada B

2014-10-15

335

P-Glycoprotein- and cytochrome P-450-mediated herbal drug interactions.  

PubMed

P-Glycoprotein (P-gp), the most extensively studied ATP-binding cassette transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of cells. Drug efflux pumps such as P-gp play a functional role in determining the pharmacokinetics of drugs administered by oral and parenteral routes. Determining the activity of drug efflux transport proteins has important implications in the identification of substrates and/or inhibitors. The significant role of the small intestine in reducing the oral bioavailability of drugs is due to metabolic enzymes and efflux transporters. The role of cytochrome P-450 3A (CYP3A) and P-gp in intestinal drug disposition has been highlighted. This review examines the structure, localisation and functional role of P-gp, the mechanism of drug efflux and drug-herb interactions. PMID:21417789

Kumar, Yamsani Shravan; Adukondalu, Devandla; Sathish, Dharani; Vishnu, Yamsani Vamshi; Ramesh, Gannu; Latha, Athukuri Bharagavi; Reddy, Palem Chinna; Sarangapani, Manda; Rao, Yamsani Madhusudan

2010-01-01

336

Cytochrome P450 119 Compounds I formed by chemical oxidation and photooxidation are the same species  

PubMed Central

Compound I from cytochrome P450 119 prepared by the photooxidation method involving peroxynitrite oxidation of the resting enzyme to Compound II followed by photooxidation to Compound I was compared to Compound I generated by m-chloroperoxybenzoic acid (MCPBA) oxidation of the resting enzyme. The two methods gave the same UV-visible spectra, the same products from oxidations of lauric acid and palmitic acid and their (?-2,?-2,?-3,?-3)-tetradeuterated analogues, and the same kinetics for oxidations of lauric acid and caprylic acid. The experimental identities between the transients produced by the two methods leave no doubt that the same Compound I speices is formed by the two methods. PMID:23108625

Su, Zhi; Horner, John H.

2014-01-01

337

Effects of genetic polymorphism of cytochrome P450 enzymes on the pharmacokinetics of benzodiazepines.  

PubMed

Pharmacogenetic studies have shown that several cytochrome P450 (CYP) enzymes exhibit genetic polymorphisms. Several benzodiazepines (BZPs) are metabolized predominantly or partly by polymorphic CYP2C19 and CYP3A4/5. The pharmacokinetics of diazepam, etizolam, quazepam and desmethylclobazam have been shown to be affected by CYP2C19 polymorphism. The CYP3A5 polymorphism has been reported to affect the pharmacokinetics of alprazolam, but its effect on midazolam kinetics has been inconclusive. For etizolam and desmethylclobazam, some data suggest that CYP2C19 deficiency leads to side-effects or toxicity. For the remaining BZPs the clinical significance of the observed pharmacokinetic changes remains unclear. Further studies on the effects of genetic polymorphisms of CYP enzymes on the pharmacokinetics and pharmacodynamics of BZPs are necessary to guide treatment individualization and optimization. PMID:17635335

Fukasawa, T; Suzuki, A; Otani, K

2007-08-01

338

Ligand-Based Site of Metabolism Prediction for Cytochrome P450 2D6  

PubMed Central

A ligand-based method based on the SMARTCyp approach that predicts the sites of cytochrome P450 2D6-mediated metabolism of druglike molecules has been developed. The method uses only two descriptors besides the reactivity from SMARTCyp: the distance to a protonated nitrogen atom and the distance to the end of the molecule. Hence, the site of metabolism is predicted directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. Testing on an independent test set gives an area under the curve value of 0.94, and a site of metabolism is found among the top two ranked atoms for 91% of the compounds. PMID:24900373

2011-01-01

339

Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations.  

PubMed

Cytochromes P450 (CYPs) belong to the superfamily of heme b containing monooxygenases with currently more than 21,000 members. These enzymes accept a vast range of organic molecules and catalyze diverse reactions. These extraordinary capabilities of CYP systems that are unmet by other enzymes make them attractive for biotechnology. However, the complexity of these systems due to the need of electron transfer from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) via redox partner proteins for the initial hydroxylation step limits a broader technical implementation of CYP enzymes. There have been several reviews during the past years tackling the potential CYPs for synthetic application. The aim of this review is to give a critical overview about possibilities and chances for application of these interesting catalysts as well as to discuss drawbacks and problems related to their use. Solutions to overcome these limitations will be demonstrated, and several selected examples of successful CYP applications under industrial conditions will be reviewed. PMID:24848420

Bernhardt, Rita; Urlacher, Vlada B

2014-07-01

340

5-HT3-receptor antagonists and the cytochrome P450 system: clinical implications.  

PubMed

Many patients with cancer receive multiple chemotherapy agents as well as other medications for coexisting medical conditions. Despite the introduction of 5-HT3 receptor antagonists, the management of nausea and vomiting following cancer treatment and after cancer surgery remains complex, particularly when patients are receiving multiple prescription medications. As a drug class, the 5-HT3 receptor antagonists have good antiemetic efficacy and an improved safety profile over conventional antiemetics. Nevertheless, pharmacologic differences exist between these agents, such as their interaction with the metabolic cytochrome P450 system. This review examines the major metabolic differences between the most frequently prescribed 5-HT3 receptor antagonists, dolasetron, granisetron, ondansetron, and tropisetron. The potential drug interactions that these differences may precipitate and key genetic interindividual variations in drug metabolism are also considered. To avoid or minimize potential drug interactions, the 5-HT3 receptor antagonist with the lowest risk of these interactions should be considered as first choice. PMID:12416899

Blower, Peter R

2002-01-01

341

Cryoradiolysis and cryospectroscopy for studies of heme-oxygen intermediates in cytochromes P450  

PubMed Central

Cryogenic radiolytic reduction is one of the most simple and convenient methods of generation and stabilization of reactive iron-oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxoferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases. PMID:22573452

Denisov, I.G.; Grinkova, Y.V.; Sligar, S.G.

2014-01-01

342

Ab Initio Modeling of Intermediate States in the Cytochrome P450 Catalytic Cycle  

NASA Astrophysics Data System (ADS)

First principles electronic structure calculations, based on Density Functional Theory, have previously been used to model the interaction between the active site of a Cytochrome P450 enzyme and ligand molecules (Segall et al., Xenobiotica (in press);. Segall et al., Phy. Rev. E. (submitted)). These enzymes are of great importance due to their participation in the metabolism of a wide range of endogenous and xenobiotic compounds. We apply the same techniques to the calculation of the energy change due to the first reduction of the active site system, finding an excellent correlation with experimental observations of the redox potential. This allows the study of the oxygen-bound active site complex, the structure of which has not been observed experimentally due to the short-lived nature of this state. The calculations were performed using the CETEP code on 64 nodes of an Hitachi SR2201 parallel supercomputer.

Segall, Matthew; Payne, Mike; Ellis, Wynne; Tucker, Geoff

1998-03-01

343

Gravity persistent signal 1 reveals a novel cytochrome P450 involved in gravitropic signal transduction  

NASA Astrophysics Data System (ADS)

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)

Wyatt, Sarah

344

Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency  

SciTech Connect

The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single (/sup 125/I)-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme.

Meier, U.T.; Meyer, U.A.

1987-12-15

345

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450  

PubMed Central

Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-01-01

346

Effect of Crude Extract of Eugenia jambolana Lam. on Human Cytochrome P450 Enzymes.  

PubMed

The fruit of Eugenia jambolana Lam. is very popular for its anti-diabetic property. Previous studies on the crude extract of E.?jambolana (EJE) have successfully explored the scientific basis for some of its traditional medicinal uses. Considering its wide use and consumption as a seasonal fruit, the present study investigates the ability of E.?jambolana to interact with cytochrome P450 enzymes. The standardized EJE was incubated with pooled human liver microsomes to assess the CYP2C9-, CYP2D6-, and CYP3A4-mediated metabolism of diclofenac, dextromethorphan, and testosterone, respectively. The metabolites formed after the enzymatic reactions were quantified by high performance liquid chromatography. EJE showed differential effect on cytochrome P450 activities with an order of inhibitory potential as CYP2C9?>?CYP3A4?>?CYP2D6 having IC50 of 76.69, 359.02, and 493.05?µg/mL, respectively. The selectivity of EJE for CYP2C9 rather than CYP3A4 and CYP2D6 led to perform the enzyme kinetics to explicate the mechanism underlying the inhibition of CYP2C9-mediated diclofenac 4'-hydroxylation. EJE was notably potent in inhibiting the reaction in a non-competitive manner with Ki of 84.85?±?5.27?µg/mL. The results revealed the CYP2C9 inhibitory potential of EJE with lower Ki value suggesting that EJE should be examined for its potential pharmacokinetic and pharmacodynamic interactions when concomitantly administered with other drugs. Copyright © 2014 John Wiley & Sons, Ltd. PMID:24590863

Chinni, Santhivardhan; Dubala, Anil; Kosaraju, Jayasankar; Khatwal, Rizwan Basha; Satish Kumar, M N; Kannan, Elango

2014-11-01

347

Genetic polymorphisms and haplotypes of por, encoding cytochrome p450 oxidoreductase, in a Japanese population.  

PubMed

Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to all microsomal cytochrome P450 (CYP) enzymes and is necessary for microsomal CYP activities. In this study, to find genetic variations and to elucidate the haplotype structures of POR, we comprehensively screened the genetic variations in the 5'-flanking region, all the exons and their flanking introns of POR for 235 Japanese subjects. Seventy-five genetic variations including 26 novel ones were found: 7 were in the 5'-flanking region, 2 in the 5'-untranslated region (5'-UTR, non-coding exon 1), 16 in the coding exons (10 nonsynonymous and 6 synonymous), 45 in the introns, 4 in the 3'-UTR and 1 in the 3'-flanking region. Of these, 4 novel nonsynonymous variations, 86C>T (T29M), 1648C>T (R550W), 1708C>T (R570C) and 1975G>A (A659T), were detected with allele frequencies of 0.002. We also detected known nonsynonymous SNPs 683C>T (P228L), 1237G>A (G413S), 1453G>A (A485T), 1508C>T (A503V), 1510G>A (G504R) and 1738G>C (E580Q) with frequencies of 0.002, 0.009, 0.002, 0.434, 0.002 and 0.002, respectively. Based on the linkage disequilibrium (LD) profiles, the analyzed region could be divided into two LD blocks. For Blocks 1 and 2, 14 and 46 haplotypes were inferred, respectively, and 2 and 6 common haplotypes found in more than 0.03 frequencies accounted for more than 81% of the inferred haplotypes. This study provides fundamental and useful information for the pharmacogenetic studies of drugs metabolized by CYPs in the Japanese population. PMID:21084761

Saito, Yoshiro; Yamamoto, Noboru; Katori, Noriko; Maekawa, Keiko; Fukushima-Uesaka, Hiromi; Sugimoto, Daisuke; Kurose, Kouichi; Sai, Kimie; Kaniwa, Nahoko; Sawada, Jun-Ichi; Kunitoh, Hideo; Ohe, Yuichiro; Yoshida, Teruhiko; Matsumura, Yasuhiro; Saijo, Nagahiro; Okuda, Haruhiro; Tamura, Tomohide

2011-01-01

348

Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism.  

PubMed Central

In experimental animals, injection of gram-negative endotoxin (LPS) decreases hepatic cytochrome P450-mediated drug metabolism. To evaluate this phenomenon in a human model of gram-negative sepsis, LPS was administered on two consecutive days to healthy male volunteers during which time a cocktail of antipyrine (AP-250 mg), hexobarbital (HB-500 mg), and theophylline (TH-150 mg) was ingested and the apparent oral clearance of each drug determined. Each subject had a control drug clearance study with saline injections. In the first experiment, six subjects received the drug cocktail 0.5 h after the first dose of LPS. In the second experiment, another six subjects received the drug cocktail 0.5 h after the second dose of LPS. In both experiments, LPS caused the expected physiologic responses of inflammation including fever with increases in serum concentrations of TNF alpha, IL-1 beta, IL-6, and acute phase reactants. In the first experiment, only minor decreases in clearances of the probe drugs were observed (7-12%). However in the second experiment, marked decreases in the clearances of AP (35, 95% CI 18-48%), HB (27, 95% CI 14-34%), and TH (22, 95% CI 12-32%) were seen. The decreases in AP clearance correlated with initial peak values of TNF alpha (r = 0.82) and IL-6 (r = 0.86). These data show that in humans the inflammatory response to even a very low dose of LPS significantly decreases hepatic cytochrome P450-mediated drug metabolism and this effect evolves over a 24-h period. It is likely that septic patients with much higher exposures to LPS have more profound inhibition of drug metabolism. PMID:7989576

Shedlofsky, S I; Israel, B C; McClain, C J; Hill, D B; Blouin, R A

1994-01-01

349

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g  

SciTech Connect

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6..beta..-hydroxyprogesterone and 16..cap alpha..-hydroxyprogesterone), two secondary metabolites (6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16..cap alpha..-hydroxyprogesterone). The K/sub m,app/ for the formation of these products from progesterone was determined to be approximately 0.5 ..mu..M, while the K/sub m,app/ for metabolism of 6..beta..- and 16..cap alpha..-hydroxyprogesterone was found to be 5-10 ..mu..M. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 ..mu..M, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-keto-16..cap alpha..-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6..beta..-hydroxyprogesterone or 16..cap alpha..-hydroxyprogesterone. Incubation of 6..beta..-hydroxyprogesterone with a reconstituted system in an atmosphere of /sup 18/I/sub 2/ resulted in > 90% incorporation of /sup 18/O in the 16..cap alpha..-position of 6..beta..,16..cap alpha..-dihydroxyprogesterone but no incorporation of /sup 18/O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O/sub 2/, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.

Swinney, D.C.; Ryan, D.E.; Thomas, P.E.; Levin, W.

1988-07-26

350

Extracellular matrix and cytochrome P450 gene expression can distinguish steatohepatitis from steatosis in mice.  

PubMed

One of the main questions regarding nonalcoholic fatty liver disease is the molecular background of the transition from simple steatosis (SS) to the inflammatory and fibrogenic condition of steatohepatitis (NASH). We examined the gene expression changes during progression from histologically normal liver to SS and NASH in models of obesity caused by hyperphagia or a high-fat diet. Microarray-based analysis revealed that the expression of 1445 and 264 probe sets was changed exclusively in SS and NASH samples, respectively, and 1577 probe sets were commonly altered in SS and NASH samples. Functional annotations indicated that transcriptome alterations that were common for NASH and SS, as well as exclusive for NASH, involved extracellular matrix (ECM)-related processes, although they differed in the type of matrix structure change. The expression of 80 genes was significantly changed in all three comparisons: SS versus control, NASH versus control and NASH versus SS. Of these genes, epithelial membrane protein 1, IKBKB interacting protein and decorin were progressively up-regulated in both SS and NASH compared to normal tissue. The molecular context of interactions of encoded 80 proteins revealed that they are highly interconnected and significantly enriched for processes involving metabolism by cytochrome P450. Validation of 10 selected mRNAs encoding genes related to ECM and cytochrome P450 with quantitative RT-PCR analysis showed consistent changes in their expression during NASH development. The expression profile of these genes has the potential to distinguish NASH from SS and normal tissue and may possibly be beneficial in the clinical diagnosis of NASH. PMID:24913135

Hennig, Ewa E; Mikula, Michal; Goryca, Krzysztof; Paziewska, Agnieszka; Ledwon, Joanna; Nesteruk, Monika; Woszczynski, Marek; Walewska-Zielecka, Bozena; Pysniak, Kazimiera; Ostrowski, Jerzy

2014-09-01

351

Effect of age on hepatic cytochrome P450 of Ross 708 broiler chickens.  

PubMed

Age has significant impact on hepatic cytochrome P450 (CYP450) systems in animals. Ross 708 broiler chicken is a breed of chicken with fast growth characteristics. Cytochrome P450 in the livers of Ross 708 broiler chicken of different ages has been investigated. The birds were raised under standard husbandry conditions. A certain number of chickens was randomly sampled weekly for liver collection from d 1 to 56 posthatch. The chicken body and liver weights were recorded. The chicken livers were processed for liver microsomes though a multiple-step procedure at low temperature. Total CYP450 content in chicken liver homogenates and liver microsomes was measured using a UV/visible spectroscopic method. The enzymatic activities of CYP450 in the chicken liver microsomes were determined through incubation of CYP450 isoform substrates followed by measurement of formation of their metabolites. The chicken showed an opposite age pattern in hepatic CYP450 content and activities compared with most mammals. The hepatic CYP450 content and activities of chicken at d 1 posthatch were higher than at other ages. The total hepatic CYP450 content in chickens at d 1 posthatch was more than twice the average hepatic value of the chickens at d 7 to 28. This high CYP450 fell quickly in the first week posthatch and slightly rose from d 28 to 56. Hepatic CYP450 activities of CYP1A, 3A, 2C, 2D, and 2H were much higher in the chicken at d 1 posthatch. The differences of these enzymatic activities between d 1 and other ages of chicken were CYP450 isoform dependent. This result suggests that embryonic development of chicken livers has a significant impact on the age profile of hepatic CYP450 content and activities of posthatch chickens. PMID:23571338

Hu, S X

2013-05-01

352

Biosynthesis of Sandalwood Oil: Santalum album CYP76F Cytochromes P450 Produce Santalols and Bergamotol  

PubMed Central

Abstract Sandalwood oil is one of the world’s most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, ?-, ?-, and epi-?-santalol and ?-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of ?-, ?-, and epi-?-santalene and ?-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests. PMID:24324844

Diaz-Chavez, Maria L.; Moniodis, Jessie; Madilao, Lufiani L.; Jancsik, Sharon; Keeling, Christopher I.; Barbour, Elizabeth L.; Ghisalberti, Emilio L.; Plummer, Julie A.; Jones, Christopher G.; Bohlmann, Jorg

2013-01-01

353

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.) 1  

PubMed Central

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 ?g/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus. Images Figure 5 Figure 7 PMID:16653087

Vetter, Hans-Peter; Mangold, Ursula; Schroder, Gudrun; Marner, Franz-Josef; Werck-Reichhart, Danielle; Schroder, Joachim

1992-01-01

354

Oxy radical formation during redox cycling of the bleomycin-iron (III) complex by NADPH-cytochrome P-450 reductase.  

PubMed

Bleomycin was aerobically incubated with FeCl3, NADPH, isolated rat-liver microsomal cytochrome P-450 reductase and methional. The conversion of methional to ethene, which indicates oxy radicals, was determined. Ethene formation depended on oxygen, NADPH, FeCl3 and the enzyme. About equimolar concentrations of bleomycin and FeCl3 resulted in optimal ethene formation. Dimethyl sulfoxide, mannitol, glycerol, glutathione and glutathione disulfide inhibited ethene formation. These results indicate that oxy radicals are formed after reduction of the bleomycin-Fe-complex by NADPH-cytochrome P-450 reductase. PMID:2412562

Mahmutoglu, I; Kappus, H

1985-09-01

355

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella).  

PubMed

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b(5) on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions. PMID:21220011

Niu, Guodong; Rupasinghe, Sanjeewa G; Zangerl, Arthur R; Siegel, Joel P; Schuler, Mary A; Berenbaum, May R

2011-04-01

356

Molecular cloning and recombinant expression of cytochrome P450 CYP6B6 from Helicoverpa armigera in Escherichia coli.  

PubMed

The cytochrome P450 s play a significant role in the detoxification of plant allelochemicals and synthetic insecticides in Lepidoptera. In the cotton bollworm Helicoverpa armigera, 2-tridecanone and quercetin can induce P450-dependent monooxygenase activity increased, to further the characterization of P450, the CYP6B6 of cotton bollworm (H. armigera) was cloned, sequenced and expressed in pMAL-p2x vector and expressed in Escherichia coli. The deduced amino acid sequences of cytochrome P450 in the midgut and fat body of H. armigera showed 98.23 and 97.84 % similarity with CYP6B6, respectively. According to nomenclature of P450 s, the P450 genes we got belong to CYP6B. Purification of recombinant protein based on the affinity of MBP for maltose was achieved by Mal-Tag magnetic beads. The purified protein was used to raise polyclonal antibody according to classical procedure. SDS-PAGE and Western blot results indicated that MBP-CYP6B6 had been successfully expressed. The ethoxycoumarin-O-deethylase activity of the purified recombinant protein was 36.5 ± 8.12 pmol of 7-hydroxycoumarin/min/mg protein, which showed the fusion MBP-CYP6B6 had the ability to o-deethylase of 7-ethoxycoumarin. PMID:23096085

Liu, Xiaoning; Zhang, Lei; Zhang, Xuetao; Xiwu, Gao

2013-02-01

357

A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution.  

PubMed Central

Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant in the host plants of this specialized herbivore. On Northern blots, mRNAs crossreactive with CYP6B1 were detected in three Papilio species that, like the black swallowtail, have high levels of xanthotoxin-metabolic P-450 activity and encounter xanthotoxin or related compounds in their host plants; in contrast, no crossreactive mRNAs were detectable in three papilinid species that never encounter xanthotoxin in their host plants and lack detectable xanthotoxin-metabolic activity. These results provide evidence that new P-450s can arise as herbivores colonize different host plants and support the hypothesis that interactions between herbivores and their toxin-producing host plants have contributed to the diversification of the P-450 superfamily. Images PMID:1279697

Cohen, M B; Schuler, M A; Berenbaum, M R

1992-01-01

358

A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution.  

PubMed

Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant in the host plants of this specialized herbivore. On Northern blots, mRNAs crossreactive with CYP6B1 were detected in three Papilio species that, like the black swallowtail, have high levels of xanthotoxin-metabolic P-450 activity and encounter xanthotoxin or related compounds in their host plants; in contrast, no crossreactive mRNAs were detectable in three papilinid species that never encounter xanthotoxin in their host plants and lack detectable xanthotoxin-metabolic activity. These results provide evidence that new P-450s can arise as herbivores colonize different host plants and support the hypothesis that interactions between herbivores and their toxin-producing host plants have contributed to the diversification of the P-450 superfamily. PMID:1279697

Cohen, M B; Schuler, M A; Berenbaum, M R

1992-11-15

359

Affinity purification of recombinant human cytochrome P450s 3A4 and 1A2 using mixed micelle systems.  

PubMed

Recombinant cytochrome P450 (CYP or P450) enzymes are useful for drug metabolism research and thereby many expression and purification systems have been developed. Here, we provide a method for the purification of human P450s 3A4 and 1A2 expressed in Escherichia coli using mixed micelles containing anionic phospholipids. This method does not require any protein-tagging system for protein isolation and has a further advantage that the purification is concomitantly conducted with reconstitution of the enzymes into a phospholipid environment, which is crucial for the catalytic activity assay of P450 enzyme. This method may also be applied to high-throughput catalytic assays of the enzymes because the purification procedures can be undertaken in a 96-well plate. PMID:24893120

Ahn, Taeho; Bae, Chun-Sik; Yun, Chul-Ho

2014-09-01

360

Cyclopropylamine inactivation of cytochromes P450: Role of metabolic intermediate complexes  

E-print Network

hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5? (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to ?-nitrosotoluene as the precursor...

Cerny, Matthew A.; Hanzlik, Robert P.

2005-04-15

361

(+)-Abscisic Acid 8?-Hydroxylase Is a Cytochrome P450 Monooxygenase1  

PubMed Central

Abscisic acid (ABA) 8?-hydroxylase catalyzes the first step in the oxidative degradation of (+)-ABA. The development of a robust in vitro assay has now permitted detailed examination and characterization of this enzyme. Although several factors (buffer, cofactor, and source tissue) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevich [1997] J Exp Bot 48: 1787–1795). (+)-ABA 8?-hydroxylase is an integral membrane protein that is localized to the microsomal fraction in suspension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, although there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)-ABA was progressively inhibited at O2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N2. (+)-ABA 8?-hydroxylase activity was inhibited by tetcyclacis (50% inhibition at 10?6 m), cytochrome c (oxidized form), and CO. The CO inhibition was reversible by light from several regions of the visible spectrum, but most efficiently by blue and amber light. These data strongly support the contention that (+)-ABA 8?-hydroxylase is a cytochrome P450 monooxygenase. PMID:9808729

Krochko, Joan E.; Abrams, Garth D.; Loewen, Mary K.; Abrams, Suzanne R.; Cutler, Adrian J.

1998-01-01

362

Characterization of a cDNA Encoding P-450 aromatase (CYP19) from Japanese eel ovary and its expression in ovarian follicles during induced ovarian development  

Microsoft Academic Search

A cDNA encoding P450 aromatase (CYP19) was isolated from a Japanese eel (Anguilla japonica) ovarian cDNA library. This cDNA contains a complete open reading frame encoding 511 amino acid residues. The deduced amino acid sequence is 59% and 65% identical to the catfish and rainbow trout forms, respectively, and 52–54% to mammalian and chicken forms. Non-steroidogenic COS-7 cells transfected with

Shigeho Ijiri; Yukinori Kazeto; P. Mark Lokman; Shinji Adachi; Kohei Yamauchi

2003-01-01

363

2,2?,3,3?,6,6?-Hexachlorobiphenyl (PCB 136) Atropisomers Interact Enantioselectively with Hepatic Microsomal Cytochrome P450 Enzymes  

PubMed Central

2,2?,3,3?,6,6?-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)- and (±)-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in vivo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with ?-naphthoflavone or 3-methylcholanthrene, phenobarbital and dexamethasone (prototypical inducers of CYP1A, CYP2B and CYP3A, respectively) were used to determine first, if the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer; and second, if P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)- or (±)-PCB 136 were added to hepatic microsomes and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PB-treated mice and rats, and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice. PMID:18494506

Kania-Korwel, Izabela; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim

2008-01-01

364

CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in Eschscholzia californica.  

PubMed

Eschscholzia californica produces various types of isoquinoline alkaloids. The structural diversity of these chemicals is often due to cytochrome P450 (P450) activities. Members of the CYP719A subfamily, which are found only in isoquinoline alkaloid-producing plant species, catalyze methylenedioxy bridge-forming reactions. In this study, we isolated four kinds of CYP719A genes from E. californica to characterize their functions. These four cDNAs encoded amino acid sequences that were highly homologous to Coptis japonica CYP719A1 and E. californica CYP719A2 and CYP719A3, which suggested that these gene products may be involved in isoquinoline alkaloid biosynthesis in E. californica, especially in methylenedioxy bridge-forming reactions. Expression analysis of these genes showed that two genes (CYP719A9 and CYP719A11) were preferentially expressed in plant leaf, where pavine-type alkaloids accumulate, whereas the other two showed higher expression in root than in other tissues. They were suggested to have distinct physiological functions in isoquinoline alkaloid biosynthesis. Enzyme assay analysis using recombinant proteins expressed in yeast showed that CYP719A5 had cheilanthifoline synthase activity, which was expected based on the similarity of its primary structure to that of Argemone mexicana cheilanthifoline synthase (deposited at DDBJ/GenBanktrade mark/EMBL). In addition, enzyme assay analysis of recombinant CYP719A9 suggested that it has methylenedioxy bridge-forming activity toward (R,S)-reticuline. CYP719A9 might be involved in the biosynthesis of pavine- and/or simple benzylisoquinoline-type alkaloids, which have a methylenedioxy bridge in an isoquinoline ring, in E. californica leaf. PMID:18854999

Ikezawa, Nobuhiro; Iwasa, Kinuko; Sato, Fumihiko

2009-01-01

365

Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects.  

PubMed

The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies significantly, and the cause has been suggested to be mainly of genetic origin but the exact molecular basis remains unknown. We present a review of the major polymorphic CYP alleles and conclude that this variability is of greatest importance for treatment with several antidepressants, antipsychotics, antiulcer drugs, anti-HIV drugs, anticoagulants, antidiabetics and the anticancer drug tamoxifen. We also present tables illustrating the relative importance of specific common CYP alleles for the extent of enzyme functionality. The field of pharmacoepigenetics has just opened, and we present recent examples wherein gene methylation influences the expression of CYP. In addition microRNA (miRNA) regulation of P450 has been described. Furthermore, this review updates the field with respect to regulatory initiatives and experience of predictive pharmacogenetic investigations in the clinics. It is concluded that the pharmacogenetic knowledge regarding CYP polymorphism now developed to a stage where it can be implemented in drug development and in clinical routine for specific drug treatments, thereby improving the drug response and reducing costs for drug treatment. PMID:18001838

Ingelman-Sundberg, Magnus; Sim, Sarah C; Gomez, Alvin; Rodriguez-Antona, Cristina

2007-12-01

366

Effect of breed upon cytochromes P450 and phase II enzyme expression in cattle liver.  

PubMed

Cattle represent an important source of animal-derived food-products; nonetheless, our knowledge about the expression of drug-metabolizing enzymes (DMEs) in present and other food-producing animals still remains superficial, despite the obvious toxicological consequences. Breed represents an internal factor that modulates DME expression and catalytic activity. In the present work, the effect of breed upon relevant phase I and phase II DMEs was investigated at the pretranscriptional and post-translational levels in male Charolais (CH), Piedmontese (PM) and Blonde d'Aquitaine (BA) cattle. Because specific substrates for cattle have not yet been identified, the breed effect upon specific cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), or glutathione S-transferase (GST) DMEs, in terms of catalytic activity, was determined by using human marker substrates. Among P450s, benzphetamine N-demethylase, 16beta-, 6beta-, and 2beta-testosterone hydroxylase, aniline and p-nitrophenol hydroxylase, and alpha-naphthol and p-nitrophenol UGT activities were significantly higher in CH; in contrast, lower levels of CYP1A1-, CYP1A2-, CYP2B6-, CYP2C9-, CYP2C18-, CYP3A4-, and UGT1A1-like mRNAs were noticed, with CH < PM < or = BA as a trend. CYP2B and CYP3A mRNA results were confirmed with immunoblotting, too. As regards conjugative DMEs, UGT1A6-like mRNA levels were consistent with respective catalytic activities. Both 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene GST activities were higher in BA, and these results agreed with GSTA1-, GSTM1-, and GSTP1-like mRNA amounts. Correlation analysis between catalytic activities and mRNAs showed either significant or uneven results, depending on the substrate. These findings confirm previous data obtained in laboratory species; however, further studies are required to ascribe this behavior to pretranscriptional or post-translational phenomena. PMID:18268077

Giantin, Mery; Carletti, Monica; Capolongo, Francesca; Pegolo, Sara; Lopparelli, Rosa Maria; Gusson, Federica; Nebbia, Carlo; Cantiello, Michela; Martin, Pascal; Pineau, Thierry; Dacasto, Mauro

2008-05-01

367

Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450  

PubMed Central

Background Cytochrome P450 monooxygenases – able to regio- and stereoselectively hydroxylate non-activated carbon atoms – are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16?-hydroxytestosterone. Since the hydroxylation at 16?-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids. Results CYP154C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), androstenedione (4), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16?-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 ?mol substrate consumed min-1 ?mol-1 CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 ?mol substrate consumed min-1 ?mol-1 CYP154C5. Conclusion CYP154C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16?-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (?mol substrate consumed ?mol-1 CYP154C5) exceeding 2000. PMID:24134652

2013-01-01

368

An evaluation of the potential for pharmacokinetic interaction between escitalopram and the cytochrome P450 3A4 inhibitor ritonavir  

Microsoft Academic Search

Background: Depression often coexists with a number of disease states, and patients with a diagnosis of depression often receive multiple medications. Thus, it is desirable to avoid coadministration of agents that have a potential for drug interactions in these patients. Although escitalopram and its metabolites are weak to negligible inhibitors of the cytochrome P450 (CYP) 3A4 isozyme and are therefore

Marcelo M. Gutierrez; Jeffrey Rosenberg; Wattanaporn Abramowitz

2003-01-01

369

Titre (franais): Effets de l'exposition aux radiofrquences de tlphone portable sur le cytochrome P450 rductase humain  

E-print Network

. The present study aimed at the effect of cell phone RF (1.9 GHz) emitted by Universal Mobile Telecommunication cytochrome P450 réductase humain Title (English): Effects of cell phone radiofrequency exposure on the human fields (EMFs) and raises serious concerns about the biological and health-related effects of RF radiation

Paris-Sud XI, Université de

370

Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules  

EPA Science Inventory

The human cytochrome P450 (CYP450) enzyme family is involved in the biotransformation of many environmental chemicals. As part of the U.S. Tox21 effort, we profiled the CYP450 activity of ~2800 chemicals predominantly of environmental concern against CYP1A2, CYP2C19, CYP2C9, CYP2...

371

Induction of cytochrome p?450 in the norway rat, rattus norvegicus, following exposure to potential environmental contaminants  

Microsoft Academic Search

Cytochrome P?450 (CYP) induction (consisting of increases in cellular RNA and protein content and associated catalytic activities) occurs predominantly in the liver, but also in small intestine, lung, kidney, and placenta, of Norway rats (Rattus norvegicus.) exposed to certain types of potential environmental contaminants. The specific isoform(s) induced in the rat and the magnitudes of the increases observed depend upon

Raymond W. Nims; Ronald A. Lubet

1995-01-01

372

Involvement of cytochrome P-450 enzyme activity in the selectivity and safening action of pyrazosulfuron-ethyl.  

PubMed

To investigate the selectivity and safening action of the sulfonylurea herbicide pyrazosulfuron-ethyl (PSE), pyrazosulfuron-ethyl O-demethylase (PSEOD) activity involving oxidative metabolism by cytochrome P-450 was studied in rice (Oryza sativa L cv Nipponbare) and Cyperus serotinus Rottb. Cytochrome P-450-dependent activity was demonstrated by the use of the inducers 1,8-naphthalic anhydride and ethanol, the herbicides PSE, bensulfuron-methyl, dimepiperate and dymron, or the inhibitor piperonyl butoxide (PBO). Growth inhibition in C serotinus seedlings was more severe than that in rice seedlings. O-Dealkylation activities of PSE were induced differently in rice and in C serotinus, with distinctly higher activity in rice seedlings. The induced PSEOD activities were slightly inhibited by PBO in rice seedlings, whereas they were strongly inhibited in C serotinus seedlings. Dimepiperate and dymron were effective safeners of rice against PSE treatment. Treatments with herbicide alone resulted in less induction of PSEOD activity compared with combined treatments of the herbicide and safener. PSEOD activity in rice seedlings induced with herbicide alone was strongly inhibited by PBO, whereas it was weakly inhibited in rice seedlings induced with combinations of PSE and two safeners. These results suggest that O-demethylation by cytochrome P-450 enzymes may be involved in the metabolism of PSE and may contribute to its selectivity and safening action. Furthermore, these results suggest the existence of a multiple form of cytochrome P-450 in plants. PMID:11455659

Yun, M S; Shim, I S; Usui, K

2001-03-01

373

LXR deficiency and cholesterol feeding affect the expression and phenobarbital-mediated induction of cytochromes P450 in mouse liver  

Microsoft Academic Search

Metabolic transformation by the superfamily of cytochromes P450 (CYPs) plays an important role in the detoxification of xenobiotics such as drugs, environmental pollutants, and food additives. Endogenous substrates of CYPs include fatty acids, sterols, steroids, and bile acids. In- duction of CYPs via transcriptional activation by substrates and other xenobiotics is an important adaptive mechanism that increases the organism's defense

Carmela Gnerre; Gertrud U. Schuster; Adrian Roth; Christoph Handschin; Lisen Johansson; Renate Looser; Paolo Parini; Michael Podvinec; Kirsten Robertsson; Jan-Åke Gustafsson; Urs A. Meyer

2005-01-01

374

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila  

Microsoft Academic Search

BACKGROUND: Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses

Chengjie Fu; Jie Xiong; Wei Miao

2009-01-01

375

Genetic polymorphism of cytochrome P450s in beagles: possible influence of CYP1A2 deficiency on toxicological evaluations  

Microsoft Academic Search

A number of human cytochrome P450 (CYP) isozymes have been shown to be genetically polymorphic, and extensive pharmaceutical studies have been conducted to characterize the clinical relevance of the polymorphism. Although the beagle is extensively used in the safety assessment studies of new drug candidates and agricultural chemicals, only a limited number of studies have been reported on the significance

H. Kamimura

2006-01-01

376

Inter-flavin electron transfer in cytochrome P450 reductase effects of solvent and pH identify hidden  

E-print Network

Inter-flavin electron transfer in cytochrome P450 reductase ­ effects of solvent and pH identify to the endoplasmic retic- ulum by a hydrophobic N-terminal membrane anchor Keywords electron transfer; pH dependence and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. p

377

Potential effects of environmental contaminants on P450 aromatase activity and DNA damage in swallows from the Rio Grande and Somerville, Texas.  

PubMed

Cliff swallows (Petrochelidon pyrrhonota) and cave swallows (P. fulva) were sampled during the breeding season at several locations in the Rio Grande, Texas, to evaluate the potential effects of environmental contaminants on P450 aromatase activity in brain and gonads and DNA damage in blood cells. The tritiated water-release aromatase assay was used to measure aromatase activity and flow cytometry was used to measure DNA damage in nucleated blood cells. There were no significant differences in brain and gonadal aromatase activities or in estimates of DNA damage (HPCV values) among cave swallow colonies from the Lower Rio Grande Valley (LRGV) and Somerville. However, both brain and gonadal aromatase activities were significantly higher (P < 0.05) in male cliff swallows from Laredo than in those from Somerville. Also, DNA damage estimates were significantly higher (P < 0.05) in cliff swallows (males and females combined) from Laredo than in those from Somerville. Contaminants of current high use in the LRGV, such as atrazine, and some of the highly persistent organochlorines, such as toxaphene and DDE, could be potentially associated with modulation of aromatase activity in avian tissues. Previous studies have indicated possible DNA damage in cliff swallows. We did not observe any differences in aromatase activity or DNA damage in cave swallows that could be associated with contaminant exposure. Also, the differences in aromatase activity and DNA damage between male cliff swallows from Laredo and Somerville could not be explained by contaminants measured at each site in previous studies. Our study provides baseline information on brain and gonadal aromatase activity in swallows that could be useful in future studies. PMID:18670880

Sitzlar, M A; Mora, M A; Fleming, J G W; Bazer, F W; Bickham, J W; Matson, C W

2009-01-01

378

Human Cytochrome P450c17: Single Step Purification and Phosphorylation of Serine 258 by Protein Kinase A  

PubMed Central

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17?-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17.