Science.gov

Sample records for hepatic p450 isoforms

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

    PubMed

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

    2016-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

    SciTech Connect

    Leoni, Claudia; Buratti, Franca M. Testai, Emanuela

    2008-12-01

    Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO{sub 3} and FMO{sub 5} was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO{sub 1} showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO{sub 1}-catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation.

  4. Increased oxidative DNA damage and hepatocyte overexpression of specific cytochrome P450 isoforms in hepatitis of mice infected with Helicobacter hepaticus.

    PubMed Central

    Sipowicz, M. A.; Chomarat, P.; Diwan, B. A.; Anver, M. A.; Awasthi, Y. C.; Ward, J. M.; Rice, J. M.; Kasprzak, K. S.; Wild, C. P.; Anderson, L. M.

    1997-01-01

    A recently discovered bacterium, Helicobacter hepaticus, infects the intrahepatic bile canaliculi of mice, causing a severe chronic hepatitis culminating in liver cancer. Thus, it affords an animal model for study of bacteria-associated tumorigenesis including H. pylori-related gastric cancer. Reactive oxygen species are often postulated to contribute to this process. We now report that hepatitis of male mice infected with H. hepaticus show significant increases in the oxidatively damaged DNA deoxynucleoside 8-hydroxydeoxyguanosine, with the degree of damage increasing with progression of the disease. Perfusion of infected livers with nitro blue tetrazolium revealed that superoxide was produced in the cytoplasm of hepatocytes, especially in association with plasmacytic infiltrates near portal triads. Contrary to expectations, Kupffer cells, macrophages, and neutrophils were rarely involved. However, levels of cytochrome P450 (CYP) isoforms 1A2 and 2A5 in hepatocytes appeared to be greatly increased, as indicated by the number of cells positive in immunohistochemistry and the intensity of staining in many cells, concomitant with severe hepatitis. The CYP2A5 immunohistochemical staining co-localized with formazan deposits resulting from nitro blue tetrazolium reduction and occurred in nuclei as well as cytoplasm. These findings suggest that CYP2A5 contributes to the superoxide production and 8-hydroxydeoxyguanosine formation, although reactive oxygen species from an unknown source in the hepatocytes leading to CYP2A5 induction or coincidental occurrence of these events are also possibilities. Three glutathione S-transferase isoforms, mGSTP1-1 (pi), mGSTA1-1 (YaYa), and mGSTA4-4, also showed striking increases evidencing major oxidative stress in these livers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9327726

  5. In vitro metabolic clearance of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome P450 isoforms

    EPA Science Inventory

    Species differences in the intrinsic clearance (CLint) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, β-cyfluthrin, cypermethrin, cis-per...

  6. Interaction of sanguinarine alkaloid, isolated from argemone oil, with hepatic cytochrome p450 in rats.

    PubMed

    Reddy, Naveen P; Das, Mukul

    2008-01-01

    ABSTRACT Prior studies have shown that argemone oil (AO), responsible for 'Epidemic dropsy', causes inhibition of catalytic activities of Cytochrome P450 (P450). In this study interaction of sanguinarine (SAN) alkaloid, isolated from AO, with rat hepatic P450 was investigated. Hepatic microsomes prepared from 3-methylcholantherene (3MC) treated rats when incubated with SAN (1-3 muM) resulted in a spectral peak at 385 nm and a trough at 415 nm, indicative of Type I binding. Incubation of SAN (50-200 muM) with hepatic microsomes prepared from phenobarbitone (PB) treated rats also showed a Type I spectra with a peak at 395 nm and a trough at 420 nm. Relative binding efficiency (DeltaA(max)/K(s)(app) factor) of SAN with P450 was found to be 1540 and 1030 absorbance units/nmol CYP/M for 3MC and PB induced microsomes, respectively. In a P450 spectral inhibition study SAN showed higher affinity towards 3MC eliciting inhibition at much lesser concentrations (0.25-5 muM) as compared to PB (100-300 muM). The IC50s of SAN with different catalytic markers of P450 isoforms, i.e. ethoxyresorufin-O-deethylase (EROD) for CYP1A1, was 2.8 muM and for methoxyresorufin-O-deethylase (MROD) for CYP1A2 was 2.2 muM in 3MC induced microsomes, while benzoyloxyresorufin-O-deethylase (BROD) for CYP 2B1/1A1 showed an IC50 of 50 muM but pentoxyresorufin-O-deethylase (PROD) for CYP2B1 showed no inhibition even at higher concentrations of SAN (> 60 muM) in PB-induced microsomes. These results indicate that higher affinity of SAN binding towards the CYP1A family may have a role in SAN toxicity.

  7. Zonation of hepatic cytochrome P-450 expression and regulation.

    PubMed Central

    Oinonen, T; Lindros, K O

    1998-01-01

    The CYP genes encode enzymes of the cytochrome P-450 superfamily. Cytochrome P-450 (CYP) enzymes are expressed mainly in the liver and are active in mono-oxygenation and hydroxylation of various xenobiotics, including drugs and alcohols, as well as that of endogenous compounds such as steroids, bile acids, prostaglandins, leukotrienes and biogenic amines. In the liver the CYP enzymes are constitutively expressed and commonly also induced by chemicals in a characteristic zonated pattern with high expression prevailing in the downstream perivenous region. In the present review we summarize recent studies, mainly based on rat liver, on the factors regulating this position-dependent expression and induction. Pituitary-dependent signals mediated by growth hormone and thyroid hormone seem to selectively down-regulate the upstream periportal expression of certain CYP forms. It is at present unknown to what extent other hormones that also affect total hepatic CYP activities, i.e. insulin, glucagon, glucocorticoids and gonadal hormones, act zone-specifically. The expression and induction of CYP enzymes in the perivenous region probably have important toxicological implications, since many CYP-activated chemicals cause cell injury primarily in this region of the liver. PMID:9405271

  8. Does Compound I Vary Significantly between Isoforms of Cytochrome P450?

    PubMed Central

    2011-01-01

    The cytochrome P450 (CYP) enzymes are important in many areas, including pharmaceutical development. Subtle changes in the electronic structure of the active species, Compound I, have been postulated previously to account partly for the experimentally observed differences in reactivity between isoforms. Current predictive models of CYP metabolism typically assume an identical Compound I in all isoforms. Here we present a method to calculate the electronic structure and to estimate the Fe–O bond enthalpy of Compound I, and apply it to several human and bacterial CYP isoforms. Conformational flexibility is accounted for by sampling large numbers of structures from molecular dynamics simulations, which are subsequently optimized with density functional theory (B3LYP) based quantum mechanics/molecular mechanics. The observed differences in Compound I between human isoforms are small: They are generally smaller than the spread of values obtained for the same isoform starting from different initial structures. Hence, it is unlikely that the variation in activity between human isoforms is due to differences in the electronic structure of Compound I. A larger difference in electronic structure is observed between the human isoforms and P450cam and may be explained by the slightly different hydrogen-bonding environment surrounding the cysteinyl sulfur. The presence of substrate in the active site of all isoforms studied appears to cause a slight decrease in the Fe–O bond enthalpy, apparently due to displacement of water out of the active site, suggesting that Compound I is less stable in the presence of substrate. PMID:21863858

  9. Effect of age on hepatic cytochrome P450 of Ross 708 broiler chickens.

    PubMed

    Hu, S X

    2013-05-01

    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.

  10. Cytochrome P450 Isoforms in the Metabolism of Decursin and Decursinol Angelate from Korean Angelica.

    PubMed

    Zhang, Jinhui; Li, Li; Tang, Suni; Hale, Thomas W; Xing, Chengguo; Jiang, Cheng; Lü, Junxuan

    2015-01-01

    We have shown that the in vitro hepatic microsomal metabolism of pyranocoumarin compound decursinol angelate (DA) to decursinol (DOH) exclusively requires cytochrome P450 (CYP) enzymes, whereas the conversion of its isomer decursin (D) to DOH can be mediated by CYP and esterase(s). To provide insight into specific isoforms involved, here we show with recombinant human CYP that 2C19 was the most active at metabolizing D and DA in vitro followed by 3A4. With carboxylesterases (CES), D was hydrolyzed by CES2 but not CES1, and DA was resistant to both CES1 and CES2. In human liver microsomal (HLM) preparation, the general CYP inhibitor 1-aminobenzotriazole (ABT) and respective competitive inhibitors for 2C19 and 3A4, (+)-N-3-benzylnirvanol (NBN) and ketoconazole substantially retarded the metabolism of DA and, to a lesser extent, of D. In healthy human subjects from a single-dose pharmacokinetic (PK) study, 2C19 extensive metabolizer genotype (2C19*17 allele) tended to have less plasma DA AUC0-48h and poor metabolizer genotype (2C19*2 allele) tended to have greater DA AUC0-48h. In mice given a single dose of D/DA, pretreatment with ABT boosted the plasma and prostate levels of D and DA by more than an order of magnitude. Taken together, our findings suggest that CYP isoforms 2C19 and 3A4 may play a crucial role in the first pass liver metabolism of DA and, to a lesser extent, that of D in humans. Pharmacogenetics with respect to CYP genotypes and interactions among CYP inhibitor drugs and D/DA should therefore be considered in designing future translation studies of DA and/or D.

  11. Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms

    PubMed Central

    Nalwalk, Julia W.; Ding, Xinxin; Scheer, Nico

    2015-01-01

    Cytochrome P450 monooxygenases (P450s), which are well-known drug-metabolizing enzymes, are thought to play a signal transduction role in µ opioid analgesia and may serve as high-affinity 3H-cimetidine (3HCIM) binding sites in the brain. 3HCIM binding sites may also be related to opioid or nonopioid analgesia. However, of the more than 100 murine P450 enzymes, the specific isoform(s) responsible for either function have not been identified. Presently, three lines of constitutive P450 gene cluster knockout (KO) mice with full-length deletions of 14 Cyp2c, 9 Cyp2d, and 7 Cyp3a genes were studied for deficiencies in 3HCIM binding and for opioid analgesia. Liver and brain homogenates from all three genotypes showed normal 3HCIM binding values, indicating that gene products of Cyp2d, Cyp3a, and Cyp2c are not 3HCIM-binding proteins. Cyp2d KO and Cyp3a KO mice showed normal antinociceptive responses to a moderate systemic dose of morphine (20 mg/kg, s.c.), thereby excluding 16 P450 isoforms as mediators of opioid analgesia. In contrast, Cyp2c KO mice showed a 41% reduction in analgesic responses following systemically (s.c.) administered morphine. However, the significance of brain Cyp2c gene products in opioid analgesia is uncertain because little or no analgesic deficits were noted in Cyp2c KO mice following intracerebroventricular or intrathecalmorphine administration, respectively. These results show that the gene products of Cyp2d and Cyp3a do not contribute to µ opioid analgesia in the central nervous system. A possible role for Cyp2c gene products in opioid analgesia requires further consideration. PMID:26109562

  12. Malathion bioactivation in the human liver: the contribution of different cytochrome p450 isoforms.

    PubMed

    Buratti, Franca M; D'Aniello, Alessandra; Volpe, Maria Teresa; Meneguz, Annarita; Testai, Emanuela

    2005-03-01

    Among organophosphorothioate (OPT) pesticides, malathion is considered relatively safe for use in mammals. Its rapid degradation by carboxylesterases competes with the cytochrome P450 (P450)-catalyzed formation of malaoxon, the toxic metabolite. However, impurities in commercial formulations are potent inhibitors of carboxylesterase, allowing a dramatic increase in malaoxon formation. Malathion desulfuration has been characterized in human liver microsomes (HLMs) with a method based on acetylcholinesterase inhibition that is able to detect nanomolar levels of oxon. The active P450 isoforms have been identified by means of a multifaceted strategy, including the use of cDNA-expressed human P450s and correlation, immunoinhibition, and chemical inhibition studies in a panel of phenotyped HLMs. HLMs catalyzed malaoxon formation with a high level of variability (>200-fold). One or two components (K(mapp1) = 53-67 microM; K(mapp2) = 427-1721 microM) were evidenced, depending on the relative specific P450 content. Results from different approaches indicated that, at low malathion concentration, malaoxon formation is catalyzed by CYP1A2 and, to a lesser extent, 2B6, whereas the role of 3A4 is relevant only at high malathion levels. These results are in line with those found with chlorpyrifos, diazinon, azynphos-methyl, and parathion, characterized by the presence of an aromatic ring in the molecule. Since malathion has linear chains as substituents at the thioether sulfur, it can be hypothesized that, independently from the chemical structure, OPTs are bioactivated by the same P450s. These results also suggest that CYP1A2 and 2B6 can be considered as possible metabolic biomarkers of susceptibility to OPT-induced toxic effects at actual human exposure levels.

  13. Prediction of cytochrome P450 isoform responsible for metabolizing a drug molecule

    PubMed Central

    2010-01-01

    Background Different isoforms of Cytochrome P450 (CYP) metabolized different types of substrates (or drugs molecule) and make them soluble during biotransformation. Therefore, fate of any drug molecule depends on how they are treated or metabolized by CYP isoform. There is a need to develop models for predicting substrate specificity of major isoforms of P450, in order to understand whether a given drug will be metabolized or not. This paper describes an in-silico method for predicting the metabolizing capability of major isoforms (e.g. CYP 3A4, 2D6, 1A2, 2C9 and 2C19). Results All models were trained and tested on 226 approved drug molecules. Firstly, 2392 molecular descriptors for each drug molecule were calculated using various softwares. Secondly, best 41 descriptors were selected using general and genetic algorithm. Thirdly, Support Vector Machine (SVM) based QSAR models were developed using 41 best descriptors and achieved an average accuracy of 86.02%, evaluated using fivefold cross-validation. We have also evaluated the performance of our model on an independent dataset of 146 drug molecules and achieved average accuracy 70.55%. In addition, SVM based models were developed using 26 Chemistry Development Kit (CDK) molecular descriptors and achieved an average accuracy of 86.60%. Conclusions This study demonstrates that SVM based QSAR model can predict substrate specificity of major CYP isoforms with high accuracy. These models can be used to predict isoform responsible for metabolizing a drug molecule. Thus these models can used to understand whether a molecule will be metabolized or not. This is possible to develop highly accurate models for predicting substrate specificity of major isoforms using CDK descriptors. A web server MetaPred has been developed for predicting metabolizing isoform of a drug molecule http://crdd.osdd.net/raghava/metapred/. PMID:20637097

  14. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

    PubMed Central

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.

    2016-01-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography–mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage–dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

  15. Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats.

    PubMed

    Sugiyama, T; Suzuki, K; Ookawara, T; Kurosawa, T; Taniguchi, N

    1989-11-01

    The Long-Evans rat with a cinnamon-like coat color (LEC rat) is a mutant strain displaying hereditary hepatitis with severe jaundice. The age related difference in microsomal dealkylation of pentoxyresorufin and ethoxyresorufin was examined. The enzyme activity levels of pentoxyresorufin O-depentylase in LEC rats were decreased to 25% of the levels in control [Long-Evans rats with an agouti coat color (LEA rats)]. In contrast, ethoxyresorufin O-deethylase exhibited a much less marked difference between the strains. In parallel with these strain differences in enzyme activities, a decrease in phenobarbital (PB) inducible P450 isozymes, mainly P450b and P450e, was observed by Western blot analysis. The level of P450PB in LEC rats was more markedly depressed than in the LEA strain. On the other hand, microsomes from uninduced LEC rat liver had more 3-methylcholanthrene (MC) inducible P450MC, mainly P450c and P450d, than microsomes from LEA rat liver and these isozymes in the LEC were markedly induced by 3-methylcholanthrene treatment. The great difference in cytochrome P450PB content of the liver microsomes between LEC and LEA rats and the maintained constitutive levels of hepatic cytochrome P450MC in the LEC rats suggest a possible role of these cytochrome isozymes in the onset of spontaneous hepatitis and hepatoma.

  16. Carbon monoxide-bound red blood cells protect red blood cell transfusion-induced hepatic cytochrome P450 impairment in hemorrhagic-shock rats.

    PubMed

    Ogaki, Shigeru; Taguchi, Kazuaki; Watanabe, Hiroshi; Otagiri, Masaki; Maruyama, Toru

    2013-01-01

    Red blood cell (RBC) transfusions for massive hemorrhage induce systemic ischemic-reperfusion and influence the disposition and pharmacological activity of drugs as a result of a reduction in the level of expression and activity of cytochrome P450s (P450). It was reported that, when organ-preserving solutions are exposed to carbon monoxide (CO), the treatment was effective in suppressing the postreperfusion reduction in renal P450 levels in cases of kidney transplantation. Therefore, we hypothesized that transfusions with RBC that contain bound CO (CO-RBC) would protect the hepatic level of rat P450 during a massive hemorrhage, compared with plasma expanders and RBC resuscitation. To achieve this, we created 40% hemorrhagic-shock model rats, followed by resuscitation, with use of recombinant human serum albumin, RBCs, and CO-RBCs. At 1 hour after resuscitation, the expressions of hepatic P450 isoforms (1A2, 2C11, 2E1, and 3A2) were significantly decreased in the RBC resuscitation group, compared with the sham group. Such alterations in hepatic P450 significantly resulted in an increase in the plasma concentrations of substrate drugs (caffeine [1A2], tolbutamide [2C11], chlorzoxazone [2E1], and midazolam [3A2]) for each P450 isoform, and thus, the hypnotic action of midazolam could be significantly prolonged. Of interest, the reductions in hepatic P450 activity observed in the RBC group were significantly suppressed by CO-RBC resuscitation, and consequently, the pharmacokinetics of substrate drugs and the pharmacological action of midazolam remained at levels similar to those under sham conditions. These results indicate that CO-RBC resuscitation has considerable potential in terms of achieving safe and useful drug therapy during massive hemorrhages.

  17. The effects of acute hydrogen sulfide poisoning on cytochrome P450 isoforms activity in rats.

    PubMed

    Wang, Xianqin; Chen, Mengchun; Chen, Xinxin; Ma, Jianshe; Wen, Congcong; Pan, Jianchun; Hu, Lufeng; Lin, Guanyang

    2014-01-01

    Hydrogen sulfide (H2S) is the second leading cause of toxin related death (after carbon monoxide) in the workplace. H2S is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression. Cocktail method was used to evaluate the influences of acute H2S poisoning on the activities of cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which were reflected by the changes of pharmacokinetic parameters of six specific probe drugs, bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide, respectively. The experimental rats were randomly divided into two groups, control group and acute H2S poisoning group (inhaling 300 ppm for 2 h). The mixture of six probes was given to rats by oral administration and the blood samples were obtained at a series of time points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. The results for acute H2S poisoning and control groups were as follows: there was a statistically significant difference in the AUC and C max for bupropion, metoprolol, phenacetin, and tolbutamide, while there was no statistical pharmacokinetic difference for midazolam and omeprazole. Acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 in rats.

  18. Immunochemical evidence for multiple steroid-inducible hepatic cytochromes P-450 in the rat.

    PubMed Central

    Hostetler, K A; Wrighton, S A; Kremers, P; Guzelian, P S

    1987-01-01

    It has been established that there are glucocorticoid-inducible hepatic cytochromes P-450 in the rat (P-450p), the rabbit (LM3c) and man (HLp) which share extensive structural, functional and regulatory features. We prepared immunochemical probes to P-450p and identified a unique monoclonal antibody, 1G8, that recognizes purified P-450p, but neither purified LM3c nor HLp, on immunoblot analysis. The N-terminal amino acid sequence of purified samples of P-450p was identical with that previously reported for P-450PCN1 [Gonzalez, Nebert, Hardwick & Kasper (1985) J. Biol. Chem. 260, 7435-7441]. Immunoblot analyses of liver microsomes from untreated male rats revealed two 1G8-reactive proteins, whereas liver microsomes from untreated females contained none. Another monoclonal antibody, 13-7-10, reacted specifically with LM3c and HLp, but not with P-450p. A single 13-7-10-reactive microsomal protein was detected in untreated male and female rats, the latter protein exhibiting a greater apparent Mr. 1G8-reactive proteins were induced to the greatest extent by triacetyloleandomycin, followed by dexamethasone, chlordane, pregnenolone-16 alpha-carbonitrile and 2,4,2',4'-tetrachlorobiphenyl. In contrast, 13-7-10-reactive proteins were most strongly induced by dexamethasone, only moderately by triacetyloleandomycin and pregnenolone-16 alpha-carbonitrile, weakly by chlordane and not at all by 2,4,2',4'-tetrachlorobiphenyl. We conclude that the P-450p family in rat liver consists of three or more proteins that are structurally related and yet appear to be under distinct regulatory control. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3311032

  19. Polar bear hepatic cytochrome P450: Immunochemical quantitation, EROD/PROD activity and organochlorines

    SciTech Connect

    Letcher, R.J.; Norstrom, R.J. |

    1994-12-31

    Polar bears (Ursus maritimus) are an ubiquitous mammal atop the arctic marine food chain and bioaccumulate lipophilic environmental contaminants. Antibodies prepared against purified rat liver cytochrome P450-1 Al, -1 A2, -2Bl and -3Al enzymes have been found to cross-react with structurally-related orthologues present in the hepatic microsomes of wild polar bears, immunochemically determined levels of P450-1 A and -2B proteins in polar bear liver relative to liver of untreated rats suggested enzyme induction, probably as a result of exposure to xenobiotic contaminants. Optical density quantitation of the most immunochemically responsive isozymes P450-I Al, -IA2 and -2Bi to polygonal rabbit anti-rat P450-IA/IA2 sera and -2BI antibodies in hepatic microsomes of 13 adult male polar bars from the Resolute Bay area of the Canadian Arctic is presented. Correlations with EROD and PROD catalytic activities and levels of organochlorines, such as polychlorinated biphenyls (PCBs), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene (p,p-DDE) and their methyl sulfone (MeSO2-) metabolites are made to determine if compound-specific enzyme induction linkages exist. Inter-species immunochemical quantitation of isozymic P450 cytochromes can serve as an indicator of exposure to biologically active contaminant.

  20. 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...

  1. Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450

    SciTech Connect

    Marinello, A.J.; Bansal, S.K.; Paul, B.; Koser, P.L.; Love, J.; Struck, R.F.; Gurtoo, H.L.

    1984-10-01

    The hepatic cytochrome P-450-mediated metabolism and metabolic activation of (chloroethyl-3H)cyclophosphamide (( chloroethyl-3H)CP) and (4-14C)cyclophosphamide (( 4-14C)CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of (14C)acrolein, a metabolite of (4-14C)CP, were also investigated. The metabolism of (chloroethyl-3H)CP and (4-14C)CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar. The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between (4-14C)CP and (chloroethyl-3H)CP metabolism. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of (chloroethyl-3H)CP to nucleic acids and almost exclusive binding of (4-14C)CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with (4-14C)CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with (14C)acrolein in the presence and the absence of NADPH. The results confirmed covalent association between (14C)acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of (14C)acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations.

  2. Responsiveness of cerebral and hepatic cytochrome P450s in rat offspring prenatally exposed to lindane

    SciTech Connect

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

    2008-08-15

    ABSTRACT: Prenatal exposure to low doses of lindane has been shown to affect the ontogeny of xenobiotic metabolizing cytochrome P450s (CYPs), involved in the metabolism and neurobehavioral toxicity of lindane. Attempts were made in the present study to investigate the responsiveness of CYPs in offspring prenatally exposed to lindane (0.25 mg/kg b. wt.; 1/350th of LD{sub 50}; p. o. to mother) when challenged with 3-methylcholanthrene (MC) or phenobarbital (PB), inducers of CYP1A and 2B families or a sub-convulsant dose of lindane (30 mg/kg b. wt., p. o.) later in life. Prenatal exposure to lindane was found to produce an increase in the mRNA and protein expression of CYP1A1, 1A2, 2B1, 2B2 isoforms in brain and liver of the offspring at postnatal day 50. The increased expression of the CYPs in the offspring suggests the sensitivity of the CYPs during postnatal development, possibly, to low levels of lindane, which may partition into mother's milk. A higher increase in expression of CYP1A and 2B isoenzymes and their catalytic activity was observed in animals pretreated prenatally with lindane and challenged with MC (30 mg/kg, i. p. x 5 days) or PB (80 mg/kg, i. p. x 5 days) when young at age (approx. 7 weeks) compared to animals exposed to MC or PB alone. Further, challenge of the control and prenatally exposed offspring with a single sub-convulsant dose of lindane resulted in an earlier onset and increased incidence of convulsions in the offspring prenatally exposed to lindane have demonstrated sensitivity of the CYPs in the prenatally exposed offspring. Our data assume significance as the subtle changes in the expression profiles of hepatic and cerebral CYPs in rat offspring during postnatal development could modify the adult response to a later exposure to xenobiotics.

  3. Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450.

    PubMed

    Wang, Z Y; Das, M; Bickers, D R; Mukhtar, H

    1988-01-01

    Green tea has been used for generations in China and Asia as an antipyretic and diuretic. Prior studies have shown that extracts of green tea inhibit the mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1. In this study, we investigated the interaction of certain flavonoid components of green tea epicatechin derivatives including (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG) with rat hepatic microsomal cytochrome P-450 (P-450). The addition of EC, EGC, ECG, and EGCG to hepatic microsomes prepared from phenobarbital (PB)-treated rats resulted in spectral changes characterized by absorbance maxima at 420 nm and minima at 380 nm, typical of modified Type II (reverse Type I) binding. Of the epicatechin derivatives, EGCG and ECG showed greater spectral change with oxidized P-450 and time- and concentration-dependent inhibition of the binding of carbon monoxide to dithionite-reduced cytochrome P-450. The addition of EC, EGC, ECG, and EGCG to microsomes prepared from control, PB- or 3-methylcholanthrene-treated rats resulted in a dose-dependent inhibition of cytochrome P-450-dependent aryl hydrocarbon hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase activities. EGCG was the most potent in this regard. Green tea polyphenols and epicatechin derivatives also significantly inhibited NADPH-cytochrome c reductase activity. An examination of the structure activity relationship of epicatechin derivatives suggests that the inhibitory effect on the microsomal enzyme system may be due to the galloyl groups or hydroxyl groups on the molecule. Our data indicate that these extracts of green tea may have potential as anticarcinogens.

  4. Cytochrome P450-mediated metabolism of triclosan attenuates its cytotoxicity in hepatic cells.

    PubMed

    Wu, Yuanfeng; Chitranshi, Priyanka; Loukotková, Lucie; Gamboa da Costa, Gonçalo; Beland, Frederick A; Zhang, Jie; Fang, Jia-Long

    2016-11-28

    Triclosan is a widely used broad-spectrum anti-bacterial agent. The objectives of this study were to identify which cytochrome P450 (CYP) isoforms metabolize triclosan and to examine the effects of CYP-mediated metabolism on triclosan-induced cytotoxicity. A panel of HepG2-derived cell lines was established, each of which overexpressed a single CYP isoform, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1. The extent of triclosan metabolism by each CYP was assessed by reversed-phase high-performance liquid chromatography with online radiochemical detection. Seven isoforms were capable of metabolizing triclosan, with the order of activity being CYP1A2 > CYP2B6 > CYP2C19 > CYP2D6 ≈ CYP1B1 > CYP2C18 ≈ CYP1A1. The remaining 11 isoforms (CYP2A6, CYP2A7, CYP2A13, CYP2C8, CYP2C9, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1) had little or no activity toward triclosan. Three metabolites were detected: 2,4-dichlorophenol, 4-chlorocatechol, and 5'-hydroxytriclosan. Consistent with the in vitro screening data, triclosan was extensively metabolized in HepG2 cells overexpressing CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP2C18, and these cells were much more resistant to triclosan-induced cytotoxicity compared to vector cells, suggesting that CYP-mediated metabolism of triclosan attenuated its cytotoxicity. In addition, 2,4-dichlorophenol and 4-chlorocatechol were less toxic than triclosan to HepG2/vector cells. Conjugation of triclosan, catalyzed by human glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also occurred in HepG2/CYP-overexpressing cells and primary human hepatocytes, with a greater extent of conjugation being associated with higher cell viability. Co-administration of triclosan with UGT or SULT inhibitors led to greater cytotoxicity in HepG2 cells and primary human hepatocytes, indicating that glucuronidation and

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

    USGS Publications Warehouse

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

    1992-01-01

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

  6. The application of hepatic P450 reductase null gpt delta mice in studying the role of hepatic P450 in genotoxic carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mutagenesis.

    PubMed

    Luan, Yang; Xing, Guozhen; Qi, Xinming; Wu, Mengjun; Li, Chenggang; Yao, Jun; Gong, Likun; Nohmi, Takehiko; Gu, Jun; Zhou, Wanhong; Zheng, Saijing; Ren, Jin

    2012-11-01

    The cytochrome P450 (P450 or CYP) is involved in both detoxification and metabolic activation of many carcinogens. In order to identify the role of hepatic P450 in the mutagenesis of genotoxic carcinogens, we generated a novel hepatic P450 reductase null (HRN) gpt delta mouse model, which lacks functional hepatic P450 on a gpt delta mouse background. In this study, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was used to treat HRN gpt delta mice and control littermates. Gene mutations in the liver and lungs were detected, and mutation spectra were analyzed. Pharmacokinetic analyses were performed, and tissue levels of NNK and metabolite were determined. NNK-induced mutant frequencies (MFs) were equivalent to spontaneous MFs in the liver, but increased more than 3 times in the lungs of HRN gpt delta mice compared to control mice. NNK-induced mutation spectra showed no difference between HRN gpt delta mice and control littermates. Toxicokinetic studies revealed reduced clearance of NNK with elevated tissue concentrations in HRN gpt delta mice. To our knowledge, these are the first data demonstrating that NNK cannot induce mutagenesis in the liver without P450 metabolic activation, but can induce mutagenesis in lungs by a hepatic P450-independent mechanism. Moreover, our data show that hepatic P450 plays a major role in the systemic clearance of NNK, thereby protecting the lungs against NNK-induced mutagenesis. Our model will be useful in establishing the role of hepatic versus extrahepatic P450-mediated mutagenesis, and the relative contributions of P450 compared to other biotransformation enzymes in the genotoxic carcinogens' activation.

  7. Low levels of hepatic cytochrome P-450 in Mexicans with uncomplicated cholelithiasis.

    PubMed

    Martínez-Zedillo, G; Amézcua, J; González Barranco, D

    1994-01-01

    The multienzymatic complex known as cytochrome P-450 represents one of the most important hemoprotein families of the liver. It participates in the metabolism of steroids, fatty acids, prostaglandins and liposoluble vitamins, and also plays a role in the bioactivation of xenobiotic compounds (generates reactive metabolites which produce acute and chronic lesions in liver tissue). This is a report on the low concentrations of total microsomal P-450 (0.093 +/- SD = 0.069 nMoles/mg protein) found in liver biopsies of 19 mexican patients diagnosed as having biliary lithiasis, as compared to an anglosaxon population with the same liver pathology (0.415 +/- 0.105 nMoles/mg protein). These low values are in agreement with the observation of a high incidence in normal mexicans (91.7%) of poor nifedipine metabolizers. Our findings justify an analyses of hepatic RNAm and cDNA of mexican individuals according to ethnic background, diet and environmental contaminants.

  8. Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants.

    PubMed Central

    Werck-Reichhart, D; Gabriac, B; Teutsch, H; Durst, F

    1990-01-01

    The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide. PMID:2241905

  9. The cytochrome P450 epoxygenase pathway regulates the hepatic inflammatory response in fatty liver disease.

    PubMed

    Schuck, Robert N; Zha, Weibin; Edin, Matthew L; Gruzdev, Artiom; Vendrov, Kimberly C; Miller, Tricia M; Xu, Zhenghong; Lih, Fred B; DeGraff, Laura M; Tomer, Kenneth B; Jones, H Michael; Makowski, Liza; Huang, Leaf; Poloyac, Samuel M; Zeldin, Darryl C; Lee, Craig R

    2014-01-01

    Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted.

  10. In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

    PubMed

    Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

    2013-01-01

    Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

  11. Effect of ergot alkaloids associated with fescue toxicosis on hepatic cytochrome P450 and antioxidant proteins

    SciTech Connect

    Settivari, Raja S.; Evans, Tim J.; Rucker, Ed; Rottinghaus, George E.; Spiers, Donald E.

    2008-03-15

    Intake of ergot alkaloids found in endophyte-infected tall fescue grass is associated with decreased feed intake and reduction in body weight gain. The liver is one of the target organs of fescue toxicosis with upregulation of genes involved in xenobiotic metabolism and downregulation of genes associated with antioxidant pathways. It was hypothesized that short-term exposure of rats to ergot alkaloids would change hepatic cytochrome P450 (CYP) and antioxidant expression, as well as reduce antioxidant enzyme activity and hepatocellular proliferation rates. Hepatic gene expression of various CYPs, selected nuclear receptors associated with the CYP induction, and antioxidant enzymes were measured using real-time PCR. Hepatic expression of CYP, antioxidant and proliferating cell nuclear antigen (PCNA) proteins were measured using Western blots. The CYP3A1 protein expression was evaluated using primary rat hepatocellular cultures treated with ergovaline, one of the major ergot alkaloids produced by fescue endophyte, in order to assess the direct role of ergot alkaloids in CYP induction. The enzyme activities of selected antioxidants were assayed spectrophotometrically. While hepatic CYP and nuclear receptor expression were increased in ergot alkaloid-exposed rats, the expression and activity of antioxidant enzymes were reduced. This could potentially lead to increased oxidative stress, which might be responsible for the decrease in hepatocellular proliferation after ergot alkaloid exposure. This study demonstrated that even short-term exposure to ergot alkaloids can potentially induce hepatic oxidative stress which can contribute to the pathogenesis of fescue toxicosis.

  12. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethan...

  13. Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): sexual dimorphism and onset of ontogenic expression.

    PubMed

    Barney, Megan L; Patil, Jawahar G; Gunasekera, Rasanthi M; Carter, Chris G

    2008-05-01

    Cytochrome P450 aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen, and therefore is thought to influence gonadal sex differentiation. In an effort to understand the role of this enzyme in ovarian differentiation, we isolated cDNA encoding the two distinct isoforms, ovarian and brain (termed cyp19a and cyp19b, respectively) of adult common carp, Cyprinus carpio. The cloned cDNA for cyp19a had an open reading frame (ORF) of 518 amino acid residues, in contrast to cyp19b with an ORF of 511 amino acids. Sequence and phylogenetic analysis showed that these CYP19 isoforms were orthologous with previously described cyp19a and cyp19b from other teleosts. Quantitative real-time PCR indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. The major aromatase expressing tissue was found to be the brain, with greatest cyp19b expression in the anterior quarter (telencephalon) in both sexes. The gonad showed sexually dimorphic expression of both genes and dimorphic expression of cyp19a was observed in the cerebellum and the liver. Ontogenic expression showed that only the ovarian aromatase transcript is inherited maternally, with lower expression observed through early larval development under warmer rearing conditions. The differential and overlapping expression suggests these two aromatase genes have different roles in reproductive physiology.

  14. Inhibitory properties of trapping agents: glutathione, potassium cyanide, and methoxylamine, against major human cytochrome p450 isoforms.

    PubMed

    Zhang, Chenghong; Wong, Susan; Delarosa, Erlie M; Kenny, Jane R; Halladay, Jason S; Hop, Cornelis E; Khojasteh-Bakht, Siamak Cyrus

    2009-04-01

    In the early stages of drug discovery, the formation of reactive metabolites is often assessed by co-incubating the drug in liver microsomes with a trapping agent in the presence of NADPH. Our group assessed the capability of commonly used trapping agents to reversibly inhibit major cytochrome P450 (CYP) isoforms. Glutathione and cyanide did not inhibit the enzymes at concentrations up to 10 mM; however methoxylamine did show inhibition, with IC(50) values of 0.53 mM for CYP1A2, 4.12 mM for CYP2C9, 2.04 mM for CYP2C19, 9.72 mM for CYP2D6, and 1.26 and >10 mM for CYP3A4/5 (for testosterone and midazolam, respectively, as substrates).

  15. 21-Aminosteroids prevent the down-regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits

    PubMed Central

    Galal, Ahmed; du Souich, Patrick

    1999-01-01

    This study was conducted to assess whether a 21-aminosteroid, U74389G, could prevent the down-regulation of hepatic cytochrome P450 (P450) induced by acute moderate hypoxia or an inflammatory reaction.The rabbits of two groups (n=6 per group) were subjected to acute moderate hypoxia (PaO2≈35 mmHg), one pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 48 h). The rabbits of two other groups received 5 ml of turpentine s.c., one of them being pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 72 h). The kinetics of theophylline (2.5 mg kg−1) were assessed to evaluate the activity of the P450. Once the rabbits were sacrificed, the P450 content and the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were estimated in the liver.Compared with control rabbits, hypoxia and inflammation increased theophylline plasma concentrations, as a result of a decrease in theophylline systemic clearance (P<0.05). Both experimental conditions reduced hepatic content of P450 by 40–50% (P<0.05) and increased the amount of hepatic TBARS by around 50% (P<0.05). Pre-treatment with U74389G prevented the hypoxia- and inflammation-induced decrease in theophylline systemic clearance, the down-regulation of hepatic P450, and the increase in liver TBARS.It is concluded that in the rabbit, U74389G prevents hepatic P450 depression produced by acute moderate hypoxia and a turpentine-induced inflammatory reaction, possibly by eliciting a radical quenching antioxidant activity. PMID:10510447

  16. A Unified Proteochemometric Model for Prediction of Inhibition of Cytochrome P450 Isoforms

    PubMed Central

    Lapins, Maris; Worachartcheewan, Apilak; Spjuth, Ola; Georgiev, Valentin; Prachayasittikul, Virapong; Nantasenamat, Chanin; Wikberg, Jarl E. S.

    2013-01-01

    A unified proteochemometric (PCM) model for the prediction of the ability of drug-like chemicals to inhibit five major drug metabolizing CYP isoforms (i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was created and made publicly available under the Bioclipse Decision Support open source system at www.cyp450model.org. In regards to the proteochemometric modeling we represented the chemical compounds by molecular signature descriptors and the CYP-isoforms by alignment-independent description of composition and transition of amino acid properties of their protein primary sequences. The entire training dataset contained 63 391 interactions and the best PCM model was obtained using signature descriptors of height 1, 2 and 3 and inducing the model with a support vector machine. The model showed excellent predictive ability with internal AUC = 0.923 and an external AUC = 0.940, as evaluated on a large external dataset. The advantage of PCM models is their extensibility making it possible to extend our model for new CYP isoforms and polymorphic CYP forms. A key benefit of PCM is that all proteins are confined in one single model, which makes it generally more stable and predictive as compared with single target models. The inclusion of the model in Bioclipse Decision Support makes it possible to make virtual instantaneous predictions (∼100 ms per prediction) while interactively drawing or modifying chemical structures in the Bioclipse chemical structure editor. PMID:23799117

  17. Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism.

    PubMed Central

    Shedlofsky, S I; Israel, B C; McClain, C J; Hill, D B; Blouin, R A

    1994-01-01

    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

  18. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    SciTech Connect

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.; Gonzalez, F.J. ); Guzelian, P.S. )

    1990-06-01

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.

  19. Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice

    PubMed Central

    Wang, Xiu Jun; Chamberlain, Mark; Vassieva, Olga; Henderson, Colin J.; Wolf, C. Roland

    2005-01-01

    Cytochrome P450 reductase is the unique electron donor for microsomal cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of cytochrome P450 reductase, hepatic cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic cytochrome P450 reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3β-hydroxy-Δ5-steroid dehydrogenase-2), Hsd3b5 (3β-hydroxy-Δ5-steroid dehydrogenase-5) and Hsd11b1 (11β-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the cytochrome system. PMID:15717863

  20. Effects of Chinese, Japanese and Western tea on hepatic P450 enzyme activities in rats.

    PubMed

    Niwattisaiwong, N; Luo, X X; Coville, P F; Wanwimolruk, S

    2004-01-01

    Previous studies have reported that green tea effectively protects against cancers caused by various dietary carcinogens. As P450 enzymes are the major system responsible for the metabolism of many carcinogens, we hypothesise that tea consumption may alter the catalytic activities of P450 enzymes. We conducted this study to screen the effects of four different teas on the activities of P450 enzymes. Tea solutions (2.5%) were prepared by adding boiling water to tea leaves and filtering. Female Wistar rats were divided into five groups (n = 4 each); each had free access to tea solutions while the control group was supplied with water for 4 weeks. Animals were sacrificed and livers were removed for preparation of microsomes. Enzyme activities were determined by incubation of liver microsomes with the appropriate CYP substrate. The activity of CYP1A1 in livers from rats receiving Oolong (Chinese) tea (185 +/- 63 pmol/mg/min), Japanese green tea (197 +/- 22 pmol/mg/min) and Earl Grey tea (228 +/- 40 pmol/mg/min) was significantly higher (p < 0.05) than in the control group (94 +/- 34 pmol/mg/min), whereas no change was observed in the activity of CYP1A2 in any of tested animals. The hepatic activity of CYP2D6 was greater only in rats drinking Earl Grey tea compared to the controls (235 +/- 37 vs 161 +/- 41 pmol/mg/min, p < 0.05). There were also significant increases (p < 0.05) in the activity of CYP3A in livers of animals given Oolong tea (653 +/- 174 vs 382 +/- 114 pmol/mg/min) and Earl Grey tea (751 +/- 202 pmol/mg/min), while Jasmine and Japanese green tea had no significant effect. These results indicate that not all types of tea cause alterations in liver CYP enzymes as some elevated activities and some did not. Further studies are needed to determine whether there is a relationship between the effect of tea on CYP activities and anti-carcinogenesis.

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

    SciTech Connect

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.; Wright, Aaron T.

    2016-07-01

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

  2. Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

    SciTech Connect

    Swinney, D.C.; Ryan, D.E.; Thomas, P.E.; Levin, W.

    1988-07-26

    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.

  3. Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

    PubMed Central

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia

    2014-01-01

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

  4. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: Evaluation of hepatic cytochrome p450 induction

    USGS Publications Warehouse

    Russell, J.S.; Halbrook, R.S.; Woolf, A.; French, J.B.; Melancon, M.J.

    2004-01-01

    We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with $-naphthoflavone ($NF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received $NF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

  5. Human hepatic cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion and diazinon.

    PubMed

    Ellison, Corie A; Tian, Yuan; Knaak, James B; Kostyniak, Paul J; Olson, James R

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  7. Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes.

    PubMed

    Yoshii, K; Kobayashi, K; Tsumuji, M; Tani, M; Shimada, N; Chiba, K

    2000-01-01

    Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.

  8. Investigating the in vitro stereoselective metabolism of m-nisoldipine enantiomers: characterization of metabolites and cytochrome P450 isoforms involved.

    PubMed

    Sun, Yupeng; Jia, Peipei; Yuan, Lin; Liu, Yanyan; Zhang, Zhiyong; Du, Yumin; Zhang, Lantong

    2015-12-01

    m-Nisoldipine, as a novel 1,4-dihydropyridine calcium ion antagonist, was presented as a couple of enantiomers [(-), (+)-m-nisoldipine]. In this report, the in vitro metabolism of m-nisoldipine enantiomers was investigated in rat liver microsomes (RLM) by the combination of two liquid chromatography mass spectrometric techniques for the first time. The metabolites were separated and assayed by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and further identified by comparison of their mass and chromatographic behaviors with reference substances. A total of 18 metabolites of (-)-m-nisoldipine and 16 metabolites of (+)-m-nisoldipine were detected, respectively, which demonstrated that (+)-m-nisoldipine is more metabolically stable than (-)-m-nisoldipine. In addition, the identified metabolic pathways of m-nisoldipine enantiomers were involved in dehydrogenation, oxidation and ester hydrolysis. Afterwards, based on high-performance liquid chromatography coupled to triple quadrupole linear ion trap mass spectrometry, various selective cytochrome P450 (CYP) enzyme inhibitors were employed to evaluate CYP isoforms. The results indicated that the inhibitors of CYP1A1/2, CYP2B1/2, 2D and 2C11 had no obvious inhibitory effects, yet the inhibitor of CYP 3A had a significant inhibitory effect on metabolism of m-nisoldipine enantiomers. This showed that CYP 3A might primarily metabolize m-nisoldipine in RLM.

  9. Selective inhibition of the cytochrome P450 isoform by hyperoside and its potent inhibition of CYP2D6.

    PubMed

    Song, Min; Hong, Miri; Lee, Min Young; Jee, Jun-Goo; Lee, You Mie; Bae, Jong-Sup; Jeong, Tae Cheon; Lee, Sangkyu

    2013-09-01

    Hyperoside, quercetin-3-O-galactoside, is a flavonoid isolated from Oenanthe javanica. In the present study, we investigated potential herb-drug inhibitory effects of hyperoside on nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) and human recombinant cDNA expressed CYP using a cocktail probe assay. Hyperoside strongly inhibited CYP2D6-catalyzed dextromethorphan O-demethylation, with IC₅₀ values of 1.2 and 0.81 μM after 0 and 15 min of preincubation, and a Ki value of 2.01 μM in HLMs, respectively. Hyperoside strongly decreased CYP2D6 activity dose-, but not time-, dependently in HLMs. In addition, the Lineweaver-Burk and Secondary plots for the inhibition of CYP2D6 in HLMs fitted a competitive inhibition mode. Furthermore, hyperoside decreased CYP2D6-catalyzed dextromethorphan O-demethylation activity of human recombinant cDNA-expressed CYP2D6, with an IC₅₀ value of 3.87 μM. However, other CYPs were not inhibited significantly by hyperoside. In conclusion, our data demonstrate that hyperoside is a potent selective CYP2D6 inhibitor in HLMs, and suggest that hyperoside might cause herb-drug interactions when co-administrated with CYP2D substrates.

  10. Cytochrome P450-2E1 promotes fast food-mediated hepatic fibrosis

    PubMed Central

    Abdelmegeed, Mohamed A.; Choi, Youngshim; Godlewski, Grzegorz; Ha, Seung-Kwon; Banerjee, Atrayee; Jang, Sehwan; Song, Byoung-Joon

    2017-01-01

    Cytochrome P450-2E1 (CYP2E1) increases oxidative stress. High hepatic cholesterol causes non-alcoholic steatohepatitis (NASH) and fibrosis. Thus, we aimed to study the role of CYP2E1 in promoting liver fibrosis by high cholesterol-containing fast-food (FF). Male wild-type (WT) and Cyp2e1-null mice were fed standard chow or FF for 2, 12, and 24 weeks. Various parameters of liver fibrosis and potential mechanisms such as oxidative and endoplasmic reticulum (ER) stress, inflammation, and insulin resistance (IR) were studied. Indirect calorimetry was also used to determine metabolic parameters. Liver histology showed that only WT fed FF (WT-FF) developed NASH and fibrosis. Hepatic levels of fibrosis protein markers were significantly increased in WT-FF. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in FF-fed WT. Serum endotoxin, TLR-4 levels, and inflammatory markers were highest in WT-FF. FAS, PPAR-α, PPAR-γ, and CB1-R were markedly altered in WT-FF. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in FF-fed WT. Indirect calorimetry showed that Cyp2e1-null-mice fed FF exhibited consistently higher total energy expenditure (TEE) than their corresponding WT. These results demonstrate that CYP2E1 is important in fast food-mediated liver fibrosis by promoting nitroxidative and ER stress, endotoxemia, inflammation, IR, and low TEE. PMID:28051126

  11. Activating nuclear xenobiotic receptors and triggering ER stress and hepatic cytochromes P450 systems in quails (Coturnix C. coturnix) during atrazine exposure.

    PubMed

    Du, Zheng-Hai; Qin, Lei; Lin, Jia; Sun, Yan-Chun; Xia, Jun; Zhang, Cong; Li, Xue-Nan; Li, Jin-Long

    2017-02-10

    Atrazine (ATR) is one of the most widely detected contaminant in the ecosystem. Nuclear xenobiotic receptors are activated by herbicides and induce the transcription of CYP450 isoforms involved in xenobiotic metabolism and transport. However, little is known about hepatic nuclear xenobiotic receptors in birds are responsible for ATR-induced hepatotoxicity via regulating the cytochrome P450 enzyme systems (CYP450s). The objective of this study was to investigate the mechanism of ATR hepatotoxicity in quails. For this purpose, male quails were dosed by oral gavage from sexual immaturity to maturity with 0, 50, 250, and 500 mg/kg/day ATR for 45 days. The results showed that ATR exposure caused the hepatotoxicity damage and endoplasmic reticulum (ER) degeneration. It suggested that ER is a target organelle of ATR toxicity in hepatocytes. ATR exposure disrupted the hepatic CYP450s homeostasis. This study also demonstrated that ATR triggered the CYP450 isoforms transcription via activating the hepatic CAR/PXR pathway. The present study provides new insights regarding the mechanism of the ATR-induced hepatotoxicity through activating nuclear xenobiotic receptors and triggering ER stress and hepatic CYP450s in quails.

  12. Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes.

    PubMed

    Ise, Ryota; Nakanishi, Yasuharu; Kohara, Sakae; Yamashita, Hiroyuki; Yoshikawa, Tsuyoshi; Iwasaki, Kazuhide; Nagata, Ryoichi; Fukuzaki, Koichiro; Utoh, Masahiro; Nakamura, Chika; Yamazaki, Hiroshi; Uno, Yasuhiro

    2012-01-01

    Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.

  13. Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

    PubMed

    Li, Albert P; Schlicht, Kari E

    2014-01-01

    A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

  14. NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes.

    PubMed

    Iba, M M; Mannering, G J

    1987-05-01

    Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.

  15. Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

    PubMed

    Diaz, Damaris S; Kozar, Michael P; Smith, Kirsten S; Asher, Constance O; Sousa, Jason C; Schiehser, Guy A; Jacobus, David P; Milhous, Wilbur K; Skillman, Donald R; Shearer, Todd W

    2008-02-01

    Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

  16. Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.

    PubMed

    Waxman, D J; Pampori, N A; Ram, P A; Agrawal, A K; Shapiro, B H

    1991-08-01

    Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.

  17. Ablation of cytochrome P450 omega-hydroxylase 4A14 gene attenuates hepatic steatosis and fibrosis.

    PubMed

    Zhang, Xiaoyan; Li, Sha; Zhou, Yunfeng; Su, Wen; Ruan, Xiongzhong; Wang, Bing; Zheng, Feng; Warner, Margaret; Gustafsson, Jan-Åke; Guan, Youfei

    2017-03-21

    Nonalcoholic fatty liver disease (NAFLD) is characterized by simple hepatic steatosis (SS), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, and cirrhosis. Dysregulated fatty acid metabolism in the liver plays a critical role in the pathogenesis of NAFLD. Cytochrome P450 omega-hydroxylase 4A14 (CYP4A14) is a homolog of human CYP4A hydroxylase that catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid in mice. The goal of this study was to determine the role of CYP4A14 in the development and the progression of NAFLD. Here, we showed that hepatic CYP4A expression was up-regulated in the livers of patients and three murine models of NAFLD. Adenovirus-mediated overexpression of CYP4A14 in the livers of C57BL/6 mice resulted in a fatty liver phenotype with a significant increase in hepatic fatty acid translocase (FAT/CD36) expression. In contrast, CYP4A14 gene-deficient mice fed a high-fat diet or a methionine and choline-deficient (MCD) diet exhibited attenuated liver lipid accumulation and reduced hepatic FAT/CD36 expression. In addition, hepatic inflammation and fibrosis was markedly ameliorated in MCD diet-fed CYP4A14-deficient mice. Collectively, CYP4A14 plays an important role in the pathogenesis of both SS and NASH and may represent a potential therapeutic target for the treatment of NAFLD.

  18. High dose of commercial products of kava (Piper methysticum) markedly enhanced hepatic cytochrome P450 1A1 mRNA expression with liver enlargement in rats.

    PubMed

    Yamazaki, Yuko; Hashida, Hiroko; Arita, Anna; Hamaguchi, Keiko; Shimura, Fumio

    2008-12-01

    Commercial products containing the kava plant (Piper methysticum), known to have the anxiolytic activity, are banned in several European countries and Canada because of the suspicion of a potential liver toxicity. In some reports, kava and kavalactones (major constituents of kava) inhibited activities of cytochrome P450 (CYP) isoforms including CYP1A2. On the other hand, a few studies showed that administration of kava to rats moderately increased CYP1A2 proteins in the liver. CYP1A isoforms are likely responsible for the metabolic activation of potent carcinogenic environmental toxins such as aflatoxins, benzo[a]pyrene, and others. On these bases, we have investigated the effects of administration of commercial kava products on gene expression of hepatic CYP1A isoforms in rats. A high dose (equivalent to approximately 380mg kavalactones/kg/day; 100 times of the suggested dosage for human use) of two different types of kava products for 8 days significantly increased liver weights. CYP1A2 mRNA expression was moderately increased (2.8-7.3 fold). More importantly, the high dose of kava markedly enhanced CYP1A1 mRNA expression (75-220 fold) as well as ethoxyresorufin O-deethylase activities and CYP1A1 immunoreactivities. Thus, no observed adverse effect levels of kavalactones would be lower than 380mg/kg/day. When the safety factor of kavalactones is assumed to be 100, a value most often used upon the risk analysis of chemicals and designed to account for interspecies and intraspecies variations, a number of kava product users likely ingest more kavalactones than acceptable daily intakes. Based on overall evidence, we should pay considerable attention to the possibility that kava products induce hepatic CYP1A1 expression in human especially in sensitive individuals.

  19. In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan.

    PubMed

    Matsumoto, Takashi; Kushida, Hirotaka; Maruyama, Takeshi; Nishimura, Hiroaki; Watanabe, Junko; Maemura, Kazuya; Kase, Yoshio

    2016-01-01

    1. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. 2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. 3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes. 4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.

  20. Enantioselective, mechanism-based inactivation of guinea pig hepatic cytochrome P450 by N-(alpha-methylbenzyl)-1-aminobenzotriazole.

    PubMed

    Sinal, C J; Hirst, M; Webb, C D; Bend, J R

    1998-07-01

    N-Aralkylated derivatives of 1-aminobenzotriazole are well-established, mechanism-based inhibitors of cytochrome P450 (CYP or P450). In this study, the kinetics of inactivation of CYP2B-dependent 7-pentoxyresorufin O-depentylation (PROD) and CYP1A-dependent 7-ethoxyresorufin O-deethylation (EROD) activities by enantiomers of N-(alpha-methylbenzyl)-1-aminobenzotriazole (alphaMB) were compared. The racemic mixture (+/-)-alphaMB, as well as the enantiomers (-)-alphaMB and (+)-alphaMB, produced a time-, concentration-, and NADPH-dependent loss of PROD and EROD activity in hepatic microsomes from phenobarbital-treated guinea pigs. The rates of PROD inactivation by (-)-alphaMB were significantly faster than for (+)-alphaMB. Consistent with this, the derived maximal kinact was also significantly greater for (-)-alphaMB than for (+)-alphaMB (0.49 vs. 0.35 min-1). In contrast, the concentrations required for the half-maximal rate of inactivation (Ki) were equivalent for (-)-alphaMB and (+)-alphaMB, whereas the degree of competitive inhibition of PROD activity was greater for (+)-alphaMB. No significant differences were found among (-)-alphaMB, (+)-alphaMB, and (+/-)-alphaMB with respect to mechanism-based inactivation (kinact = 0.18, 0.16, and 0.17 min-1, respectively) or competitive inhibition of EROD activity. No differences were found for the maximal extent of PROD or EROD inhibition or the loss of spectral P450 after an extended 30-min incubation with the inhibitors. We conclude that mechanism-based inactivation of guinea pig CYP2B, but not CYP1A, isozymes by alphaMB occurs in a stereoselective manner, most likely as a result of a difference in the balance between metabolic activation and deactivation for the alphaMB enantiomers.

  1. Bromopropylate: induction of hepatic cytochromes P450 and absence of covalent binding to DNA in mouse liver.

    PubMed

    Thomas, H; Sagelsdorff, P; Molitor, E; Skripsky, T; Waechter, F

    1994-11-01

    Oral administration of benzilic acid ester-based acaricide bromopropylate at daily doses of 3, 15, 100, and 300 mg/kg body wt to young adult male Tif:MAGf mice for 14 days caused slightly increased liver weights in the high-dose group. A dose-dependent increase of the microsomal cytochrome P450 content was accompanied by elevated ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, and total testosterone hydroxylase activities. When compared with mice treated in parallel with the model compounds for hepatic xenobiotic metabolizing enzyme induction, phenobarbitone, and 3-methylcholanthrene, the enzyme activity changes observed with bromopropylate largely equalled those expressed in phenobarbitone-treated mice. Immunochemical studies with monoclonal antibodies against rat liver cytochrome P450 isoenzymes of the gene families 1A, 2B, 3A, and 4A confirmed that bromopropylate is a phenobarbitone-type inducer in the mouse liver. Titration of liver microsomal suspensions with bromopropylate yielded Type I substrate binding spectra. The specific amplitude was increased 1.5-fold when microsomes from bromopropylate-treated mice (300 mg/kg body wt) were used instead of control microsomes, indicating the induction of cytochromes P450 catalyzing the oxidative metabolism of the test compound. Single oral administration of 300 mg/kg body wt [14C]bromopropylate to male mice, without or following pretreatment for 14 days with 300 mg/kg body wt unlabeled bromopropylate, gave no indication for DNA binding of the test compound in the liver. This excludes a genotoxic potential via covalent DNA modification. The results suggest that, in analogy to phenobarbitone, bromopropylate acts as a tumor promotor rather than a tumor initiator in the mouse liver.

  2. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    PubMed Central

    Ye, Qinyuan; Lian, Fuzhi; Chavez, Pollyanna R.G.; Chung, Jayong; Ling, Wenhua; Qin, Hua; Seitz, Helmut K.

    2012-01-01

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2'-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats. PMID:23543859

  3. Induction of rat hepatic cytochromes P450 by toxic ingredients in plants: lack of correlation between toxicity and inductive activity.

    PubMed

    Yamada, H; Nakamura, T; Oguri, K

    1998-12-01

    "Animal-Plant Warfare" is one of the hypotheses for the evolution of drug-metabolizing P450s. To address the validity of this hypothesis, we examined the induction of xenobiotic-metabolizing P450s by 12 plant toxins in rats, using hepatic activity for testosterone metabolism as the index. The compounds tested were aconitine, morphine, tubocurarine, physostigmine, pilocarpine, muscarine, cocaine, atropine, amygdalin, digitonin, nicotine and solanine. Drinking water containing a test compound was given to rats for 4 days, and the hepatic activity of testosterone metabolism was determined together with monitoring body weight gain and liver weight as the indices of toxicity. The results showed that while cocaine and nicotine have a minor ability to increase testosterone 16 beta-hydroxylase activity, a marker activity for the CYP2B1 and 2, all other compounds did not have any such effect. No correlation was observed between a change in 16 beta-hydroxylase and toxicity caused by toxins. Therefore, these results did not support the idea that the inducibility of the CYP2B subfamily in animals is acquired through "Animal-Plant Warfare". Several compounds examined here increased or decreased hepatic activities of testosterone 2 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylation and 17-oxidation, indicating a possible effect on the CYP2A, 2C and 3A subfamily. Of these effects, a moderate correlation (r < 0.49) was observed in the changes in the activities of 2 alpha-/16 alpha-hydroxylation and 17-oxidation vs. that in toxicity. It is therefore suggested that inhibition or suppression of the expression of CYP2C11 is one of the mechanisms in the toxicity of plant toxins for rats, although it comes from an examination using limited numbers of compounds.

  4. Hepatic cytochrome P450 3A drug metabolism is reduced in cancer patients who have an acute-phase response

    PubMed Central

    Rivory, L P; Slaviero, K A; Clarke, S J

    2002-01-01

    Inflammatory disease states (infection, arthritis) are associated with reduced drug oxidation by the cytochrome P450 3A system. Many chemotherapy agents are metabolised through this pathway, and disease may therefore influence inter-individual differences in drug pharmacokinetics. The purpose of this study was to assess cytochrome P450 3A function in patients with advanced cancer, and its relation to the acute-phase response. We evaluated hepatic cytochrome P450 3A function in 40 patients with advanced cancer using the erythromycin breath test. Both the traditional C20min measure and the recently proposed 1/TMAX values were estimated. The marker of acute-phase response, C-reactive protein and the pro-inflammatory cytokines IL-6, IL-1β, TNFα and IL-8 were measured in serum or plasma at baseline. Cancer patients with an acute phase response (C-reactive protein >10 mg l−1, n=26) had reduced metabolism as measured with the erythromycin breath test 1/TMAX (Kruskal–Wallis Anova, P=0.0062) as compared to controls (C-reactive protein ⩽10 mg l−1, n=14). Indeed, metabolism was significantly associated with C-reactive protein over the whole concentration range of this acute-phase marker (r=−0.64, Spearman Rank Correlation, P<0.00001). C-reactive protein serum levels were significantly correlated with those of IL-6 (Spearman coefficient=0.58, P<0.0003). The reduction in cytochrome P450 3A function with acute-phase reaction was independent of the tumour type and C-reactive protein elevation was associated with poor performance status. This indicates that the sub-group of cancer patients with significant acute-phase response have compromised drug metabolism, which may have implications for the safety of chemotherapy in this population. British Journal of Cancer (2002) 87, 277–280. doi:10.1038/sj.bjc.6600448 www.bjcancer.com © 2002 Cancer Research UK PMID:12177794

  5. Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

    PubMed

    Akingbasote, James A; Foster, Alison J; Wilson, Ian; Sarda, Sunil; Jones, Huw B; Kenna, J Gerry

    2016-04-01

    Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.

  6. Hierarchical cluster analysis of environmental pollutants through P450 induction in cultured hepatic cells.

    PubMed

    Dubois, M; Plaisance, H; Thomé, J P; Kremers, P

    1996-08-01

    Environmental pollutants are classically associated with increased drug metabolism. Cultures of rat hepatocytes, quail hepatocytes, and human hepatoma (Hep G2) cells were used to study the effects of pesticides on drug-metabolizing enzymes. Membrane integrity and mitochondrial activity were evaluated and induction of ethoxycoumarin-O-deethylase and ethoxyresorufin-O-deethylase activities were measured. Induced P450s were identified by immunoblotting. Pentachlorophenol and lindane appeared as the strongest inducers. On the immunoblots, specific antibodies revealed induced CYP1A1 in fetal rat hepatocytes, CYP2B in quail hepatocytes, and CYP3A7 in Hep G2 cells. Pesticide effects on these different activities in each type of cultured cells were compared by cluster analysis. Results obtained under similar conditions with reference inducers phenobarbital (PB) and benzo[a]anthracene and other environmental pollutants (polychlorobiphenyls) were added to previous data prior to multivariate analysis. The tested products fell into four major groups: a first group with pentachlorophenol, identified as a CYP3A inducer; a second group containing the methylcholanthrene-type inducers that increase CYP1A-related activities; a third class represented by dieldrin, a PB-type inducer; a fourth group including inert compounds or weak inducers. Lindane shares the criteria of the second and third groups and seems to induce both CYP1A and CYP2B activities. The current study results highlight the advantage of using several types of cultured hepatocytes to evaluate the short-term toxicity of environmental pollutants in vitro and constitute a useful model for predicting the potential toxicity of pesticides in humans (Hep G2 cells) and wildlife (fetal quail hepatocytes).

  7. Hepatic cytochrome P450 enzymes belonging to the CYP2C subfamily from an Australian marsupial, the koala (Phascolarctos cinereus).

    PubMed

    Jones, Brett R; El-Merhibi, Adaweyah; Ngo, Suong N T; Stupans, Ieva; McKinnon, Ross A

    2008-09-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported that the obligate Eucalyptus feeder koala (Phascolarctos cinereus) exhibits a higher hepatic CYP2C activity as compared to non-Eucalyptus feeders human or rat, with stimulation of CYP2C activity by cineole. In the present study, we examine CYP2C expression by immunohistochemistry and describe the identification and cloning of koala CYP2Cs. Utilising anti-rat CYP2C6 antibody, the expression of CYP2C was found to be uniform across the hepatic sections, being consistent with that observed in human and rat. Two 1647 and 1638 bp koala liver CYP2C complete cDNAs, designated CYP2C47 and CYP2C48 respectively, were cloned by cDNA library screening. The koala CYP2C cDNAs encode a protein of 495 amino acids. Three additional partial CYP2C sequences were also identified from the koala, indicating the multiplicity of the CYP2C subfamily in this unique marsupial species. The results of this study demonstrate the presence of koala hepatic CYP2Cs that share several common features with other published CYP2Cs; however CYP2C47 and CYP2C48 contain four extra amino acid residues at the NH2-terminal, a transmembrane anchor which was reported being a fundamentally conserved structure core of all eukaryote CYP enzymes.

  8. Two azole fungicides (carcinogenic triadimefon and non-carcinogenic myclobutanil) exhibit different hepatic cytochrome P450 activities in medaka fish.

    PubMed

    Lin, Chun-Hung; Chou, Pei-Hsin; Chen, Pei-Jen

    2014-07-30

    Conazoles are a class of imidazole- or triazole-containing drugs commonly used as fungicides in agriculture and medicine. The broad application of azole drugs has led to the contamination of surface aquifers receiving the effluent of municipal or hospital wastewater or agricultural runoff. Several triazoles are rodent carcinogens; azole pollution is a concern to environmental safety and human health. However, the carcinogenic mechanisms associated with cytochrome P450 enzymes (CYPs) of conazoles remain unclear. We exposed adult medaka fish (Oryzias latipes) to continuous aqueous solutions of carcinogenic triadimefon and non-carcinogenic myclobutanil for 7 to 20 days at sub-lethal or environmentally relevant concentrations and assessed hepatic CYP activity and gene expression associated with CYP-mediated toxicity. Both triadimefon and myclobutanil induced hepatic CYP3A activity, but only triadimefon enhanced CYP1A activity. The gene expression of cyp3a38, cyp3a40, pregnane x receptor (pxr), cyp26b, retinoid acid receptor γ1 (rarγ1) and p53 was higher with triadimefon than myclobutanil. As well, yeast-based reporter gene assay revealed that 4 tested conazoles were weak agonists of aryl hydrocarbon receptor (AhR). We reveal differential CYP gene expression with carcinogenic and non-carcinogenic conazoles in a lower vertebrate, medaka fish. Liver CYP-enzyme induction may be a key event in conazole-induced tumorigenesis. This information is essential to evaluate the potential threat of conazoles to human health and fish populations in the aquatic environment.

  9. Induction of hepatic cytochrome P-450 activity in wild cotton rats (Sigmodon hispidus) by phenobarbital and 3-methylcholanthrene

    SciTech Connect

    Elangbam, C.S.; Qualls, C.W.,Jr.; Bauduy, M. )

    1989-05-01

    Wild cotton rats (Sigmodon hispidus) are ubiquitous throughout the Southeast quadrant of the United States, easy to capture, have a generation interval of less than one year and a limited range of movement (less than one hectare). This species may prove to be an excellent model for monitoring environmental contamination. Traditionally, cytochrome P-450 inducing agents are grouped into two classes. One, represented by phenobarbital, induces P-450b and P-450e; the other, represented by 3-methylcholanthrene, induces P-450c and P-450d isoenzymes. The types and amounts of cytochrome P-450 vary among species, organs, health status, sex, and stress of the animal. If the levels of cytochrome P-450 of wild cotton rats are to be used in monitoring environmental pollution, it is necessary to characterize the inducibility and concentration of cytochrome P-450 in this species. This study was designed to determine the concentration and inducibility of cytochrome P-450 in the livers of cotton rats after intraperitoneal (ip) administration of phenobarbital and 3-methylcholanthrene.

  10. INDUCTION AND POST-TRANSCRIPTIONAL SUPPRESSION OF HEPATIC CYTOCHROME P450 1A1 BY 3,3',4,4'-TETRACHLOROBIPHENYL. (R827102)

    EPA Science Inventory

    Abstract

    3,3',4,4'-Tetrachlorobiphenyl (TCB) can induce and inhibit cytochrome P450 1A1 (CYP1A1) in vertebrates. TCB may also suppress CYP1A1 protein levels, but the mechanism is unknown. This study examined transcriptional and translational aspects of hepatic ...

  11. High-throughput screening of inhibitory effects of Bo-yang-hwan-o-tang on human cytochrome P450 isoforms in vitro using UPLC/MS/MS.

    PubMed

    Lee, Miran; Park, Jeonghyeon; Lim, Mi-sun; Seong, Sook Jin; Lee, Joomi; Seo, Jeong Ju; Park, Yong-Ki; Lee, Hae Won; Yoon, Young-Ran

    2012-01-01

    Bo-yang-hwan-o-tang (BHT) is an oriental herbal medicine for treating brain disorders such as cerebral ischemia. The objective of this study was to develop an economically feasible and time-saving high-throughput screening method to monitor the potential inhibitory effects of BHT on human cytochrome P450 (CYP) enzymes in vitro. Two cocktail sets were used for incubation of human liver microsomes: Cocktail A: 6 probe substrates for CYP1A2, CYP2A6, CYP2C8, CYP2C19, CYP2D6, CYP3A4; Cocktail B: 3 for CYP2B6, CYP2C9, CYP2E1. The concentrations of the substrate metabolites were simultaneously analyzed using UPLC/MS/MS. The BHT extract had almost negligible inhibitory effects on the nine human CYP isoforms tested, with the half-maximal inhibitory concentration value ranged from 3624.99 to 45412.44 μg/ml. The results suggest that BHT extract has no inhibitory effects on CYP isoforms within the clinically recommended dosage range. We conclude that BHT might be free of drug-herb interactions when co-administered with other medicines. However, more in vivo human studies are needed to confirm these results. The high-throughput screening method can be a useful tool for drug discovery and for understanding drug interactions.

  12. Pharmacokinetic changes in drugs during protein-calorie malnutrition: correlation between drug metabolism and hepatic microsomal cytochrome P450 isozymes.

    PubMed

    Lee, Joo Hyun; Suh, Ok Kyung; Lee, Myung Gull

    2004-07-01

    The rats with protein-calorie malnutrition (PCM, 5% casein diet for a period of 4-week) were reported to exhibit 60 and 80% suppression in the hepatic microsomal cytochrome P450 (CYP) 1A2 and CYP2C11 levels, respectively, and 40-50% decreases in CYP2E1 and CYP3A1/2 levels compared to control (23% casein diet for a period of 4-week) based on Western blot analysis. In addition, Northern blot analysis showed that CYP1A2, CYP2E1, CYP2C11, and CYP3A1/2 mRNAs decreased in the state of PCM as well. Hence, pharmacokinetic changes of the drugs in rats with PCM [especially the area under the plasma concentration-time curve from time zero to time infinity (AUC) changes of metabolite(s)] reported from literatures were tried to explain in terms of CYP isozyme changes in the rats. Otherwise, the time-averaged nonrenal clearance (CL NR) of parent drug was compared. Pharmacokinetic changes of the drugs in other types of malnutritional state, such as kwashiorkor and marasmus, in both human and animal models were also compared. The drugs reviewed are as follows: diuretics, antibiotics, anticancer agents, antiepileptics, antiarrythmics, analgesics, xanthines, antimalarials, and miscellaneous.

  13. Effects of acetone, acetonitrile, ethanol, methanol and DMSO on cytochrome P450 in rainbow trout (Oncorhynchus mykiss) hepatic microsomes.

    PubMed

    Sakalli, Sidika; Burkina, Viktoriia; Zlabek, Vladimir; Zamaratskaia, Galia

    2015-01-01

    In vitro impacts of five organic solvents on cytochrome P450 (CYP450) enzyme activity were investigated using hepatic microsomes of rainbow trout. The rates of several CYP450-mediated reactions were investigated at solvent concentrations ranging from 0.01% to 3%. The solvents greatly affected all tested reactions. In at least 0.8% ethanol, 2% methanol or acetone, 1% acetonitrile or 3% dimethyl sulfoxide (DMSO), 7-ethoxyresorufin-O-deethylase (EROD) activity decreased and at 3% acetonitrile or ethanol, it was undetected. At 3%, all tested solvents except methanol reduced 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylase (BFCOD) activity, but at low concentrations of ethanol (2% and lower) or DMSO (1% and lower), it was induced. This was not seen with the inclusion of a pre-incubation step. p-Nitrophenolhydroxylase (PNPH) activity was not affected at concentrations below 1% DMSO, and at 2% acetonitrile it was reduced, as it was above 1% methanol or 0.5% ethanol. Acetone did not affect PNPH activity with or without a pre-incubation step. In general, the degree of inhibition was similar with and without the pre-incubation step. We conclude that the concentration of organic solvent for solubilizing the substrate and inhibitor in in vitro microsomal studies should be minimized.

  14. Effect of antipsychotic drugs on human liver cytochrome P-450 (CYP) isoforms in vitro: preferential inhibition of CYP2D6.

    PubMed

    Shin, J G; Soukhova, N; Flockhart, D A

    1999-09-01

    The ability of antipsychotic drugs to inhibit the catalytic activity of five cytochrome P-450 (CYP) isoforms was compared using in vitro human liver microsomal preparations to evaluate the relative potential of these drugs to inhibit drug metabolism. The apparent kinetic parameters for enzyme inhibition were determined by nonlinear regression analysis of the data. All antipsychotic drugs tested competitively inhibited dextromethorphan O-demethylation, a selective marker for CYP2D6, in a concentration-dependent manner. Thioridazine and perphenazine were the most potent, with IC(50) values (2.7 and 1.5 microM) that were comparable to that of quinidine (0.52 microM). The estimated K(i) values for CYP2D6-catalyzing dextrorphan formation were ranked in the following order: perphenazine (0.8 microM), thioridazine (1.4 microM), chlorpromazine (6.4 microM), haloperidol (7.2 microM), fluphenazine (9.4 microM), risperidone (21.9 microM), clozapine (39.0 microM), and cis-thiothixene (65.0 microM). No remarkable inhibition of other CYP isoforms was observed except for moderate inhibition of CYP1A2-catalyzed phenacetin O-deethylation by fluphenazine (K(i) = 40.2 microM) and perphenazine (K(i) = 65.1). The estimated K(i) values for the inhibition of CYP2C9, 2C19, and 3A were >300 microM in almost all antipsychotics tested. These results suggest that antipsychotic drugs exhibit a striking selectivity for CYP2D6 compared with other CYP isoforms. This may reflect a remarkable commonality of structure between the therapeutic targets for these drugs, the transporters, and metabolic enzymes that distribute and eliminate them. Clinically, coadministration of these medicines with drugs that are primarily metabolized by CYP2D6 may result in significant drug interactions.

  15. Effect of four environmental toxicants on plasma Ca and estradiol 17[beta] and hepatic P450 in laying hens

    SciTech Connect

    Chen, S.W.; Dziuk, P.J.; Francis, B.M. . Dept. of Animal Sciences)

    1994-05-01

    In a previous study, the authors found that administration of phenobarbital to laying hens was associated with an increase in content of liver cytochrome P450 and a reduction of estradiol (E2) in serum. Thus, the authors hypothesized that other xenobiotics such as environmental toxicants that affect P450 might also affect E2 in laying hens. In experiment 1, the authors examined the effect of four environmental pollutants, three of which induced different isoenzymes of P450 and one inhibitor, on circulating E2 and related reproductive functions. Aroclor 1254 (PCB), 20 mg/d; dichlorodiphenyltrichloroethane (DDT), 40 mg/d; or benzo[a]pyrene (BZ), 5 mg/d, was administered for 5 d. An inhibitor, lead acetate, was injected for 2 d. Controls received corn oil or sodium acetate. No significant difference was observed due to administration of lead. Treatment with PCB or DDT decreased the concentration of E2 and increased P450. Only PCB significantly decreased plasma total calcium and egg lay. Therefore in experiment 2, the authors determined the dose-response effect of PCB. The PCB was given orally at doses of 0, 5, 10, and 25 mg in corn oil for 5 d. The depression of concentrations of E2 was associated with the induction of P450 in a dose-dependent manner. Egg production and plasma total calcium were reduced by the two highest doses, but eggshell thickness was not different from control in all regimens. Plasma E2 and plasma total calcium were negatively correlated with induction of P450. BZ is not a strong inducer of P450 and had no effect on E2 or reproduction, whereas DDT and PCB had a profound effect on P450 with consequent depression of circulating E2. These data indicate that the effects of environmental pollutants on reproduction in birds can be mediated through increased P450, thereby increasing the metabolism of steroid hormones and depressing concentration in circulation.

  16. Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay.

    PubMed

    Liu, Xidong; Hu, Lianghai; Ge, Guangbo; Yang, Bo; Ning, Jing; Sun, Shixin; Yang, Ling; Pors, Klaus; Gu, Jingkai

    2014-08-01

    Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

  17. Cytochrome p450 mRNA expression in the rodent brain: species-, sex-, and region-dependent differences.

    PubMed

    Stamou, Marianna; Wu, Xianai; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-02-01

    Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity.

  18. Immunohistochemical analysis of expressions of hepatic cytochrome P450 in F344 rats following oral treatment with kava extract.

    PubMed

    Clayton, Natasha P; Yoshizawa, Katsuhiko; Kissling, Grace E; Burka, Leo T; Chan, Po-Chuen; Nyska, Abraham

    2007-01-01

    Kava (Piper methysticum), used for relaxation and pain relief, has been one of the leading dietary supplements and several reports linking hepatic functional disturbances and liver failure to kava have resulted in a ban on sales in Europe and Canada and the issuance of warnings by the US FDA. The National Toxicology Program conducted 14-week rat studies to characterize the toxicology of kava exposure in Fischer 344 rats [National Toxicity Program. 90 day gavage toxicity studies of KAVA KAVA EXTRACT in Fischer rats and B6C3F1 mice. Research Triangle Park, NC; 2005a; National Toxicity Program. Testing status of agents at NTP (KAVA KAVA EXTRACT M990058). Research Triangle Park, NC; 2005b. (http://ntp.niehs.nih.gov/index.cfm?objectid=071516E-C6E1-7AAA-C90C751E23D14C1B)]. Groups of 10 male and 10 female rats were administered kava extract by gavage at 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg/day. Increased gamma-glutamyl-transpeptidase (GGT) activities were observed in the 2.0 g/kg males and 1.0 and 2.0 g/kg females, as well as increased serum cholesterol levels in males and females at 0.5 g/kg and higher. Increases in incidence and severity of hepatocellular hypertrophy (HP) were noted in males at 1.0 g/kg and females at 0.5 g/kg and higher, as well as increased liver weights. Immunohistochemical analyses of the expression of cytochrome-P450 (CYP) enzymes in liver of the control and 1.0- and 2.0-g/kg-treated groups indicated decreased expression of CYP2D1 (human CYP2D6 homolog) in 2.0 g/kg females and increased expression of CYP1A2, 2B1, and 3A1 in 1.0 and 2.0 g/kg groups of both sexes. The no observed adverse effect levels were decided as 0.25 g/kg in both genders, based on neurotoxic effects, increases in GGT, cholesterol, liver weight, and HP and decreases in body weight. Kava-induced hepatic functional changes in the F344 rat might be relevant to human clinical cases of hepatotoxicity following exposure.

  19. Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes.

    PubMed

    Shi, Rong; Ma, Bingliang; Wu, Jiasheng; Wang, Tianming; Ma, Yueming

    2015-10-01

    The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high-throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin-O deethylation, coumarin-7 hydroxylation, bupropion hydroxylation, taxol-6 hydroxylation, omeprazole-5 hydroxylation, dextromethorphan-O demethylation, tolbutamide-4 hydroxylation, chlorzoxazone-6 hydroxylation, testosterone-6β hydroxylation, and midazolam-1 hydroxylation in rat liver microsomes.

  20. An investigation into the hepatic cytochrome P-450 catalysed metabolism of the anaesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether).

    PubMed

    Marsh, J A; Ivanetich, K M; Bradshaw, J J; Harrison, G G; Webber, B L; Kaminsky, L S

    1975-01-01

    The role of the different cytochromes P-450 in the metabolism of the anaesthetic agent fluroxene, and the mechanism of production of toxic effects seen after pre-treatment of the animals with pehnobarbital prior to anaesthesia, have been investigated. Male rats were anaesthetized with fluroxene, or with 2,2,2-trifluroethyl ethyl ether, or with ethyl vinyl ether in an attempt to ascertain the in vivo toxic effects of the three anaesthetic agents. The resultant hepatic histology is reported. A study of the binding and metabolism of fluroxene by isolated rat hepatic microsomes was also made. We conclude that it is elevated levels of cytochrome P-450 which potentiate the toxicity of fluroxene anaesthesia in phenobarbital treated animals and that cytochrome P-448 does not bind or metabolize fluroxene. The potential toxicity of the fluroxene molecule is considered to reside in the trifluoroethyl moiety, while the vinyl group of fluroxene appears to play a role in the observed liver damage.

  1. Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta and pre-implantation blastocyst. All catalyze estrogen synthesis, but the “gonadal” type enzyme is unique in also synthesizing a nonaromat...

  2. Catalytic and Immunochemical Detection of Hepatic and Extrahepatic Microsomal Cytochrome P450 1A1 (CYP1A1) in White-sided Dolphin (Lagenorhynchus acutus)

    PubMed Central

    Wilson, Joanna Y.; Moore, Michael J.; Stegeman, John J.

    2009-01-01

    We have characterized microsomal systems and measured the levels of microsomal cytochrome P450 1A1 (CYP1A1) and ethoxyresorufin-O-deethylase activity in multiple internal organs of male and female white-sided dolphin (Lagenorhynchus acutus) from the northwest Atlantic Ocean. Internal organs were sampled within 24 hours of death, sometimes in a period of hours, collection times which are significantly less than usually seen for marine mammals. Tissue autolysis, as assessed by histological analysis of liver, was minimal to none in all individuals. Total P420 did not correlate with time from death to sampling, suggesting that it is a poor indicator of P450 degradation in cetacean tissues where perfusion isn’t practical. The total hepatic microsomal P450 content, cytochrome b5 content, and NADPH-cytochrome c (P450) reductase (CPR) activity averaged 0.29 nmol mg−1, 0.12 nmol mg−1, and 238 nmol mg−1 min−1, respectively. Microsomal CPR activity in liver was higher than that in lung and kidney, and was higher than that reported in liver of most other cetacean species. Immunodetected CYP1A1 content was low in all organs, less than 3 pmoles CYP1A equivalents mg−1. EROD activity ranged from 9 – 376 pmoles mg−1 min−1 and was greater in liver than in other tissues. Hepatic microsomal EROD activity and CYP1A1 content did not correlate. However, hepatic EROD activity, but not CYP1A1 protein content, was well correlated with both total PCB and Σmono-ortho PCB concentrations in blubber. Length, as a proxy for age, did not correlate with hepatic EROD activity or CYP1A1 protein levels, and sex did not influence the relationship between EROD and contaminant concentrations. We cannot easily control for the extent of tissue degradation in cetacean studies nor do we have a complete history of these animals. Therefore, other factors such as degradation or hormonal state may have a role in the observed relationships. Yet, as in other mammals, hepatic tissues appear to be

  3. Impact of six fruits--banana, guava, mangosteen, pineapple, ripe mango and ripe papaya--on murine hepatic cytochrome P450 activities.

    PubMed

    Chatuphonprasert, Waranya; Jarukamjorn, Kanokwan

    2012-12-01

    The effects of six Thai fruits, namely banana, guava, mangosteen, pineapple, ripe mango and ripe papaya, on cytochrome P450 (P450) activities were investigated. The median inhibitory concentrations (IC(50) ) of each of the fruit juices on CYP1A1, CYP1A2, CYP2E1 and CYP3A11 activities were determined. Pineapple juice showed the strongest inhibitory effect against all the evaluated P450 isozyme activities in mouse hepatic microsomes, followed by mangosteen, guava, ripe mango, ripe papaya and banana. The study was further performed in male ICR mice given pineapple juice intragastrically at doses of 10, 20 and 40 mg kg(-1) per day for 7 or 28 days. In a concentration-dependent fashion, the pineapple juice raised ethoxyresorufin O-deethylase, aniline hydroxylase and erythromycin N-demethylase activities, which are marker enzymatic reactions responsible for CYP1A1, CYP2E1 and CYP3A11, respectively. The effect of pineapple juice on the expression of CYP1A1, CYP2E1 and CYP3A11 mRNAs corresponded to their enzymatic activities. However, the pineapple juice significantly decreased methoxyresorufin O-demethylase activity. These observations supported that the six Thai fruits were a feasible cause of food-drug interaction or adverse drug effects owing to their potential to modify several essential P450 activities. Individuals consuming large quantities of pineapple for long periods of time should be cautioned of these potential adverse effects.

  4. Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

    SciTech Connect

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

    2007-12-15

    Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD{sub 50}) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.

  5. Cytochrome P450 CYP3A in marsupials: cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Crittenden, Tamara A; Marchant, Ceilidh L; Stupans, Ieva; McKinnon, Ross A

    2011-11-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610 bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP

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

    PubMed

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

    1999-09-01

    1 To investigate the effect of moderate hypoxia alone or combined with an inflammatory reaction or after 3-methylcholanthrene (3MC) pre-treatment on cytochrome P450 (P450), conscious rabbits were exposed for 24 h to a fractional concentration of inspired O2 of 10% (mean PaO2 of 34 mmHg). Hypoxia decreased theophylline metabolic clearance (ClM) from 1.73+/-0.43 to 1.48+/-0.13 ml min-1 kg-1 (P<0. 05), and reduced (P<0.05) the formation clearance of theophylline metabolites, 3-methylxanthine (3MX), 1-methyluric acid (1MU) and 1,3-dimethyluric acid (1,3DMU). Hypoxia reduced the amount of CYP1A1 and 1A2 but increased CYP3A6 proteins. 2 Turpentine-induced inflammatory reaction reduced (P<0.05) the formation clearance of 3MX, 1MU, and 1,3DMU, and diminished the amount of CYP1A1, 1A2 and 3A6 proteins. However, when combined with hypoxia, inflammation partially prevented the decrease in ClM, especially by impeding the reduction of 1,3DMU. The amount of CYP1A1 and 1A2 remained reduced but the amount of CYP3A6 protein returned to normal values. 3 Pre-treatment with 3MC augmented the ClM by 114% (P<0.05) due to the increase in the formation clearance of 3MX, 1MU and 1,3DMU. 3MC treatment increased the amount of CYP1A1 and 1A2 proteins. Pre-treatment with 3MC prevented the hypoxia-induced decrease in amount and activity of the P450. 4 It is concluded that acute moderate hypoxia and an inflammatory reaction individually reduce the amount and activity of selected apoproteins of the P450. However, the combination of hypoxia and the inflammatory reaction restores P450 activity to near normal values. On the other hand, pre-treatment with 3MC prevents the hypoxia-induced depression of the P450.

  7. In vitro characterization of the cytochrome P450 isoforms involved in the metabolism of 6-methoxy-2-napthylacetic acid, an active metabolite of the prodrug nabumetone.

    PubMed

    Matsumoto, Kaori; Nemoto, Eiichi; Hasegawa, Tetsuya; Akimoto, Masayuki; Sugibayashi, Kenji

    2011-01-01

    The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis-Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the K(m) and V(max) values for the formation of 6-HNA in humans and rats were 640.0 ± 30.9 and 722.9 ± 111.7 µM, and 1167.5 ± 33.0 and 1312.7 ± 73.8 pmol min⁻¹ mg protein⁻¹, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.

  8. Effects of colupulone, a component of hops and brewers yeast, and chromium on glucose tolerance and hepatic cytochrome P450 in nondiabetic and spontaneously diabetic mice.

    PubMed

    Mannering, G J; Shoeman, J A; Shoeman, D W

    1994-05-16

    Brewers yeast contains factors that increase and decrease glucose tolerance. Hop components (lupulones) that adhere to yeast during the brewing process elicit a variety of biological effects including the induction of hepatic cytochrome P4503A. Colupulone was tested for its effects on glucose tolerance and cytochrome P450. Serum glucose levels 30 min after the injection of glucose were lowered by colupulone in nondiabetic Swiss-Webster mice, elevated in diabetic C57B1/KSJ-db/db mice, and unaffected in nondiabetic C57B1/KSJ+m/+m mice. Colupulone lowered hemoglobin glycation slightly in +m/+m mice but not in db/db mice. The cytochrome P450 system was highly induced by colupulone in both db/db and +m/+m mice. Chromium, which acts in concert with the factor in yeast that enhances glucose tolerance, had little or no effect on the plasma glucose level or the cytochrome P450 system in either +m/+m or db/db mice.

  9. [Effect of Siwu decoction and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats].

    PubMed

    Liang, Miao; Ma, Zeng-Chun; Yi, Jian-Feng; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Tang, Xiang-Lin; Li, Hua; Shen, Guo-Lin; Gao, Yue

    2013-11-01

    To study the effect of Siwu decoction (SWD) compound and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats. Rats were orally administered with SWD and water decoction combined with other medicines for two weeks, and then sacrificed. Their livers were perfused with normal saline to prepare liver micrisomes. Mixed probe and liver microsome in vitro incubation method were adopted to detect the effect of SWD on hepatic cytochrome P450. The real-time quantitative polymerase chain reaction (Q-PCR) was used to detect the effect of SWD on the expression of hepatic cytochrome P450. Compared with the control group, the SWD compound group showed higher CYP1A2 enzymatic activity (P < 0.05); Rehmanniae-paeoniae, angelicae-paeoniae, angelicae-rhizome, paeoniae-rhizome groups had lower CYP1A2 and CYP2C19 enzymatic activities (P < 0.05); And the compound group, the single component group and the combination group showed lower CYP2B6 enzymatic activities (P < 0.05). The compound could up-regulated the mRNA expression of CYP2B1 (P < 0.05); And the four single components could down-regulated the mRNA expression of CYP2B1 (P < 0.05). SWD compound had the effect in inducing CYP1A2 enzymatic activity. The rehmanniae-paeoniae group and the angelicae-paeoniae group had identical enzymatic activity with the control group, but significant down-regulation in CYP1A2 enzymatic activity after being combined with paeoniae. The compound and its combined administration showed the inhibitory effect on CYP2B6 enzymatic activity, particularly being combined with angelicae. The compound showed identical effect with the four single components in terms of CYP1A2 mRNA expression and enzymatic activity.

  10. Hepatic cytochrome P450 deficiency in mouse models for intrahepatic cholestasis predispose to bile salt-induced cholestasis.

    PubMed

    Kunne, Cindy; de Graaff, Marijke; Duijst, Suzanne; de Waart, Dirk R; Oude Elferink, Ronald P J; Paulusma, Coen C

    2014-10-01

    Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.

  11. Cytochrome P450-dependent metabolism of midazolam in hepatic microsomes from chickens, turkeys, pheasant and bobwhite quail.

    PubMed

    Cortright, K A; Craigmill, A L

    2006-12-01

    In vitro putative cytochrome P450 3A mediated activity, and inhibition thereof, were measured in four avian species using midazolam (MDZ) as a substrate and ketoconazole as an inhibitor. All species produced 1-hydroxymidazolam (1-OH MDZ) to a much greater extent than 4-hydroxymidazolam (4-OH MDZ). Calculated Vmaxapparent values for formation of 1-OH MDZ were 117+/-17, 239+/-108, 437+/-168, and 201+/-55 pmol/mg protein*min and Kmapparent values were 2.1+/-0.8, 2.4+/-1.6, 6.7+/-5.1 and 3.2+/-2.1 microm for chicken, turkey, pheasant and bobwhite quail, respectively. For the formation of 4-OH MDZ the Vmaxapparent values were 21+/-10, 94+/-46, 144+/-112, and 68+/-30 pmol/mg protein*min and Kmapparent values for 4-OH MDZ formation were 12.4+/-10.1, 18.0+/-10.8, 38.6+/-34.7 and 29.1+/-10.1 microm for chicken, turkey, pheasant and bobwhite quail, respectively. In all four species, ketoconazole inhibited the production of both major metabolites of MDZ, with 4-OH MDZ formation more sensitive to inhibition than 1-OH MDZ. Pheasant and bobwhite quail appeared most sensitive to ketoconazole inhibition.

  12. Potential Contribution of Cytochrome P450 2B6 to Hepatic 4-Hydroxycyclophosphamide Formation In Vitro and In VivoS⃞

    PubMed Central

    Raccor, Brianne S.; Claessens, Adam J.; Dinh, Jean C.; Park, Julie R.; Hawkins, Douglas S.; Thomas, Sushma S.; Makar, Karen W.; McCune, Jeannine S.

    2012-01-01

    Results from retrospective studies on the relationship between cytochrome P450 (P450) 2B6 (CYP2B6) genotype and cyclophosphamide (CY) efficacy and toxicity in adult cancer patients have been conflicting. We evaluated this relationship in children, who have faster CY clearance and receive different CY-based regimens than adults. These factors may influence the P450s metabolizing CY to 4-hydroxycyclophosphamide (4HCY), the principal precursor to CY's cytotoxic metabolite. Therefore, we sought to characterize the in vitro and in vivo roles of hepatic CYP2B6 and its main allelic variants in 4HCY formation. CYP2B6 is the major isozyme responsible for 4HCY formation in recombinant P450 Supersomes. In human liver microsomes (HLM), 4HCY formation correlated with known phenotypic markers of CYP2B6 activity, specifically formation of (S)-2-ethyl-1,5-dimethyl-3,3-diphenyl pyrrolidine and hydroxybupropion. However, in HLM, CYP3A4/5 also contributes to 4HCY formation at the CY concentrations similar to plasma concentrations achieved in children (0.1 mM). 4HCY formation was not associated with CYP2B6 genotype at low (0.1 mM) or high (1 mM) CY concentrations potentially because CYP3A4/5 and other isozymes also form 4HCY. To remove this confounder, 4HCY formation was evaluated in recombinant CYP2B6 enzymes, which demonstrated that 4HCY formation was lower for CYP2B6.4 and CYP2B6.5 compared with CYP2B6.1. In vivo, CYP2B6 genotype was not directly related to CY clearance or ratio of 4HCY/CY areas under the curve in 51 children receiving CY-based regimens. Concomitant chemotherapy agents did not influence 4HCY formation in vitro. We conclude that CYP2B6 genotype is not consistently related to 4HCY formation in vitro or in vivo. PMID:21976622

  13. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    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

  14. Detection of human lung cytochromes P450 that are immunochemically related to cytochrome P450IIE1 and cytochrome P450IIIA.

    PubMed

    Wheeler, C W; Wrighton, S A; Guenthner, T M

    1992-07-07

    We have used monoclonal antibodies that were prepared against and specifically recognize human hepatic cytochromes P450 as probes for solid phase radioimmunoassay and Western immunoblotting to directly demonstrate the presence in human lung microsomes of cytochromes P450 immunochemically related to human liver cytochromes P450IIE1 (CYP2E1) and P450IIIA (CYP3A). The detected levels of these cytochromes are much lower than levels in human liver microsomes, but similar to the levels seen in microsomes from untreated baboon lung. Proteins immunochemically related to two other constitutive hepatic cytochromes P450, cytochrome P450IIC8 (CYP2C8) and cytochrome P450IIC9 (CYP2C9), were not detectable in lung microsomes.

  15. Comparative modelling of cytochromes P450.

    PubMed

    Kirton, Stewart B; Baxter, Carol A; Sutcliffe, Michael J

    2002-03-31

    The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.

  16. Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs.

    PubMed

    Nishimuta, Haruka; Nakagawa, Tetsuya; Nomura, Naruaki; Yabuki, Masashi

    2013-11-01

    1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds. 2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A. 3. Hepatic intrinsic clearance (CLint) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CLint values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CLint values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans. 4. In conclusion, dogs showed the most similar metabolic activity to humans.

  17. Hepatic cytochrome P450 and UDP-glucuronosyl transferase are affected by five sources of dietary fiber in germ-free rats.

    PubMed

    Nugon-Baudon, L; Roland, N; Flinois, J P; Beaune, P

    1996-02-01

    The influence of dietary fiber on xenobiotic-metabolizing enzymes (XME) was assessed using germ-free rats fed inulin and other sources of fiber (wheat bran, carrot, cocoa and oat). The consumption of cocoa fiber greatly modified the hepatic cytochrome P450 isoenzymatic profile, causing a strong enhancement of 1A2 and 2B1/B2 forms, concomitant with a significant decrease of the constitutive form 2C11, compared with all of the other types of fiber. Moreover, rats fed the cocoa fiber diet had a higher specific activity of hepatic UDP-glucuronosyl transferase than their carrot fiber- and wheat bran-fed counterparts. Intestinal UDP-glucuronosyl transferase was unaffected by the type of ingested fiber. Diet composition also did not alter the specific activity of glutathione-S-transferase in the liver, small intestine, or colon. Using earlier results obtained in heteroxenic rats, we show that intestinal microflora plays a key role in some of the effects of fiber on XME, although this is not a necessary prerequisite for all of the liver alterations.

  18. Caged and wild fish: Induction of hepatic cytochrome P-450 (CYP1A1) as an environmental biomonitor

    SciTech Connect

    Haasch, M.L.; Lech, J.J. ); Prince, R.; Cooper, K.R. ); Wejksnora, P.J. )

    1993-05-01

    Hepatic monooxygenase activity can be induced by many different environmental chemical contaminants, and measurement of this activity has been proposed as an environmental biomonitor. Using in situ caged catfish and largemouth bass, and collected wild killifish, environmental induction of hepatic CYP1A1 was investigated using catalytic enzyme assays, regiospecific metabolism, immunodetection, and nucleic acid hybridization. The purpose of these studies was to evaluate these techniques for detection of CYP1A1 induction as a potential environmental biomonitor of environmental chemical contamination. Exposure of catfish in cages to polyaromatic hydrocarbon- (PAH-) and polychlorinated biphenyl- (PCB-) contaminated river water for two, four, or six weeks resulted in fourfold increases in ethoxyresorufin-O-deethylase (EROD) activity and three- and fivefold increases in immunoreactive CYP1A1 protein and hybridizable CYP1A1 mRNA, respectively, when compared to laboratory water control values. Hybridizable CYP1A1 mRNA in caged largemouth bass increased 5.1-fold at 1 d of exposure. Caged largemouth bass had 5-, 1.4-, and 0.8-fold increases at 3 d and 6-, 2.4-, 0.4-fold increases at 7 d of river water exposure in EROD, immunoreactive CYP1A1 protein, and CYP1A1 mRNA, respectively, when compared to laboratory water control values. Liver of killifish from a 2,3,7,8-tetrachlorodibenzo-p-dioxin-contaminated area had threefold higher EROD activity and similarly elevated immunoreactive CYP1A1 protein, a two- to fourfold increase in CYP1A1 mRNA, and a four- to eightfold increase in 6[beta]-hydroxyprogesterone activity, when compared to killifish livers sampled from a clean site.

  19. Cytochrome P450 2E1 Gene Polymorphisms/Haplotypes and Anti-Tuberculosis Drug-Induced Hepatitis in a Chinese Cohort

    PubMed Central

    Tang, Shaowen; Lv, Xiaozhen; Zhang, Yuan; Wu, Shanshan; Yang, Zhirong; Xia, Yinyin; Tu, Dehua; Deng, Peiyuan; Ma, Yu; Chen, Dafang; Zhan, Siyan

    2013-01-01

    Objective The pathogenic mechanism of anti-tuberculosis (anti-TB) drug-induced hepatitis is associated with drug metabolizing enzymes. No tagging single-nucleotide polymorphisms (tSNPs) of cytochrome P450 2E1(CYP2E1) in the risk of anti-TB drug-induced hepatitis have been reported. The present study was aimed at exploring the role of tSNPs in CYP2E1 gene in a population-based anti-TB treatment cohort. Methods and Design A nested case-control study was designed. Each hepatitis case was 14 matched with controls by age, gender, treatment history, disease severity and drug dosage. The tSNPs were selected by using Haploview 4.2 based on the HapMap database of Han Chinese in Beijing, and detected by using TaqMan allelic discrimination technology. Results Eighty-nine anti-TB drug-induced hepatitis cases and 356 controls were included in this study. 6 tSNPs (rs2031920, rs2070672, rs915908, rs8192775, rs2515641, rs2515644) were genotyped and minor allele frequencies of these tSNPs were 21.9%, 23.0%, 19.1%, 23.6%, 20.8% and 44.4% in the cases and 20.9%, 22.7%, 18.9%, 23.2%, 18.2% and 43.2% in the controls, respectively. No significant difference was observed in genotypes or allele frequencies of the 6 tSNPs between case group and control group, and neither of haplotypes in block 1 nor in block 2 was significantly associated with the development of hepatitis. Conclusion Based on the Chinese anti-TB treatment cohort, we did not find a statistically significant association between genetic polymorphisms of CYP2E1 and the risk of anti-TB drug-induced hepatitis. None of the haplotypes showed a significant association with the development of hepatitis in Chinese TB population. PMID:23460870

  20. A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone.

    PubMed

    Mkrtchian, S L; Andersson, K K

    1990-01-30

    Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.

  1. Characterization of cytochrome P450 isoforms involved in sequential two-step bioactivation of diclofenac to reactive p-benzoquinone imines.

    PubMed

    den Braver, Michiel W; den Braver-Sewradj, Shalenie P; Vermeulen, Nico P E; Commandeur, Jan N M

    2016-06-24

    Idiosyncratic drug-induced lever injury (IDILI) is a rare but severe side effect of diclofenac (DF). Several mechanisms have been proposed as cause of DF-induced toxicity including the formation of protein-reactive diclofenac-1',4'-quinone imine (DF-1',4'-QI) and diclofenac-2,5-quinone imine (DF-2,5-QI). Formation of these p-benzoquinone imines result from two-step oxidative metabolism involving aromatic hydroxylation to 4'-hydroxydiclofenac and 5-hydroxydiclofenac followed by dehydrogenation to DF-1',4'-QI and DF-2,5-QI, respectively. Although the contribution of individual cytochrome P450s (CYPs) in aromatic hydroxylation of DF is well studied, the enzymes involved in the dehydrogenation reactions have been poorly characterized. The results of the present study show that both formation of 4'-hydroxydiclofenac and it subsequent bioactivation to DF-1',4'-QI is selectively catalyzed by CYP2C9. However, the two-step bioactivation to DF-2,5-QI appears to be catalyzed with highest activity by two different CYPs: 5-hydroxylation of DF is predominantly catalyzed by CYP3A4, whereas its subsequent bioactivation to DF-2,5-QI is catalyzed with 14-fold higher intrinsic clearance by CYP2C9. The fact that both CYPs involved in two-step bioactivation of DF show large interindividual variability may play a role in different susceptibility of patients to DF-induced IDILI. Furthermore, expression levels of these enzymes and protective enzymes might be important factors determining sensitivity of in vitro models for hepatotoxicity.

  2. Hepatic cytochrome P450 1A as a biomarker for hydrocarbon and oxidant pollutants in fish: Relevance of identification of specific mechanism(s) of induction and potential effects on homeostasis

    SciTech Connect

    Zacharewski, T.R.; McCallum, G.P.; Bend, J.R.; Stegeman, J.J.

    1996-12-31

    The induction of hepatic and extrahepatic P450 1A isozymes in fish and mammals is used as a biomarker for certain classes of toxic environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs), planar polychlorinated (PCBs) and polybrominated (PBBs) biphenyl congeners, polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). However, recent data from the laboratory have shown that rat pulmonary P450 1A1 can also be induced by arsenite or liver transplantation (via a stress mechanism), raising the possibility that regulation of this biomarker can occur by mechanisms other than Ah receptor-mediated increases of CYP1A gene transcription.

  3. Dioxin-like and perfluorinated compounds in pigs in an Indian open waste dumping site: toxicokinetics and effects on hepatic cytochrome P450 and blood plasma hormones.

    PubMed

    Watanabe, Michio X; Kunisue, Tatsuya; Tao, Lin; Kannan, Kurunthachalam; Subramanian, Annamalai; Tanabe, Shinsuke; Iwata, Hisato

    2010-07-01

    Dioxins and related compounds (DRCs) and perfluorinated compounds were measured in the livers of pigs (Sus scrofa) collected from an open waste dumping site in South India. Hepatic concentrations of DRCs and perfluorooctanesulfonate (PFOS; up to 200 ng/g wet wt) were significantly higher in male and female pigs, respectively, collected from the dumping site than in those from a reference site. Results suggest that dumping sites are a source of DRCs and PFOS. Hepatic concentrations of DRCs in piglets were higher than in mothers, especially for the congeners with molecular weights in the range of 360 to 400, implying congener-specific maternal transfer of DRCs in swine. Concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans and some non-ortho dioxin-like polychlorinated biphenyls (PCBs) in the liver of pigs were higher than those in the adipose fat and muscle of the same specimens. In addition, the liver-to-adipose concentration ratios for each congener had a significant positive correlation with the levels of hepatic cytochrome P450 (CYP)1A-like protein, suggesting congener-specific and CYP1A-dependent hepatic sequestration of DRCs in the swine. Total hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs; 8.9-350 pg/g fat wt) had a significant positive correlation with CYP1A-like protein expression (r=0.56, p=0.012), suggesting the induction of CYP1A by DRCs. However, the total TEQs had a significant negative correlation with CYP4A-like protein (r=-0.49, p=0.029), suggesting repression of peroxisome proliferator-activated receptor-alpha (PPARalpha)-mediated signaling pathway by DRCs. Decreases in plasma total thyroxine (T4), free T4, and immunoglobulin (Ig) G were also found in pigs from the dumping site compared with those from the reference site. This study provides insight into the toxicological impacts of DRCs and perfluorinated compounds in wild animals from open waste dumping sites.

  4. Stimulatory effect of sesamin on hepatic cytochrome P450 activities in Atlantic salmon (Salmo salar L.) is not directly associated with expression of genes related to xenobiotic metabolism.

    PubMed

    Zlabek, Vladimir; Vestergren, AnnaLotta Schiller; Trattner, Sofia; Wagner, Liane; Pickova, Jana; Zamaratskaia, Galia

    2015-01-01

    1. This study examined hepatic cytochrome P450 (CYP450) response to dietary sesamin in combination with different n-6/n-3 fatty acid ratios in fish diet. Over a period of 4 months, fish were fed seven different experimental diets an n-6/n-3 FA ratio of either 0.5 or 1.0 in combination with two sesamin levels: low sesamin = 1.16 g/kg feed and high sesamin = 5.8 g/kg feed. Control diets did not contain sesamin. 2. The CYP450-associated activities of ethoxyresorufin O-deethylase (EROD), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD), pentoxyresorufin O-depentylase (PROD), coumarin hydroxylase (COH), methoxyresorufin O-deethylase (MROD) and p-nitrophenol hydroxylase (PNPH) were significantly induced by dietary sesamin in a dose-related manner. 3. Expressions of the genes CYP1A1, CYP1A3, CYP3A, AhR1α, AhR2β, AhR2δ and PXR involved in the regulation of CYP450 activities, was not the primary source of this induction.

  5. Hepatic cytochrome p450-2A and phosphoribosylpyrophosphate synthetase-associated protein mRNA are induced in gerbils after consumption of isoflavone-containing protein.

    PubMed

    Mezei, Orsolya; Chou, Chris N; Kennedy, Kathleen J; Tovar-Palacio, Claudia; Shay, Neil F

    2002-09-01

    Soy intake reduces cholesterol levels, but neither the exact component in soy causing this reduction nor the mechanism by which cholesterol is reduced is known with certainty. In this study, a genetic screen was performed to identify hepatic mRNA in gerbils regulated by soy or soy isoflavones. Gerbils were fed casein, an alcohol-washed soy-based diet (containing low levels of isoflavones), and the soy-based diet supplemented with an isoflavone-containing soy extract. After feeding for 28 d, gerbils were killed, hepatic RNA was isolated, and genes that were differentially expressed in any of the three dietary conditions were identified. Fifteen different mRNA were originally selected, including two mRNA that were studied further and shown to be highly regulated. Messenger RNA levels for both cytochrome P450-2A and phosphoribosylpyrophosphate synthetase-associated protein were up-regulated in a dose-dependent manner when soy replaced casein in the diet at 0, 33, 67 and 100% of original casein levels. A subsequent experiment used purified amino acid mixtures resembling the percentage amino acid composition of soy and casein to ensure that isoflavone-free protein sources could be tested. Using these mixtures, a 2 x 2 x 2 design tested: natural vs. synthetic protein sources, casein- vs. soy-based diets, and isoflavone extract-supplemented or supplement-free diets. This design demonstrated that these two mRNA were again significantly up-regulated more than twofold (P < 0.05) in gerbils fed all diets containing isoflavones. Induction of these two mRNA by soy may be due to the aryl hydrocarbon receptor element in the promoter region of both genes.

  6. Organohalogens and their hydroxylated metabolites in the blood of pigs from an open waste dumping site in south India: association with hepatic cytochrome P450.

    PubMed

    Mizukawa, Hazuki; Nomiyama, Kei; Kunisue, Tatsuya; Watanabe, Michio X; Subramanian, Annamalai; Iwata, Hisato; Ishizuka, Mayumi; Tanabe, Shinsuke

    2015-04-01

    The concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and their hydroxylated metabolites (OH-PCBs and OH-PBDEs) were measured in the blood of Eurasian wild pigs (Sus scrofa) from a municipal waste open dumping site (DS) and a reference site (RS) in South India. We showed that contamination with OH-PCBs was higher in female pigs from the DS than in all other adult pigs. The highest OH-PCB concentrations were found in piglets from the DS. Moreover, the hepatic expression levels of CYP1A and CYP2B were higher in piglets than in their dam, implying metabolism of PCBs by cytochrome P450 (CYP) enzymes. The OH-PCB congener profiles differed according to sex and collection sites, possibly because of variations in the expression levels of phase I and phase II enzymes among individual pigs, differences in the exposure sources, and maternal transfer of parent PCBs. The hepatic CYP1A expression levels were positively correlated with the blood concentrations of 4OH-CB107, 4OH-CB162, and 4OH-CB187, implying CYP1A-dependent formation of these OH-PCBs in the pig liver. We found no significant correlations between the blood concentrations of OH-PCBs and thyroid hormones (THs); however, the thyroxin (T4) levels were lower in pigs from the DS than in pigs from the RS. Our limited dataset suggest that induced CYP enzymes accelerate the metabolism of xenobiotics and endogenous molecules in pigs. Thus, besides parental compounds, the risk of hydroxylated metabolites entering wildlife and humans living in and around municipal open waste dumping sites should be considered.

  7. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

    PubMed Central

    Moskaleva, Natalia; Moysa, Alexander; Novikova, Svetlana; Tikhonova, Olga; Zgoda, Victor; Archakov, Alexander

    2015-01-01

    Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine. PMID:26561010

  8. Mechanism-based inactivation of hepatic cytochrome P450 2C6 and P450 3A1 following in vivo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine to rats: differences from previously observed in vitro results.

    PubMed

    Kimmett, S M; McNamee, J P; Marks, G S

    1994-04-01

    Using progesterone 21-hydroxylase as a selective substrate for P450 2C6 in phenobarbital-treated male rats, and androstenedione and progesterone 6 beta-hydroxylases as well as erythromycin N-demethylase as selective markers for P450 3A1 in dexamethasone-treated female rats, we have shown that these P450 isozymes undergo mechanism-based inactivation after in vivo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC). These results differ from our previous studies where no inactivation was observed after in vitro administration of 4-ethyl DDC to rat hepatic microsomes. We show that the differences between the in vivo and in vitro effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) analogues are due to the presence of residual 4-ethyl DDC in the in vitro experiments causing time-independent competitive inhibition and obscuring observation of mechanism-based inactivation.

  9. INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

    EPA Science Inventory

    Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

  10. CROSS-SPECIES COMPARISON OF CONAZOLE FUNGICIDE METABOLITES USING RAT AND RAINBOW TROUT (ONCHORHYNCHUS MYKISS) HEPATIC MICROSOMES AND PURIFIED HUMAN CYTOCHROME P450 3A4

    EPA Science Inventory

    Conazoles represent a unique class of azole-containing fungicides that are widely used in both pharmaceutical and agriculture applications. The antifungal property of conazoles occurs via complexation with cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell...

  11. Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax).

    PubMed

    Vaccaro, E; Giorgi, M; Longo, V; Mengozzi, G; Gervasi, P G

    2003-01-10

    Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

  12. Hepatic microsomal cytochrome p450s and chlorinated hydrocarbons in largha and ribbon seals from Hokkaido, Japan: differential response of seal species to Ah receptor agonist exposure.

    PubMed

    Chiba, Issei; Sakakibara, Akihito; Iwata, T Hisato; Ishizuka, Mayumi; Tanabe, Shinsuke; Akahori, Fumiaki; Kazusaka, Akio; Fujita, Shoichi

    2002-04-01

    From 16 largha seals (Phoca largha) and 15 ribbon seals (Phoca fasciata) in the coastal waters of Hokkaido, Japan, blubber chlorinated hydrocarbon (CHC) levels and hepatic cytochrome P450 (CYP) catalytic activities and their immunochemically detected protein content levels were measured. Concentrations of DDTs (2,2-bis(4-chlorophenyl)-1,1-dichloroethylene,p,p'-DDE; 2,2-bis(4-chlorophenyl)-1,1-dichloroethane, p,p'-DDD; dichlorodiphenyltrichloroethane, p,p'-DDT), polychlorinated biphenyl congeners (PCBs), and chlordane compounds (oxychlordane, chlordanes, and nonachlors) in both species were in the range of 290 to 5,300, 420 to 4,000, and 130 to 1,500 ng/g lipid weight, respectively. Aryl hydrocarbon receptor (AhR) agonists, non-ortho (IUPAC 77 and 126) and mono-ortho (IUPAC 105, 118, and 156) coplanar PCB congeners, were also detected, and the 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) toxic equivalents (TEQs) were 4.9 to 120 pg TEQ/g lipid weight. Cross-reactive proteins with polyclonal antibodies against rat CYP1A1 and CYP3A2 were notably detected in seal liver microsomes. Interestingly, a polyclonal antibody against rat CYP2B1 recognized proteins only at trace levels. In largha seals, both levels of alkoxyresorufin- (methoxy-, ethoxy-, pentoxy-, and benzyloxyresorufin) O-dealkylase (AROD) activities and proteins detected by polyclonal antibodies against rat CYP1A1 were significantly correlated with the concentrations of individual coplanar PCB congeners, total TEQs, and total PCBs. Threshold concentrations for TEQs in blubber of the largha seal to induce hepatic CYP1A protein and EROD activity were estimated to be 8.5 and 19 pg TEQ/g fat weight, respectively. In ribbon seals, similar correlations were not detected, although the TEQ levels were not significantly lower than those in largha seals. These results suggest that AROD activity and CYP1A1 protein in the liver of the largha seal could be a biomarker for the exposure to AhR agonists such as coplanar PCB

  13. Kinetics and selectivity of mechanism-based inhibition of guinea pig hepatic and pulmonary cytochrome P450 by N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole.

    PubMed

    Sinal, C J; Bend, J R

    1996-09-01

    The time dependence for mechanism-based inactivation of cytochrome P450 (P450)-dependent 7-pentoxyresorufin O-depentylation (PROD), 7-ethoxyresorufin O-deethylation (EROD), and 7-methoxyresorufin O-demethylation (MROD) activities by N-benzyl-1-aminobenzotriazole (BBT) and N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB) was investigated in hepatic and pulmonary microsomes from phenobarbital-treated guinea pigs. In the presence of NADPH, both compounds inhibited P450-dependent catalytic activity in a time- and concentration-dependent manner. Inactivation of hepatic PROD activity was more rapid (t1/2 = 13.2 vs. 155 min) for 0.1 microM alpha MB when compared with equimolar BBT. On the other hand, hepatic EROD inactivation was more rapid (t1/2 = 8.1 vs. 11 min) with 0.1 microM BBT, compared with equimolar alpha MB. Inactivation of pulmonary PROD activity was the most rapid and potent, with an apparent half-life for inactivation of t1/2 = 0.94 and 32.2 min for 0.025 microM alpha MB and BBT, respectively. Incubation of hepatic microsomes for 45 min in the presence of NADPH and 10 microM BBT or alpha MB resulted in > 90% inhibition of PROD, EROD, and MROD activities. After washing by repeated sedimentation and resuspension, inhibition of PROD (78%; 93% for BBT and alpha MB, respectively), EROD (80% and 50%), and MROD (15% and 3%) activities was reversed to varying degrees. We conclude that BBT and alpha MB are rapidly metabolized to products that inhibit individual P450 isozymes by both mechanism-based (P4502B and P4501A1) and reversible (P4501A2) mechanisms. Of the two inhibitors, alpha MB is relatively more potent and selective for guinea pig lung P4502B isozyme(s).

  14. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  15. The role of human hepatic cytochrome P450 isozymes in the metabolism of racemic 3,4-methylenedioxy-methamphetamine and its enantiomers.

    PubMed

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

    2008-11-01

    The entactogen, 3,4-methylenedioxy-methamphetamine (MDMA), is a chiral drug that is mainly metabolized by N-demethylation and demethylenation. The involvement of cytochrome P450 (P450) isozymes in these metabolic steps has been studied by inhibition assays with human liver microsomes and, in part, with heterologously expressed human P450 isozymes. However, a comprehensive study on the involvement of all relevant human P450s has not been published yet. In addition, the chirality of this drug was not considered in these in vitro studies. The aim of the present work was to study the contribution of human P450 isozymes in the N-demethylation and demethylenation of racemic MDMA and its single enantiomers. MDMA and its enantiomers were incubated using heterologously expressed human P450s, and the metabolites were quantified by gas chromatography-mass spectrometry after derivatization with S-heptafluorobutyrylprolyl chloride. The highest contribution for the N-demethylation as calculated from the enzyme kinetic data, were obtained for CYP2B6 (R,S-MDMA), CYP1A2 (R-MDMA), and CYP2B6 (S-MDMA). In the case of the demethylenation, the isozyme with the highest contribution to net clearance for R,S-MDMA, R-MDMA, and S-MDMA was CYP2D6. For the first time, marked enantioselectivity was observed for N-demethylation and demethylenation by CYP2C19 with a preference for the S-enantiomers. In addition, CYP2D6 showed preference for S-MDMA in the case of demethylenation. None of the other isozymes showed major preferences for certain enantiomers. In conclusion, therefore, the different pharmacokinetic properties of the MDMA enantiomers may be caused by enantioselective metabolism by CYP2C19 and CYP2D6.

  16. Metazoan cytochrome P450 evolution.

    PubMed

    Nelson, D R

    1998-11-01

    There are 37 cytochrome P450 families currently identified in animals. The concept of higher order groupings of P450 families called P450 CLANS is introduced. The mammalian CYP3 and CYP5 families belong to the same clan as insect CYP6 and CYP9. All mitochondrial P450s seem to belong to the same clan. Lack of mitochondrial P450s in C. elegans suggests that mitochondrial P450s probably arose from the mistargeting of a microsomal P450 after the coelomates diverged from acoelomates and pseudocoelomates. Different taxonomic groups appear to have recruited different ancestral P450s for expansion as they evolved, since each major taxon seems to have one large cluster of P450s. In insects, this cluster derives from the ancestor to the CYP4 family. Vertebrates and C. elegans may have used the same ancestor independently to generate the CYP1, 2, 17, and 21 families in vertebrates and a large distinctive clan with 45 genes in C. elegans.

  17. Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations

    NASA Astrophysics Data System (ADS)

    Ye, Meiling; Tang, Ling; Luo, Mengjun; Zhou, Jing; Guo, Bin; Liu, Yangyuan; Chen, Bo

    2014-11-01

    Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under in vitro incubations of human liver microsomes (HLMs) with tannic acid (TA)-stabilized AuNPs in the size range of 5 to 100 nm. It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV-vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the

  18. Inducible cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Raza, H.; Shayiq, F.M.; Avadhani, N.G.

    1987-05-01

    In the present study they have purified US -naphthoflavone (BNF, which induces isotypes similar to 3-MC) and PB induced mitochondrial isoforms. They have been able to purify two isoforms with molecular weights of 54 Kd and 52 Kd from BNF induced mitochondria. Only the 54 KD form, but not the 52 KD species reacts with the polyclonal antibody to microsomal P-450c, though, both show arylhydrocarbon hydroxylase activity in an in vitro system reconstituted with adrenodoxin and adrenodoxin-reductase. Fingerprint analyses, N-terminal sequencing and use of monoclonal antibody probes show that the two mitochondrial isoforms are different from the microsomal P-450c. Further, the 54 Kd mitochondrial isoform is not detected in control mitochondria indicating that it is truly an induced form. Similarly, a PB induced mitochondrial form which exhibits physical, immunochemical and enzymatic properties different from the microsomal P-450b has also been purified.

  19. Novel extrahepatic cytochrome P450s

    SciTech Connect

    Karlgren, Maria . E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-09-01

    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.

  20. Identification of human hepatic cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus lupulus L.).

    PubMed

    Guo, Jian; Nikolic, Dejan; Chadwick, Lucas R; Pauli, Guido F; van Breemen, Richard B

    2006-07-01

    The female flowers of hops (Humulus lupulus L.) are used in the brewing of beer and are under investigation for use in dietary supplements for the management of menopausal symptoms in women. Hop extracts contain the weakly estrogenic compound isoxanthohumol (IX), proestrogenic xanthohumol, and the potent estrogen 8-prenylnaringenin (8PN). Because IX can be metabolized in the human liver to form 8PN, the specific cytochrome P450 (P450) enzymes responsible for this O-demethylation reaction were identified. In addition, the enzymes that convert IX and 8PN to their most abundant metabolites were identified because these metabolic pathways might also affect the estrogenicity of hop preparations. Specifically, the P450 enzymes that catalyze the oxidation of the prenyl side chains of IX and 8PN into trans- or cis-alcohols were investigated. Human liver microsomes and monoclonal antibodies that inhibit specific P450 enzymes were used in combination with liquid chromatography/mass spectrometry to identify the enzymes responsible for these transformations. CYP2C19 was found to catalyze the formation of both cis- and trans-alcohols of the prenyl side chain of 8PN with K(m) values of 14.8 +/- 3.2 and 16.6 +/- 4.6 microM, respectively. CYP2C8 converted 8PN regioselectively to the trans-alcohol of the prenyl group with a K(m) of 3.7 +/- 0.9 microM. Finally, CYP1A2 was found to catalyze the O-demethylation of IX to generate 8PN, with a K(m) value of 17.8 +/- 3.7 microM. These results suggest that the estrogenicity of hop constituents in vivo will depend in part on metabolic conversion that may show individual variation.

  1. Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus).

    PubMed

    Li, Hongxia; Boon, Jan P; Lewis, Wilma E; van den Berg, Martin; Nyman, Madeleine; Letcher, Robert J

    2003-03-01

    Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory

  2. Interactions of pharmaceuticals and other xenobiotics on hepatic pregnane X receptor and cytochrome P450 3A signaling pathway in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Wassmur, Britt; Gräns, Johanna; Kling, Peter; Celander, Malin C

    2010-10-01

    The pregnane X receptor (PXR) belongs to the nuclear hormone receptor (NR) superfamily and is commonly described as a xenophore or a pharmacophore, as it can be activated by a wide array of xenobiotics, including numerous pharmaceuticals and other environmental pollutants. The PXR regulates expression of e.g. cytochrome P450 3A (CYP3A) and the P-glycoprotein (P-gp) that are involved in excretion of lipophilic xenobiotics and endobiotics. A full length PXR cDNA was isolated from rainbow trout liver and it was expressed in a descending order of magnitude in liver>intestine>kidney>heart. A rainbow trout PXR reporter assay was developed and a suite of pharmaceuticals and other xenobiotics were screened. However, no specific activation of rainbow trout PXR was observed with the substances tested. Interactions of prototypical PXR agonists on PXR signaling in rainbow trout were further investigated in cells of hepatic origin exposed in vitro and in juvenile rainbow trout exposed in vivo. The rainbow trout hepatoma cell line (RTH-149), displayed 600 times lower expression of CYP3A mRNA compared to primary cultures of hepatocytes, and did not respond to treatment with either pregnenolone 16α-carbonitrile (PCN), ketoconazole (KCZ) or rifampicin (RIF), which implies a non-functional PXR in this cell line. Exposure of hepatocytes to PCN and lithocholic acid (LA), resulted in a weak concentration-dependent induction of CYP3A and P-gp mRNA levels, though, exposure to the higher concentration of LA (50 μM) decreased PXR mRNA levels. Exposure to dexamethasone (DEX) resulted in a decrease in PXR mRNA, without affecting CYP3A mRNA levels in hepatocytes in vitro. Injections of rainbow trout in vivo with 1 mg LA/kg fish resulted in a slight (albeit not significant) increase in CYP3A mRNA levels without affecting PXR mRNA levels. Although, injection with 10mg omeprazole (OME)/kg fish had no effect on PXR and CYP3A mRNA levels, a 60% inhibition of CYP3A enzyme activities was evident

  3. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  4. Artificial neural network cascade identifies multi-P450 inhibitors in natural compounds

    PubMed Central

    Li, Zhangming; Li, Yan; Sun, Lu; Tang, Yun; Liu, Lanru

    2015-01-01

    Substantial evidence has shown that most exogenous substances are metabolized by multiple cytochrome P450 (P450) enzymes instead of by merely one P450 isoform. Thus, multi-P450 inhibition leads to greater drug-drug interaction risk than specific P450 inhibition. Herein, we innovatively established an artificial neural network cascade (NNC) model composed of 23 cascaded networks in a ladder-like framework to identify potential multi-P450 inhibitors among natural compounds by integrating 12 molecular descriptors into a P450 inhibition score (PIS). Experimental data reporting in vitro inhibition of five P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were obtained for 8,148 compounds from the Cytochrome P450 Inhibitors Database (CPID). The results indicate significant positive correlation between the PIS values and the number of inhibited P450 isoforms (Spearman’s ρ = 0.684, p < 0.0001). Thus, a higher PIS indicates a greater possibility for a chemical to inhibit the enzyme activity of at least three P450 isoforms. Ten-fold cross-validation of the NNC model suggested an accuracy of 78.7% for identifying whether a compound is a multi-P450 inhibitor or not. Using our NNC model, 22.2% of the approximately 160,000 natural compounds in TCM Database@Taiwan were identified as potential multi-P450 inhibitors. Furthermore, chemical similarity calculations suggested that the prevailing parent structures of natural multi-P450 inhibitors were alkaloids. Our findings show that dissection of chemical structure contributes to confident identification of natural multi-P450 inhibitors and provides a feasible method for virtually evaluating multi-P450 inhibition risk for a known structure. PMID:26719820

  5. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans.

  6. Hepatic and intestinal cytochrome P-450, glutathione-S-transferase and UDP-glucuronosyl transferase are affected by six types of dietary fiber in rats inoculated with human whole fecal flora.

    PubMed

    Roland, N; Nugon-Baudon, L; Flinois, J P; Beaune, P

    1994-09-01

    The effects of six different sources of dietary fiber (inulin, wheat brain, carrot, cocoa, pea and oat fiber) on hepatic and intestinal cytochrome P-450 (EC 1.14.14.1), glutathione-S-transferase (GSH-T, EC 2.5.1.18) and UDP-glucuronosyl transferase (UDPG-T, EC 2.4.1.17) were studied using germ-free F344 rats subsequently inoculated with a human whole fecal flora. In the liver, the total concentration of P-450 was significantly lower in the wheat bran-fed group than in the carrot-fed group. The 2E1 form of P-450, involved in nitrosamine metabolism, was enhanced in the carrot-fed group compared with those fed most other types of fiber. Compared with the pea-fed group, rats fed cocoa had a lower constitutive 2C11 form and a higher 1A2 form. A very high concentration of small intestinal 1A1 form--involved in "toxication" reactions--was observed in rats fed cocoa. The specific activity of hepatic GSH-T was significantly higher in rats fed inulin than in all other groups, except the carrot-fed group. In the colon, GSH-T specific activity was twice as high in the oat-fed group as in the wheat bran-fed counterpart. Small intestinal GSH-T activity and hepatic and intestinal UDPG-T activities were unaffected by diet. Results are discussed in relation to potential health benefits.

  7. Induction time course of cytochromes P450 by phenobarbital and 3-methylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis.

    PubMed

    Ertl, R P; Stegeman, J J; Winston, G W

    1998-05-01

    Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48-72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

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

    PubMed

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

    2009-10-08

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

  9. Cytochrome P450 CYP3A in marsupials: cloning and identification of the first CYP3A subfamily member, isoform 3A70 from Eastern gray kangaroo (Macropus giganteus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Marchant, Ceilidh L; Height, Tamara A; Stupans, Ieva; McKinnon, Ross A

    2012-09-15

    Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well-studied eutherian mammals. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of both xenobiotics and endogenous substrates. In this study we have cloned and characterized CYP3A70, the first identified member of the CYP3A gene subfamily from Eastern gray kangaroo (Macropus giganteus). A 1665 base pair kangaroo hepatic CYP3A complete cDNA, designated CYP3A70, was cloned by reverse transcription-polymerase chain reaction approaches, which encodes a protein of 506 amino acids. The CYP3A70 cDNA shares approximately 71% nucleotide and 65% amino acid sequence homology to human CYP3A4 and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Transfection of the CYP3A70 cDNAs into 293T cells resulted in stable cell lines expressing a CYP3A immuno-reactive protein that was recognized by a goat anti-human CYP3A4 polyclonal antibody. The anti-human CYP3A4 antibody also detected immunoreactive proteins in liver microsomes from all test marsupials, including the kangaroo, koala, wallaby, and wombat, with multiple CYP3A immunoreactive bands observed in kangaroo and wallaby tissues. Relatively, very low CYP catalytic activity was detected for the kangaroo CYP3A70 cDNA-expressed proteins (19.6 relative luminescent units/μg protein), which may be due to low protein expression levels. Collectively, this study provides primary molecular data regarding the Eastern kangaroo hepatic CYP3A70 gene and enables further functional analyses of CYP3A enzymes in marsupials.

  10. Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

    PubMed

    Takaya, N; Suzuki, S; Kuwazaki, S; Shoun, H; Maruo, F; Yamaguchi, M; Takeo, K

    1999-12-15

    Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

  11. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  12. Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

    EPA Science Inventory

    EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

  13. Propiconazole increases reactive oxygen species levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450 enzyme mediated process

    EPA Science Inventory

    Propiconazole induces hepatocarcinomas and hepatoadenomas in mice and is a rat liver tumor promoter. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicate that ...

  14. Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

    PubMed

    Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

    2014-01-01

    The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

  15. Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice.

    PubMed

    Hernandez, Juan P; Chapman, Laura M; Kretschmer, Xiomara C; Baldwin, William S

    2006-10-15

    Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16alpha-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.

  16. Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

    SciTech Connect

    Hernandez, Juan P.; Chapman, Laura M.; Kretschmer, Xiomara C.; Baldwin, William S. . E-mail: wbaldwin@utep.edu

    2006-10-15

    Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16{alpha}-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.

  17. Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock.

    PubMed

    Ogaki, Shigeru; Taguchi, Kazuaki; Maeda, Hitoshi; Watanabe, Hiroshi; Ishima, Yu; Otagiri, Masaki; Maruyama, Toru

    2015-10-01

    Red blood cell (RBC) transfusions for controlling hemorrhaging induce systemic ischemia reperfusion, resulting in a decrease in hepatic cytochrome P450 (CYP) levels. Carbon monoxide (CO), when bound to red blood cells (CO-RBC) has the potential to protect the hepatic CYP protein to produce a resuscitative effect in a hemorrhagic shock rat model. The aim of this study was to investigate the mechanism by which CO-RBC resuscitation from a massive hemorrhage protects against a decrease in hepatic CYP. In the early phase (∼1h) after a hemorrhage and RBC resuscitation, hepatic CYP protein levels were significantly decreased with increasing hepatic free heme levels, but were maintained by a pre-treatment of gadolinium chloride (GdCl3), a Kupffer cell inhibitor, and Trolox, an anti-oxidant agent, as well as CO-RBC resuscitation. Under these conditions, the production of reactive oxygen species (ROS) derived from activated Kupffer cells was increased, but this increase was suppressed by CO-RBC resuscitation. At a late phase (6∼24h), CYP mRNA levels decreased after hemorrhage and RBC resuscitation, but not in the case of CO-RBC resuscitation. The increases in plasma IL-6 and TNF-α levels were decreased by CO-RBC resuscitation via the suppression of the toll-like receptor-4 (TLR-4) and the expression of the high mobility group box-1 (HMGB-1). Hepatic CYP protection after a hemorrhage and CO-RBC resuscitation can be attributed to the inactivation of Kupffer cells, resulting in the suppression of ROS production in the early phase and the suppression of inflammatory cytokine production via the TLR-4/HMGB-1signal pathway in the late phase.

  18. Krüppel-like factor 9 promotes hepatic cytochrome P450 2D6 expression during pregnancy in CYP2D6-humanized mice.

    PubMed

    Koh, Kwi Hye; Pan, Xian; Zhang, Wei; McLachlan, Alan; Urrutia, Raul; Jeong, Hyunyoung

    2014-12-01

    Cytochrome P450 2D6 (CYP2D6), a major drug-metabolizing enzyme, is responsible for metabolism of approximately 25% of marketed drugs. Clinical evidence indicates that metabolism of CYP2D6 substrates is increased during pregnancy, but the underlying mechanisms remain unclear. To identify transcription factors potentially responsible for CYP2D6 induction during pregnancy, a panel of genes differentially expressed in the livers of pregnant versus nonpregnant CYP2D6-humanized (tg-CYP2D6) mice was compiled via microarray experiments followed by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) verification. As a result, seven transcription factors-activating transcription factor 5 (ATF5), early growth response 1 (EGR1), forkhead box protein A3 (FOXA3), JUNB, Krüppel-like factor 9 (KLF9), KLF10, and REV-ERBα-were found to be up-regulated in liver during pregnancy. Results from transient transfection and promoter reporter gene assays indicate that KLF9 itself is a weak transactivator of CYP2D6 promoter but significantly enhances CYP2D6 promoter transactivation by hepatocyte nuclear factor 4 (HNF4α), a known transcriptional activator of CYP2D6 expression. The results from deletion and mutation analysis of CYP2D6 promoter activity identified a KLF9 putative binding motif at -22/-14 region to be critical in the potentiation of HNF4α-induced transactivation of CYP2D6. Electrophoretic mobility shift assays revealed a direct binding of KLF9 to the putative KLF binding motif. Results from chromatin immunoprecipitation assay showed increased recruitment of KLF9 to CYP2D6 promoter in the livers of tg-CYP2D6 mice during pregnancy. Taken together, our data suggest that increased KLF9 expression is in part responsible for CYP2D6 induction during pregnancy via the potentiation of HNF4α transactivation of CYP2D6.

  19. Induction of hepatic cytochrome P450 isozymes, benzo(a)pyrene metabolism and DNA binding following exposure to polycyclic aromatic hydrocarbon residues generated during repeated fish fried oil in rats

    SciTech Connect

    Pandey, Manoj K.; Yadav, Sanjay; Parmar, Devendra; Das, Mukul . E-mail: mditrc@rediffmail.com

    2006-06-01

    In the present study the effect of repeated fish fried oil (RFFO) and its extract (RFFE) on hepatic cytochrome P450 (CYP) isozymes, benzo(a)pyrene (BP) metabolism and DNA adduct formation was undertaken. HPLC analysis of RFFO showed the presence of several polycyclic aromatic hydrocarbons. CYP in microsomes from control and RFFO-treated animals showed a peak at 450 nm; however, a shift of 2 nm in the SORET region along with significant induction was observed in microsomes prepared from 3-methylcholanthrene (MC)- and RFFE-treated animals. Activities of hepatic ethoxyresorufin-O-deethylase, methoxyresorufin-O-deethylase, aryl hydrocarbon hydroxylase and erythromycin-N-demethylase were found to be significantly (P < 0.05) induced following exposure of RFFE, whereas none of these enzymes were altered in RFFO-treated group. Immunoblot analysis revealed that RFFE and MC were potent inducers of CYP1A1, 1A1/2 and 3A1 isozymes, where as RFFO showed no change in these protein levels. RT-PCR analysis showed induction of cDNA of CYP1A1 and CYP3A1 by RFFE treatment. Hepatic microsomes prepared from RFFE exposed animals enhanced BP metabolism with a concomitant increase in the relative proportion of BP 7,8-diol. Hepatic microsomes prepared from animals pretreated with RFFE and MC significantly enhanced the binding of [{sup 3}H]-BP to calf thymus DNA. The overall results suggest that exposure to RFFE may induce hepatic CYP isozymes thereby producing enhanced reactive metabolites with a potential to bind with DNA that may result in cancer.

  20. Cytochrome P450-activated prodrugs

    PubMed Central

    Ortiz de Montellano, Paul R

    2013-01-01

    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

  1. Cytochrome P450 (CYP450) Tests

    MedlinePlus

    ... By Mayo Clinic Staff Your doctor may use cytochrome P450 (CYP450) tests to help determine how your body processes (metabolizes) a drug. The human body contains P450 enzymes to process medications. Because of inherited (genetic) traits ...

  2. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.

    PubMed

    Wortham, Matthew; Czerwinski, Maciej; He, Lin; Parkinson, Andrew; Wan, Yu-Jui Yvonne

    2007-09-01

    Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

  3. Metabolism of sesamin by cytochrome P450 in human liver microsomes.

    PubMed

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2010-12-01

    Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

  4. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

    Krest, Courtney M.; Onderko, Elizabeth L.; Yosca, Timothy H.; Calixto, Julio C.; Karp, Richard F.; Livada, Jovan; Rittle, Jonathan; Green, Michael T.

    2013-01-01

    Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

  5. The environmental pollutant and carcinogen 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone are potent inducers of rat hepatic cytochromes P450 1A1 and -1A2 and NAD(P)H:quinone oxidoreductase.

    PubMed

    Stiborová, Marie; Dracínská, Helena; Hájková, Jana; Kaderábková, Pavla; Frei, Eva; Schmeiser, Heinz H; Soucek, Pavel; Phillips, David H; Arlt, Volker M

    2006-08-01

    3-Nitrobenzanthrone (3-NBA), a suspected human carcinogen occurring in diesel exhaust and air pollution, and its human metabolite 3-aminobenzanthrone (3-ABA) were investigated for their ability to induce biotransformation enzymes in rat liver and the influence of such induction on DNA adduct formation by the compounds. Rats were treated (i.p.) with 0.4, 4, or 40 mg/kg body weight 3-NBA or 3-ABA. When hepatic cytosolic fractions from rats treated with 40 mg/kg body weight 3-NBA or 3-ABA were incubated with 3-NBA, DNA adduct formation, measured by 32P-postlabeling analysis, was 10-fold higher in incubations with cytosols from pretreated rats than with controls. The increase in 3-NBA-derived DNA adduct formation corresponded to a dose-dependent increase in protein levels and enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is the major enzyme reducing 3-NBA in human and rat livers. Incubations of 3-ABA with hepatic microsomes of rats treated with 3-NBA or 3-ABA (40 mg/kg body weight) led to as much as a 12-fold increase in 3-ABA-derived DNA adduct formation compared with controls. The observed stimulation of DNA adduct formation by both compounds was attributed to their potential to induce protein expression and enzymatic activity of cytochromes P450 1A1 and/or -1A2 (CYP1A1/2), the major enzymes responsible for 3-ABA activation in human and rat livers. Collectively, these results demonstrate for the first time, to our knowledge, that by inducing hepatic NQO1 and CYP1A1/2, both 3-NBA and 3-ABA increase the enzymatic activation of these two compounds to reactive DNA adduct-forming species, thereby enhancing their own genotoxic potential.

  6. Tissue-specific contaminant accumulation and associated effects on hepatic serum analytes and cytochrome P450 enzyme activities in hooded seals (Cystophora cristata) from the Gulf of St. Lawrence.

    PubMed

    Wolkers, Hans; Boily, France; Fink-Gremmels, Johanna; van Bavel, Bert; Hammill, Mike O; Primicerio, Raul

    2009-02-01

    The current study aims to assess contaminant levels and tissue burdens in hooded seal (Cystophora cristata) blubber, liver, and blood in association with cytochrome P450 (CYP) enzymes (CYP1A and -3A) and serum analytes (hepatic enzymes like alanine aminotransferase [ALT], aspartate aminotransferase, alkaline phosphatase [AP], and gamma-glutamyltransferase [GGT], serum proteins, and creatine kinase). Contaminant accumulation levels and patterns of polychlorinated biphenyls, chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs) differed between tissues and seal groups, with the highest levels in liver. Pups showed higher liver contaminant levels, especially for PBDEs, than adults. These high levels might be associated with the ingestion of large amounts of contaminated milk and subsequent accumulation in the liver. Adult males and females mainly differed in PBDE levels, which were higher in females, possibly due to a sex-specific diet. The association between blubber contaminant burdens and the diagnostic enzymes ALT, GGT, and AP, and serum albumin, was inconclusive. In contrast, several CYP isoenzymes showed a clear positive relationship with the overall blubber contaminant burden, indicating enzyme induction following exposure to polyhalogenated hydrocarbons. Therefore, liver CYP isoenzymes may serve as a sensitive biomarker for long-term exposure to polyhalogenated hydrocarbons.

  7. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

    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

  8. Cytochrome P450s and molecular epidemiology

    NASA Astrophysics Data System (ADS)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

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

  9. Microsomal monooxygenase as a multienzyme system: the role of P450-P450 interactions

    PubMed Central

    Davydov, Dmitri R.

    2011-01-01

    Introduction There is increasing evidence of physical interactions (association) among cytochromes P450 in the membranes of the endoplasmic reticulum. Functional consequences of these interactions are often underestimated. Areas covered This article provides a comprehensive overview of available experimental material regarding P450-P450 interactions. Special emphasis is given to the interactions between different P450 species and to the functional consequences of homo- and heterooligomerization. Expert opinion Recent advances provide conclusive evidence for a substantial degree of P450 oligomerization in membranes. Interactions between different P450 species resulting in the formation of mixed oligomers with altered activity and substrate specificity have been demonstrated clearly. There are important indications that oligomerization of cytochromes P450 impedes electron flow to a fraction of the P450 population, which render some P450 species non-functional. Functional consequences of P450-P450 interactions make the integrated properties of the microsomal monooxygenase remarkably different from a simple summation of the properties of the individual P450 species. This complexity compromises the predictive power of the current in vitro models of drug metabolism and warrants an urgent need for development of new model systems that consider the interactions of multiple P450 species. PMID:21395496

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

    PubMed

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

    2017-03-01

    Cytochrome P450 (CYP450) drug biotransformation enzymes and multidrug resistance (MDR) proteins may influence drug disposition processes. The first part of the study aimed to evaluate the effect of mycotoxins deoxynivalenol (DON) and/or fumonisins (FBs), at contamination levels approaching European Union guidance levels, on intestinal and hepatic CYP450 enzymes and MDR proteins gene expression in broiler chickens. mRNA expression of genes encoding CYP450 enzymes (CYP3A37, CYP1A4 and CYP1A5) and drug transporters (MDR1/ABCB1 and MRP2/ABCC2) was determined using qRT-PCR. A significant up-regulation of CYP1A4 (P = 0.037) and MDR1 (P = 0.036) was observed in the jejunum of chickens fed a diet contaminated with FBs. The second part of this study aimed to investigate the impact of feeding a FBs contaminated diet on the oral absorption of enrofloxacin (10 mg/kg BW), a MDR1 substrate. A significant (P = 0.045), however small, decreased area under the plasma concentration-time curve (AUC0-48 h, mean ± SD) was observed for enrofloxacin in chickens fed the FBs contaminated diet compared to the control group, 16.28 ± 1.82 h μg/mL versus 18.27 ± 1.79 h μg/mL. These findings suggest that concurrent administration of drugs with FBs contaminated feed might alter the pharmacokinetic characteristics of CYP1A4 substrate drugs and MDR1 substrates, such as enrofloxacin.

  11. Role of cytochrome P450 in drug interactions

    PubMed Central

    Bibi, Zakia

    2008-01-01

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. PMID:18928560

  12. Male specific expression of a cytochrome P450 (Cyp312a1) in Drosophila melanogaster.

    PubMed

    Kasai, Shinji; Tomita, Takashi

    2003-01-24

    Using cDNA array techniques, the transcriptional levels of Drosophila cytochrome P450 (P450) genes were compared between male and female flies. Of the 86 P450s, 15 isoforms were picked up and the levels of transcription were confirmed by the real time quantitative RT-PCR. Cyp315a1 and Cyp302a1, which had been reported as P450s involved in the ecdysteroid biosynthesis, were included in the 15 isoforms and expression of these P450s was 8.6- and 7.9-fold higher in females than in males, respectively. In addition, we confirmed that expression of Cyp312a1 was 82-fold higher in adult males than females. This gene expression was observed mostly in the abdomen and its transcription level gradually increased from pupal stage and peaked in the 5-day-old adult. Furthermore, the male specific expression of Cyp312a1 was universally observed in three Drosophila strains originated from the USA (Oregon R), China (Canton S), and Japan (HKJ), suggesting possible involvement of this P450 in significant endogenous catalytic reaction(s). This is the first report of a P450 enzyme being predominantly expressed in male Drosophila.

  13. Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b/sub 5/ and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

    SciTech Connect

    Klasing, S.A.; Mora, M.I.; Wilson, W.C.; Fahey, G.C. Jr.; Garst, J.E.

    1985-09-01

    Experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b/sub 5/, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose, or 6.0% arenaceous flour. Chicks fed 12.0% wood molasses had a higher cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic, had a higher cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.

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

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    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

  15. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes.

    PubMed

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-07-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members' duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes.

  16. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  17. Unsaturated fatty acid regulation of cytochrome P450 expression via a CAR-dependent pathway

    PubMed Central

    Finn, Robert D.; Henderson, Colin J.; Scott, Claire L.; Wolf, C. Roland

    2008-01-01

    The liver is responsible for key metabolic functions, including control of normal homoeostasis in response to diet and xenobiotic metabolism/detoxification. We have shown previously that inactivation of the hepatic cytochrome P450 system through conditional deletion of POR (P450 oxidoreductase) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triacylglycerol (triglyceride) homoeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR (constitutive androstane receptor) and, to a lesser degree, PXR (pregnane X receptor). To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homoeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals. PMID:18778245

  18. Nerval influences on liver cytochrome P450.

    PubMed

    Klinger, W; Karge, E; Danz, M; Krug, M

    1995-09-01

    In male young adult Wistar rats the influences of nucleus raphe electrocoagulation, spinal cord dissection (cordotomy between C7 and Th1), vagotomy and denervation of liver hilus by phenol on liver cytochrome P450-system (cytochrome P450 concentration, ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities, hexobarbitone sleeping time) were investigated. In general the influences were small or negligible when compared with sham operated controls, only after vagotomy the depressing effect of sham operation was abolished. In all cases sham operation had a depressing effect until up to five weeks after operation.

  19. Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen.

    PubMed Central

    Lewis, D F; Ioannides, C; Parke, D V

    1998-01-01

    The importance of cytochrome P450 isoforms to species differences in the metabolism of foreign compounds and activation of procarcinogens has been identified. The possible range of P450 isozymes in significant variations in toxicity exhibited by experimental rodent species may have a relevance to chemical risk assessment, especially as human P450s are likely to show changes in the way they metabolize xenobiotics. Consequently, in the safety evaluation of chemicals, we should be cautious in extrapolating results from experimental animal models to humans. This paper focuses on examples in which species differences in P450s lead to significant alterations in carcinogenic response, and includes a discussion of the current procedures for toxicity screening, with an emphasis on short-term tests. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9755138

  20. Pharmacokinetics of Lipophilic Agents Following Preexposure: Non-Cytochrome P-450 Mediated Mechanisms

    DTIC Science & Technology

    1990-05-30

    FOLLOWING PREEXPOSUR G - AFOSR-87- 185 U’) NON -CYTOCHROME P-450 MEDIATED MECHAN’ICS PE - 61102F (NJ . AUTHOKS) PR - 2312 N TA - A5 Dr Lawrence R Curtis, Dr...Z39- I*. PHARMACOKINETICS OF LIPOPHILIC AGENTS FOLLOWING PREEXPOSURE: NON -CYTOCHROME P-450 MEDIATED MECHANISMS Air Force Grant No. 87-0185 Hillary M...amounts located in non -hepatic tissues. These studies showed that, despite the differences in PDR, the systems involved respond in a dose related manner

  1. Activities of cytochrome P450 1A2, N-acetyltransferase 2, xanthine oxidase, and cytochrome P450 2D6 are unaltered in children with cystic fibrosis.

    PubMed

    Kennedy, Mary Jayne; Scripture, Charity D; Kashuba, Angela D M; Scott, Christy S; Gaedigk, Andrea; Kearns, Gregory L

    2004-03-01

    The activities of hepatic cytochrome P450 (CYP) 1A2, N-acetyltransferase 2 (NAT-2), xanthine oxidase (XO), and CYP2D6 were evaluated in 12 young children (aged 3-8 years) with mild cystic fibrosis (CF) and 12 age-matched healthy control subjects by use of standard caffeine and dextromethorphan phenotyping methods. Subjects were given 4 oz of Coca-Cola (approximately 35 mg caffeine) (The Coca-Cola Company, Atlanta, Ga) and a single 0.5-mg/kg dose of dextromethorphan. Urine was collected for 8 hours after biomarker administration, and enzyme activity was assessed by use of previously validated caffeine and dextromethorphan molar ratios. CYP2D6 genotyping was also performed in 10 of 12 subjects with CF and 11 of 12 control subjects. There were no significant differences in the urinary molar ratios for any of the enzyme systems evaluated. These data suggest that CF does not alter the activities of CYP1A2, NAT-2, XO, and CYP2D6. Altered biotransformation of drugs in this patient population is likely enzyme- and isoform-specific and thus is apparent for only selected compounds that are substrates for enzymes other than CYP1A2, NAT-2, XO, and CYP2D6.

  2. P450 AND METABOLISM IN TOXICOLOGY

    EPA Science Inventory

    The cytochromes P450 catalyze the initial phase of detoxification of many environmental chemicals, xenobiotic, drugs and the secondary metabolic product of plants. Plant secondary chemicals can be highly toxic, and they evolved in a coevolving plant - animal warfare - the plants ...

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

    PubMed

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

    2015-11-01

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

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

    SciTech Connect

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

    1986-05-01

    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.

  5. Cytochrome P450 structure, function and clinical significance: A review.

    PubMed

    Palrasu, Manikandan; Nagini, Siddavaram

    2017-01-25

    The cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. Induction or inhibition of CYP enzymes is a major mechanism that underlies drug-drug interactions. CYP enzymes can be transcriptionally activated by various xenobiotics and endogenous substrates through receptor-dependent mechanisms. CYP enzyme inhibition is a principal mechanism for metabolism-based drug-drug interactions. Many chemotherapeutic drugs can cause drug interactions due to their ability to either inhibit or induce the CYP enzyme system. Predictions based on in silico analyses followed by validation have identified several microRNAs that regulate CYPs. Genetic polymorphisms and epigenetic changes in CYP genes may be responsible for inter-individual and inter-ethnic variations in disease susceptibility and the therapeutic efficacy of drugs. Knowledge about the substrates, inducers, inhibitors of CYP isoforms, and the polymorphisms of CYP enzymes may be used as an aid by clinicians to determine therapeutic strategy, and treatment doses for drugs that are metabolized by CYP gene products. The present review is a comprehensive compilation of cytochrome P450 structure, function, pharmacogenetics, and pharmacoepigenetics and clinical significance.

  6. CYP51--the omnipotent P450.

    PubMed

    Lepesheva, Galina I; Waterman, Michael R

    2004-02-27

    Sterol 14 alpha-demethylase (CYP51) is the single cytochrome P450 (CYP) required for sterol biosynthesis in different phyla, and it is the most widely distributed P450 gene family being found in all biological kingdoms. It catalyzes the first step following cyclization in sterol biosynthesis such as removal of the 14 alpha-methyl group from lanosterol in the cholesterol biosynthetic pathway, leading to formation of the initial substrate in steroid hormone biosynthesis. CYP51 from different phyla have low sequence similarity across kingdoms and contain only about 40 conserved amino acid residues in the whole family. An attempt to predict the possible role of these conserved residues is being made by a combination of the results of site-directed mutagenesis and information from the known crystal structure of sterol 14 alpha-demethylase from Mycobacterium tuberculosis.

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

    PubMed Central

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

    2016-01-01

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

  8. A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

    PubMed

    Miyakawa, Kazuhisa; Albee, Ryan; Letzig, Lynda G; Lehner, Andreas F; Scott, Michael A; Buchweitz, John P; James, Laura P; Ganey, Patricia E; Roth, Robert A

    2015-08-01

    Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

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

    SciTech Connect

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

    1992-01-01

    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.

  10. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s

    PubMed Central

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E.; Nelson, David R.; Tuszynski, Jack A.; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-01-01

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s. PMID:27616185

  11. Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing.

    PubMed

    Wiseman, Alan

    2003-04-01

    Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).

  12. Flower colour and cytochromes P450.

    PubMed

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

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

  13. P450monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium

    PubMed Central

    Syed, Khajamohiddin; Yadav, Jagjit S

    2012-01-01

    Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system (LDS), mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosporium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed. PMID:22624627

  14. P450 enzymes of estrogen metabolism.

    PubMed

    Martucci, C P; Fishman, J

    1993-01-01

    Endogenous and exogenous estrogens undergo extensive oxidative metabolism by specific cytochrome P450 enzymes. Certain drugs and xenobiotics have been found to be potent inducers of estrogen hydroxylating enzymes with C-2 hydroxylase induction being greater than that of C-16 hydroxylase. Oxygenated estrogen metabolites have different biological activities, with C-2 metabolites having limited or no activity and C-4 and C-16 metabolites having similar potency to estradiol. Pathophysiological roles for some of the oxygenated estrogen metabolites have been proposed, e.g. 16 alpha-hydroxyestrone and 4-hydroxyestrone. These reactive estrogens are capable of damaging cellular proteins and DNA and may be carcinogenic in specific cells.

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

    SciTech Connect

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

    1993-09-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene 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.

  16. Interspecies comparison and role of human cytochrome P450 and flavin-containing monooxygenase in hepatic metabolism of L-775,606, a potent 5-HT(1D) receptor agonist.

    PubMed

    Prueksaritanont, T; Lu, P; Gorham, L; Sternfeld, F; Vyas, K P

    2000-01-01

    1. Quantitative species differences and human liver enzymes involved in the metabolism of L-775,606, a potent and selective 5-HT1D receptor agonist developed for the acute treatment of migraine headache, have been investigated in vitro. 2. In human, monkey, dog and rat liver microsomes, formation of the hydroxylated M1 and the N-dealkylated M2 was mediated by enzyme(s) of high-affinity (apparent Km approximately 1-6 microM), and that of the two N-oxide isomers (M3) was catalysed by those of low affinity (apparent Km approximately 50-110 microM). In dog, M3 constituted a major pathway (approximately 40%), whereas in all other species it was a minor metabolite (< 5%). 3. In human liver microsomes, a marked inhibition (> or =80%) of M1 and M2 formation was observed by SKF525-A, troleandomycin, ketoconazole and anti-CYP3A antibodies, whereas the inhibition was modest (approximately 20-40%) with quercetin. Of seven cDNA-expressed human P450 tested, only CYP3A4 and CYP2C8 were capable of oxidizing L-775,606, resulting primarily in M1 and M2. However, CYP3A4 possessed much higher affinity (> or = 20-fold) and much higher intrinsic activity (> 100-fold) than CYP2C8. 4. In contrast, N-oxidation was not inhibited by any inhibitors of P450 tested, but rather was reduced significantly by heat treatment and methimazole, and was increased substantially with an incubation pH>7.4. Human flavin-containing monooxygenase form 3 (FMO3) catalysed exclusively the N-oxidation to M3, with apparent Km and optimum pH comparable with those observed in human liver microsomes. 5. These results demonstrated quantitative interspecies differences in the metabolism of L-775,606. In human, metabolism of L-775,606 to the principal metabolites, M1 and M2, was mediated primarily by CYP3A4 with minimal contribution from CYP2C8, whereas the minor N-oxidative pathway was catalysed mainly by FMO3.

  17. Cytochrome P450 expression in oesophageal cancer.

    PubMed Central

    Murray, G I; Shaw, D; Weaver, R J; McKay, J A; Ewen, S W; Melvin, W T; Burke, M D

    1994-01-01

    The cytochrome P450 superfamily of enzymes play a central part in the metabolism of carcinogens and anti-cancer drugs. The expression, cellular localisation, and distribution of different forms of P450 and the functionally associated enzymes epoxide hydrolase and glutathione S-transferases have been investigated in oesophageal cancer and non-neoplastic oesophageal tissue using immunohistochemistry. Expression of the different enzymes was confined to epithelial cells in both non-neoplastic samples and tumour samples except the CYP3A was also identified in mast cells and glutathione S-transferase pi was present in chronic inflammatory cells. CYP1A was present in a small percentage of non-neoplastic samples but both CYP2C and CYP3A were absent. Epoxide hydrolase was present in half of the non-neoplastic samples and the different classes of glutathione S-transferase were present in a low number of samples. In carcinomas CYP1A, CYP3A, epoxide hydrolase, and glutathione S-transferase pi were expressed in at least 60% of samples. The expression of glutathione S-transferases alpha and mu were significantly less in adenocarcinoma compared with squamous carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:8200549

  18. Inhibition of human cytochrome P450 enzymes by Bacopa monnieri standardized extract and constituents.

    PubMed

    Ramasamy, Seetha; Kiew, Lik Voon; Chung, Lip Yong

    2014-02-24

    Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP) enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL), CYP2C9 (36.49/12.5 µg/mL), CYP1A2 (52.20/25.1 µg/mL); competitive inhibition of CYP3A4 (83.95/14.5 µg/mL) and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL). However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day), B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%). These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  19. Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw; Chi, Chin-Wen; Ho, Li-Kang

    2004-08-01

    Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1'-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n = 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 +/- 0.7 nmol/min/mg protein. In human liver (n = 3) microsomes, the mean K(m) and V(max) values of SOH were 5.7 +/- 1.2 mM and 0.14 +/- 0.03 micromol/min/nmol P450, respectively. The mean intrinsic clearance (V(max)/K(m)) was 25.3 +/- 2.3 microL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r = 0.569) and chlorzoxazone hydroxylation (r = 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH.

  20. P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia.

    PubMed

    Flück, Christa E; Pandey, Amit V; Huang, Ningwu; Agrawal, Vishal; Miller, Walter L

    2008-01-01

    Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

  1. 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...

  2. 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...

  3. Molecular modeling of cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Szklarz, Grazyna D.; Halpert, James R.

    1997-05-01

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

  4. Physical Studies of P450P450 Interactions: Predicting Quaternary Structures of P450 Complexes in Membranes from Their X-ray Crystal Structures

    PubMed Central

    Reed, James R.; Backes, Wayne L.

    2017-01-01

    Cytochrome P450 enzymes, which catalyze oxygenation reactions of both exogenous and endogenous chemicals, are membrane bound proteins that require interaction with their redox partners in order to function. Those responsible for drug and foreign compound metabolism are localized primarily in the endoplasmic reticulum of liver, lung, intestine, and other tissues. More recently, the potential for P450 enzymes to exist as supramolecular complexes has been shown by the demonstration of both homomeric and heteromeric complexes. The P450 units in these complexes are heterogeneous with respect to their distribution and function, and the interaction of different P450s can influence P450-specific metabolism. The goal of this review is to examine the evidence supporting the existence of physical complexes among P450 enzymes. Additionally, the review examines the crystal lattices of different P450 enzymes derived from X-ray diffraction data to make assumptions regarding possible quaternary structures in membranes and in turn, to predict how the quaternary structures could influence metabolism and explain the functional effects of specific P450P450 interactions. PMID:28194112

  5. Flower colour and cytochromes P450

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

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

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

    PubMed Central

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

    2011-01-01

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  8. Crystal Structure of Cindoxin, the P450cin Redox Partner

    PubMed Central

    2015-01-01

    The crystal structure of the flavin mononucleotide (FMN)-containing redox partner to P450cin, cindoxin (Cdx), has been determined to 1.3 Å resolution. The overall structure is similar to that of the FMN domain of human cytochrome P450 reductase. A Brownian dynamics–molecular dynamics docking method was used to produce a model of Cdx with its redox partner, P450cin. This Cdx–P450cin model highlights the potential importance of Cdx Tyr96 in bridging the FMN and heme cofactors as well P450cin Arg102 and Arg346. Each of the single-site Ala mutants exhibits ∼10% of the wild-type activity, thus demonstrating the importance of these residues for binding and/or electron transfer. In the well-studied P450cam system, redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex. In sharp contrast, Cdx does not shift P450cin to a low-spin state, although the stability of oxy-P450cin is decreased 10-fold in the presence of Cdx. This indicates that Cdx may have a modest effect on the open–closed equilibrium in P450cin compared to that in P450cam. It has been postulated that part of the effector role of Pdx on P450cam is to promote a significant structural change that makes available a proton relay network involving Asp251 required for O2 activation. The structure around the corresponding Asp in P450cin, Asp241, provides a possible structural reason for why P450cin is less dependent on its redox partner for functionally important structural changes. PMID:24533927

  9. Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.

    PubMed

    Yoon, Yune-Jung; Kim, Kwon-Bok; Kim, Hyunmi; Seo, Kyung-Ah; Kim, Ho-Sook; Cha, In-June; Kim, Eun-Young; Liu, Kwang-Hyeon; Shin, Jae-Gook

    2007-09-01

    Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.

  10. 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 ...

  11. Modulation of P450-dependent ifosfamide pharmacokinetics: a better understanding of drug activation in vivo.

    PubMed

    Brain, E G; Yu, L J; Gustafsson, K; Drewes, P; Waxman, D J

    1998-06-01

    , and demonstrate the potential for modulation of IF's alternative metabolic pathways in a therapeutically useful manner. These studies also highlight several clinically relevant drug interactions that may occur during concomitant administration of IF with drugs and other compounds that modulate hepatic P450 enzyme levels.

  12. Repurposing Resveratrol and Fluconazole To Modulate Human Cytochrome P450-Mediated Arachidonic Acid Metabolism.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2016-04-04

    Cytochrome P450 (P450) enzymes metabolize arachidonic acid (AA) to several biologically active epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Repurposing clinically-approved drugs could provide safe and readily available means to control EETs and HETEs levels in humans. Our aim was to determine how to significantly and selectively modulate P450-AA metabolism in humans by clinically-approved drugs. Liquid chromatography-mass spectrometry was used to determine the formation of 15 AA metabolites by human recombinant P450 enzymes, as well as human liver and kidney microsomes. CYP2C19 showed the highest EET-forming activity, while CYP1B1 and CYP2C8 showed the highest midchain HETE-forming activities. CYP1A1 and CYP4 showed the highest subterminal- and 20-HETE-forming activity, respectively. Resveratrol and fluconazole produced the most selective and significant modulation of hepatic P450-AA metabolism, comparable to investigational agents. Monte Carlo simulations showed that 90% of human population would experience a decrease by 6-22%, 16-39%, and 16-35% in 16-, 18-, and 20-HETE formation, respectively, after 2.5 g daily of resveratrol, and by 22-31% and 14-23% in 8,9- and 14,15-EET formation after 50 mg of fluconazole. In conclusion, clinically-approved drugs can provide selective and effective means to modulate P450-AA metabolism, comparable to investigational drugs. Resveratrol and fluconazole are good candidates to be repurposed as new P450-based treatments.

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

    USGS Publications Warehouse

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

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

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

    USGS Publications Warehouse

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

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

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

    SciTech Connect

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

    1987-05-01

    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.

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

    USGS Publications Warehouse

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

    1996-01-01

    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.

  17. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

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

    SciTech Connect

    VandenBerg, M.; Bosveld, A.T.C.

    1995-12-31

    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.

  19. Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases.

    PubMed

    Chiapella, C; Radovan, R D; Moreno, J A; Casares, L; Barbé, J; Llagostera, M

    2000-10-31

    To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.

  20. Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals.

    PubMed

    Darwish, W; Ikenaka, Y; Eldaly, E; Ishizuka, M

    2010-01-01

    The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using alpha-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione-S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals.

  1. Cytochrome P450 gene polymorphism and cancer.

    PubMed

    Agundez, Jose A G

    2004-06-01

    Human cytochrome P450 (CYP) enzymes play a key role in the metabolism of drugs and environmental chemicals. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. Phenotyping analyses revealed an association between CYP enzyme activity and the risk to develop several forms of cancer. Research carried out in the last decade demonstrated that several CYP enzymes are polymorphic due to single nucleotide polymorphisms, gene duplications and deletions. As genotyping procedures became available for most human CYP, an impressive number of association studies on CYP polymorphisms and cancer risk were conducted. Here we review the findings obtained in these studies regarding CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP8A1 and CYP21 gene polymorphisms. Consistent evidences for association between CYP polymorphisms and lung, head and neck, and liver cancer were reported. Controversial findings suggest that colorectal and prostate cancers may be associated to CYP polymorphisms, whereas no evidences for a relevant association with breast or bladder cancers were reported. We summarize the available information related to the association of CYP polymorphisms with leukaemia, lymphomas and diverse types of cancer that were investigated only for some CYP genes, including brain, esophagus, stomach, pancreas, pituitary, cervical epithelium, melanoma, ovarian, kidney, anal and vulvar cancers. This review discusses on causes of heterogeneity in the proposed associations, controversial findings on cancer risk, and identifies topics that require further investigation. In addition, some recommendations on study design, in order to obtain more conclusive findings in further studies, are provided.

  2. Cytochrome P450 1 family and cancers.

    PubMed

    Go, Ryeo-Eun; Hwang, Kyung-A; Choi, Kyung-Chul

    2015-03-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to xenobiotics response element (XREs), and then starts the expressions of downstream genes including cytochrome P450 (CYP) 1 family members: CYP1A1, CYP1A2 and CYP1B1. Role of CYP1 family is involved in the metabolism of endogenous hormones, xenobiotics and drug. The expression of CYP1 family is regulated by estradiol (E2) or xenobiotics in diverse cancers. In breast cancers expressing estrogen receptors (ERs), level of CYP1B1 is increased by E2 and reversed by an estrogen receptor antagonist, ICI 182,780 or 4-hydrotamoxifen, which indicates that the expression of CYP1 family in downstream region of AhR is regulated by an activation of ERα. In metabolic pathways, E2 is converted into 4-hydroxyestradiol by CYP1B1, which can be converted into mainly estradiol-3,4-quinone, a potential carcinogen, by peroxidase. Increased expression of CYP1 family indicates the possibility of carcinogenesis by exposure of xenobiotics in endometrial and ovarian cancers. Apart from roles of CYP1 family in relation with ER pathway, CYP1 family is over-expressed in ER independent cancers. CYP1A1 exhibits hydroxylase activity in oxidation of arachidonic acid, which has been transformed to 12(R)-hydrxyeicosatetraenoic (HETEs), a potent activator of AhR activity. On the basis of results, phytoestrogens and dexamethasone are provided as cancer therapy regulating the expression of CYP1 family. Thus, this review focuses on the role(s) of CYP1 family in ER-dependent or ER-independent cancers and the potential for cancer therapy to target CYP1 family in these cancers.

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

    USGS Publications Warehouse

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

    1992-01-01

    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.

  4. Epoxidation Activities of Human Cytochromes P450c17 and P450c21

    PubMed Central

    2015-01-01

    Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon–carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes. PMID:25386927

  5. Hepatic expression patterns of aryl hydrocarbon receptor, pregnane X receptor, two cytochrome P450s and five phase II metabolism genes responsive to 17alpha-methyltestosterone in rare minnow Gobiocypris rarus.

    PubMed

    Gao, Jiancao; Liu, Shaozhen; Zhang, Yingying; Yuan, Cong; Yang, Yanping; Wang, Zaizhao

    2014-05-01

    17Alpha-methyltestosterone (MT), a synthetic androgen, is widely used in aquaculture. Aquatic organisms can receive continuous exposure to residual MT throughout their lives. Aiming to evaluate the effects of MT on genes involved in biotransformation pathway, meanwhile attempting to unravel the MT metabolic pathway at the transcriptional level in fish, here we isolated the cDNAs of previously unreported AHR2, Sult1 st1, Ugt2a1 and Ugt2b6 in rare minnow, and predominantly investigated the hepatic transcriptional patterns of AHR2, PXR and five biotransformation genes after MT exposure in both genders adult rare minnow Gobiocypris rarus. The present findings suggest that AHR2 and PXR should play important roles in regulating biotransformation enzymes related to MT catabolism, moreover, CYP1A, CYP3A, SULT1 ST4, SULT1 ST6 and UGT2A1 may play certain roles in catabolism of MT in adult G. rarus. Additionally, UGT2A1 may make greater contribution than SULT1 ST4 and SULT1 ST6 in MT catabolism in males.

  6. Thiomers: Inhibition of cytochrome P450 activity.

    PubMed

    Iqbal, Javed; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

    2011-08-01

    The aim of the present study was to investigate the potential of different thiolated polymers (thiomers) on the catalytic activity of CYP450s on one hand and to explore new inhibitors for CYP activity on the other hand. Several thiolated polymers including poly(acrylic acid)-cysteine (PAA-cysteine), chitosan-thioglycolic acid (chitosan-TGA), and thiolated PEG-g-PEI copolymer along with brij 35, myrj 52 and the well-established CYPP450 inhibitor verapamil were screened for their CYP3A4 and CYP2A6 inhibitory activity, and their IC(50) values were determined. Both enzyme inhibition assays were performed in 96-well microtiter plates. 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and 7-hydroxycoumarin (7-HC) were used as fluorescent substrates in order to determine CYP3A4 and CYP2A6 catalytic activity, respectively. All investigated compounds inhibited CYP3A4 as well as CYP2A6 activity. All tested (thiolated) polymers were found to be more potent inhibitors of CYP3A4 than of CYP2A6 catalytic activity. Apart from verapamil that is a known CYP3A4 inhibitor, brij 35 and myrj 52 were explored as potent inhibitors of CYP3A4 and CYP2A6 catalytic activity. Among the tested polymers, the rank order for CYP3A4 inhibition was PAA-cysteine (100 kDa)>brij 35>thiolated PEG-g-PEI copolymer (16 kDa)>myrj 52>PAA (100 kDa)>PAA-cysteine (450 kDa)>verapamil>PAA (450 kDa)>chitosan-TGA (150 kDa)>chitosan (150 kDa). On the other hand, the rank order of CYP2A6 inhibition was brij 35>PAA-cysteine (100kDa)>chitosan-TGA (150 kDa)>PAA (100 kDa)>thiolated PEG-g-PEI copolymer (16 kDa)>PAA-cysteine (450 kDa)>chitosan (150 kDa)>verapamil>PAA (450 kDa)>myrj 52. Thus, this study suggests that (thiolated) polymers display a promising potential to inhibit cytochrome P450s activity and might turn out to be potentially valuable tools for improving the oral bioavailability of actively secreted compounds by avoiding intestinal metabolism.

  7. Oxidation of tolualdehydes to toluic acids catalyzed by cytochrome P450-dependent aldehyde oxygenase in the mouse liver.

    PubMed

    Watanabe, K; Matsunaga, T; Yamamoto, I; Yashimura, H

    1995-02-01

    Mouse hepatic microsomal enzymes catalyzed the oxidation of o-, m-, and p-tolualdehydes, intermediate metabolites of xylene, to the corresponding toluic acids. Cofactor requirement for the catalytic activity indicates that the microsomes contain NAD- and NADPH-dependent enzymes for this reaction. GC/MS analyses of the carboxylic acids formed by incubation under oxygen-18 gas indicate that the mechanism for this oxidation is an oxygenation and a dehydrogenation for the NADPH- and NAD-dependent reaction. Vmax/Km (nmol/min/mg protein) ratios indicate that the NADPH-dependent activity is more pronounced than the NAD-dependent activity. These results suggest that the NADPH-dependent reaction is mainly responsible for the microsomal oxidation of tolualdehydes. The NADPH-dependent activity was significantly inhibited by SKF 525-A, disulfiram and menadione, inhibitors of cytochrome P450 (P450), suggesting the involvement of P450 in the reaction. In a reconstituted system, P450 MUT-2 (CYP2C29) purified from mouse hepatic microsomes catalyzed the oxidation of o-, m-, and p-tolualdehydes to the carboxylic acids, and the specific activities (nmol/min/nmol P450) were 1.44, 2.81, and 2.32, respectively. Rabbit antibody raised against P450 MUT-2 significantly inhibited the NADPH-dependent oxidation of tolualdehydes to toluic acids by 88% (o-), 63% (m-), and 62% (p-) using mouse hepatic microsomes. The present study demonstrated that a mouse hepatic microsomal aldehyde oxygenase, P450 MUT-2, catalyzed the most of oxidative activity of tolualdehydes to toluic acids in the microsomes.

  8. CCAAT/enhancer-binding protein alpha (C/EBPalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha) synergistically cooperate with constitutive androstane receptor to transactivate the human cytochrome P450 2B6 (CYP2B6) gene: application to the development of a metabolically competent human hepatic cell model.

    PubMed

    Benet, Marta; Lahoz, Agustín; Guzmán, Carla; Castell, José V; Jover, Ramiro

    2010-09-10

    The transcription of tissue-specific and inducible genes is usually subject to the dynamic control of multiple activators. Dedifferentiated hepatic cell lines lose the expression of tissue-specific activators and many characteristic hepatic genes, such as drug-metabolizing cytochrome P450. Here we demonstrate that by combining adenoviral vectors for CCAAT/enhancer-binding protein alpha (C/EBPalpha), hepatocyte nuclear factor 4alpha (HNF4alpha), and constitutive androstane receptor, the CYP2B6 expression and inducibility by CITCO are restored in human hepatoma HepG2 cells at levels similar to those in cultured human hepatocytes. Moreover, several other phase I and II genes are simultaneously activated, which suggests that this is an effective approach to endow dedifferentiated human hepatoma cells with a particular metabolic competence and response to inducers. In order to gain insight into the molecular mechanism, we examined the cooperation of these three transcription factors on the CYP2B6 5'-flanking region. We show new CYP2B6-responsive sequences for C/EBPalpha and HNF4alpha and a novel synergistic regulatory mechanism whereby C/EBPalpha, HNF4alpha, and constitutive androstane receptor bind and cooperate through proximal and distal response elements to confer a maximal level of expression. The results obtained from human liver also suggest that important differences in the expression and binding of C/EBPalpha and HNF4alpha could account for the large interindividual variability of the hepatic CYP2B6 enzyme, which metabolizes commonly used drugs.

  9. The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme

    PubMed Central

    Murai, Takahiro; Reilly, Christopher R.; Ward, Robert M.; Yost, Garold S.

    2010-01-01

    Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5 and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with KI, kinact and partition ratio of 16 μM, 0.027 min-1 and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 μM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs. PMID:20707410

  10. A three-dimensional model of aromatase cytochrome P450.

    PubMed Central

    Graham-Lorence, S.; Amarneh, B.; White, R. E.; Peterson, J. A.; Simpson, E. R.

    1995-01-01

    P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate. PMID:7549871

  11. Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

    PubMed Central

    2015-01-01

    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes. PMID:25133307

  12. Role of protein-protein interactions in cytochrome P450-mediated drug metabolism and toxicity.

    PubMed

    Kandel, Sylvie E; Lampe, Jed N

    2014-09-15

    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein-protein interactions play a critical role in this process. Historically, the study of CYP-protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein-protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein-protein interactions with CYP enzymes.

  13. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  14. Inactivation of cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a thiophene-containing anticancer drug.

    PubMed

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Hollenberg, Paul F; Johnson, William W

    2011-02-01

    An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b(5) and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b(5). The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a K(I) of 24 μM and a k(inact) of 0.04 min(-1). This K(I) is significantly greater than the clinical OSI-930 C(max) of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site

  15. Purification of the pyrazole-inducible cytochrome P-450 isozyme

    SciTech Connect

    Palakodety, R.; Clejan, L.; Krikun, G.; Feierman, D.; Cederbaum, A.I.

    1987-05-01

    The alcohol dehydrogenase inhibitor, pyrazole, appears to induce a cytochrome P-450 isozyme with properties similar to the ethanol-inducible P-450. The pyrazole-inducible P-450 isozyme was purified from the liver microsomes of rats treated with pyrazole essentially by the procedure of Ryan et al and also by chromatofocussing. The final preparation appeared homogenous by SDS-PAGE with an apparent molecular weight of 52,000, had a specific content of 11 nmoles P-450 per mg protein, showed very high activity of low K/sub m/ dimethylnitrosamine demethylase and produced a type II binding spectrum with dimethylsulfoxide. The enzyme was also active with aniline and aminopyrine as substrates. Pyrazole itself served as an excellent substrate with 4-hydroxy pyrazole being the product. An antibody against the pyrazole-inducible P-450 raised in chickens recognized a protein with mol.wt of about 52,000 in control microsomes. This band was highly enriched in microsomes from rats treated with pyrazole, 4-methyl-pyrazole, ethanol or acetone, but not phenobarbital or 3-methylcholanthrene. In summary, the pyrazole-inducible P-450 has been purified and appears to be identical in its catalytic and immunological properties to the alcohol-inducible P-450.

  16. Conformational selectivity in cytochrome P450 redox partner interactions

    PubMed Central

    Hollingsworth, Scott A.; Batabyal, Dipanwita; Nguyen, Brian D.; Poulos, Thomas L.

    2016-01-01

    The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis. PMID:27439869

  17. Evolving P450pyr Monooxygenase for Regio- and Stereoselective Hydroxylations.

    PubMed

    Yang, Yi; Li, Zhi

    2015-01-01

    P450pyr monooxygenase from Sphingomonas sp. HXN-200 catalysed the regio- and stereoselective hydroxylation at a non-activated carbon atom, a useful but challenging reaction in classic chemistry, with unique substrate specificity for a number of alicyclic compounds. New P450pyr mutants were developed by directed evolution with improved catalytic performance, thus significantly extending the application of the P450pyr monooxygenase family in biohydroxylation to prepare useful and valuable chiral alcohols. Directed evolution of P450pyr created new enzymes with improved S-enantioselectivity or R-enantioselectivity for the hydroxylation of N-benzyl pyrrolidine, enhanced regioselectivity for the hydroxylation of N-benzyl pyrrolidinone, and increased enantioselectivity for the hydroxylation of N-benzyl piperidinone, respectively. Directed evolution of P450pyr generated also mutants with fully altered regioselectivity (from terminal to subterminal) and newly created excellent S-enantioselectivity for the biohydroxylation of n-octane and propylbenzene, respectively, providing new opportunities for the regio- and enantioselective alkane functionalization. New P450pyr mutants were engineered as the first catalyst for highly selective terminal hydroxylation of n-butanol to 1,4-butanediol. Several novel, accurate, sensitive, simple, and HTS assays based on colorimetric or MS detection for measuring the enantio- and/or regioselectivity of hydroxylation were developed and proven to be practical in directed evolution. The P450pyr X-ray structure was obtained and used to guide the evolution. In silico modelling and substrate docking provided some insight into the influence of several important amino acid mutations of the engineered P450pyr mutants on the altered or enhanced regio- and enantioselectivity as well as new substrate acceptance. The obtained information and knowledge is useful for further engineering of P450pyr for other hydroxylations and oxidations.

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

    PubMed Central

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

    2013-01-01

    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

  19. In vitro interaction of emerging contaminants with the cytochrome p450 system of Mediterranean deep-sea fish.

    PubMed

    Ribalta, Carla; Solé, Montserrat

    2014-10-21

    The interactions of emerging contaminants with the xenobiotic and endogenous metabolizing system of deep-sea fish were compared. The drugs diclofenac, fluoxetine, and gemfibrozil belong to different pharmaceutical classes with diverse mechanistic actions, and the personal care products triclosan, galaxolide, and nonylphenol are representative of antibacterial agents, nitro-musks, and surfactants, respectively. The fish compared are representative of the middle and lower slope of deep-sea habitats. The species were adults of Trachyrynchus scabrus, Mora moro, Cataetix laticeps, and Alepocehalus rostratus. The hepatic metabolic system studied were the activities associated with several cytochrome P450 isoforms (CYPs): 7-ethoxyresorufin-O-deethylase (EROD), benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD), and 7-ethoxycoumarin-O-deethylase (ECOD). Results showed differences in baseline activities and sensitivity to chemicals which were species, chemical, and pathway dependent. T. scabrous was the most sensitive species to chemical interactions with the xenobiotic and endogenous metabolizing (EROD and BFCOD) systems, especially in the case of diclofenac interference with BFCOD activity (IC50 = 15.7 ± 2.2 μM). Moreover, T. scabrous and A. rostratus possessed high basal ECOD activity, and this was greatly affected by in vitro exposure to diclofenac in T. scabrous also (IC50 = 6.86 ± 1.4 μM). These results highlight the sensitivity of marine fish to emerging contaminants and propose T. scabrous (middle slope) and A. rostratus (lower slope) as sentinels and the inclusion of ECOD activity as a sensitive biomarker to these exposures.

  20. Metabolism of bilirubin by human cytochrome P450 2A6

    SciTech Connect

    Abu-Bakar, A'edah; Arthur, Dionne M.; Wikman, Anna S.; Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu; Ng, Jack C.; Lang, Matti A.

    2012-05-15

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic CYP2

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

    SciTech Connect

    Abdulla, Dalya; Goralski, Kerry B.; Renton, Kenneth W. . E-mail: Ken.Renton@dal.ca

    2006-10-01

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

  2. Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450.

    PubMed

    Yoshida, Y; Kumaoka, H

    1975-10-01

    A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast.

  3. Cytochrome P450 down-regulation by serum from humans with a viral infection and from rabbits with an inflammatory reaction.

    PubMed

    Bleau, A M; Fradette, C; El-Kadi, A O; Côté, M C; du Souich, P

    2001-07-01

    Serum from humans with an upper respiratory viral infection (HS(URVI)) and from rabbits with a turpentine-induced acute inflammatory reaction (RS(TIAR)) reduces the activity of hepatic cytochrome P450 (P450) following 4 h of incubation. The aim of the present study was to assess the effect of HS(URVI) and RS(TIAR) on P450 activity and expression following 24 h of incubation with hepatocytes from control (H(CONT)) and rabbits with a TIAR (H(INFLA)). RS(TIAR) incubated with H(CONT) for 24 h reduced P450 content and activity, and CYP3A6 by 45%, without changing CYP1A1 and 1A2; when incubated with H(INFLA), RS(TIAR) decreased P450 content and activity without affecting CYP1A1 or 1A2. HS(URVI) incubated for 4 h with H(CONT) decreased P450 activity without affecting the amounts of CYP1A1, 1A2, or 3A6, although when incubated for 24 h, P450 activity and CYP3A6 amount decreased. HS(URVI) incubated with H(INFLA) for 4 h reduced P450 content and activity, and incubated for 24 h reduced activity, P450 content, and amount of CYP1A1 and 1A2 proteins. The present study demonstrates that 1) the effect of RS(TIAR) and HS(URVI) depends upon the susceptibility of the hepatocyte, i.e., H(CONT) or primed H(INFLA); 2) P450 down-regulation is preceded by a decrease in P450 activity; 3) the nature of the inflammatory reaction determines the repercussions on P450 activity and expression; and 4) CYP3A6 is more vulnerable than CYP1A1 and 1A2 to the down-regulation provoked by an inflammatory challenge.

  4. Genetics Home Reference: cytochrome P450 oxidoreductase deficiency

    MedlinePlus

    ... hormones, which are needed for normal development and reproduction. The hormonal changes associated with cytochrome P450 oxidoreductase ... which are essential for normal sexual development and reproduction; corticosteroids, which are involved in the body's response ...

  5. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Halpert, James R.

    2015-01-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  6. The contribution of atom accessibility to site of metabolism models for cytochromes P450.

    PubMed

    Rydberg, Patrik; Rostkowski, Michal; Gloriam, David E; Olsen, Lars

    2013-04-01

    Three different types of atom accessibility descriptors are investigated in relation to site of metabolism predictions. To enable the integration of local accessibility we have constructed 2DSASA, a method for the calculation of the atomic solvent accessible surface area that is independent of 3D coordinates. The method was implemented in the SMARTCyp site of metabolism prediction models and improved the results by up to 4 percentage points for nine cytochrome P450 isoforms. The final models are made available at http://www.farma.ku.dk/smartcyp.

  7. Special issue: Cytochrome P450 structure and function: introduction.

    PubMed

    Munro, Andrew W; Leys, David

    2012-05-01

    The 17th International Conference on Cytochrome P450 Biochemistry, Biophysics and Structure was held in Manchester, UK from 26-30 June 2011. This issue of FEBS J. contains review and primary research articles reflecting the breadth of science covered at this conference, and reflecting the impact of P450-related research in fields as diverse as steroid metabolism, plant biochemistry, structural biology and biotechnology.

  8. Immunochemical evidence for an ethanol-inducible form of liver microsomal cytochrome P-450 in rodents and primates

    SciTech Connect

    Lasker, J.M.; Ardies, C.M.; Bloswick, B.P.; Lieber, C.S.

    1986-05-01

    Polyclonal antibodies against cytochrome P-450-4, a major liver microsomal P-450 isozyme purified from ethanol (E)-treated hamsters, were used to probe for immunochemically-related hemeproteins in other species. Liver microsomes (LM) were obtained from naive and E-treated rats, deermice, hamsters, and baboons. Baboon liver 9000 x g supernatant (S-9) was prepared from needle biopsy samples. LM and S-9 proteins were resolved by SDS-PAGE, then transferred to nylon membranes. Immunodetection was performed on the Western blots using rabbit anti P-450-4 IgG, anti-rabbit IgG-alk. phos., and an appropriate chromagen. Control LM from all species contained a cross-reacting protein of mol. wt. similar to P-450-4 (54k). The amount of this cross-reacting protein as reflected by staining intensity, was much higher in LM from E-treated animals. This protein was also detected in S-9 from E-treated baboons. In contrast, no increase in phenobarbital-inducible P-450-2 related LM protein (assessed using anti P-450-2) was observed after E treatment. Increased P-450-4 related protein in LM from E-treated animals was associated with enhanced oxidation of ethanol and aniline by these LM when compared to controls. In conclusion, LM from rats, deermice, and baboons contain a protein immunochemically homologous to hamster liver P-450-4. As observed in hamsters, the amount of this hepatic protein increases in these other species after E treatment.

  9. Enhanced expression of cytochrome P450 in stomach cancer.

    PubMed Central

    Murray, G. I.; Taylor, M. C.; Burke, M. D.; Melvin, W. T.

    1998-01-01

    The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9569036

  10. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    PubMed

    Steuck, Maryvonne; Hellhake, Stefan; Schebb, Nils Helge

    2016-11-30

    The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC50 value of 4.6 μM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 μM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.

  11. Pharmacogenetics of P450 oxidoreductase: implications in drug metabolism and therapy.

    PubMed

    Hu, Lei; Zhuo, Wei; He, Yi-Jing; Zhou, Hong-Hao; Fan, Lan

    2012-11-01

    The redox reaction of cytochrome P450 enzymes (CYP) is an important physiological and biochemical reaction in the human body, as it is involved in the oxidative metabolism of both endogenous and exogenous substrates. Cytochrome P450 oxidoreductase (POR) is the only obligate electron donor for all of the hepatic microsomal CYP enzymes. It plays a crucial role in drug metabolism and treatment by not only acting as an electron donor involved in drug metabolism mediated by CYP enzymes but also by directly inducing the transformation of some antitumor precursors. Studies have found that the gene encoding human POR is highly polymorphic, which is of considerable clinical significance as it affects the metabolism and curative effects of clinically used drugs. This review aims to discuss the effect of POR and its genetic polymorphisms on drug metabolism and therapy, as well as the potential mechanisms of POR pharmacogenetics.

  12. MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

    PubMed

    Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

    2017-01-01

    An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

  13. MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication

    PubMed Central

    Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

    2017-01-01

    An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses. PMID:28056087

  14. Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar1

    PubMed Central

    Ro, Dae-Kyun; Ehlting, Jürgen; Douglas, Carl J.

    2002-01-01

    NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa × Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue. PMID:12481067

  15. Cytochrome P450 1A2 Detoxicates Aristolochic Acid in the Mouse

    PubMed Central

    Einolf, Heidi J.; Dickman, Kathleen G.; Wang, Lai; Smith, Amanda; Grollman, Arthur P.

    2010-01-01

    Aristolochic acids (AAs) are plant-derived nephrotoxins and carcinogens responsible for chronic renal failure and associated urothelial cell cancers in several clinical syndromes known collectively as aristolochic acid nephropathy (AAN). Mice provide a useful model for study of AAN because the renal histopathology of AA-treated mice is strikingly similar to that of humans. AA is also a potent carcinogen in mice with a tissue spectrum somewhat different from that in humans. The toxic dose of AA in mice is higher than that in humans; this difference in susceptibility has been postulated to reflect differing rates of detoxication between the species. Recent studies in mice have shown that the hepatic cytochrome P450 system detoxicates AA, and inducers of the arylhydrocarbon response protect mice from the nephrotoxic effects of AA. The purpose of this study was to determine the role of specific cytochrome P450 (P450) enzymes in AA metabolism in vivo. Of 18 human P450 enzymes we surveyed only two, CYP1A1 and CYP1A2, which were effective in demethylating 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) to the nontoxic derivative 8-hydroxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAIa). Kinetic analysis revealed similar efficiencies of formation of AAIa by human and rat CYP1A2. We also report here that CYP1A2-deficient mice display increased sensitivity to the nephrotoxic effects of AAI. Furthermore, Cyp1a2 knockout mice accumulate AAI-derived DNA adducts in the kidney at a higher rate than control mice. Differences in bioavailability or hepatic metabolism of AAI, expression of CYP1A2, or efficiency of a competing nitroreduction pathway in vivo may explain the apparent differences between human and rodent sensitivity to AAI. PMID:20164109

  16. Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    PubMed Central

    Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-01-01

    Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported. PMID:23443101

  17. [Cytochromes P450 and formation of reactive metabolites. Role in hepatotoxicity of drugs].

    PubMed

    Pessayre, D

    1993-01-01

    During the millennia of evolution, animals have been subjected to a relentless biological warfare mounted by the plants that they ingested. By duplication of an ancestral gene, divergent evolution of these 2 genes, and so forth, surviving animals have been endowed with multiple cytochromes P450s which can metabolize (and thus eliminate) a multitude of environmental liposoluble xenobiotics. A disadvantage of this system (fortunately limited by the concomitant installation of several protective systems) is that cytochrome P450 transforms some of these xenobiotics into chemically reactive metabolites. These free radicals or electrophilic metabolites attack tissue constituents, and may lead to mutation, cancer or tissue necrosis. Tissue necrosis affect mainly the liver, whose content in cytochrome P450 is particularly high. Indeed, reactive metabolites are usually extremely unstable, and react mainly in situ, in the same organ that forms them. When the formation of reactive metabolites is extensive, protective mechanisms are overwhelmed, extensive alterations of diverse hepatic constituents occur, and toxic hepatitis ensues. When the formation of reactive metabolites is moderate, severe toxic lesions do not occur. However, the covalent binding of reactive metabolites to hepatic proteins modifies the self of the subject. In some subjects, the presence of this modified self triggers immunization, and leads to immunoallergic hepatitis. The immune response may be directed either against protein (or peptide) epitopes modified by the presence of a reactive metabolite (reaction against modified self) and/or against normal, unmodified, epitopes of proteins (autoimmune reaction against the self, triggered by the modified self). Both metabolic factors, and the HLA phenotype, appear to modulate the likelihood of immunization.

  18. Sequential metabolism of sesamin by cytochrome P450 and UDP-glucuronosyltransferase in human liver.

    PubMed

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Munetsuna, Eiji; Ohta, Miho; Sakaki, Toshiyuki

    2011-09-01

    Our previous study revealed that CYP2C9 played a central role in sesamin monocatecholization. In this study, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 (P450) or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its antioxidant activity, whereas glucuronidation strongly reduces its antioxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. In contrast, in rat liver microsomes, catecholization is predominant over glucuronidation. In addition, rat liver produced two isomers of the glucuronide, whereas human liver produced only one glucuronide. These results suggest a significant species-based difference in the metabolism of sesamin between humans and rats. Kinetic studies using recombinant human UGT isoforms identified UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol. In addition, a good correlation was observed between the glucuronidation activity and UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes. These results strongly suggest that UGT2B7 plays an important role in glucuronidation of sesamin monocatechol. Interindividual difference among the 10 human liver microsomes is approximately 2-fold. These results, together with our previous results on the metabolism of sesamin by human P450, suggest a small interindividual difference in sesamin metabolism. We observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. On the basis of these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver.

  19. Cytochrome P450-mediated metabolism of vitamin D

    PubMed Central

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

    2014-01-01

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

  20. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

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

    2010-11-16

    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.

  1. Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.

    PubMed

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

    1995-06-01

    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.

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

    ClinicalTrials.gov

    2016-10-24

    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

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

    SciTech Connect

    Park, Young Joo; Lee, Eun Kyung; Lee, Yoon Kwang; Park, Do Joon; Jang, Hak Chul; Moore, David D.

    2012-09-01

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

  4. Technology evaluation: MetXia-P450, Oxford Biomedica.

    PubMed

    Hunt, S

    2001-12-01

    Oxford BioMedica is developing gene therapies for treating various forms of cancer. The therapies comprise the transfer of several anticancer genes at a time using a recombinant retrovirus approach based on the company's proprietary LTR Deleted Vector and Accelerated Vector Evolution technologies [238147]. MetXia-P450 is a gene therapy construct containing the cytochrome P450 gene CYP2B6, and is designed to be injected directly into tumors to convert them into 'drug factories'. This is achieved because CYP2B6 converts the inactive produg form cyclophosphamide into the active cytotoxic drug. MetXia-P450 is in phase I/II trials for breast cancer [339582].

  5. Repellents Inhibit P450 Enzymes in Stegomyia (Aedes) aegypti

    PubMed Central

    Jaramillo Ramirez, Gloria Isabel; Logan, James G.; Loza-Reyes, Elisa; Stashenko, Elena; Moores, Graham D.

    2012-01-01

    The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s) underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES) arm-in cage assay with Stegomyia (Aedes) aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils. PMID:23152795

  6. Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450.

    PubMed

    Kokkinakis, D M; Koop, D R; Scarpelli, D G; Coon, M J; Hollenberg, P F

    1985-02-01

    approximately 90% inhibition of the metabolism of NNDM. These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.

  7. Hepatic Farnesoid X-Receptor Isoforms α2 and α4 Differentially Modulate Bile Salt and Lipoprotein Metabolism in Mice

    PubMed Central

    Boesjes, Marije; Bloks, Vincent W.; Hageman, Jurre; Bos, Trijnie; van Dijk, Theo H.; Havinga, Rick; Wolters, Henk; Jonker, Johan W.; Kuipers, Folkert; Groen, Albert K.

    2014-01-01

    The nuclear receptor FXR acts as an intracellular bile salt sensor that regulates synthesis and transport of bile salts within their enterohepatic circulation. In addition, FXR is involved in control of a variety of crucial metabolic pathways. Four FXR splice variants are known, i.e. FXRα1-4. Although these isoforms show differences in spatial and temporal expression patterns as well as in transcriptional activity, the physiological relevance hereof has remained elusive. We have evaluated specific roles of hepatic FXRα2 and FXRα4 by stably expressing these isoforms using liver-specific self-complementary adeno-associated viral vectors in total body FXR knock-out mice. The hepatic gene expression profile of the FXR knock-out mice was largely normalized by both isoforms. Yet, differential effects were also apparent; FXRα2 was more effective in reducing elevated HDL levels and transrepressed hepatic expression of Cyp8b1, the regulator of cholate synthesis. The latter coincided with a switch in hydrophobicity of the bile salt pool. Furthermore, FXRα2-transduction caused an increased neutral sterol excretion compared to FXRα4 without affecting intestinal cholesterol absorption. Our data show, for the first time, that hepatic FXRα2 and FXRα4 differentially modulate bile salt and lipoprotein metabolism in mice. PMID:25506828

  8. A Quantitative High-Throughput 96-well plate Fluorescence Assay for Mechanism-Based Inactivators of Cytochromes P450 Exemplified using CYP2B6

    PubMed Central

    Kenaan, Cesar; Zhang, Haoming; Hollenberg, Paul F.

    2010-01-01

    Mechanism-based inactivators such as bergamottin are useful chemical tools for identifying the roles of specific active-site amino acid residues in the reactions catalyzed by the cytochromes P450 (CYPs or P450s) that are responsible for the metabolism of a wide variety of drugs and endogenous substrates. In clinical settings mechanism-based inactivation of P450s involved in xenobiotic metabolism has the potential to lead to adverse drug-drug interactions and assays to identify and characterize drug candidates as P450 inactivators are important in drug discovery and development. Here we present a quantitative high-throughput protocol for investigating cytochrome P450 mechanism-based inactivators using the example of CYP2B6 and bergamottin to illustrate the finer points of this protocol. This protocol details the adaptation of a 7-ethoxytrifluoromethyl coumarin (7-EFC) O-deethylation fluorescence activity assay to a 96-well microtiter plate format and uses a plate-reader to detect the fluorescence of the product. Compared to previous methods, this protocol requires less P450 and takes significantly less time while greatly increasing throughput. The protocol as written takes approximately two hours to complete. The principles and procedures outlined in this protocol can be easily adapted to other inactivators, P450 isoforms, substrates and plate-readers. PMID:20885377

  9. Cytochrome P450 1A induction in sea ducks inhabiting nearshore areas of Prince William Sound, Alaska

    USGS Publications Warehouse

    Trust, Kimberly A.; Esler, Daniel N.; Woodin, Bruce R.; Stegeman, John J.

    2000-01-01

    Following the Exxon-Valdez oil spill, hepatic rates of EROD activity and thus, P450 1A expression, were significantly higher in harlequin ducks (Histrionicus histrionicus) and Barrow’s goldeneyes (Bucephala islandica) from oiled areas of Prince William Sound, Alaska when compared to birds from unoiled sites. Polychlorinated biphenyl exposure did not account for areal differences in P450 1A induction in harlequin ducks. Background hydrocarbon levels in Prince William Sound were negligible prior to the 1989 oil spill, but remnant Exxon-Valdez oil was still present in nearshore habitats of the spill zone coincident with our study. We conclude that P450 1A induction in sea ducks from areas oiled during the Exxon-Valdez oil spill was likely due to exposure to residual oil. We speculate that biochemical and physiological changes in individuals chronically exposed to oil may be constraining population recovery of some sea duck species affected by the spill.

  10. Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse.

    PubMed

    Tydén, E; Olsén, L; Tallkvist, J; Tjälve, H; Larsson, P

    2008-08-01

    Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).

  11. Reactive Intermediates Produced from Metabolism of the Vanilloid Ring of Capsaicinoids by P450 Enzymes

    PubMed Central

    Reilly, Christopher A.; Henion, Fred; Bugni, Tim S.; Ethirajan, Manivannan; Stockmann, Chris; Pramanik, Kartick C.; Srivastava, Sanjay K.; Yost, Garold S.

    2012-01-01

    This study characterized electrophilic and radical products derived from metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin, were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5’-Dicapsaicin, presumably arising from bi-molecular coupling of free radical intermediates, was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated, quinone methide, and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems. PMID:23088752

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

    USGS Publications Warehouse

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

    1999-01-01

    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.

  13. Spectroscopic quantitation of cytochrome P-450 in human lung microsomes.

    PubMed

    Wheeler, C W; Guenthner, T M

    1990-01-01

    The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.

  14. Virtual Experiments Enable Exploring and Challenging Explanatory Mechanisms of Immune-Mediated P450 Down-Regulation

    PubMed Central

    Petersen, Brenden K.; Ropella, Glen E. P.; Hunt, C. Anthony

    2016-01-01

    Hepatic cytochrome P450 levels are down-regulated during inflammatory disease states, which can cause changes in downstream drug metabolism and hepatotoxicity. Long-term, we seek sufficient new insight into P450-regulating mechanisms to correctly anticipate how an individual’s P450 expressions will respond when health and/or therapeutic interventions change. To date, improving explanatory mechanistic insight relies on knowledge gleaned from in vitro, in vivo, and clinical experiments augmented by case reports. We are working to improve that reality by developing means to undertake scientifically useful virtual experiments. So doing requires translating an accepted theory of immune system influence on P450 regulation into a computational model, and then challenging the model via in silico experiments. We build upon two existing agent-based models—an in silico hepatocyte culture and an in silico liver—capable of exploring and challenging concrete mechanistic hypotheses. We instantiate an in silico version of this hypothesis: in response to lipopolysaccharide, Kupffer cells down-regulate hepatic P450 levels via inflammatory cytokines, thus leading to a reduction in metabolic capacity. We achieve multiple in vitro and in vivo validation targets gathered from five wet-lab experiments, including a lipopolysaccharide-cytokine dose-response curve, time-course P450 down-regulation, and changes in several different measures of drug clearance spanning three drugs: acetaminophen, antipyrine, and chlorzoxazone. Along the way to achieving validation targets, various aspects of each model are falsified and subsequently refined. This iterative process of falsification-refinement-validation leads to biomimetic yet parsimonious mechanisms, which can provide explanatory insight into how, where, and when various features are generated. We argue that as models such as these are incrementally improved through multiple rounds of mechanistic falsification and validation, we will

  15. Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion.

    PubMed

    Legraverend, C; Mode, A; Westin, S; Ström, A; Eguchi, H; Zaphiropoulos, P G; Gustafsson, J A

    1992-02-01

    The onset of the sexually dimorphic pattern of GH secretion and increased hepatic GH-binding capacity in rats at puberty is temporally correlated with the developmental induction of three hepatic cytochrome P-450s with steroid hydroxylase activity, P-450 IIC11, P-450 IIC12, and P-450 IIC13, and one cytochrome P-450 with vitamin A hydroxylase activity, P-450 IIC7. In this study we demonstrate that expression of the 2C11, 2C12, and 2C13 genes is modulated by GH at the level of transcriptional initiation both in vivo and in primary cultures of adult hepatocytes. In an effort to define the minimum sequence responsible for the inductive effects of GH, we have analyzed the ability of a 0.7-kilobase fragment isolated from the 5'-flank of the 2C12 gene, including the natural promoter, to drive transcription of a 320-basepair G-less cassette in vitro. We were unable to detect any substantial difference in RNA polymerase-II-dependent transcriptional efficiency toward the 2C12 promoter between liver nuclear extracts from normal and hypophysectomized rats of both sexes. This observation supports the assumption that the sequence information contained between bases -700 and 1 is sufficient to support basal transcription of the 2C12 gene. Sequence information residing 5' or 3' of the 0.7-kilobase 5'-flank or a higher ordered chromatin structure may be necessary for the sex-specific transcriptional activation of the 2C12 gene.

  16. Genomewide annotation and comparative genomics of cytochrome P450 monooxygenases (P450s) in the polypore species Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora.

    PubMed

    Syed, Khajamohiddin; Nelson, David R; Riley, Robert; Yadav, Jagjit S

    2013-01-01

    Genomewide annotation of cytochrome P450 monooxygenases (P450s) in three white-rot species of the fungal order Polyporales, namely Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora, revealed a large contingent of P450 genes (P450ome) in their genomes. A total of 199 P450 genes in B. adusta and 209 P450 genes each in Ganoderma sp. and P. brevispora were identified. These P450omes were classified into families and subfamilies as follows: B. adusta (39 families, 86 subfamilies), Ganoderma sp. (41 families, 105 subfamilies) and P. brevispora (42 families, 111 subfamilies). Of note, the B. adusta genome lacked the CYP505 family (P450foxy), a group of P450-CPR fusion proteins. The three polypore species revealed differential enrichment of individual P450 families in their genomes. The largest CYP families in the three genomes were CYP5144 (67 P450s), CYP5359 (46 P450s) and CYP5344 (43 P450s) in B. adusta, Ganoderma sp. and P. brevispora, respectively. Our analyses showed that tandem gene duplications led to expansions in certain P450 families. An estimated 33% (72 P450s), 28% (55 P450s) and 23% (49 P450s) of P450ome genes were duplicated in P. brevispora, B. adusta and Ganoderma sp., respectively. Family-wise comparative analysis revealed that 22 CYP families are common across the three Polypore species. Comparative P450ome analysis with Ganoderma lucidum revealed the presence of 143 orthologs and 56 paralogs in Ganoderma sp. Multiple P450s were found near the characteristic biosynthetic genes for secondary metabolites, namely polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), terpene cyclase and terpene synthase in the three genomes, suggesting a likely role of these P450s in secondary metabolism in these Polyporales. Overall, the three species had a richer P450 diversity both in terms of the P450 genes and P450 subfamilies as compared to the model white-rot and brown-rot polypore species Phanerochaete chrysosporium and Postia placenta.

  17. The role of cytochrome P450s in polycyclic aromatic hydrocarbon carcinogenesis

    SciTech Connect

    Polzer, R.J.

    1993-01-01

    Metabolic activation of polycyclic aromatic hydrocarbons (PAH) to carcinogenic diol epoxides has been determined to be a critical step in tumor initiation by PAH. The key enzyme(s) involved in the metabolic activation are members of the cytochrome P450 superfamily. Two distinct isoforms of cytochrome P450 have been determined to be induced upon treatment of cells in culture with benzo(a)pyrene (B(a)P) by use of Immobilized Artificial Membrane Column High Performance Liquid Chromatography, Western blotting, Northern blotting, and in vitro metabolism studies. Cytochrome P4501A is involved in the metabolism of PAH in the human hepatoma cell line, HepG2; the human mammary carcinoma cell line, MCF-7; and the mouse hepatoma cell line; Hepa-1; whereas cytochrome P450EF is involved in this metabolism in both secondary hamster and mouse embryo cell cultures. Induction of cytochrome P450s by B(a)P generally leads to an increased metabolism of tritiated B(a)P, DMBA, and DB(a,1)P to water-soluble metabolities and to the formation of PAH-DNA adducts, suggesting that induction by B(a)P alters the metabolism of PAH to metabolic activation. DMBA induction of cytochrome P450s leads to various changes in metabolism and PAH-DNA binding and these changes were both cell and PAH specific. These results suggest that DMBA can shift metabolism of certain PAH towards metabolic activation in some cells, while in other cells DMBA or one of its metabolities can compete with other PAH for metabolic activation. UDP-glucuronosyl-transferase and epoxide hydrase do not have significant roles in detoxifying proximate or ultimate carcinogenic PAH metabolites, however, sulfotransferase and glutathione-S-transferase do detoxify proximate and ultimate carcinogenic metabolities in the HepG2 cell line. Finally, attempts to inhibit B(a)P metabolism and DNA-binding in intact cells in culture through conjugation of inhibitory cytochrome P4501A1 antibodies to insulin or folic acid were examined.

  18. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

    PubMed Central

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L.; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s−1. POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  19. In Vitro Cytochrome P450 Formation of a Mono-Hydroxylated Metabolite of Zearalenone Exhibiting Estrogenic Activities: Possible Occurrence of This Metabolite in Vivo

    PubMed Central

    Bravin, Frederique; Duca, Radu C.; Balaguer, Patrick; Delaforge, Marcel

    2009-01-01

    The mycoestrogen zearalenone (ZEN), as well as its reduced metabolites, which belong to the endocrine disruptor bio-molecule family, are substrates for various enzymes involved in steroid metabolism. In addition to its reduction by the steroid dehydrogenase pathway, ZEN also interacts with hepatic detoxification enzymes, which convert it into hydroxylated metabolites (OH-ZEN). Due to their structures to that of estradiol, ZEN and its derived metabolites bind to the estrogen receptors and are involved in endocrinal perturbations and are possibly associated with estrogen-dependent cancers. The primary aim of this present study was to identify the enzymatic cytochrome P450 isoforms responsible for the formation of the most abundant OH-ZEN. We thus studied its in vitro formation using hepatic microsomes in a range of animal model systems including man. OH-ZEN was also recovered in liver and urine of rats treated orally with ZEN. Finally we compared the activity of ZEN and its active metabolites (α-ZAL and OH-ZEN) on estrogen receptors using HeLa ER-α and ER-β reporter cell lines as reporters. OH-ZEN estrogenic activities were revealed to be limited and not as significant as those of ZEN or α-ZAL. PMID:19468341

  20. P450 GENETIC VARIATION: IMPLICATIONS FOR ENVIRONMENTAL AND WORKPLACE EXPOSURE

    EPA Science Inventory

    The Cytochrome P450 array detoxifies many chemicals by catalyzing the conversion of mostly hydrophobic chemicals into more hydrophilic forms that can subsequently be excreted by the body. Human genetic variation in the genes for these enzymes produces wide variations in the abili...

  1. Cytochrome P450 arachidonic acid metabolism in bovine corneal epithelium

    SciTech Connect

    Masferrer, J.; Schwartzman, M.L.; Abraham, N.G.; Dunn, M.W.; McGiff, J.C.

    1986-03-01

    The presence of the cytochrom P450 system and its involvement in the metabolism of AA was studied in the corneal epithelium. This tissue contains cytochrome P450 as assessed directly by measurement of the carbon monoxide reduced spectrum (specific activity of 161 pmol/10 mg protein) and indirectly by measuring the activity of aryl hydrocarbon hydroxylase (AHH) - a cytochrome P450-dependent enzyme (11-39 pmol 3-OH benzopyrene/mg protein/10 min). When corneal epithelial microsomes were incubated with /sup 14/C-arachidonic acid, 30-50% of the total radioactivity was converted to two peaks, I and II. Further separation using high performance liquid chromatography has shown that each peak contains two metabolites, A,B and C,D. Metabolite formation was dependent on the addition of NADPH (1 mM) and inhibited by carbon monoxide and SKF-525A (100 ..mu..M) suggesting a cytochrome P450-dependent mechanism. Compound C (5-10 ..mu..M) inhibited the activity of corneal epithelial Na-K-ATPase by 30-60%, being 100-fold more potent than ouabain. Compound D (10-100 ng) induced a dose dependent relaxation of the rat caudal artery. Compound D also inhibited corneal Na-K-ATPase activity but less potently than compound C. These compounds may be important to transport processes of ocular epithelia and participate in the control of the ocular circulation and aqueous humor dynamics.

  2. Bimodal action of miroestrol and deoxymiroestrol, phytoestrogens from Pueraria candollei var. mirifica, on hepatic CYP2B9 and CYP1A2 expressions and antilipid peroxidation in mice.

    PubMed

    Udomsuk, Latiporn; Juengwatanatrakul, Thaweesak; Putalun, Waraporn; Jarukamjorn, Kanokwan

    2012-01-01

    Miroestrol and deoxymiroestrol are phytoestrogens isolated from Pueraria candollei var. mirifica. The influence of miroestrol and dexoymirosestrol on hepatic cytochrome P450 (P450) enzymes and antioxidative activity in brain was examined in C57BL/6 mice compared with that of a synthetic female sex hormone estradiol. We hypothesized that miroestrol and deoxymiroestrol would induce CYP2B9 expression, whereas CYP1A2 expression would be suppressed compared with estradiol. Miroestrol and deoxymiroestrol treatment significantly increased uterus weight and volume. In addition, both of these phytoestrogens induced the expression of CYP2B9 and suppressed the expression of CYP1A2, as expected. Hepatic P450 activities correspondingly showed that both compounds increased benzyloxyresorufin O-dealkylase activity, whereas methoxyresorufin O-dealkylase activity was reduced. These observations suggested that miroestrol and deoxymiroestrol might affect hepatic P450 enzymes, including the CYP2B9 and CYP1A2 P450 isoforms. Assessment of lipid peroxidation demonstrated that miroestrol and deoxymiroestrol markedly decreased levels of malondialdehyde formation in the mouse brain. This is the first report suggesting miroestrol and deoxymiroestrol as potential alternative medicines to estradiol because of their distinctive ability to regulate mouse hepatic P450 expression and their beneficial antioxidative activities.

  3. Cytochromes P450 in the bioactivation of chemicals.

    PubMed

    Ioannides, Costas; Lewis, David F V

    2004-01-01

    The initial view that the cytochrome P450 enzyme system functions simply in the deactivation of xenobiotics is anachronistic on the face of mounting evidence that this system can also transform many innocuous chemicals to toxic products. However, not all xenobiotic-metabolising cytochrome P450 subfamilies show the same propensity in the bioactivation of chemicals. For example, the CYP2C, 2B and 2D subfamilies play virtually no role in the bioactivation of toxic and carcinogenic chemicals, whereas the CYP1A, 1B and 2E subfamilies are responsible for the bioactivation of the majority of xenobiotics. Electronic and molecular structural features of organic chemicals appear to predispose them to either bioactivation by one cytochrome P450 enzyme or deactivation by another. Consequently, the fate of a chemical in the body is largely dependent on the cytochrome P450 profile at the time of exposure. Any factor that modulates the enzymes involved in the metabolism of a certain chemical will also influence its toxicity and carcinogenicity. For example, many chemical carcinogens bioactivated by CYP1, on repeated administration, selectively induce this family, thus exacerbating their carcinogenicity. CYP1 induction potency by chemicals appears to be determined by a combination of their molecular shape and electron activation. The function of cytochromes P450 in the bioactivation of chemicals is currently being exploited to design systems that can be used clinically to facilitate the metabolic conversion of prodrugs to their biologically-active metabolites in cells that poorly express them, such as tumour cells, in the so-called gene-directed prodrug therapy.

  4. Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates, metoprolol, bufuralol and dextromethorphan.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Hagihira, Yuya; Murayama, Norie; Shimizu, Makiko; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2015-01-01

    1. Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient. 2. A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83-86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat. 3. Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting. 4. By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1'-hydroxylation. 5. These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.

  5. Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

    PubMed Central

    Ueng, Yune-Fang; Chen, Chien-Chih; Chung, Yu-Ting; Liu, Tsung-Yun; Chang, Yu-Ping; Lo, Wei-Sheng; Murayama, Norie; Yamazaki, Hiroshi; Souček, Pavel; Chau, Gar-Yang; Chi, Chin-Wen; Chen, Ruei-Ming; Li, Ding-Tzai

    2011-01-01

    BACKGROUND AND PURPOSE Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC50 value decreased by 98%). This time-dependent inactivation (kinact 0.044 min−1; KI 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice. PMID:21418183

  6. Expression of P450c17 in the human fetal nervous system.

    PubMed

    Schonemann, Marcus D; Muench, Marcus O; Tee, Meng Kian; Miller, Walter L; Mellon, Synthia H

    2012-05-01

    P450c17 catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. P450c17 is expressed in human fetal and postnatal adrenals and gonads and in the developing mouse nervous system, but little is known about its expression in the human nervous system. We obtained portions of 9-, 10-, and 11-wk gestation human fetuses and delineated the pattern of expression of P450c17 in their peripheral nervous systems by immunocytochemistry using the P450c17 antiserum previously used to characterize P450c17 in the mouse brain. P450c17 was readily detected in the dorsal root ganglia (DRG) and spinal cord. Neural structures were identified with antisera to the cytoskeletal protein neural cell adhesion molecule; DRG were identified with antisera to the neuronal transcription factor BRN3A and neurotrophin receptor tropomyosin-receptor-kinase B. The identification of P450c17 was confirmed using commercial antisera directed against different domains of P450c17 and by using antisera immunodepleted with authentic human P450c17. We also found expression of the P450 cholesterol side-chain cleavage enzyme (P450scc) in the spinal cord and DRG. Expression of P450scc is limited to cell bodies; unlike P450c17, we never detected P450scc in fiber tracts. Catalysis by P450c17 requires electron donation from P450 oxidoreductase (POR). Dual-label immunohistochemistry detected P450c17 and POR colocalized in DRG bundles, but some fibers containing P450c17 lacked POR. These data suggest that neurosteroids synthesized via these two enzymes may act in the developing human nervous system. The expression of P450c17 in structures lacking POR means that P450c17 may not be steroidogenic in those locations, suggesting that P450c17 may have additional functions that do not require POR.

  7. 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

    Meier, U.T.; Meyer, U.A.

    1987-12-15

    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.

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

    PubMed

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

    2015-12-25

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

  9. Effect of prenatal exposure of deltamethrin on the ontogeny of xenobiotic metabolizing cytochrome P450s in the brain and liver of offsprings

    SciTech Connect

    Johri, Ashu; Dhawan, Alok; Lakhan Singh, Ram; Parmar, Devendra . E-mail: parmar_devendra@hotmail.com

    2006-08-01

    Prenatal exposure to low doses (0.25 or 0.5 or 1.0 mg/kg, p.o.) of deltamethrin, a type II pyrethroid insecticide, to pregnant dams from gestation days 5 to 21 (GD5-21) produced dose-dependent alterations in the ontogeny of xenobiotic metabolizing cytochrome P450 (CYP) isoforms in brain and liver of the offsprings. RT-PCR analysis revealed dose-dependent increase in the mRNA expression of cerebral and hepatic CYP1A1, 1A2, 2B1, 2B2, and 2E1 isoenzymes in the offsprings exposed prenatally to deltamethrin. Similar increase in the activity of the marker enzymes of these CYP isoforms has indicated that placental transfer of the pyrethroid, a mixed type of CYP inducer, even at these low doses may be sufficient to induce the CYPs in brain and liver of the offsprings. Our data have further revealed persistence in the increase in expression of xenobiotics metabolizing CYPs up to adulthood in brain and liver of the exposed offsprings, suggesting the potential of deltamethrin to imprint the expression of CYPs in brain and liver of the offsprings following its in utero exposure. Furthermore, though the levels of CYPs were several fold lower in brain, almost equal magnitude of induction in cerebral and hepatic CYPs has further suggested that brain CYPs are responsive to the induction by environmental chemicals. The present data indicating alterations in the expression of xenobiotic metabolizing CYPs during development following prenatal exposure to deltamethrin may be of significance as these CYP enzymes are not only involved in the neurobehavioral toxicity of deltamethrin but have a role in regulating the levels of ligands that modulate growth, differentiation, and neuroendocrine functions.

  10. NADPH-Cytochrome P450 Oxidoreductase: Prototypic Member of the Diflavin Reductase Family

    PubMed Central

    Iyanagi, Takashi; Xia, Chuanwu; Kim, Jung-Ja P.

    2012-01-01

    NADPH-cytochrome P450 oxidoreductase (CYPOR) and nitric oxide synthase (NOS), two members of the diflavin oxidoreductase family, are multi-domain enzymes containing distinct FAD and FMN domains connected by a flexible hinge. FAD accepts a hydride ion from NADPH, and reduced FAD donates electrons to FMN, which in turn transfers electrons to the heme center of cytochrome P450 or NOS oxygenase domain. Structural analysis of CYPOR, the prototype of this enzyme family, has revealed the exact nature of the domain arrangement and the role of residues involved in cofactor binding. Recent structural and biophysical studies of CYPOR have shown that the two flavin domains undergo large domain movements during catalysis. NOS isoforms contain additional regulatory elements within the reductase domain that control electron transfer through Ca2+-dependent calmodulin (CaM) binding. The recent crystal structure of an iNOS Ca2+/CaM-FMN construct, containing the FMN domain in complex with Ca2+/CaM, provided structural information on the linkage between the reductase and oxgenase domains of NOS, making it possible to model the holo iNOS structure. This review summarizes recent advances in our understanding of the dynamics of domain movements during CYPOR catalysis and the role of the NOS diflavin reductase domain in the regulation of NOS isozyme activities. PMID:22982532

  11. Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines.

    PubMed

    Degawa, M; Agatsuma, T; Hashimoto, Y

    1990-12-01

    The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.

  12. Development of cytochromes P450 in avian species as a biomarker for environmental contaminant exposure and effect: Procedures and baseline values

    USGS Publications Warehouse

    Melancon, M.J.; Bengston, David A.; Henshel, Diane S.

    1996-01-01

    As in mammals and fish, birds respond to many environmental contaminants with induction of hepatic cytochromes P450. In order to monitor cytchromes P450 in specific avian species, for assessing the status of that species or the habitat it utilizes, it is necessary to have background information on the appropriate assay conditions and the responsiveness of cytochrome P450 induction in that species. Assay of four monooxygenases which give resorufin as product using a fluorescence microwell plate scanner has proven to be an effective approach. Information is provided on the incubation conditions and baseline activity for twenty avian species at ages ranging from pipping embryo to adult. Induction responsiveness is presented for sixteen of them. This information can serve as a guide for those who wish to utilize cytochrome P450 as a biomarker for contaminant exposure and effect to aid in selection of appropriate species, age, and monooxygenase assay(s).

  13. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    NASA Astrophysics Data System (ADS)

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover

  14. Valence tautomerism in synthetic models of cytochrome P450

    PubMed Central

    Das, Pradip Kumar; Samanta, Subhra; McQuarters, Ashley B.; Lehnert, Nicolai

    2016-01-01

    CytP450s have a cysteine-bound heme cofactor that, in its as-isolated resting (oxidized) form, can be conclusively described as a ferric thiolate species. Unlike the native enzyme, most synthetic thiolate-bound ferric porphyrins are unstable in air unless the axial thiolate ligand is sterically protected. Spectroscopic investigations on a series of synthetic mimics of cytP450 indicate that a thiolate-bound ferric porphyrin coexists in organic solutions at room temperature (RT) with a thiyl-radical bound ferrous porphyrin, i.e., its valence tautomer. The ferric thiolate state is favored by greater enthalpy and is air stable. The ferrous thiyl state is favored by entropy, populates at RT, and degrades in air. These ground states can be reversibly interchanged at RT by the addition or removal of water to the apolar medium. It is concluded that hydrogen bonding and local electrostatics protect the resting oxidized cytP450 active site from degradation in air by stabilizing the ferric thiolate ground state in contrast to its synthetic analogs. PMID:27302948

  15. Human cytochromes P450 in health and disease

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

    Kaipainen, Arja; Greene, Emily R.; Huang, Sui

    2010-01-01

    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: ω-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

  17. Regulation of cytochrome P450 expression in Drosophila: Genomic insights

    PubMed Central

    Giraudo, Maeva; Unnithan, G. Chandran; Le Goff, Gaëlle; Feyereisen, René

    2009-01-01

    Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action. PMID:20582327

  18. Non-Michaelis-Menten kinetics in cytochrome P450-catalyzed reactions.

    PubMed

    Atkins, William M

    2005-01-01

    The cytochrome P450 monooxygenases (CYPs) are the dominant enzyme system responsible for xenobiotic detoxification and drug metabolism. Several CYP isoforms exhibit non-Michaelis-Menten, or "atypical," steady state kinetic patterns. The allosteric kinetics confound prediction of drug metabolism and drug-drug interactions, and they challenge the theoretical paradigms of allosterism. Both homotropic and heterotropic ligand effects are now widely documented. It is becoming apparent that multiple ligands can simultaneously bind within the active sites of individual CYPs, and the kinetic parameters change with ligand occupancy. In fact, the functional effect of any specific ligand as an activator or inhibitor can be substrate dependent. Divergent approaches, including kinetic modeling and X-ray crystallography, are providing new information about how multiple ligand binding yields complex CYP kinetics.

  19. Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    SciTech Connect

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W. . Patuxent Environmental Science Center); Tillitt, D.E. . Midwest Science Center)

    1994-11-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P450-associated mono-oxygenates and cytochrome P450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r[sup 2] often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the AH receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  20. Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black- crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P450-associated monooxygenases and cytochrome P450 proteins, induced up to 85- fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r super(2) often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah- active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  1. Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  2. Integrated in vitro analysis for the in vivo prediction of cytochrome P450-mediated drug-drug interactions.

    PubMed

    McGinnity, Dermot F; Waters, Nigel J; Tucker, James; Riley, Robert J

    2008-06-01

    Unbound IC(50) (IC(50,u)) values of 15 drugs were determined in eight recombinantly expressed human cytochromes P450 (P450s) and human hepatocytes, and the data were used to simulate clinical area under the plasma concentration-time curve changes (deltaAUC) on coadministration with prototypic CYP2D6 substrates. Significant differences in IC(50,u) values between enzyme sources were observed for quinidine (0.02 microM in recombinant CYP2D6 versus 0.5 microM in hepatocytes) and propafenone (0.02 versus 4.1 microM). The relative contribution of individual P450s toward the oxidative metabolism of clinical probes desipramine, imipramine, tolterodine, propranolol, and metoprolol was estimated via determinations of intrinsic clearance using recombinant P450s (rP450s). Simulated deltaAUC were compared with those observed in vivo via the ratios of unbound inhibitor concentration at the entrance to the liver to inhibition constants determined against rP450s ([I](in,u)/K(i)) and incorporating parallel substrate elimination pathways. For this dataset, there were 20% false negatives (observed deltaAUC >or= 2, predicted deltaAUC < 2), 77% correct predictions, and 3% false positives. Thus, the [I](in,u)/K(i) approach appears relatively successful at estimating the degree of clinical interactions and can be incorporated into drug discovery strategies. Using a Simcyp ADME (absorption, metabolism, distribution, elimination) simulator (Simcyp Ltd., Sheffield, UK), there were 3% false negatives, 94% correct simulations, and 3% false positives. False-negative predictions were rationalized as a result of mechanism-based inhibition, production of inhibitory metabolites, and/or hepatic uptake. Integrating inhibition and reaction phenotyping data from automated rP450 screens have shown applicability to predict the occurrence and degree of in vivo drug-drug interactions, and such data may identify the clinical consequences for candidate drugs as both "perpetrators" and "victims" of P450

  3. Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo

    PubMed Central

    Wright, Aaron T.; Cravatt, Benjamin F.

    2007-01-01

    The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class. PMID:17884636

  4. Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology.

    PubMed

    Girvan, Hazel M; Munro, Andrew W

    2016-04-01

    Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenase enzymes with enormous potential for synthetic biology applications. Across Nature, their substrate range is vast and exceeds that of other enzymes. The range of different chemical transformations performed by P450s is also substantial, and continues to expand through interrogation of the properties of novel P450s and by protein engineering studies. The ability of P450s to introduce oxygen atoms at specific positions on complex molecules makes these enzymes particularly valuable for applications in synthetic biology. This review focuses on the enzymatic properties and reaction mechanisms of P450 enzymes, and on recent studies that highlight their broad applications in the production of oxychemicals. For selected soluble bacterial P450s (notably the high-activity P450-cytochrome P450 reductase enzyme P450 BM3), variants with a multitude of diverse substrate selectivities have been generated both rationally and by random mutagenesis/directed evolution approaches. This highlights the robustness and malleability of the P450 fold, and the capacity of these biocatalysts to oxidise a wide range of chemical scaffolds. This article reviews recent research on the application of wild-type and engineered P450s in the production of important chemicals, including pharmaceuticals and drug metabolites, steroids and antibiotics. In addition, the properties of unusual members of the P450 superfamily that do not follow the canonical P450 catalytic pathway are described.

  5. Microsomal cytochrome P450 as a target for drug discovery and repurposing.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2017-02-01

    Cytochrome P450 (P450) enzymes are ancient electron-transfer-chain system of remarkable biological importance. Microsomal P450 enzymes are the P450 attached to endoplasmic reticulum, which, in humans, are critical for body's defenses against xenobiotics by mediating their metabolism, and cell signaling by mediating arachidonic acid (AA) transformation to several potent bioactive molecules. Only recently, modulating P450-mediated AA metabolism has risen as a promising new drug target. This review presents the therapeutic potential of finding effective, selective and safe treatments targeting P450-mediated AA metabolism, and the several approaches that have been used to find these treatments; among which, our focus was on modulators of P450 activities. We detailed the efforts done to develop new molecular entities designed to modulate P450, and the more recent efforts tried to employ our previous knowledge on drug metabolism to repurpose old drugs with the capacity to alter P450-mediated drug metabolism to target AA metabolism. Because of the long recognition of P450 role in xenobiotic metabolism, several clinically approved agents were identified to alter P450 activity. Repurposing old drugs as P450 modulators can facilitate bringing treatments targeting P450-mediated AA metabolism to clinical trials. However, the capacity of the modulation of P450-derived AA metabolites of clinically approved drugs has to be systematically investigated and validated for their new use in humans.

  6. Cytochrome P450 induction in mallard duck (MD), black-crowned night heron (BCNH) and Fisher-344 rat

    USGS Publications Warehouse

    Melancon, M.J.; Rattner, B.A.; Stegeman, John J.

    1991-01-01

    P450 induction was studied in adult and pipping MDs, pipping BCNHs, and rats. Adult MDs and rats received i.p. injection of corn oil, 3-methylcholanthrene (MC) in corn oil (20 mg/kg), saline or phenobarbital (PB) in saline (80 mg/kg) for 3 days. MD and BCNH embryos received MC and PB by injection into the aircell approximately 2 days before pipping and were sacrificed at pipping. Hepatic microsomes were assayed for protein, arylhydrocarbon hydroxylase (AHH), benzphetamine-N-demethylase (BEND), ethoxy-resorufin-O-dealkylase (EROD), pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD), ethoxycoumarin-O-dealkylase (ECOD), and by SDS-PAGE with western blot using a polyclonal anti-P4S0IIB antibody and a monoclonal anti-P450IA antibody (MAb 1-12-3). Although species and age caused substantial differences in responses, all treated groups showed an increase in one or more monooxygenase assays. All animals treated with MC showed a strong induction of a protein recognized by anti-P450IA, and all those treated with PB showed strong induction of a band recognized by anti-P450IIB.

  7. Effects of acetone and fasting on cytochrome P-450 and xenobiotic metabolism in intact and hypophysectomized rats

    SciTech Connect

    Williams, M.T.; Simonet, L.

    1987-05-01

    Hypophysectomized and intact male and female rats were fasted for 24-48 hrs or given acetone (5ml/kg body weight) in order to evaluate the effects of these treatments on hepatic microsomal cytochrome P-450 and xenobiotic metabolism. Fasting and acetone treatment resulted in a significant increase (p < 0.05) in total P-450 in intact female rats. However, there was no significant changes in P-450 in microsomes from fasted or acetone-treated hypophysectomized rats. Fasting and acetone treatment resulted in significant increases in nitrosamine metabolism in intact rats. This effect was markedly reduced in the hypophysectomized rat. When intact male rats were fasted or treated with acetone there was a significant increase in P-450 in microsomes from acetone treated rats. Aryl hydrocarbon hydroxylase activity was significantly increased in both intact and hypophysectomized male and female rats treated with acetone. These results suggest that the pituitary gland or some product markedly influences acetone-stimulated nitrosamine metabolism.

  8. The Effects of Milk Thistle (Silybum marianum) on Human Cytochrome P450 Activity

    PubMed Central

    Kawaguchi-Suzuki, Marina; Frye, Reginald F.; Zhu, Hao-Jie; Brinda, Bryan J.; Chavin, Kenneth D.; Bernstein, Hilary J.

    2014-01-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. PMID:25028567

  9. Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita.

    PubMed Central

    Ropp, J D; Gunsalus, I C; Sligar, S G

    1993-01-01

    Cytochrome P-450lin catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-450lin gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-450lin on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content of linC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-450lin was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an O2-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-450lin and all other P-450 sequences has shown that P-450lin is the first member of the CYP111 P-450 gene family. PMID:8376348

  10. Coupled motions direct electrons along human microsomal P450 Chains.

    PubMed

    Pudney, Christopher R; Khara, Basile; Johannissen, Linus O; Scrutton, Nigel S

    2011-12-01

    Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex

  11. Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450.

    PubMed

    Chen, C; Gurka, D P

    1985-04-01

    9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.

  12. Cytochromes P450 for terpene functionalisation and metabolic engineering.

    PubMed

    Pateraki, Irini; Heskes, Allison Maree; Hamberger, Björn

    2015-01-01

    Plants have evolved the capacity to produce a striking array of specialised metabolites. Terpenoids are the oldest and most diverse class of such compounds and have attracted interest for industrial and pharmaceutical applications. The development of biotechnological alternatives for their production is the focus of intense research. Photosynthetic systems provide new strategies for autotrophic metabolic engineering. Focusing on cytochromes P450, involved in the functionalisation of the core terpene molecules, this review highlights the latest approaches in this field and looks towards recent discoveries that have the potential to shape the future of terpenoid bioengineering.

  13. The cytochrome P-450 active site. Regiospecificity of prosthetic heme alkylation by olefins and acetylenes.

    PubMed

    Kunze, K L; Mangold, B L; Wheeler, C; Beilan, H S; Ortiz de Montellano, P R

    1983-04-10

    Hepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats is inactivated during the metabolism of linear olefins (ethylene, propene, and octene) and acetylenes (acetylene, propyne, and octyne). As expected from previous work, the inactivation is due to N-alkylation of the prosthetic heme group by the substrate. The N-alkyl group in each adduct is formally obtained by addition of a porphyrin nitrogen to the terminal carbon and of an oxygen atom (as a hydroxyl function) to the internal carbon of the pi-bond. The oxygen is shown here by 18O studies to be catalytically introduced by the enzyme. The olefins exclusively alkylate the nitrogen of pyrrole ring D, but the acetylenes alkylate that of pyrrole ring A. Acetylene is an exception in that it reacts with more than one nitrogen. Circular dichroism studies of the ethylene adduct and of the ring D regioisomer of N-ethylprotoporphyrin IX obtained by alkylation of the prosthetic heme of hemoglobin have been used to determine which face of cytochrome P-450 heme is alkylated by the unsaturated substrates. These results implicate an active site that is sterically encumbered in the region over pyrrole ring B and has a lipophilic binding site that accommodates chains of at least six carbon atoms over pyrrole ring C.

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

    USGS Publications Warehouse

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

    1995-01-01

    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 (P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

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

    SciTech Connect

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

    1995-12-31

    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.

  16. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    PubMed Central

    Elenewski, Justin E.; Hackett, John C

    2015-01-01

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis. PMID:25681906

  17. Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450*

    PubMed Central

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

    2014-01-01

    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

  18. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    SciTech Connect

    Elenewski, Justin E.; Hackett, John C

    2015-02-14

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis.

  19. A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 aromatase).

    PubMed

    Koymans, L M; Moereels, H; Vanden Bossche, H

    1995-06-01

    In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the "I-helix model" by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (-)-R-vorozole, (-)-S-fadrozole, R-liarozole and (-)-R-aminoglutethimide. It is found that (+)-S-vorozole, (-)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu302 and Asp309) and C-terminus (Ser478 and His480). The weak activity of (-)-R-aminoglutethimide might be attributed to a lack of interaction with the C-terminus.

  20. Relationships among cytochromes P450 and dioxin equivalents in pipping heron embryos from Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillett, D.E.

    1993-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from undisturbed (Chincoteague National Wildlife Refuge, VA) and industrialized (Cat Island, Green Bay, WI; San Francisco Bay, CA) locations. Hepatic P450 associated monooxygenases (AHH, EROD, BROD, ECOD) and P450 proteins (CYP1A, CYP2B) were induced up to 85-fold, and were associated with burdens of total PCBs and 11 AHH-active PCB congeners. Dioxin equivalents (TCDD-EQs) of sample extracts, derived by bioassay (H4I1E rat hepatoma cell) and mathematically (product of PCB congener concentration and relative TCDD potency), revealed greatest TCDD-EQs in Cat Island samples. TCDD-EQs were associated with P450s, especially BROD, EROD and CYP1A (r2 = 0.35 to 0.66). TCDD-EQs derived by bioassay were highly correlated with TCDD-EQs derived mathematically (r2 = 0.58 to 0.67) . Multiple regressions were also performed to investigate relationships among P450s and PCB congeners. In summary, these data demonstrate that hepatic P450s of heron embryos are biomarkers of exposure to dioxin-like compounds and provide further evidence that this species has considerable value for assessing wetland and estuarine contamination.

  1. Utility of intersystem extrapolation factors in early reaction phenotyping and the quantitative extrapolation of human liver microsomal intrinsic clearance using recombinant cytochromes P450.

    PubMed

    Chen, Yuan; Liu, Liling; Nguyen, Khanh; Fretland, Adrian J

    2011-03-01

    Reaction phenotyping using recombinant human cytochromes P450 (P450) has great utility in early discovery. However, to fully realize the advantages of using recombinant expressed P450s, the extrapolation of data from recombinant systems to human liver microsomes (HLM) is required. In this study, intersystem extrapolation factors (ISEFs) were established for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 using 11 probe substrates, based on substrate depletion and/or metabolite formation kinetics. The ISEF values for CYP2C9, CYP2D6, and CYP3A4 determined using multiple substrates were similar across substrates. When enzyme kinetics of metabolite formation for CYP1A2, 2C9, 2D6, and 3A4 were used, the ISEFs determined were generally within 2-fold of that determined on the basis of substrate depletion. Validation of ISEFs was conducted using 10 marketed drugs by comparing the extrapolated data with published data. The major isoforms responsible for the metabolism were identified, and the contribution of the predominant P450s was similar to that of previously reported data. In addition, phenotyping data from internal compounds, extrapolated using the rhP450-ISEF method, were comparable to those obtained using an HLM-based inhibition assay approach. Moreover, the intrinsic clearance (CL(int)) calculated from extrapolated rhP450 data correlated well with measured HLM CL(int). The ISEF method established in our laboratory provides a convenient tool in early reaction phenotyping for situations in which the HLM-based inhibition approach is limited by low turnover and/or unavailable metabolite formation. Furthermore, this method allows for quantitative extrapolation of HLM intrinsic clearance from rhP450 phenotyping data simultaneously to obtaining the participating metabolizing enzymes.

  2. Combining in silico protein stability calculations with structure-function relationships to explore the effect of polymorphic variation on cytochrome P450 drug metabolism.

    PubMed

    Arendse, Lauren; Blundell, Tom L; Blackburn, Jonathan

    2013-09-01

    We carried out an in silico structural analysis of 348 non-synonymous single nucleotide polymorphisms, found across nine of the major human drug metabolising cytochrome P450 isoforms, to determine the effects of mutations on enzyme structure and function. Previous functional studies in our group have delineated regions of the cytochrome P450 structure important for substrate recognition, substrate and product access and egress from the active site and interaction with the cytochrome P450 reductase. Here we combine the information from those studies with new in silico calculations on the effect of mutations on protein stability and we compare our results to experimental data in order to establish the likely causes of altered drug metabolism observed for cytochrome P450 variants in functional assays to date, in the process creating a cytochrome P450 polymorphic variant map. Using the computational tool Site Directed Mutator we predicted destabilising mutations that result in altered enzyme function in vitro with a specificity of 83%. We found that 75% of all cytochrome P450 mutations that show altered activity in vitro are either predicted to be destabilising to protein structure or are found within regions predicted to be important for catalytic activity. Furthermore, we found that 70% of the mutations that showed similar activity to the wild-type enzyme in in vitro studies lie outside of functional regions important for catalytic activity and are predicted to have no effect on protein stability. Our resultant cytochrome P450 polymorphic variant map should therefore find utility in predicting the likely functional effect of uncharacterised variants on drug metabolism.

  3. The Interaction of Microsomal Cytochrome P450 2B4 with its Redox Partners, Cytochrome P450 Reductase and Cytochrome b5

    PubMed Central

    Im, Sang-Choul; Waskell, Lucy

    2010-01-01

    1 Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, catalysis (product formation) is ∼ 10 to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼ 15% the coupling of NADPH consumption to product formation. Cytochrome b5 has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochome b5 to cytochrome P450 reductase, cytochrome b5 inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase. PMID:21055385

  4. Cytochrome P450 Enzyme Metabolites in Lead Discovery and Development

    PubMed Central

    Kandel, Sylvie E.; Wienkers, Larry C.; Lampe, Jed N.

    2015-01-01

    The cytochrome P450 (CYP) enzymes are a versatile superfamily of heme-containing monooxygenases, perhaps best known for their role in the oxidation of xenobiotic compounds. However, due to their unique oxidative chemistry, CYPs are also important in natural product drug discovery and in the generation of active metabolites with unique therapeutic properties. New tools for the analysis and production of CYP metabolites, including microscale analytical technologies and combinatorial biosynthesis, are providing medicinal chemists with the opportunity to use CYPs as a novel platform for lead discovery and development. In this review, we will highlight some of the recent examples of drug leads identified from CYP metabolites and the exciting possibilities of using CYPs as catalysts for future drug discovery. PMID:25797999

  5. Enzyme kinetics of oxidative metabolism: cytochromes P450.

    PubMed

    Korzekwa, Ken

    2014-01-01

    The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

  6. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    PubMed

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping.

  7. Identification of small-molecule scaffolds for p450 inhibitors.

    PubMed

    von Kries, Jens P; Warrier, Thulasi; Podust, Larissa M

    2010-02-01

    Mycobacterium tuberculosis cytochrome P450 enzymes (CYP) attract ongoing interest for their pharmacological development potential, driving direct screening efforts against potential CYP targets with the ultimate goal of developing potent CYP-specific inhibitors and/or molecular probes to address M. tuberculosis biology. The property of CYP enzymes to shift the ferric heme Fe Soret band in response to ligand binding provides the basis for an experimental platform for high-throughput screening (HTS) of compound libraries to select chemotypes with high binding affinities to the target. Promising compounds can be evaluated in in vitro assays or in vivo disease models and further characterized by x-ray crystallography, leading to optimization strategies to assist drug design. Protocols are provided for compound library screening, analysis of inhibitory potential, and co-crystallization with the target CYP, as well as expression and purification of soluble CYP enzymes.

  8. Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

    PubMed

    Cheng, Xingguo; Zhang, Youcai; Klaassen, Curtis D

    2014-10-01

    NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.

  9. Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

    PubMed Central

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

    2013-01-01

    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

  10. Relevance of cytochrome P450s in plants: also one of Ron Estabrook's research interests.

    PubMed

    Shet, Manjunath S

    2007-01-01

    I worked with Dr. Ronald Estabrook for nearly 10 years at The University of Texas Southwestern Medical Center in Dallas, Texas. In Ron's lab, when I joined I was initially involved in the isolation, purification, and characterization of cytochrome P450s and NADPH-P450(c) reductase(s) from plants, which was his new exploratory project at the time. We developed methods for the isolation, solubilization, and purification of P450s and NADPH-P450(c) reductase from plant tissue microsomes. We carried out number of in vitro experiments to study the involvement P450s and NADPH-P450(c) reductase in the biosynthesis of number of phytoalexins. We successfully isolated, purified, and cloned NADPH-P450(c) reductase from etiolated mung bean (Vigna radiate) seedlings. In addition, a series of studies were undertaken to show that purified mung bean NADPH-P450(c) reductase was able to catalyze P450-supported reactions for mammalian and bacterial P450s. My stay in Ron's lab was very educational and productive. He provided the necessary support and led the way through the maze in different research projects in the lab, which allowed me to understand the roles of P450s in humans, animals, plants, and microorganisms. He liked to teach and discover new things everyday in the lab. He is a great scientist, as well as loving and caring mentor.

  11. Monooxygenation of small hydrocarbons catalyzed by bacterial cytochrome p450s.

    PubMed

    Shoji, Osami; Watanabe, Yoshihito

    2015-01-01

    Cytochrome P450s (P450s) catalyze the NAD(P)H/O2-dependent monooxygenation of less reactive organic molecules under mild conditions. The catalytic activity of bacterial P450s is very high compared with P450s isolated from animals and plants, and the substrate specificity of bacterial P450s is also very high. Accordingly, their catalytic activities toward nonnative substrates are generally low especially toward small hydrocarbons. However, mutagenesis approaches have been very successful for engineering bacterial P450s for the hydroxylation of small hydrocarbons. On the other hand, "decoy" molecules, whose structures are very similar to natural substrates, can be used to trick the substrate recognition of bacterial P450s, allowing the P450s to catalyze oxidation reactions of nonnative substrates without any substitution of amino acid residues in the presence of decoy molecules. Thus, the hydroxylation of small hydrocarbons such as ethane, propane, butane and benzene can be catalyzed by P450BM3, a long-alkyl-chain hydroxylase, using substrate misrecognition of P450s induced by decoy molecules. Furthermore, a number of H2O2-dependent bacterial P450s can catalyze the peroxygenation of a variety of nonnative substrates through a simple substrate-misrecognition trick, in which catalytic activities and enantioselectivity are dependent on the structure of decoy molecules.

  12. Expression of cytochrome P450 genes in CD34+ hematopoietic stem and progenitor cells.

    PubMed

    Soucek, Pavel; Anzenbacher, Pavel; Skoumalová, Ivana; Dvorák, Michal

    2005-10-01

    Expression of major cytochrome P450 forms (P450) was followed in preparation of purified hematopoietic CD34+ stem and progenitor cells. Levels of transcripts as well as mature proteins were traced by quantitative real-time polymerase chain reaction and by Northern and Western blotting. P450 1B1 and P450 2E1 proteins and respective mRNAs were found in all cases. On the other hand, no expression of P450 3A4, P450 3A7, and P450 2C9 was found. The results showed that expression of various P450 enzymes starts at different stages of cell differentiation. Both P450 forms found are known to be connected with cancer cells and with activation of procarcinogens (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and solvents). Hence, cells at the early stage of differentiation already may be influenced by interaction with xenobiotics. This fact should also be taken into consideration when hematopoietic cell transplant therapy is applied.

  13. tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

    PubMed Central

    Zhang, Haoming; Lin, Hsia-lien; Walker, Vyvyca J.; Hamdane, Djemel

    2009-01-01

    We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4. PMID:19720728

  14. In Silico Prediction of Cytochrome P450-Drug Interaction: QSARs for CYP3A4 and CYP2C9

    PubMed Central

    Nembri, Serena; Grisoni, Francesca; Consonni, Viviana; Todeschini, Roberto

    2016-01-01

    Cytochromes P450 (CYP) are the main actors in the oxidation of xenobiotics and play a crucial role in drug safety, persistence, bioactivation, and drug-drug/food-drug interaction. This work aims to develop Quantitative Structure-Activity Relationship (QSAR) models to predict the drug interaction with two of the most important CYP isoforms, namely 2C9 and 3A4. The presented models are calibrated on 9122 drug-like compounds, using three different modelling approaches and two types of molecular description (classical molecular descriptors and binary fingerprints). For each isoform, three classification models are presented, based on a different approach and with different advantages: (1) a very simple and interpretable classification tree; (2) a local (k-Nearest Neighbor) model based classical descriptors and; (3) a model based on a recently proposed local classifier (N-Nearest Neighbor) on binary fingerprints. The salient features of the work are (1) the thorough model validation and the applicability domain assessment; (2) the descriptor interpretation, which highlighted the crucial aspects of P450-drug interaction; and (3) the consensus aggregation of models, which largely increased the prediction accuracy. PMID:27294921

  15. Effects of zedoary turmeric oil on P450 activities in rats with liver cirrhosis induced by thioacetamide.

    PubMed

    Cheng, Jing-Jing; Yang, Nai-Bin; Wu, Liang; Lin, Jia-Le; Dai, Ge-Xin; Zhu, Jia-Yin

    2014-01-01

    The aim of this study was to elucidate the effects of zedoary turmeric oil (ZTO) on P450 activities (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) in rats with liver cirrhosis induced by thioacetamide (TAA). For the induction of liver cirrhosis, rats were given TAA in their drinking water at a concentration of 0.03% for consecutive 5 weeks and then 0.04% for the next consecutive 5 weeks throughout the establishment of cirrhosis. Then the cirrhotic rats were ip given saline, ZTO 100, 200 and 400 mg/kg, respectively, once daily for 2 weeks. When cirrhosis model was established at week 10, all rats of five groups were administered intragastrically with 15 mg/kg phenacetin, 0.6 mg/kg tolbutamide, 15 mg/kg omeprazole, 15 mg/kg bupropion, 15 mg/kg metoprolol, and 10 mg/kg midazolam. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The degree of liver cirrhosis was assessed by HE staining. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from the model group increased by approximately 4-fold, and a decreased level of albumin (Alb) was also observed, as compared to the control group (P < 0.05). However, ZTO was found to reverse those changes of serum levels observed in the model group, and the 200 mg/kg ZTO treatment group showed the most obvious reverse tendency with significantly decreased ALT, AST and increased Alb levels (P < 0.05). The results indicated that ZTO with the dose of 100 mg/kg could inhibit the activities of CYP450 isoforms CYP2C9 and CYP2D6 in vivo in cirrhotic rats induced by TAA, while ZTO with the dose of 400 mg/kg could induce the activity of CYP2C19 in vivo in cirrhotic rats induced by TAA. However, ZTO showed no influence on cirrhotic rat hepatic CYP1A2, CYP2B6 and CYP3A4 activity in vivo. This has certain guiding significance to clinical treatment.

  16. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1)

    PubMed Central

    Yoshimoto, Francis K.; Auchus, Richard J.

    2014-01-01

    The steroid hydroxylation and carbon-carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F-G loop and the B′-helix to explain the 16α-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized. PMID:25482340

  17. An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

    PubMed

    Agnew, Christopher R J; Warrilow, Andrew G S; Burton, Nicholas M; Lamb, David C; Kelly, Steven L; Brady, R Leo

    2012-01-01

    CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 μM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 μM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

  18. Human plasma concentrations of cytochrome P450 probes extrapolated from pharmacokinetics in cynomolgus monkeys using physiologically based pharmacokinetic modeling.

    PubMed

    Shida, Satomi; Utoh, Masahiro; Murayama, Norie; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-01-01

    1. Cynomolgus monkeys are widely used in preclinical studies as non-human primate species. Pharmacokinetics of human cytochrome P450 probes determined in cynomolgus monkeys after single oral or intravenous administrations were extrapolated to give human plasma concentrations. 2. Plasma concentrations of slowly eliminated caffeine and R-/S-warfarin and rapidly eliminated omeprazole and midazolam previously observed in cynomolgus monkeys were scaled to human oral biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Results of the simplified human PBPK models were consistent with reported experimental PK data in humans or with values simulated by a fully constructed population-based simulator (Simcyp). 3. Oral administrations of metoprolol and dextromethorphan (human P450 2D probes) in monkeys reportedly yielded plasma concentrations similar to their quantitative detection limits. Consequently, ratios of in vitro hepatic intrinsic clearances of metoprolol and dextromethorphan determined in monkeys and humans were used with simplified PBPK models to extrapolate intravenous PK in monkeys to oral PK in humans. 4. These results suggest that cynomolgus monkeys, despite their rapid clearance of some human P450 substrates, could be a suitable model for humans, especially when used in conjunction with simple PBPK models.

  19. Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids.

    PubMed

    Furuya, Toshiki; Shibata, Daisuke; Kino, Kuniki

    2009-11-01

    Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE_2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids.

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

    PubMed Central

    Ning, Daliang; Wang, Hui

    2012-01-01

    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

  1. Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.

    PubMed Central

    Bozak, K R; Yu, H; Sirevåg, R; Christoffersen, R E

    1990-01-01

    The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family members. Further evidence that this polypeptide represents a cytochrome P-450 oxidase comes from the recent isolation and characterization of a cytochrome P-450 from ripe avocado mesocarp [O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141-1149]. The N terminus of the predicted polypeptide in the cDNAs is identical to the N terminus of the purified avocado P-450. Gel blot analysis of RNA from fruit at various stages of ripening showed the accumulation of an 1800-nucleotide P-450 mRNA that hybridized to the P-450 cDNA. The P-450 protein predicted by the avocado cDNA sequence shares less than 40% positional identity with any known P-450 gene family. We propose therefore that it be placed in a separate family, P450LXXI, and that the corresponding gene from avocado be named cyp71A1. Images PMID:1692626

  2. Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

    PubMed Central

    Stark, Katarina; Dostalek, Miroslav; Guengerich, F. Peter

    2016-01-01

    Summary Cytochrome P450 (P450) 4X1 is one of the so-called “orphan” P450s without assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5α cells (<100 nmol P450/L). The highest level of expression (300-450 nmol P450/L culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b5. Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala, and basal ganglia), in addition to expression in human heart, liver, prostate, and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain. PMID:18549450

  3. Identification of N-(hydroxymethyl) norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.

    PubMed

    Brown, Kathryn M; von Weymarn, Linda B; Murphy, Sharon E

    2005-12-01

    Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primary pathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and its glucuronide conjugate account for up to 50% of the nicotine metabolites excreted by smokers. Minor metabolites of cotinine excreted by smokers include norcotinine and cotinine N-oxide, each of which account for <5% of the nicotine dose. It has been reported that P450 2A6 is the catalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human metabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and its stability under the conditions of the enzyme reactions was confirmed. The products of P450 2A6-catalyzed [5-3H]cotinine metabolism were quantified by radioflow HPLC. The identification of N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on three unique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14 and 8% of the total cotinine metabolites, respectively. N-(Hydroxymethyl)norcotinine was a product of cotinine metabolism by the extrahepatic P450, 2A13, but it was a minor one. The major product of P450 2A13-catalyzed cotinine metabolism was 5'-hydroxycotinine, which was formed at twice the rate of trans-3'-hydroxycotinine. The identification of all cotinine metabolites formed by both enzymes was confirmed by LC/MS/MS analysis. Kinetic parameters for cotinine metabolism were determined for P450 2A6 and P450 2A13. This work has confirmed that the major metabolite of cotinine in smokers, trans-3'-hydroxycotinine, is only a minor metabolite of P450 2A6-catalyzed cotinine metabolism.

  4. High-throughput mass spectrometric cytochrome P450 inhibition screening.

    PubMed

    Lim, Kheng B; Ozbal, Can C; Kassel, Daniel B

    2013-01-01

    We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.

  5. First principles calculation of the activity of cytochrome P450

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

    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.

  6. Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer

    PubMed Central

    Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.

    2015-01-01

    Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909

  7. Oxidation of Acenaphthene and Acenaphthylene by Human Cytochrome P450 Enzymes

    PubMed Central

    Shimada, Tsutomu; Takenaka, Shigeo; Murayama, Norie; Yamazaki, Hiroshi; Kim, Joo-Hwan; Kim, Donghak; Yoshimoto, Francis K.; Guengerich, F. Peter; Komori, Masayuki

    2016-01-01

    Acenaphthene and acenaphthylene, two known environmental polycyclic aromatic hydrocarbon (PAH) pollutants, were incubated at 50 µM concentrations in a standard reaction mixture with human P450s 2A6, 2A13, 1B1, 1A2, 2C9, and 3A4 and the oxidation products were determined using HPLC and LC-MS. HPLC analysis showed that P450 2A6 converted acenaphthene and acenaphthylene to several mono- and di-oxygenated products. LC-MS analysis of acenaphthene oxidation by P450s indicated the formation of 1-acenaphthenol as a major product, with turnover rates of 6.7, 4.5, and 3.6 nmol product formed/min/nmol P450 for P450 2A6, 2A13, and 1B1, respectively. Acenaphthylene oxidation by P450 2A6 showed the formation of 1,2-epoxyacenaphthene as a major product (4.4 nmol epoxide formed/min/nmol P450) and also several mono- and di-oxygenated products. P450 2A13, 1B1, 1A2, 2C9, and 3A4 formed 1,2-epoxyacenaphthene at rates of 0.18, 5.3 2.4, 0.16, and 3.8 nmol/min nmol P450, respectively. 1-Acenaphthenol, which induced Type I binding spectra with P450 2A13, was further oxidized by P450 2A13 but not P450 2A6. 1,2-Epoxyacenaphthene induced Type I binding spectra with P450 2A6 and 2A13 (Ks 1.8 and 0.16 µM, respectively) and was also oxidized to several oxidation products by these P450s. Molecular docking analysis suggested different orientations of acenaphthene, acenaphthylene, 1-acenaphthenol, and 1,2-epoxyacenaphthene in their interactions with P450 2A6 and 2A13. Neither these four PAHs induced umu gene expression in a Salmonella typhimurium NM tester strain. These results suggest, for the first time, that acenaphthene and acenaphthylene are oxidized by human P450s 2A6 and 2A13 and other P450s to form several mono- and di-oxygenated products. The results are of use in considering the biological and toxicological significance of these environmental PAHs in humans. PMID:25642975

  8. Cytochrome P450IA1 induction and localization in endothelium of vertebrate (teleost) heart.

    PubMed

    Stegeman, J J; Miller, M R; Hinton, D E

    1989-11-01

    Previous studies have shown that high levels of cytochrome P450 can occur in cardiac microsomes of vertebrates [Mol. Pharmacol. 21:517-526, (1982)]. Here we identify the dominant cardiac P450 in the marine fish scup as P450E, a teleost representative of P450IA1, and we describe its restricted cellular localization in the heart. Treatment of scup with beta-naphthoflavone produced an unusually strong (10-fold) induction of spectrally measured P450 in cardiac microsomes, with specific content reaching levels (0.5 nmol/mg) similar to those induced in scup liver. Microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities, catalytic functions of scup P450E, were induced in parallel with P450 content. Similar induction was seen in both atrium and ventricle. Immunoblot analysis with monoclonal antibody 1-12-3, specific to scup P450E and other vertebrate P450IA1 proteins, showed that this hydrocarbon-inducible P450 is the dominant and possibly sole P450 form in heart microsomes of experimentally induced animals. Immunohistochemical analysis of scup heart sections (2-4-microns) with monoclonal antibody 1-12-3 revealed that P450E was detectable only in endothelial cells of the endocardium and of the coronary vasculature. A similar endothelial cell localization of the monoclonal antibody 1-12-3 epitope was observed in heart of rainbow trout, induced with beta-naphthoflavone, indicating a general nature for the endothelial localization of induced cardiac P450. Morphometric analysis showed that endothelium could constitute 8-9% of the volume of teleost heart, from which we calculate that P450IA1 could account for as much as 25% of the endothelial cell microsomal protein. Heart microsomes of untreated animals from contaminated environments also contained high levels of P450E, indicating that induction like that caused by beta-naphthoflavone could occur with chemicals in the environment. Strongly induced P450E (P450IA1) in endothelium could play a critical

  9. Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology.

    PubMed

    Gillam, E M; Guengerich, F P

    2001-12-01

    The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

  10. Ruthenium-containing P450 inhibitors for dual enzyme inhibition and DNA damage.

    PubMed

    Zamora, Ana; Denning, Catherine A; Heidary, David K; Wachter, Erin; Nease, Leona A; Ruiz, José; Glazer, Edith C

    2017-02-14

    Cytochrome P450s are key players in drug metabolism, and overexpression in tumors is associated with significant resistance to many medicinal agents. Consequently, inhibition of P450s could serve as a strategy to restore drug efficacy. However, the widespread expression of P450s throughout the human body and the critical roles they play in various biosynthetic pathways motivates the development of P450 inhibitors capable of controlled local administration. Ruthenium complexes containing P450 inhibitors as ligands were synthesized in order to develop pro-drugs that can be triggered to release the inhibitors in a spatially and temporally controlled fashion. Upon light activation the compounds release ligands that directly bind and inhibit P450 enzymes, while the ruthenium center is able to directly damage DNA.

  11. Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis.

    PubMed

    Polic, Vanja; Auclair, Karine

    2014-10-15

    P450 enzymes (P450s) are well known for their ability to oxidize unactivated CH bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications.

  12. Targeting Steroidogenic Cytochromes P450 (CYPs) with 6-Substituted 1-Imidazolylmethylxanthones.

    PubMed

    Gobbi, Silvia; Hu, Qingzhong; Zimmer, Christina; Belluti, Federica; Rampa, Angela; Hartmann, Rolf W; Bisi, Alessandra

    2016-08-19

    Abnormally high corticosteroid levels are responsible for the onset of serious hormone-related diseases, and the inhibition of their biosynthesis by targeting cytochrome P450 (CYP) isoforms CYP11B1 and CYP11B2 has emerged as a promising strategy to restore healthy physiological levels of corticosteroids. With the aim of exploiting the xanthone scaffold as a privileged structure in medicinal chemistry and to further explore the chemical space of inhibitors of these CYPs, a small library of imidazolylmethylxanthones was designed based on the results of a previously described compound series. Assuming the capacity for an additional interaction with these enzymes, a properly selected substituent was introduced at position 6 of the xanthone core, maintaining the key imidazolylmethyl moiety at position 1. The 6-fluoro and 6-nitro derivatives [1-(1H-imidazol-1-yl)methyl-6-fluoro-9H-xanthen-9-one (1 a) and 1-(1H-imidazol-1-yl)methyl-6-nitro-9H-xanthen-9-one (1 d), respectively] proved to be active in the low nanomolar range, showing selectivity toward the related steroidogenic enzymes CYP19 and CYP17, even if the problem of selectivity between the two CYP11B isoforms remains unsolved. On the other hand, the 6-chloro derivative 1-(1H-imidazol-1-yl)methyl-6-chloro-9H-xanthen-9-one (1 b) was found to be a fairly potent and somewhat selective CYP19 inhibitor, confirming the versatility of the scaffold.

  13. Pyruvate Kinase Isoform Switching and Hepatic Metabolic Reprogramming by the Environmental Contaminant 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

    PubMed Central

    Nault, Rance; Fader, Kelly A.; Kirby, Mathew P.; Ahmed, Shaimaa; Matthews, Jason; Jones, A. Daniel; Lunt, Sophia Y.; Zacharewski, Timothy R.

    2016-01-01

    The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits dose-dependent hepatotoxicity that includes fat accumulation, inflammation, and fibrosis that may progress to hepatocellular carcinoma. To further investigate these effects, RNA-Seq data were integrated with computationally identified putative dioxin response elements, and complementary targeted metabolomic and aryl hydrocarbon receptor (AhR) ChIP-Seq data from female C57BL/6 mice gavaged with TCDD every 4 days for 28 days. Data integration using CytoKEGG with manual curation identified dose-dependent alterations in central carbon and amino acid metabolism. More specifically, TCDD increased pyruvate kinase isoform M2 (PKM2) gene and protein expression. PKM2 has lower catalytic activity resulting in decreased glycolytic flux and the accumulation of upstream intermediates that were redirected to the pentose phosphate pathway and serine/folate biosynthesis, 2 important NADPH producing pathways stemming from glycolysis. In addition, the GAC:KGA glutaminase (GLS1) protein isoform ratio was increased, consistent with increases in glutaminolysis which serves an anaplerotic role for the TCA cycle and compensates for the reduced glycolytic flux. Collectively, gene expression, protein, and metabolite changes were indicative of increases in NADPH production in support of cytochrome P450 activity and ROS defenses. This AhR-mediated metabolic reprogramming is similar to the Warburg effect and represents a novel advantageous defense mechanism to increase anti-oxidant capacity in normal differentiated hepatocytes. PMID:26582802

  14. Pyruvate Kinase Isoform Switching and Hepatic Metabolic Reprogramming by the Environmental Contaminant 2,3,7,8-Tetrachlorodibenzo-p-Dioxin.

    PubMed

    Nault, Rance; Fader, Kelly A; Kirby, Mathew P; Ahmed, Shaimaa; Matthews, Jason; Jones, A Daniel; Lunt, Sophia Y; Zacharewski, Timothy R

    2016-02-01

    The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits dose-dependent hepatotoxicity that includes fat accumulation, inflammation, and fibrosis that may progress to hepatocellular carcinoma. To further investigate these effects, RNA-Seq data were integrated with computationally identified putative dioxin response elements, and complementary targeted metabolomic and aryl hydrocarbon receptor (AhR) ChIP-Seq data from female C57BL/6 mice gavaged with TCDD every 4 days for 28 days. Data integration using CytoKEGG with manual curation identified dose-dependent alterations in central carbon and amino acid metabolism. More specifically, TCDD increased pyruvate kinase isoform M2 (PKM2) gene and protein expression. PKM2 has lower catalytic activity resulting in decreased glycolytic flux and the accumulation of upstream intermediates that were redirected to the pentose phosphate pathway and serine/folate biosynthesis, 2 important NADPH producing pathways stemming from glycolysis. In addition, the GAC:KGA glutaminase (GLS1) protein isoform ratio was increased, consistent with increases in glutaminolysis which serves an anaplerotic role for the TCA cycle and compensates for the reduced glycolytic flux. Collectively, gene expression, protein, and metabolite changes were indicative of increases in NADPH production in support of cytochrome P450 activity and ROS defenses. This AhR-mediated metabolic reprogramming is similar to the Warburg effect and represents a novel advantageous defense mechanism to increase anti-oxidant capacity in normal differentiated hepatocytes.

  15. Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450

    PubMed Central

    2012-01-01

    A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type. PMID:22697884

  16. Resveratrol stimulates cortisol biosynthesis by activating SIRT-dependent deacetylation of P450scc.

    PubMed

    Li, Donghui; Dammer, Eric B; Sewer, Marion B

    2012-07-01

    In the human adrenal cortex, cortisol is synthesized from cholesterol by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings that activation of ACTH signaling changes the ratio of nicotinamide adenine dinucleotide (NAD) phosphate to reduced NAD phosphate in adrenocortical cells, we hypothesized that pyridine nucleotide metabolism may regulate the activity of the mitochondrial NAD(+)-dependent sirtuin (SIRT) deacetylases. We show that resveratrol increases the protein expression and half-life of P450 side chain cleavage enzyme (P450scc). The effects of resveratrol on P450scc protein levels and acetylation status are dependent on SIRT3 and SIRT5 expression. Stable overexpression of SIRT3 abrogates the cellular content of acetylated P450scc, concomitant with an increase in P450scc protein expression and cortisol secretion. Mutation of K148 and K149 to alanine stabilizes the expression of P450scc and results in a 1.5-fold increase in pregnenolone biosynthesis. Finally, resveratrol also increases the protein expression of P450 11β, another mitochondrial enzyme required for cortisol biosynthesis. Collectively, this study identifies a role for NAD(+)-dependent SIRT deacetylase activity in regulating the expression of mitochondrial steroidogenic P450.

  17. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

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

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached 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.

  18. Pharmacophore modeling and in silico screening for new P450 19 (aromatase) inhibitors.

    PubMed

    Schuster, Daniela; Laggner, Christian; Steindl, Theodora M; Palusczak, Anja; Hartmann, Rolf W; Langer, Thierry

    2006-01-01

    Cytochrome P450 19 (P450 19, aromatase) constitutes a successful target for the treatment of breast cancer. This study analyzes chemical features common to P450 19 inhibitors to develop ligand-based, selective pharmacophore models for this enzyme. The HipHop and HypoRefine algorithms implemented in the Catalyst software package were employed to create both common feature and quantitative models. The common feature model for P450 19 includes two ring aromatic features in its core and two hydrogen bond acceptors at the ends. The models were used as database search queries to identify active compounds from the NCI database.

  19. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    SciTech Connect

    Liu, Senyan; Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin; Mei, Changlin; Gu, Jun

    2013-10-01

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity.

  20. Cytochrome P450-dependent metabolism of caffeine in Drosophila melanogaster.

    PubMed

    Coelho, Alexandra; Fraichard, Stephane; Le Goff, Gaëlle; Faure, Philippe; Artur, Yves; Ferveur, Jean-François; Heydel, Jean-Marie

    2015-01-01

    Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone--an inhibitor of CYP enzymes--showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects.

  1. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  2. CYP94A1, a plant cytochrome P450-catalyzing fatty acid omega-hydroxylase, is selectively induced by chemical stress in Vicia sativa seedlings.

    PubMed

    Benveniste, Irène; Bronner, Roberte; Wang, Yong; Compagnon, Vincent; Michler, Pierre; Schreiber, Lukas; Salaün, Jean-Pierre; Durst, Francis; Pinot, Franck

    2005-08-01

    CYP94A1 is a cytochrome P450 (P450) catalyzing fatty acid (FA) omega-hydroxylation in Vicia sativa seedlings. To study the physiological role of this FA monooxygenase, we report here on its regulation at the transcriptional level (Northern blot). Transcripts of CYP94A1, as those of two other P450-dependent FA hydroxylases (CYP94A2 and CYP94A3) from V. sativa, are barely detectable during the early development of the seedlings. CYP94A1 transcripts, in contrast to those of the two other isoforms, are rapidly (less than 20 min) and strongly (more than 100 times) enhanced after treatment by clofibrate, an hypolipidemic drug in animals and an antiauxin (p-chlorophenoxyisobutyric acid) in plants, by auxins (2,4-dichlorophenoxyacetic acid and indole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid. All these compounds activate CYP94A1 transcription only at high concentrations (50-500 microM range). In parallel, these high levels of clofibrate and auxins modify seedling growth and development. Therefore, the expression of CYP94A1 under these conditions and the concomitant morphological and cytological modifications would suggest the implication of this P450 in a process of plant defense against chemical injury.

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

    PubMed Central

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

    2016-01-01

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

  4. Cytochrome P450 enzyme mediated herbal drug interactions (Part 2)

    PubMed Central

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the

  5. Cytochrome P450 enzyme mediated herbal drug interactions (Part 1)

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

    It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John’s wort, may have critical clinical consequences. St. John’s wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good

  6. Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes

    PubMed Central

    Baj-Rossi, Camilla; De Micheli, Giovanni; Carrara, Sandro

    2012-01-01

    We report on the electrochemical detection of anti-cancer drugs in human serum with sensitivity values in the range of 8–925 nA/μM. Multi-walled carbon nanotubes were functionalized with three different cytochrome P450 isoforms (CYP1A2, CYP2B6, and CYP3A4). A model used to effectively describe the cytochrome P450 deposition onto carbon nanotubes was confirmed by Monte Carlo simulations. Voltammetric measurements were performed in phosphate buffer saline (PBS) as well as in human serum, giving well-defined current responses upon addition of increasing concentrations of anti-cancer drugs. The results assert the capability to measure concentration of drugs in the pharmacological ranges in human serum. Another important result is the possibility to detect pairs of drugs present in the same sample, which is highly required in case of therapies with high side-effects risk and in anti-cancer pharmacological treatments based on mixtures of different drugs. Our technology holds potentials for inexpensive multi-panel drug-monitoring in personalized therapy. PMID:22778656

  7. Forster Distances of Ligand-Heme Pairs in Cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Fern, Joel; Guengerich, F. Peter; Marsch, Glenn A.

    2003-04-01

    Cytochrome P450 3A4 is a protein in the human intestine and liver which oxidizes over half of drugs in use today. Cytochrome P450 3A4 has proven resistant to structure determination by NMR or x-ray crystallography. Fluorescence Resonance Energy Transfer (FRET) studies of P450 3A4 can be used to compute distances between fluorophores in the protein, providing information on the structure of the protein. For a ligand to be suitably used as a probe its fluorescence must not be completely quenched by the heme cofactor in P450 3A4. By using quantum yields, fluorescence, and the absorption spectra of six P450 ligands, the following Forster distances between each ligand and the P450 heme moiety were obtained: pyrene 4.6 nm, aflatoxin B2 5.7 nm, alpha-naphthoflavone 3.7 nm, indinavir 2.6 nm, quinidine 3.5 nm, and terfenadine 2.8 nm. Having these distances should yield a better low-resolution cytochrome P450 3A4 structure. Using the Forster distances, FRET experiments on inter-ligand placement in P450 3A4 will be undertaken soon.

  8. Cytochrome P450 of wood-rotting basidiomycetes and biotechnological applications.

    PubMed

    Ichinose, Hirofumi

    2013-01-01

    Wood-rotting basidiomycetes possess superior metabolic functions to degrade woody biomass, and these activities are indispensable for the carbon cycle of the biosphere. As well as basic studies of the biochemistry of basidiomycetes, many researchers have been focusing on utilizing basidiomycetes and/or their enzymes in the biotechnology sector; therefore, the unique activities of their extracellular and intracellular enzymes have been widely demonstrated. A rich history of applied study has established that basidiomycetes are capable of metabolizing a series of endogeneous and exogeneous compounds using cytochrome P450s (P450s). Recently, whole genome sequence analyses have revealed large-scale divergences in basidiomycetous P450s. The tremendous variation in P450s implies that basidiomycetes have vigorously diversified monooxygenase functions to acquire metabolic adaptations such as lignin degradation, secondary metabolite production, and xenobiotics detoxification. However, fungal P450s discovered from genome projects are often categorized into novel families and subfamilies, making it difficult to predict catalytic functions by sequence comparison. Experimental screening therefore remains essential to elucidate the catalytic potential of individual P450s, even in this postgenomic era. This paper archives the known metabolic capabilities of basidiomycetes, focusing on their P450s, outlines the molecular diversity of basidiomycetous P450s, and introduces new functions revealed by functionomic studies using a recently developed, rapid, functional screening system.

  9. 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 ...

  10. Transcriptional Regulation of Grape Cytochrome P450 Gene Expression in Response to Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  11. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

    PubMed Central

    Chun, Young-Jin; Kim, Donghak

    2016-01-01

    Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

  12. Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis

    PubMed Central

    2016-01-01

    Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products. PMID:27119279

  13. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  14. The active site of cytochrome P-450 nifedipine oxidase: a model-building study.

    PubMed

    Ferenczy, G G; Morris, G M

    1989-12-01

    A model of the active site of cytochrome P-450 nifedipine oxidase is built on the basis of sequence homology with cytochrome P-450CAM. Substrates are docked into the binding pocket, and molecular mechanical energy minimization is performed to analyze the forces between the substrates and the enzyme.

  15. cDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

    PubMed Central

    Khani, S C; Zaphiropoulos, P G; Fujita, V S; Porter, T D; Koop, D R; Coon, M J

    1987-01-01

    Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily. Images PMID:3027695

  16. Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity.

    PubMed

    Hanna, I H; Krauser, J A; Cai, H; Kim, M S; Guengerich, F P

    2001-10-26

    Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.

  17. Evaluation of cytochrome P450{sub BS{beta}} reactivity against polycyclic aromatic hydrocarbons and drugs

    SciTech Connect

    Torres, Eduardo; Hayen, Heiko; Niemeyer, Christof M.; E-mail: christof.niemeyer@uni-dortmund.de

    2007-03-30

    The oxidation of 10 polycyclic aromatic hydrocarbons (PAH) by cytochrome P450{sub BS{beta}} using three different electron acceptors is reported. Three PAH were found to be substrates for the oxidation by P450{sub BS{beta}}, namely anthracene, 9-methyl-anthracene and azulene. The respective oxidation products were identified by reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry. In addition, 10 drug-like compounds were investigated for their effects on the catalytic activity of P450{sub BS{beta}} by carrying out inhibition studies. The stability of P450{sub BS{beta}} against hydrogen peroxide, cumene, and ter-butyl hydroperoxide was determined. Overall, the results of this study suggested that the P450{sub BS{beta}} enzyme represents a powerful catalyst in terms of the catalytic activity and operational stability.

  18. Inhibition of cytochrome P450 3A: relevant drug interactions in gastroenterology.

    PubMed

    Sagir, A; Schmitt, M; Dilger, K; Häussinger, D

    2003-01-01

    Cytochrome P450 3A (CYP3A) is involved in biotransformation of more than half of all drugs currently available. Drug interactions by inhibition of CYP3A are of major interest in patients receiving combinations of drugs. Some interactions with CYP3A inhibitors also involve inhibition of the multidrug export pump, P-glycoprotein. An increasing number of adverse drug reactions might be avoided on the basis of knowledge about CYP3A substrates and inhibitors. This article summarizes some examples of such interactions relevant to gastroenterologists. Serious cases by coadministration of CYP3A inhibitors resulting in acute hepatitis, hypotension, rhabdomyolyis, torsade de pointes, sedation, or ergotism are presented: interactions with azole antifungals (ketoconazole, itraconazole, fluconazole), HIV protease inhibitors (ritonavir, indinavir, saquinavir, nelfinavir), macrolide antibiotics (clarithromycin, erythromycin), and grapefruit juice. In addition, 1 case is reported who presented the highest trough levels of the CYP3A substrate budesonide in serum ever measured. Practitioners have to be aware of the high potential of metabolic drug interactions when they prescribe a CYP3A inhibitor. It is wise to check carefully comedication in patients complaining of side effects with substrates of CYP3A.

  19. Brevetoxin: tissue distribution and effect on cytochrome P450 enzymes in fish.

    PubMed

    Washburn, B S; Baden, D G; Gassman, N J; Walsh, P J

    1994-07-01

    As part of our investigation of the metabolism of brevetoxin (PbTx) in fish, we initiated a two-part study to determine the toxin's tissue distribution and its ability to induce xenobiotic metabolizing enzymes. In the first study, gulf toadfish (Opsanus beta) were administered 14C-PbTx-3 orally in a fishmeal slurry and sacrificed 72 hr later. Radioactivity was greatest in the hepatobiliary system (40% of body burden), representing the key role this system plays in the detoxification and elimination of brevetoxin. Muscle tissue contained 27%, followed by gastrointestinal tract (25%). To investigate the effects of PbTx on xenobiotic metabolizing enzymes, immature redfish (Scianops ocellatus) were given two doses of brevetoxin (1.5 or 2.5 micrograms/100 g body weight) or cod liver oil in a fishmeal slurry by gavage. The activities of two hepatic P450 enzymes, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD), as well as the cytosolic enzyme, glutathione S-transferase (GST), were measured. At the higher dose, PbTx significantly increased EROD activity. These results suggest that brevetoxin is capable of inducing an important xenobiotic metabolizing enzyme (EROD).

  20. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    PubMed

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.

  1. Sex difference in the principal cytochrome P-450 for tributyltin metabolism in rats

    SciTech Connect

    Ohhira, Shuji . E-mail: s-ohhira@dokkyomed.ac.jp; Enomoto, Mitsunori; Matsui, Hisao

    2006-01-15

    Tributyltin is metabolized by cytochrome P-450 (CYP) system enzymes, and its metabolic fate may contribute to the toxicity of the chemical. In the present study, it is examined whether sex differences in the metabolism of tributyltin exist in rats. In addition, the in vivo and in vitro metabolism of tributyltin was investigated using rat hepatic CYP systems to confirm the principal CYP involved. A significant sex difference in metabolism occurred both in vivo and in vitro, suggesting that one of the CYPs responsible for tributyltin metabolism in rats is male specific or predominant at least. Eight cDNA-expressed rat CYPs, including typical phenobarbital (PB)-inducible forms and members of the CYP2C subfamily, were tested to determine their capability for tributyltin metabolism. Among the enzymes studied, a statistically significant dealkylation of tributyltin was mediated by CYP2C6 and 2C11. Furthermore, the sex difference in metabolism disappeared in vitro after anti-rat CYP2C11 antibody pretreatment because CYP2C11 is a major male-specific form in rats. These results indicate that CYP2C6 is the principal CYP for tributyltin metabolism in female rats, whereas CYP2C11 as well as 2C6 is involved in tributyltin metabolism in male rats, and it is suggested that CYP2C11 is responsible for the significant sex difference in the metabolism of tributyltin observed in rats.

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

    SciTech Connect

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

    2012-02-17

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

  3. [Overexpression, homology modeling and coenzyme docking studies of the cytochrome P450nor2 from Cylindrocarpon tonkinense].

    PubMed

    Li, N; Zhang, Y Z; Li, D D; Niu, Y H; Liu, J; Li, S X; Yuan, Y Z; Chen, S L; Geng, H; Liu, D L

    2016-01-01

    Cytochrome P450nor catalyzes an unusual reaction that transfers electrons from NADP/NADPH to bound heme directly. To improve the expression level of P450nor2 from Cylindrocarpon tonkinense (C.P450nor2), Escherichia coli system was utilized to substitute the yeast system we constructed for expression of the P450nor2 gene, and the protein was purified in soluble form using Ni(+)-NTA affinity chromatography. In contrast to P450nor from Fusarium oxysporum (F.P450nor) and P450nor1 from Cylindrocarpon tonkinense (C.P450nor1), C.P450nor2 shows a dual specificity for using NADH or NADPH as electron donors. The present study developed a computational approach in order to illustrate the coenzyme specificity of C.P450nor2 for NADH and NADPH. This study involved homology modeling of C.P450nor2 and docking analyses of NADH and NADPH into the crystal structure of F.P450nor and the predictive model of C.P450nor2, respectively. The results suggested that C.P450nor2 and F.P450nor have different coenzyme specificity for NADH and NADPH; whilst the space around the B'-helix of the C.P450nor2, especially the Ser79 and Gly81, play a crucial role for the specificity of C.P450nor2. In the absence of the experimental structure of C.P450nor2, we hope that our model will be useful to provide rational explanation on coenzyme specificity of C.P450nor2.

  4. Xenobiotic biotransformation in unicellular green algae. Involvement of cytochrome P450 in the activation and selectivity of the pyridazinone pro-herbicide metflurazon.

    PubMed Central

    Thies, F; Backhaus, T; Bossmann, B; Grimme, L H

    1996-01-01

    The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determined for two unicellular green algae, Chlorella sorokiniana and Chlorella fusca; however, the latter is 25 to 80 times more sensitive to metflurazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotransformation assay. Apparent K(m) values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is characterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established inhibitors of cytochrome P450-mediated reactions, including piperonylbutoxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1yl)-4-hydroxy-5,5-dimethylhexane++ +, and tetcyclacis, as well as cinnamic acid, a potential endogenous substrate, inhibited the N-demethylation of metflurazon. The results suggest that the N-demethylation of metflurazon by both algae is mediated by a cytochrome P450 monooxygenase. The determination of antigenic cross-reactivity of algal proteins with heterologous polyclonal antibodies originally raised against plant P450s, anti-cinnamic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or those of bacterial origin, CYP105A1 and CYP105B1, suggests the presence of distinct P450 isoforms in both algae. PMID:8819332

  5. Comparative Analysis of P450 Signature Motifs EXXR and CXG in the Large and Diverse Kingdom of Fungi: Identification of Evolutionarily Conserved Amino Acid Patterns Characteristic of P450 Family

    PubMed Central

    Syed, Khajamohiddin; Mashele, Samson Sitheni

    2014-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research. PMID:24743800

  6. Mechanism-Based Inactivation of Cytochrome P450 2B6 by Methadone through Destruction of Prosthetic Heme

    PubMed Central

    Amunugama, Hemali T.; Zhang, Haoming

    2012-01-01

    Methadone is a μ-opioid receptor agonist widely used in the treatment of narcotic addiction and chronic pain conditions. Methadone is metabolized predominantly in the liver by cytochromes P450 to its pharmacologically inactive primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. Initial in vitro data suggested that CYP3A4 is the major isoform responsible for the in vivo clearance of methadone in humans. However, recent clinical data have indicated that CYP2B6 is actually the major isoform responsible for methadone metabolism and clearance in vivo. In this study, methadone was shown to act as a mechanism-based inactivator of CYP2B6. Methadone inactivates CYP2B6 in a time-, concentration-, and NADPH-dependent manner with a KI = 10.0 μM and kinact = 0.027 min−1. The loss of CYP2B6 activity in the presence of methadone and NADPH occurred with concomitant loss of the reduced CO spectrum of the P450. Moreover, there was good correlation between the loss of CYP2B6 activity and the loss of the CO-binding spectrum. High-performance liquid chromatography analysis of the native heme of the inactivated CYP2B6 demonstrated that approximately 75% loss of heme was accompanied by comparable inactivation of CYP2B6. Liquid chromatography-mass spectrometry analysis did not reveal the formation of a protein adduct during the inactivation. The evidence strongly suggests that destruction of prosthetic heme is the underlying mechanism leading to the inactivation of CYP2B6 by methadone. PMID:22685215

  7. Mechanism-based inactivation of cytochrome P450 2B6 by methadone through destruction of prosthetic heme.

    PubMed

    Amunugama, Hemali T; Zhang, Haoming; Hollenberg, Paul F

    2012-09-01

    Methadone is a μ-opioid receptor agonist widely used in the treatment of narcotic addiction and chronic pain conditions. Methadone is metabolized predominantly in the liver by cytochromes P450 to its pharmacologically inactive primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. Initial in vitro data suggested that CYP3A4 is the major isoform responsible for the in vivo clearance of methadone in humans. However, recent clinical data have indicated that CYP2B6 is actually the major isoform responsible for methadone metabolism and clearance in vivo. In this study, methadone was shown to act as a mechanism-based inactivator of CYP2B6. Methadone inactivates CYP2B6 in a time-, concentration-, and NADPH-dependent manner with a K(I) = 10.0 μM and k(inact) = 0.027 min⁻¹. The loss of CYP2B6 activity in the presence of methadone and NADPH occurred with concomitant loss of the reduced CO spectrum of the P450. Moreover, there was good correlation between the loss of CYP2B6 activity and the loss of the CO-binding spectrum. High-performance liquid chromatography analysis of the native heme of the inactivated CYP2B6 demonstrated that approximately 75% loss of heme was accompanied by comparable inactivation of CYP2B6. Liquid chromatography-mass spectrometry analysis did not reveal the formation of a protein adduct during the inactivation. The evidence strongly suggests that destruction of prosthetic heme is the underlying mechanism leading to the inactivation of CYP2B6 by methadone.

  8. Curcumin Prevents Aflatoxin B1 Hepatoxicity by Inhibition of Cytochrome P450 Isozymes in Chick Liver

    PubMed Central

    Zhang, Ni-Ya; Qi, Ming; Zhao, Ling; Zhu, Ming-Kun; Guo, Jiao; Liu, Jie; Gu, Chang-Qin; Rajput, Shahid Ali; Krumm, Christopher Steven; Qi, De-Sheng; Sun, Lv-Hui

    2016-01-01

    This study was designed to establish if Curcumin (CM) alleviates Aflatoxin B1 (AFB1)-induced hepatotoxic effects and to determine whether alteration of the expression of cytochrome P450 (CYP450) isozymes is involved in the regulation of these effects in chick liver. One-day-old male broilers (n = 120) were divided into four groups and used in a two by two factorial trial in which the main factors included supplementing AFB1 (< 5 vs. 100 μg/kg) and CM (0 vs. 150 mg/kg) in a corn/soybean-based diet. Administration of AFB1 induced liver injury, significantly decreasing albumin and total protein concentrations and increasing alanine aminotransferase and aspartate aminotransferase activities in serum, and induced hepatic histological lesions at week 2. AFB1 also significantly decreased hepatic glutathione peroxidase, catalase, and glutathione levels, while increasing malondialdehyde, 8-hydroxydeoxyguanosine, and exo-AFB1-8,9-epoxide (AFBO)-DNA concentrations. In addition, the mRNA and/or activity of enzymes responsible for the bioactivation of AFB1 into AFBO—including CYP1A1, CYP1A2, CYP2A6, and CYP3A4—were significantly induced in liver microsomes after 2-week exposure to AFB1. These alterations induced by AFB1 were prevented by CM supplementation. Conclusively, dietary CM protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of increased antioxidant capacities and inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO. PMID:27834912

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

    PubMed

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

    2012-12-01

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

  10. Mammalian Cytochrome P450-Dependent Metabolism of Polychlorinated Dibenzo-p-dioxins and Coplanar Polychlorinated Biphenyls

    PubMed Central

    Inui, Hideyuki; Itoh, Toshimasa; Yamamoto, Keiko; Ikushiro, Shin-Ichi; Sakaki, Toshiyuki

    2014-01-01

    Polychlorinated dibenzo-p-dioxins (PCDDs) and coplanar polychlorinated biphenyls (PCBs) contribute to dioxin toxicity in humans and wildlife after bioaccumulation through the food chain from the environment. The authors examined human and rat cytochrome P450 (CYP)-dependent metabolism of PCDDs and PCBs. A number of human CYP isoforms belonging to the CYP1 and CYP2 families showed remarkable activities toward low-chlorinated PCDDs. In particular, human CYP1A1, CYP1A2, and CYP1B1 showed high activities toward monoCDDs, diCDDs, and triCDDs but no detectable activity toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetraCDD). Large amino acids located at putative substrate-recognition sites and the F-G loop in rat CYP1A1 contributed to the successful metabolism of 2,3,7,8-tetraCDD. Rat, but not human, CYP1A1 metabolized 3,3',4,4',5-pentachlorobiphenyl (CB126) to two hydroxylated metabolites. These metabolites are probably less toxic than is CB126, due to their higher solubility. Homology models of human and rat CYP1A1s and CB126 docking studies indicated that two amino acid differences in the CB126-binding cavity were important for CB126 metabolism. In this review, the importance of CYPs in the metabolism of dioxins and PCBs in mammals and the species-based differences between humans and rats are described. In addition, the authors reveal the molecular mechanism behind the binding modes of dioxins and PCBs in the heme pocket of CYPs. PMID:25123135

  11. [Heterologous expression of functionally active human cytochrome P-450s. Cytochrome P-450IIIA4 catalyzes the biotransformation of the anabolic steroid hormone methandrostenolone].

    PubMed

    Krynetskiĭ, E Iu; Kovaleva, I E; Luzikov, V N

    1994-02-01

    The expression of the cytochrome P450IIIA4 gene in the Saccharomyces cerevisiae yeast using the shuttle vector pYeDP1-8/2 has been carried out. The microsomal fraction isolated from the transformed yeast cells was used for biotransformation of the anabolic steroid hormone-methandrostenolone (MA). The microsomal oxidation products were analyzed by HPLC and two-dimensional TLC. It was shown that microsomes of the yeasts expressing human cytochrome P450IIIA4 catalyze the MA conversion into its 6 beta-hydroxy derivative. An identical product is formed via a reaction catalyzed by human liver microsomes. The use of the heterological system of cytochrome P450IIIA4 expression has made it possible to establish its role in MA metabolism. The experimental system simulates the first phase of the drug biotransformation in liver cells.

  12. [Activity of cytochromes P-450p and P-450h in liver microsomes and blood corticosteroid levels in experimental animals under the action of physical factors].

    PubMed

    Zolotareva, T A; Gorchakova, G A; Konovalenko, V L; Konovalenko, L N; Grishanova, A Iu; Guliaeva, L F; Liakhovich, V V

    1992-05-01

    In experiments on male Wistar rats it has been found that physical factors applied in medicine (laser radiation of low intensity with wave length 0.89 microns, microwaves of centimeter range of 2450 MHz, and ultrasound of low intensity 880 KHz) changed catalytic activity of liver microsomal and rostenedione 16 alpha- and 6 beta-hydroxylating cytochromes P-450h and P-450p and blood corticosteroids level. Activities of these two steroid-metabolizing cytochromes decreased under ultrasonic skin application on liver region and increased under microwave and laser action. Contents of physiologically inactive form of corticosterone were not changed by the physical factors action while level of active hormone was increased under ultrasonic and microwave action. These findings suggest association of the activity of liver steroid-metabolizing cytochromes P-450 and level of physiologically active form of corticosterone in blood under physical factors skin application on liver region.

  13. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    SciTech Connect

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

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the target tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.

  14. Toxaphene detoxification and acclimation in Daphnia magna: do cytochrome P-450 enzymes play a role?

    PubMed

    Kashian, Donna R

    2004-01-01

    Toxaphene is a persistent environmental contaminant that has been shown to alter male production in Daphnia magna and to induce P-450 activity in mammals. Cytochrome P-450-mediated metabolism may lead to xenobiotic detoxification resulting in acclimation. To determine if D. magna acclimate to toxaphene via P-450 pathways, chronic and acute toxicity tests were conducted with D. magna exposed to toxaphene in the presence and absence of piperonyl butoxide (PBO), an inhibitor of cytochrome P-450 enzymes. Toxaphene exposure increased male production in acute but not chronic assays, indicating that D. magna may acclimate to chronic toxaphene exposure. Upon co-administration of toxaphene and PBO in chronic tests, D. magna exhibited a decline in growth rate, fecundity and survival. The observed toxaphene acclimation in chronic tests, along with its increased toxicity in the presence of a P-450 suppressor, suggests that P-450 enzymes may contribute to detoxification and subsequent acclimation of D. magna to chronic toxaphene exposure. Additional chronic toxicity tests indicated that toxaphene acclimation occurs between 7 and 12 days following initial exposure, at which time sex determination is no longer affected. Thus, sublethal toxaphene toxicity effects such as reproductive impairments may be detectable with acute but not chronic tests, potentially due to the upregulation of P-450 isozymes.

  15. Construction of a 3D model of cytochrome P450 2B4.

    PubMed

    Chang, Y T; Stiffelman, O B; Vakser, I A; Loew, G H; Bridges, A; Waskell, L

    1997-02-01

    A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

  16. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

    PubMed

    Beyer, Nina; Kulig, Justyna K; Bartsch, Anette; Hayes, Martin A; Janssen, Dick B; Fraaije, Marco W

    2017-03-01

    To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

  17. Characterization of maize cytochrome P450 monooxygenases induced in response to safeners and bacterial pathogens.

    PubMed

    Persans, M W; Wang, J; Schuler, M A

    2001-02-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens.

  18. Characterization of Maize Cytochrome P450 Monooxygenases Induced in Response to Safeners and Bacterial Pathogens1

    PubMed Central

    Persans, Michael W.; Wang, Jian; Schuler, Mary A.

    2001-01-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens. PMID:11161067

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

    PubMed Central

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

    2013-01-01

    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

  20. Function and membrane topology of wild-type and mutated cytochrome P-450c21.

    PubMed Central

    Hu, M C; Hsu, L C; Hsu, N C; Chung, B C

    1996-01-01

    We have studied membrane topology of cytochrome P-450c21 (P450c21) using the approaches of mutagenesis and protease digestion. P450c21 is located at the cytoplasm with an N-terminal hydrophobic domain integrated into microsomal membranes. When this hydrophobic domain was replaced by a secretory signal peptide, P450c21 was translocated into the lumen and lost enzymic activity. No other topogenic sequence was detected in the bulk of the P450c21 peptide. A mutant protein with Pro-30 replaced by Leu (L30) corresponding to the mutation found in the diseased state was created. L30 protein lost 90% of enzymic activity, while a double mutant (L30R32) with an additional Leu-32 to Arg mutation had slightly higher residual enzymic activity. Apart from lower activity, L30 was also present in the cell at a lower level than wild-type P450c21. This lower level is probably due to increased degradation, as L30 is synthesized at a normal rate. Both L30 and L30R32 proteins, however, were integrated into membranes normally. Therefore the Pro-30 --> Leu mutation did not affect membrane integration, but affected the abundance and enzymic activity of P450c21. PMID:8645225

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

    PubMed Central

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

    1995-01-01

    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

  2. Novel Marmoset Cytochrome P450 2C19 in Livers Efficiently Metabolizes Human P450 2C9 and 2C19 Substrates, S-Warfarin, Tolbutamide, Flurbiprofen, and Omeprazole.

    PubMed

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

    2015-10-01

    The common marmoset (Callithrix jacchus), a small New World monkey, has the potential for use in human drug development due to its evolutionary closeness to humans. Four novel cDNAs, encoding cytochrome P450 (P450) 2C18, 2C19, 2C58, and 2C76, were cloned from marmoset livers to characterize P450 2C molecular properties, including previously reported P450 2C8. The deduced amino acid sequence showed high sequence identities (>86%) with those of human P450 2Cs, except for marmoset P450 2C76, which has a low sequence identity (∼70%) with any human P450 2Cs. Phylogenetic analysis showed that marmoset P450 2Cs were more closely clustered with those of humans and macaques than other species investigated. Quantitative polymerase chain reaction analysis showed that all of the marmoset P450 2C mRNAs were predominantly expressed in liver as opposed to the other tissues tested. Marmoset P450 2C proteins were detected in liver by immunoblotting using antibodies against human P450 2Cs. Among marmoset P450 2Cs heterologously expressed in Escherichia coli, marmoset P450 2C19 efficiently catalyzed human P450 2C substrates, S-warfarin, diclofenac, tolbutamide, flurbiprofen, and omeprazole. Marmoset P450 2C19 had high Vmax and low Km values for S-warfarin 7-hydroxylation that were comparable to those in human liver microsomes, indicating warfarin stereoselectivity similar to findings in humans. Faster in vivo S-warfarin clearance than R-warfarin after intravenous administration of racemic warfarin (0.2 mg/kg) to marmosets was consistent with the in vitro kinetic parameters. These results indicated that marmoset P450 2C enzymes had functional characteristics similar to those of humans, and that P450 2C-dependent metabolic properties are likewise similar between marmosets and humans.

  3. mRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

    PubMed Central

    Stark, Katarina; Wu, Zhong-Liu; Bartleson, Cheryl J.; Guengerich, F. Peter

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3–4 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  4. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans.

    PubMed

    Danielson, P B

    2002-12-01

    Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.

  5. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  6. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism.

    PubMed

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Li, Wei; Szczesniewski, Andre; Tuckey, Robert C

    2005-08-01

    We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized.

  7. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism

    PubMed Central

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Li, Wei; Szczesniewski, Andre; Tuckey, Robert C.

    2008-01-01

    We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized. PMID:16098191

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

    PubMed Central

    Cheng, Qian

    2011-01-01

    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

  9. Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

    PubMed

    Conley, A J; Corbin, C J; Browne, P; Mapes, S M; Place, N J; Hughes, A L; Glickman, S E

    2007-07-01

    At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to

  10. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

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

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

    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

  12. High-throughput fluorescence assay of cytochrome P450 3A4

    PubMed Central

    Cheng, Qian; Sohl, Christal D; Guengerich, F Peter

    2013-01-01

    Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less. PMID:19661996

  13. Cytochrome P450 Family 1 Inhibitors and Structure-Activity Relationships

    PubMed Central

    Liu, Jiawang; Sridhar, Jayalakshmi; Foroozesh, Maryam

    2014-01-01

    With the widespread use of O-alkoxyresorufin dealkylation assays since the 1990’s, thousands of inhibitors of cytochrome P450 family 1 enzymes (P450s 1A1, 1A2, and 1B1) have been identified and studied. Generally, planar polycyclic molecules such as polycyclic aromatic hydrocarbons, stilbenoids, and flavonoids are considered to potentially be effective inhibitors of these enzymes. However, the details of structure-activity relationships and selectivity of these inhibitors are still ambiguous. In this review, we thoroughly discuss the selectivity of many representative P450 family 1 inhibitors reported in the past 20 years through a meta-analysis. PMID:24287985

  14. P450 inhibitors of use in medical treatment: focus on mechanisms of action.

    PubMed

    Vanden Bossche, H; Koymans, L; Moereels, H

    1995-01-01

    A number of cytochrome P450s are targets for compounds that are clinically used or under clinical evaluation for treatment of patients with mycotic infections, such as dermatophytosis, superficial and systemic candidiasis, cryptococcosis and aspergillosis, with skin diseases, such as psoriasis or ichthyosis, and other retinoid-sensitive malignancies, e.g., neuro-ectodermal glioma. Some of the P450 inhibitors are candidates for the treatment of hirsutism or prostate cancer, others are potent inhibitors of the P450 isomerase involved in the synthesis of thromboxane A2, a potent platelet aggregation inducer and vasoconstrictor.

  15. Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo.

    PubMed

    Henderson, Colin J; McLaughlin, Lesley A; Scheer, Nico; Stanley, Lesley A; Wolf, C Roland

    2015-04-01

    The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

  16. A newly developed DNA microarray is useful to assess induction of cytochromes p450 in the cynomolgus monkey.

    PubMed

    Ise, Ryota; Uehara, Shotaro; Akiyama, Hideo; Kondo, Satoshi; Iwasaki, Kazuhide; Nagata, Ryoichi; Nobumasa, Hitoshi; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-06-01

    Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

  17. Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

    PubMed

    Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Padala, Nagasurya Prakash; Boggavarapu, Rajesh Kumar; Bhyrapuneni, Gopinadh; Ajjala, Devender Reddy; Nirogi, Ramakrishna

    2017-04-01

    The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the Km values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values.

  18. Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support.

    PubMed

    Tan, Cheau Yuaan; Hirakawa, Hidehiko; Suzuki, Risa; Haga, Tomoaki; Iwata, Fumiya; Nagamune, Teruyuki

    2016-11-21

    Bacterial cytochrome P450s (P450s), which catalyze regio- and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost-effective industrial manufacturing. However, immobilization of P450s is difficult because electron-transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein-mediated co-immobilization of a bacterial P450, and its electron-transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co-immobilized enzymes catalyzed monooxygenation because the electron-transfer protein fused to PCNA via a single peptide linker retained its electron-transport function.

  19. Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations.

    PubMed

    Zilly, Felipe E; Taglieber, Andreas; Schulz, Frank; Hollmann, Frank; Reetz, Manfred T

    2009-12-14

    A light-driven deazaflavin-dependent direct enzyme regeneration system has been developed for a P450-BM3 catalyzed CH-activating hydroxylation, thereby avoiding the need for the expensive NADPH cofactor.

  20. Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

    PubMed Central

    Sanglard, D; Käppeli, O; Fiechter, A

    1984-01-01

    In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells. PMID:6690424

  1. [Establishment of prokaryotic expression and optimization ox expression conditions of Eleutherococcus senticosus P450 gene].

    PubMed

    Wu, Peng; Xiu, Le-shan; Li, Fei-fei; Xing, Zhao-bin

    2015-04-01

    According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.

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

    PubMed Central

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

    2016-01-01

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

  3. Structure and Function of Cytochromes P450 2B: From Mechanism-Based Inactivators to X-Ray Crystal Structures and Back

    PubMed Central

    2011-01-01

    This article reviews work from the author dating back to 1978 and focuses on the structural basis of cytochrome P450 (P450) function using available contemporary techniques. Early studies used mechanism-based inactivators that bound to the protein moiety of hepatic P450s to try to localize the active site. Subsequent studies used cDNA cloning, heterologous expression, site-directed mutagenesis, and homology modeling based on multiple bacterial P450 X-ray crystal structures to predict the active sites of CYP2B enzymes with considerable accuracy. Breakthroughs in engineering and expression of mammalian P450s enabled us to determine our first X-ray crystal structure of ligand-free rabbit CYP2B4. To date, we have solved 11 CYP2B4 and three human CYP2B6 structures, which represent four significantly different conformations. The plasticity of CYP2B4 has been confirmed by deuterium exchange mass spectrometry and is substantiated by molecular dynamics simulations. In addition to major movement of secondary structure elements, more subtle reorientation of active site side chains, especially Phe206, Phe297, and Glu301, contributes to the ability of CYP2B enzymes to bind various ligands. Isothermal titration calorimetry has proven to be a useful tool for studying the thermodynamics of ligand binding to CYP2B4 and CYP2B6, and NMR has enabled study of ligand binding orientation in solution as an adjunct to X-ray crystallography. A major challenge remains to harness the power of the various approaches to facilitate prediction of CYP2B specificity and inhibition. PMID:21502194

  4. Microbial P450 enzymes in bioremediation and drug discovery: Emerging potentials and challenges.

    PubMed

    Bhattacharya, Sukanta S; Yadav, Jagjit S

    2016-11-21

    Cytochrome P450 enzymes are a structurally conserved but functionally diverse group of heme-containing mixed function oxidases found across both prokaryotic and eukaryotic forms of the microbial world. Microbial P450s are known to perform diverse functions ranging from the synthesis of cell wall components to xenobiotic/drug metabolism to biodegradation of environmental chemicals. Conventionally, many microbial systems have been reported to mimic mammalian P450-like activation of drugs and were proposed as the in-vitro models of mammalian drug metabolism. Recent reports suggest that native or engineered forms of specific microbial P450s from these and other microbial systems could be employed for desired specific biotransformation reactions toward natural and synthetic (drug) compounds underscoring their emerging potential in drug improvement and discovery. On the other hand, microorganisms particularly fungi and actinomycetes have been shown to possess catabolic P450s with unusual potential to degrade toxic environmental chemicals including persistent organic pollutants (POPs). Wood-rotting basidiomycete fungi in particular have revealed the presence of exceptionally large P450 repertoire (P450ome) in their genomes, majority of which are however orphan (with no known function). Our pre- and post-genomic studies have led to functional characterization of several fungal P450s inducible in response to exposure to several environmental toxicants and their potential in bioremediation of these chemicals. This review is an attempt to summarize the post-genomic unveiling of this versatile enzyme superfamily in microbial systems and investigation of their potential to synthesize new drugs and degrade persistent pollutants, among other biotechnological applications.

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

    SciTech Connect

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

    1984-09-28

    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.

  6. Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450.

    PubMed

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

    2015-06-20

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

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

    PubMed Central

    2015-01-01

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-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 ω-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 12-2H-substituted lauric acid. However, considerable “metabolic switching” to 11-hydroxylation was observed with [12-2H3]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 [12-3H]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 ω-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 ω-oxidation. PMID:25203493

  8. Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.

    PubMed

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

    2014-10-07

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-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 ω-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 ω-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 ω-oxidation.

  9. A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity.

    PubMed

    Zvelebil, M J; Wolf, C R; Sternberg, M J

    1991-02-01

    A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

  10. The Molecular Evolution of Cytochrome P450 Genes within and between Drosophila Species

    PubMed Central

    Good, Robert T.; Gramzow, Lydia; Battlay, Paul; Sztal, Tamar; Batterham, Philip; Robin, Charles

    2014-01-01

    We map 114 gene gains and 74 gene losses in the P450 gene family across the phylogeny of 12 Drosophila species by examining the congruence of gene trees and species trees. Although the number of P450 genes varies from 74 to 94 in the species examined, we infer that there were at least 77 P450 genes in the ancestral Drosophila genome. One of the most striking observations in the data set is the elevated loss of P450 genes in the Drosophila sechellia lineage. The gain and loss events are not evenly distributed among the P450 genes—with 30 genes showing no gene gains or losses whereas others show as many as 20 copy number changes among the species examined. The P450 gene clades showing the fewest number of gene gain and loss events tend to be those evolving with the most purifying selection acting on the protein sequences, although there are exceptions, such as the rapid rate of amino acid replacement observed in the single copy phantom (Cyp306a1) gene. Within D. melanogaster, we observe gene copy number polymorphism in ten P450 genes including multiple cases of interparalog chimeras. Nonallelic homologous recombination (NAHR) has been associated with deleterious mutations in humans, but here we provide a second possible example of an NAHR event in insect P450s being adaptive. Specifically, we find that a polymorphic Cyp12a4/Cyp12a5 chimera correlates with resistance to an insecticide. Although we observe such interparalog exchange in our within-species data sets, we have little evidence of it between species, raising the possibility that such events may occur more frequently than appreciated but are masked by subsequent sequence change. PMID:24751979

  11. Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli.

    PubMed

    Ba, Lina; Li, Pan; Zhang, Hui; Duan, Yan; Lin, Zhanglin

    2013-07-01

    P450sca-2 is an industrially important enzyme that stereoselectively converts mevastatin into pravastatin. However, little information or engineering efforts have been reported for this enzyme or its redox partner. In this study, we successfully reconstituted the P450sca-2 activity in Escherichia coli by co-expression with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from the Pseudomonas putida cytochrome P450cam system. With an HPLC-based screening assay, random mutagenesis was applied to yield a mutant (R8-5C) with a pravastatin yield of the whole-cell biotransformation 4.1-fold that of the wild type. P450sca-2 wild-type and R8-5C were characterized in terms of mevastatin binding and hydroxylation, electron transfer, and circular dichroism spectroscopy. R8-5C showed an active P450 expression level that was 3.8-fold that of the wild type, with relatively smaller changes in the apparent k(cat)/K(M) with respect to the substrate mevastatin (1.3-fold) or Pdx (1.5-fold) compared with the wild type. Thus, the increase in the pravastatin yield of the whole-cell biotransformation primarily came from the improved active P450 expression, which has resulted largely from better heme incorporation, although none of the six mutations of R8-5C are located near the heme active site. These results will facilitate further engineering of this P450sca-2 system and provide useful clues for improving other hybrid P450 systems.

  12. Multiple Structural and Functional Abnormalities in the P450 Aromatase Expressing Transgenic Male Mice Are Ameliorated by a P450 Aromatase Inhibitor

    PubMed Central

    Li, Xiangdong; Strauss, Leena; Mäkelä, Sari; Streng, Tomi; Huhtaniemi, Ilpo; Santti, Risto; Poutanen, Matti

    2004-01-01

    The present study was undertaken to analyze the effect of a P450 aromatase inhibitor (finrozole) on 4-month-old transgenic mice expressing human P450 aromatase (P450arom) under the human ubiquitin C promoter (AROM+). AROM+ mice present several dysfunctions, such as adrenal and pituitary hyperplasia, cryptorchidism, Leydig cell hypertrophy and hyperplasia, and gynecomastia. The present study demonstrates that these abnormalities were efficiently treated by administration of a P450arom inhibitor, finrozole. The treatment normalized the reduced intratesticular and serum testosterone levels, while those of estradiol were decreased. The body weight and several affected organ weights were normalized with the treatment. Histological analysis revealed that both the pituitary and adrenal hyperplasia were diminished. Furthermore, the cryptorchid testes present in the untreated AROM+ males descended to scrotum, 4 to 15 days after inhibitor treatment. In addition, the disrupted spermatogenesis was recovered and qualitatively complete spermatogenesis appeared with the inhibitor treatment. This was associated with normalized structure of the interstitial tissue, as analyzed by immunohistochemical staining for Leydig cells and macrophages. One of the features was that the Leydig cell hypertrophy was markedly diminished in the treated mice. AROM+ mice also present with severe gynecomastia, while the development and differentiation of the mammary gland in AROM+ males was markedly diminished with the inhibitor treatment. Interestingly, the mammary gland involution was associated with the induction of androgen receptor in the epithelial cells, while estrogen receptors were still detectable in the epithelium. The data show that AROM+ mouse model is a novel tool to further analyze the use of P450arom inhibitors in the treatment of the dysfunctions in males associated with misbalanced estrogen to androgen ratio, such as pituitary adenoma, testicular dysfunction, and gynecomastia. PMID

  13. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    PubMed

    Qhanya, Lehlohonolo Benedict; Matowane, Godfrey; Chen, Wanping; Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

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

    PubMed Central

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

    2014-01-01

    Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our

  15. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

    PubMed Central

    Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccini