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Sample records for hepatic p450 isoforms

  1. Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

    PubMed

    Renwick, A B; Surry, D; Price, R J; Lake, B G; Evans, D C

    2000-10-01

    1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 micro

  2. In vitro metabolism and interaction of cilostazol with human hepatic cytochrome P450 isoforms.

    PubMed

    Abbas, R; Chow, C P; Browder, N J; Thacker, D; Bramer, S L; Fu, C J; Forbes, W; Odomi, M; Flockhart, D A

    2000-03-01

    1. Cilostazol (OPC-13013) undergoes extensive hepatic metabolism. The hydroxylation of the quinone moiety of cilostazol to OPC-13326 was the predominant route in all the liver preparations studies. The hydroxylation of the hexane moiety to OPC-13217 was the second most predominant route in vitro. 2. Ketoconazole (1 microM) was the most potent inhibitor of both quinone and hexane hydroxylation. Both the CYP2D6 inhibitor quinidine (0.1 microM) and the CYP2C19 inhibitor omeprazole (10 microM) failed to consistently inhibit metabolism of cilostazol via either of these two predominant routes. 3. Data obtained from a bank of pre-characterized human liver microsomes demonstrated a stronger correlation (r2=0.68, P < 0.01) between metabolism of cilostazol to OPC-13326 and metabolism of felodipine, a CYP3A probe, that with probes for any other isoform. Cimetidine demonstrated concentration-dependent competitive inhibition of the metabolism of cilostazol by both routes. 4. Kinetic data demonstrated a Km value of 101 microM for cilostazol, suggesting a relatively low affinity of cilostazol for CYP3A. While recombinant CYP1A2, CYP2D6 and CYP2C19 were also able to catalyze formation of specific cilostazol metabolites, they did not appear to contribute significantly to cilostazol metabolism in whole human liver microsomes.

  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. Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formation by various P450 isoforms.

    PubMed

    Hu, Yiding; Kupfer, David

    2002-12-01

    mono-OH-M into catechol-M and bis-OH-M into tris-OH-M was primarily by 3A4 and was not enantioselective. In conclusion, enantiomeric abundance of methoxychlor metabolites depends on the relative catalytic activity of the hepatic p450 isoforms.

  7. Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver

    PubMed Central

    Zhang, Hai-Feng; Li, Zhi-Hui; Liu, Jia-Yu; Liu, Ting-Ting; Wang, Ping; Fang, Yan; Zhou, Jun; Cui, Ming-Zhu; Gao, Na; Tian, Xin; Gao, Jie; Wen, Qiang; Jia, Lin-Jing

    2016-01-01

    Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism. PMID:27271371

  8. Cytochrome P450 isoforms in the Metabolism of Decursin and Decursinol Angelate from Korean Angelica

    PubMed Central

    ZHANG, Jinhui; LI, Li; TANG, Suni; HALE, Thomas W.; XING, Chengguo; JIANG, Cheng; LÜ, Junxuan

    2016-01-01

    We have shown that the in vitro hepatic microsomal metabolism of pyranocoumarin compound decursinol angelate (DA) to decursinol (DOH) exclusively requires cytochrome P450 enzymes (CYP) 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 preparation, general CYP inhibitor 1-aminobenzotriazole (ABT) and respective competitive inhibitors for 2C19 and 3A4, (+)-N-3-benzylnirvanol and ketoconazole, substantially retarded the metabolism of DA and, to a lesser extent, of D. In healthy human subjects from a single-dose pharmacokinetic 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. PMID:26394652

  9. 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.

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

    EPA Science Inventory

    1. This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague-Dawley rats. Rats were dosed by gavage for 1...

  11. Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex.

    PubMed

    Ishii, Yuji; Iwanaga, Megumi; Nishimura, Yoshio; Takeda, Shuso; Ikushiro, Shin-Ichi; Nagata, Kiyoshi; Yamazoe, Yasushi; Mackenzie, Peter I; Yamada, Hideyuki

    2007-10-01

    The interaction between cytochrome P450s (CYP, P450) and UDP-glucuronosyltransferases (UGTs) was studied by co-immunoprecipitation. P450 isoform-selective antibody was used as a probe to co-precipitate UGTs with the P450s from solubilized rat liver microsomes. Antibodies toward CYP3A2, CYP2B2, CYP2C11/13 and CYP1A2 co-precipitated UGTs with corresponding P450s. However, calnexin, a type-I membrane protein, in the endoplasmic reticulum was not co-precipitated by anti-P450 antibodies. UGT activity toward 4-methylumbelliferone was detected in all co-precipitates, suggesting that UGT in the complex with P450s is functionally active. Repeated washing of co-immunoprecipitates revealed differences among P450 isoforms with regard to the affinity for UGT. Larger amounts of UGT1A1 and UGT1A6, compared with UGT2B1, were washed out from UGTs-CYP2C11/13 co-precipitates, whereas UGT-CYP3A2 and UGT-CYP2Bs complexes were resistant to thorough washing. Thus, CYP2C11/13 could associate with UGTs, but the affinity is assumed to be weaker than that of CYP2B/3As. These results suggest that there is isoform specificity in the interaction between P450s and UGTs.

  12. 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

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

    PubMed

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

    2016-07-01

    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

  14. Assessment of inhibition of porcine hepatic cytochrome P450 enzymes by 48 commercial drugs.

    PubMed

    Hu, Steven X; Mazur, Chase A; Feenstra, Kenneth L; Lorenz, Julie K; Merritt, Dawn A

    2016-05-01

    Drug interactions due to inhibition of hepatic cytochrome P450 (CYP450) enzymes are not well understood in veterinary medicine. Forty-eight commercial porcine medicines were selected to evaluate their potential inhibition on porcine hepatic CYP450 enzymes at their commercial doses and administration routes. Those drugs were first assessed through a single point inhibitory assay at 3 µM in porcine liver microsomes for six specific CYP450 metabolisms (phenacetin o-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorozoxazone 6-hydroxylation and midazolam 1'-hydroxylation). When the inhibition was > 10% in the single point inhibitory assay, IC50 values (inhibitory concentrations that decrease biotransformation of selected substrate by 50%) were determined. Overall, 17 drugs showed in vitro inhibition on one or more porcine hepatic CYP450 metabolisms with different IC50 values. The potential in vivo porcine hepatic CYP450 inhibition by those drugs was assessed by combining the in vitro data and in vivo Cmax (maximum plasma concentrations from pharmacokinetic studies of the porcine medicines at their commercial doses and administration routes). Three drugs showed high potential inhibition to one or two porcine hepatic CYP450 isoforms at their commercial doses and administration routes, while seven drugs had medium risk and seven had low risk of such in vivo inhibition. These data are useful to prevent potential drug interactions in veterinary medical practice.

  15. Expression and inducibility of cytochrome P450 isoforms in 1-year-old intrasplenic liver cell transplants in rats.

    PubMed

    Lupp, Amelie; Danz, Manfred; Müller, Dieter; Klinger, Wolfgang

    2002-03-01

    Syngenic fetal liver tissue suspensions were transplanted into the spleens of 60- to 90-day-old male Fischer 344 inbred rats. Transplant recipients were compared with age-matched control rats. One year after surgery, the animals were treated orally with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the respective solvents 24 or 48 h before being killed. Expression of cytochrome P450 (P450) isoforms in spleens and orthotopic livers was assessed by immunohistochemistry and P450-dependent monooxygenase functions by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD) and ethylmorphine N-demethylation (EMND). Spleens of control animals displayed almost no expression of P450 isoforms and P450-mediated monooxygenase functions. Similar to liver, in the transplanted hepatocytes no P450 1A1 but distinct P450 2B1 and 3A2 expression was observed. Furthermore, the transplant-containing spleens displayed significant EROD, ECOD, PROD and EMND activities. Similar to normal liver, BNF treatment enhanced P450 1A1 and 2B1, PB induced P450 2B1 and 3A2, and DEX induced P450 3A2 expression in the transplanted hepatocytes. Correspondingly, in the transplant-containing spleens EROD, ECOD and PROD activities were significantly enhanced following BNF treatment, EROD, ECOD, PROD and EMND activities after PB administration, and EMND activity by DEX treatment. These results demonstrate that hepatocytes originating from fetal liver tissue suspensions can survive in the spleen at least for 1 year. They have differentiated into adult hepatocytes and even 1 year after transplantation express different P450 isoforms which are inducible by BNF, PB and DEX, corresponding to normal adult liver.

  16. Hepatic metabolism of cyclodiene insecticides by constitutive forms of cytochrome P-450 from lower vertebrates.

    PubMed

    Ronis, M J; Walker, C H; Peakall, D

    1987-01-01

    1. Multiple forms of cytochrome P-450 were separated from the hepatic microsomes of untreated male rats, pigeons (Columbia livia), razorbills (Alca torda), puffins (Fratercula arctica), and rainbow trout (Salmo gairdnerii), using anion exchange chromatography and DEAE-cellulose. 2. In some cases cytochrome P-450 forms were further purified on hydroxylapatite and carboxymethyl-sephadex columns. 3. Considerable differences in the distribution of forms between these five species were evident from elution profiles on DEAE cellulose, and on analysis of the cytochrome P-450 containing pools by SDS-PAGE. 4. The metabolism of two organochlorine compounds, aldrin and the dieldrin analogue HCE, were studied in (a) intact microsomes and (b) reconstituted systems containing cytochrome P-450, from each of the five species. 5. In spite of their close structural similarity, significant differences were found between the two substrates in the distribution of catalytic activity between the cytochrome P-450 isozymes of each species. PMID:2888582

  17. Influence of recipient gender on cytochrome P450 isoforms expression in intrasplenic fetal liver tissue transplants in rats.

    PubMed

    Lupp, Amelie; Hugenschmidt, Sabine; Danz, Manfred; Müller, Dieter

    2003-06-30

    Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern which is regulated by a differential profile of growth hormone (GH) secretion. The aim of the present study was to elucidate whether liver cell transplants at an ectopic site are also subject to this influence. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant recipients and age-matched controls were treated with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the solvents and sacrificed 24 or 48 h thereafter. Livers and intrasplenic transplants were evaluated for the expression of the P450 subtypes 1A1, 2B1, 2E1, 3A2 and 4A1 by means of immunohistochemistry. The livers of both male and female rats displayed nearly no P450 1A1, but a distinct P450 2B1, 2E1, 3A2 and 4A1 expression. Whereas no sex differences were seen in the P450 1A1 expression, the immunostaining for P450 2B1, 3A2 and 4A1 was stronger in males and that for P450 2E1 in females. Similarly, in the intrasplenic liver cell transplants almost no P450 1A1, but a noticeable P450 2B1, 2E1, 3A2 and 4A1 expression was observed. Like in the respective livers, the immunostaining for P450 2B1, 3A2 and 4A1 was stronger in the transplants hosted by male than by female rats, whereas the opposite was the case for the P450 2E1 expression. Both in livers and transplants with some sex-specific differences P450 1A1 and 2E1 expression was induced by BNF, that of P450 2B1 by BNF and PB, and that of P450 3A2 by PB and DEX. These results indicate that the P450 system of ectopically transplanted liver cells is influenced by the gender of the recipient organism like that of the orthotopic livers.

  18. Evaluation of the assumptions of an ontogeny model of rat hepatic cytochrome P450 activity.

    PubMed

    Alcorn, Jane; Elbarbry, Fawzy A; Allouh, Mohammed Z; McNamara, Patrick J

    2007-12-01

    We previously reported an ontogeny model of hepatic cytochrome P450 (P450) activity that predicts in vivo P450 elimination from in vitro intrinsic clearance. The purpose of this study was to conduct investigations into key assumptions of the P450 ontogeny model using the developing rat model system. We used two developmentally dissimilar enzymes, CYP2E1 and CYP1A2, and male rats (n = 4) at age groups representing critical developmental stages. Total body and liver weights and hepatic microsomal protein contents were measured. Following high-performance liquid chromatography analysis, apparent K(M) and V(max) estimates were calculated using nonlinear regression analysis for CYP2E1- and CYP1A2-mediated chlorzoxazone 6-hydroxylation and methoxyresorufin O-dealkylation, and V(max) estimates for p-nitrophenol and phenacetin hydroxylations, respectively. Hepatic scaling factors and V(max) values provided estimates for infant scaling factors (ISF). The data show microsomal protein contents increased with postnatal age and reached adult values after postnatal day (PD) 7. Apparent K(M) values were similar at all developmental stages except at < or =PD7. Developmental increases in probe substrate V(max) values did not correlate with the biphasic increase in immunoquantifiable P450. The activity of two different probe substrates for each P450 covaried as a function of age. A plot of observed ISF values as a function of age reflected the developmental pattern of rat hepatic P450. In summation, these observations diverge from several of the model's assumptions. Further investigations are required to explain these inconsistencies and to investigate whether the developing rat may provide a predictive paradigm for pediatric risk assessment for P450-mediated elimination processes.

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

    SciTech Connect

    Stiborova, Marie Arlt, Volker M.; Henderson, Colin J.; Wolf, C. Roland; Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri; Phillips, David H.; Frei, Eva

    2008-02-01

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

  20. HEPATIC CYTOCHROME P450 UBIQUITINATION: CONFORMATIONAL PHOSPHODEGRONS FOR E2/E3 RECOGNITION?

    PubMed Central

    Correia, Maria Almira; Wang, YongQiang; Kim, Sung-Mi; Guan, Shenheng

    2014-01-01

    Hepatic endoplasmic reticulum (ER) integral cytochromes P450 (P450s) are monooxygenases engaged in the biotransformation and elimination of endo- as well as xenobiotics. Of the human liver P450s, CYP3A4 is the major and most dominant catalyst, responsible for the biotransformation of over 50% of clinically prescribed drugs. CYP2E1 metabolizes smaller molecular weight compounds (EtOH), carcinogens, environmental toxins and endobiotics, and is justly implicated in various toxigenic/pathogenic mechanisms of human disease. Both P450s are notorious for their potential to generate pathogenic reactive oxygen species (ROS) during futile oxidative cycling and/or oxidative uncoupling. Such ROS not only oxidatively damage the P450 catalytic cage, but on their escape into the cytosol, also the P450 outer surface and any surrounding cell organelles. Given their ER-monotopic topology coupled with this high potential to acquire oxidative lesions in their cytosolic (C) domain, not surprisingly these P450 proteins exhibit shorter lifespans and are excellent prototype substrates of ER-associated degradation (“ERAD-C”) pathway. Indeed, we have shown that both CYP3A4 and CYP2E1 incur ERAD-C, during which they are first phosphorylated by protein kinases A and C, which greatly enhance/accelerate their ubiquitination by UBC7/gp78 and UbcH5a/CHIP/Hsp70/Hsp40 E2/E3 ubiquitin ligase complexes. Such P450 phosphorylation occurs on Ser/Thr residues within linear sequences as well as spatially clustered acidic (Asp/Glu) residues. We propose that such S/T phosphorylation within these clusters creates a negatively charged patch i.e. conformational phosphodegrons, for interaction with positively charged E2/E3 domains. Such P450 S/T phosphorylation we posit serves as a switch to turn on its ubiquitination and ERAD-C. PMID:24488826

  1. Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450

    SciTech Connect

    Wyman, J.F.; Gollan, J.L.; Settle, W.; Farrell, G.C.; Correia, M.A.

    1986-01-01

    After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglogin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that heamoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of (tritium) haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic free haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.

  2. 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.

  3. 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...

  4. 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.

  5. Cytochrome P450 isoforms catalyze formation of catechol estrogen quinones that react with DNA.

    PubMed

    Zhang, Yan; Gaikwad, Nilesh W; Olson, Kevin; Zahid, Muhammad; Cavalieri, Ercole L; Rogan, Eleanor G

    2007-07-01

    Accumulating evidence suggests that specific metabolites of estrogens, namely, catechol estrogen quinones, react with DNA to form adducts and generate apurinic sites, which can lead to the mutations that induce breast cancer. Oxidation of estradiol (E(2)) produces 2 catechol estrogens, 4-hydroxyestradiol (4-OHE(2)) and 2-OHE(2) among the major metabolites. These, in turn, are oxidized to the quinones, E(2)-3,4-quinone (E(2)-3,4-Q) and E(2)-2,3-Q, which can react with DNA. Oxidation of E(2) to 2-OHE(2) is mainly catalyzed by cytochrome P450 (CYP) 1A1, and CYP3A4, whereas oxidation of E(2) to 4-OHE(2) in extrahepatic tissues is mainly catalyzed by CYP1B1 as well as some CYP3As. The potential involvement of CYP isoforms in the further oxidation of catechols to semiquinones and quinones has, however, not been investigated in detail. In this project, to identify the potential function of various CYPs in oxidizing catechol estrogens to quinones, we used different recombinant human CYP isoforms, namely, CYP1A1, CYP1B1, and CYP3A4, with the scope of oxidizing the catechol estrogens 2-OHE(2) and 4-OHE(2) to their respective estrogen quinones, which then reacted with DNA. The depurinating adducts 2-OHE(2)-6-N3Ade, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua were observed in the respective reaction systems by ultraperformance liquid chromatography/tandem mass spectrometry. Furthermore, more than 100-fold higher levels of estrogen-glutathione (GSH) conjugates were detected in the reactions. Glutathione conjugates were observed, in much smaller amounts, when control microsomes were used. Depurinating adducts, as well as GSH conjugates, were obtained when E(2)-3,4-Q was incubated with CYP1B1 or control microsomes in a 30-minute reaction, further demonstrating that GSH is present in these recombinant enzyme preparations. These experiments demonstrated that CYP1A1, CYP1B1, and CYP3A4 are able to oxidize catechol estrogens to their respective quinones, which can further react with GSH

  6. 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.

  7. Methodologies for investigating drug metabolism at the early drug discovery stage: prediction of hepatic drug clearance and P450 contribution.

    PubMed

    Emoto, Chie; Murayama, Norie; Rostami-Hodjegan, Amin; Yamazaki, Hiroshi

    2010-10-01

    /or liver microsomes is an integral part to predicting hepatic clearance; therefore, the prediction methods for protein binding have been addressed in terms of automation and in silico prediction. The approach to reaction phenotyping using recombinant P450 microsome data are reviewed as this approach enables combining the drug depletion method with appropriate scaling factors to predict clearance values. CYP3A enzymes have broad substrate specificities and are responsible for the oxidative metabolism of more than 50% of clinically used drugs. Although CYP3A4 is the most abundant CYP3A isoform in adult human liver, CYP3A5 may contribute more to CYP3A-mediated drug oxidation by human liver microsomes than CYP3A4 does, especially in Japanese subjects, who typically have a relatively high frequency of genetic CYP3A5 expression. Lack of efficacy and presence of serious side effects in some sub-group of patients remain the biggest sources of drug failure at late stage of drug development. Advances in appreciation of inter-individual variabilities in ADME, by creation of virtual individuals and use of appropriate information from early discovery may lead to a better anticipation of variable clinical and toxicological outcome following administration of any new drug candidate. Thus may also help with dosing strategies which minimize the potential side effects and maximize the clinical benefits. Accordingly, front-loading of efforts for characterizing the candidate drugs at early stages of discovery is recommended.

  8. 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.

  9. Strain differences in hepatic cytochrome P450 1A and 3A expression between Sprague-Dawley and Wistar rats.

    PubMed

    Kishida, Tomoyuki; Muto, Shin-ichi; Hayashi, Morimichi; Tsutsui, Masaru; Tanaka, Satoru; Murakami, Makoto; Kuroda, Junji

    2008-10-01

    Expression of hepatic cytochrome P450 (CYP) isoforms was compared in Sprague-Dawley (SD) and Wistar (WI) rats, which are commonly used strains in preclinical studies. Basal CYP1A1, CYP1A2, and CYP3A2 mRNA levels were higher in WI rats than in SD rats (by 8-, 3- and 2-fold, respectively). Treatment with phenobarbital, a potent CYP inducer, increased the predominance of expression of these three mRNAs in WI rats (by 26-, 4-, and 2-fold, respectively) along with the predominance of increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular hepatocytes. CYP1A enzymatic activity was also higher in WI rats than in SD rats. No strain differences were observed in phenobarbital induction of CYP2B1/2, CYP2C6, or CYP3A1. CYP3A2 mRNA was more strongly induced by dexamethasone, a typical inducer of CYP3A, together with CYP3A1 mRNA, in WI rats than in SD rats (by 2-fold), whereas the CYP1A1 and CYP1A2 mRNA expression induced by beta-naphtoflavone, a typical inducer of CYP1A, did not differ between the two strains. Furthermore, WI rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor mRNAs, responsible for CYP1A or CYP3A induction, with phenobarbital or dexamethasone induction. In conclusion, significant, predominant expression of hepatic CYP1A and CYP3A mRNAs in WI rats was observed, possibly related to nuclear receptor-mediated induction. Considering the pharmacokinetic and toxicological importance of CYP1A and CYP3A, different outcomes might arise depending on the rat strains used in preclinical studies of drugs metabolized typically or mainly by both isoforms.

  10. 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

  11. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1.

    PubMed

    Aoyama, T; Yamano, S; Guzelian, P S; Gelboin, H V; Gonzalez, F J

    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 B1 to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B1 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 B1 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, IIIA5, and IVB1, did not significantly activate aflatoxin B1 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 B1 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 B1 in human liver involves the contribution of multiple forms of P450. PMID:2162057

  12. TLR4-Dependent and -Independent Regulation of Hepatic Cytochrome P450 in Mice with Chemically-Induced Inflammatory Bowel Disease

    PubMed Central

    Chaluvadi, Madhusudana R.; Nyagode, Beatrice A.; Kinloch, Ryan D.; Morgan, Edward T.

    2010-01-01

    The transcription and protein expression of many cytochrome P450 (P450) genes are down-regulated in animal models of inflammation and infection. We determined previously that hepatic P450 mRNAs are selectively regulated in a mouse model of enteropathogenic bacterial infection, and that this regulation was not dependent on the lipopolysaccharide (LPS) receptor protein toll-like receptor 4 (TLR4). In the dextran sulfate sodium (DSS) model of chemically-induced inflammatory bowel disease (IBD), the reduction in activities of several hepatic P450 enzymes were concluded to be partially dependent on LPS from commensal bacteria (Masubuchi Y and Horie T (2004) Drug Metab. Dispos. 32: 437–441). In the present study, we sought to determine whether colitis induced by LPS regulates hepatic P450 mRNA and protein expression similarly to infectious colitis, and to determine the role of TLR4 in the response to DSS colitis. The role of LPS in the response to DSS was further examined by comparison with the effects of injected LPS. We demonstrate that administration of DSS results in the down-regulation of multiple P450 enzymes in mouse liver. However, there are discernable differences in the pattern of P450 expression in the two models. Some effects of DSS-induced colitis are TLR4-dependent, and others are not. In contrast, the effects of injected LPS on hepatic P450 mRNA expression are entirely TLR4-dependent. Thus, our results indicate that the pattern of hepatic P450 expression, and the mechanism of regulation, during inflammation of the bowel depend on the etiology of the disease. PMID:19027721

  13. Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos.

    PubMed

    Joo, Hyun; Choi, Kyoungju; Rose, Randy L; Hodgson, Ernest

    2007-01-01

    Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.

  14. 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.

  15. Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes.

    PubMed

    Lehman-McKeeman, L D; Caudill, D; Vassallo, J D; Pearce, R E; Madan, A; Parkinson, A

    1999-12-20

    The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-Mc-Keeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264-271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

  16. Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

    PubMed

    Stringer, Rowan A; Ferreira, Suzie; Rose, Jonathan; Ronseaux, Sebastien

    2016-08-01

    The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor.

  17. Downregulation of Mouse Hepatic CYP3A Protein by 3-Methylcholanthrene Does Not Require Cytochrome P450-Dependent Metabolism

    PubMed Central

    Lee, Chunja; Ding, Xinxin

    2013-01-01

    The aryl hydrocarbon receptor (AHR)–dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity. PMID:23846873

  18. Coleus forskohlii extract induces hepatic cytochrome P450 enzymes in mice.

    PubMed

    Virgona, Nantiga; Yokotani, Kaori; Yamazaki, Yuko; Shimura, Fumio; Chiba, Tsuyoshi; Taki, Yuko; Yamada, Shizuo; Shinozuka, Kazumasa; Murata, Masatsune; Umegaki, Keizo

    2012-03-01

    Coleus forskohlii root extract (CFE) is popular for use as a weight loss dietary supplement. In this study, the influence of standardized CFE containing 10% active component forskolin on the hepatic drug metabolizing system was investigated to evaluate the safety through its drug interaction potential. Male ICR mice were fed AIN93G-based diets containing 0-5% CFE or 0.05% pure forskolin for 2-3 weeks. Intake of two different sources of 0.5% CFE significantly increased the relative liver weight, total content of hepatic cytochrome P450 (CYP) and induced CYPs (especially 2B, 2C, 3A types) and glutathione S-transferase (GST) activities. CFE significantly increased mRNA expression of CYPs and GST with dose related responses. However, unlike the CFE, intake of 0.05% pure forskolin was found to be associated with only weak induction in CYP3A and GST activities with no significant increases in relative liver weight, total hepatic content or other CYPs activities. The inductions of CYPs and GST by CFE were observed at 1 week of feeding and rapidly recovered by discontinuation of CFE. These results indicated the induction potential of CFE on CYPs, and that this effect was predominantly due to other, as yet unidentified constituents, and not forskolin contained in CFE. PMID:22178802

  19. 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.

  20. Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whale (Delphinapterus leucas).

    PubMed

    White, R D; Hahn, M E; Lockhart, W L; Stegeman, J J

    1994-05-01

    Understanding the effects of environmental contaminants on cetaceans and other marine mammals will require information on the biochemistry of xenobiotic metabolism in these species. We characterized the hepatic microsomal cytochrome P450 system in beluga whales (Delphinapterus leucas) from the Canadian Arctic. The content of native P450 averaged 0.203 and 0.319 nmol/mg microsomal protein, cytochrome b5 content averaged 0.199 and 0.236 nmol/mg, and rates of NADPH-cytochrome c reductase were 79 and 76 nmol/min/mg, for females and males respectively. Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), and benzo[a]pyrene (BP) hydroxylase (AHH) activities were significantly greater in males than in females, and were highly correlated with one another (r2 between 0.853 and 0.912). HPLC analysis of in vitro BP metabolites revealed benzo-ring (7,8- and 9,10-) dihydrodiols, consistent with activation of this compound, as well as 4,5-dihydrodiol,3-OH-, 7-OH-, and 9-OH-BP and 1,6- and 3,6-quinones. Estradiol 2-hydroxylase activity did not differ between sexes, and rates did not correlate with those of the other activities. Antibodies against scup P450B (an apparent teleost CYP2B) and rat CYP2B1 did not recognize proteins in beluga liver microsomes, but there was a protein detected by antibodies to PB-inducible rabbit CYP2B4. Antibodies to ethanol and ketone-inducible rat CYP2E1 reacted with two proteins in beluga liver microsomes. Antibodies specific to hydrocarbon-inducible CYP1A1 and/or CYP1A2 forms showed a single protein band, apparently more closely related to CYP1A1. The content of CYP1A was fivefold greater in male than in female beluga. CYP1A content was highly correlated with EROD, PROD, and AHH activities, suggesting that this P450 form is a primary catalyst for these reactions in beluga. CYP1A content and activity were highly correlated with the concentrations in blubber of non-ortho and mono-ortho PCB congeners, compounds that induce CYP1A

  1. Species differences in intestinal metabolic activities of cytochrome P450 isoforms between cynomolgus monkeys and humans.

    PubMed

    Nishimuta, Haruka; Sato, Kimihiko; Mizuki, Yasuyuki; Yabuki, Masashi; Komuro, Setsuko

    2011-06-01

    The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans. PMID:21383522

  2. Hepatic expression of cytochrome P450 in Zucker diabetic fatty rats.

    PubMed

    Park, So Young; Kim, Chung Hyeon; Lee, Ji Yoon; Jeon, Jang Su; Kim, Min Ju; Chae, Song Hee; Kim, Hyoung Chin; Oh, Soo Jin; Kim, Sang Kyum

    2016-10-01

    In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1'- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.

  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. Regulation of porcine hepatic cytochrome p450 - implication for boar taint.

    PubMed

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia

    2014-09-01

    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

  5. Fasting-Induced Changes in Hepatic P450 Mediated Drug Metabolism Are Largely Independent of the Constitutive Androstane Receptor CAR

    PubMed Central

    de Vries, E. M.; Lammers, L. A.; Achterbergh, R.; Klümpen, H-J; Mathot, R. A. A.; Boelen, A.; Romijn, J. A.

    2016-01-01

    Introduction Hepatic drug metabolism by cytochrome P450 enzymes is altered by the nutritional status of patients. The expression of P450 enzymes is partly regulated by the constitutive androstane receptor (CAR). Fasting regulates the expression of both P450 enzymes and CAR and affects hepatic drug clearance. We hypothesized that the fasting-induced alterations in P450 mediated drug clearance are mediated by CAR. Methods To investigate this we used a drug cocktail validated in humans consisting of five widely prescribed drugs as probes for specific P450 enzymes: caffeine (CYP1A2), metoprolol (CYP2D6), omeprazole (CYP2C19), midazolam (CYP3A4) and s-warfarin (CYP2C9). This cocktail was administered to wild type (WT, C57Bl/6) mice or mice deficient for CAR (CAR-/-) that were either fed ad libitum or fasted for 24 hours. Blood was sampled at predefined intervals and drug concentrations were measured as well as hepatic mRNA expression of homologous/orthologous P450 enzymes (Cyp1a2, Cyp2d22, Cyp3a11, Cyp2c37, Cyp2c38 and Cyp2c65). Results Fasting decreased Cyp1a2 and Cyp2d22 expression and increased Cyp3a11 and Cyp2c38 expression in both WT and CAR-/- mice. The decrease in Cyp1a2 was diminished in CAR-/- in comparison with WT mice. Basal Cyp2c37 expression was lower in CAR-/- compared to WT mice. Fasting decreased the clearance of all drugs tested in both WT and CAR-/- mice. The absence of CAR was associated with an decrease in the clearance of omeprazole, metoprolol and midazolam in fed mice. The fasting-induced reduction in clearance of s-warfarin was greater in WT than in CAR-/-. The changes in drug clearance correlated with the expression pattern of the specific P450 enzymes in case of Cyp1a2-caffeine and Cyp2c37-omeprazole. Conclusion We conclude that CAR is important for hepatic clearance of several widely prescribed drugs metabolized by P450 enzymes. However the fasting-induced alterations in P450 mediated drug clearance are largely independent of CAR. PMID

  6. Effects of portal vein ligation on sex hormone metabolism in male rats: relationship to lowered hepatic cytochrome P450 levels.

    PubMed

    Farrell, G C; Koltai, A; Zaluzny, L; Murray, M

    1986-02-01

    Hepatic cytochrome P450 levels in male rats fall after portal vein ligation, a procedure that produces total hepatic bypass of portal blood. The present study was undertaken to examine whether changes in sex hormone metabolism could account for these lowered cytochrome P450 levels. Portal vein ligation resulted in testicular atrophy and low serum testosterone concentrations. Serum luteinizing hormone levels were also reduced, suggesting that testicular atrophy was secondary to suppression of the hypothalamic-pituitary-gonadal axis. Serum estrone and estradiol concentrations were significantly increased after portal vein ligation, while the magnitude and delayed onset of increases in urinary total estrogen excretion suggested that this was due largely to increased estrogen production. In male rats, both castration (at 12 wk) and exogenous estrogen administration resulted in changes in hepatic cytochrome P450 levels and ethylmorphine N-demethylase activity that were qualitatively similar to those seen after portal vein ligation. In female and castrated male rats, however, cytochrome P450 was not affected by portal vein ligation. Testosterone supplementation corrected the changes of cytochrome P450 levels in castrated male rats but did not have this effect in portal vein-ligated male rats. It is concluded that changes in sex hormone metabolism do occur after portal vein ligation and may contribute to alterations in cytochrome P450 and drug-metabolizing enzyme activity. Decreased levels of serum testosterone, however, do not alone account for the changes in hepatic drug metabolism in this model, and suppression of a hypothalamic-pituitary factor appears to be important.

  7. Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s.

    PubMed

    Lai, T S; Chiang, J Y

    1990-01-01

    We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test. PMID:2126562

  8. Absence of hepatic cytochrome P450bufI causes genetically deficient debrisoquine oxidation in man

    SciTech Connect

    Zanger, U.M.; Vilbois, F.; Hardwick, J.P.; Meyer, U.A.

    1988-07-26

    The common genetic deficiency of drug oxidation known as debrisoquine/sparteine-type polymorphism was investigated with bufuralol as prototype substrate. In human liver microsomes the 1'-hydroxylation of bufuralol is catalyzed by two functionally distinct P-450 isozymes, the high-affinity/highly stereoselective P450bufI and the low-affinity/nonstereoselective P450bufII. The authors demonstrate that P450bufI is unique in hydroxylating bufuralol in a cumene hydroperoxide (CuOOH) mediated reaction whereas P450bufII is active only in the classical NADPH- and O/sub 2/-supported monooxygenation. In microsomes of liver biopsies of in vivo phenotyped poor metabolizers of debrisoquine or sparteine, the CuOOH-mediated activity was drastically reduced. Rabbit antibodies against a rat P-450 isozyme with high bufuralol 1'-hydroxylase activity (P450db1) precipitated exclusively P450bufI-type activity from solubilized microsomes. Western blotting of microsomes with these antibodies revealed a close correlation between the immunoreactive protein and CuOOH-mediated (+)-bufuralol 1'-hydroxylation. No immunoreactive protein was detected in liver microsomes of in vivo phenotyped poor metabolizers. These data provide evidence for a specific deficiency of P450bufI and are consistent with the complete or almost complete absence of this protein in the liver of poor metabolizers.

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

    EPA Science Inventory

    Abstract

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

  10. 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.

  11. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

    PubMed

    Russell, Julie S; Halbrook, Richard S; Woolf, Alan; French, John B; Melancon, Mark J

    2004-08-01

    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 beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF 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. PMID:15352474

  12. Competition between hydrocarbon and barbiturate for spectral binding to hepatic cytochrome P-450

    SciTech Connect

    Backes, W.L.; Means, M.; Canady, W.J.

    1984-08-25

    The substrates hexobarbital and ethylbenzene have been shown to compete for the spectral binding site of phenobarbital-induced rat hepatic microsomal cytochrome P-450. Over the concentration ranges studied, there is no change in the availability of the enzyme as a result of substrate addition; the difference in ..delta..Abs/sub max/ apparently being due to varying abilities of different substrates to bring about a spin shift in the enzyme. Evidence is presented to indicate that differences between enzymes from untreated male rats and phenobarbital-treated male rats are attributable to differences in the enzyme itself and not to changes in the nature of the membrane brought about by phenobarbital administration, at least insofar as heat entropy compensation is concerned. The enthalpy-entropy compensation observed in the binding of a homologous series of barbiturates to the microsomal membrane as determined from the membrane concentration dependence of their binding constants is shown to agree surprisingly well with the direct determination performed by Sitar and Mannering.

  13. Effect of cefixime and cefdinir, oral cephalosporins, on cytochrome P450 activities in human hepatic microsomes.

    PubMed

    Niwa, Toshiro; Shiraga, Toshifumi; Hashimoto, Tomoko; Kagayama, Akira

    2004-01-01

    The effects of two kinds of oral cephalosporins, cefixime and cefdinir, on cytochrome P450 (CYP) activities in human hepatic microsomes were investigated. Both cefixime and cefdinir at 2 mM concentration neither inhibited nor stimulated CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, or CYP3A4-mediated testosterone 6beta-hydroxylation. The free fractions of cefixime and cefdinir in the incubation mixture, which were measured by ultracentrifugation, were 86.1-93.8% and 94.1-97.8%, respectively. These results suggest that both cefixime and cefdinir would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.

  14. Inhibitory effect of several fluoroquinolones on hepatic microsomal cytochrome P-450 1A activities in dogs.

    PubMed

    Regmi, N L; Abd El-Aty, A M; Kuroha, M; Nakamura, M; Shimoda, M

    2005-12-01

    We examined inhibitory effects of ofloxacin (OFX), orbifloxacin (OBFX), ciprofloxacin (CFX), enrofloxacin (EFX) and norfloxacin (NFX) on cytochrome P-450 1A (CYP1A) activities using hepatic microsomes from four beagle dogs. Ethoxyresorufin O-de-ethylation was referred as CYP1A activities. All the fluoroquinolones inhibited the reaction in a noncompetitive manner. The determined inhibitory constants were the followings; 10.1 +/- 3.8 mM for OFX, 6.43 +/- 2.01 mM for OBFX, 0.726 +/- 0.134 mM for CFX, 4.06 +/- 1.19 mM for EFX and 4.75 +/- 1.63 mM for NFX respectively. As these values are >100-fold of plasma concentrations after a clinical single dose of the fluoroquinolones, it is suggested that the inhibitory effect on CYP1A activities is not so high to elicit drug-drug interaction with CYP1A substrates, when these fluoroquinolones are co-administered. Mechanism based inhibition was also examined in this study. Of the five fluoroquinolones examined, OFX, OBFX and CFX had this inhibition manner. As this inhibition is irreversible, inhibitory effects of the three fluoroquinolones may accumulate, when they are repeatedly administered. Therefore, OFX, OBFX and CFX may result in substantial drug-drug interaction with a CYP1A substrate even in clinical states. As EFX is metabolized to CFX in the body, it may also have the same possibility.

  15. Identification of cytochrome P450 isoform involved in the metabolism of YM992, a novel selective serotonin re-uptake inhibitor, in human liver microsomes.

    PubMed

    Noguchi, K; Mera, A; Watanabe, T; Higuchi, S; Chiba, K

    2000-05-01

    1. In vitro studies were conducted to identify the hepatic cytochrome P450 isoform involved in the metabolism of YM992, ((S)-2-[[(fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride), a novel serotonin re-uptake inhibitor, in human liver microsomes. 2. Microsomes prepared from yeast expressing CYP1A1, CYP1A2 and CYP2D6 effectively metabolized YM992. A significant correlation was observed between the rate of YM992 metabolism and 7-ethoxyresorufin O-deethylation, CYP1A1/2 specific activity, in liver microsomes from 16 individual donors (r2 = 0.628, p < 0.001). Alpha-naphtoflavone and isosafrole, CYP1A1/2 inhibitors, suppressed the metabolism of YM992 in human liver microsomes in a concentration-dependent manner. 3. The metabolism of YM992 in human liver microsomes was inhibited by approximately 95% by antibodies which recognize both CYP1A1 and CYP1A2 whereas antibodies specific for CYP1A1 did not show inhibitory effects. 4. The same major metabolites, M6 and M7, were generated from YM992 after incubation with human liver microsomes and recombinant human CYP1A2. 5. These results suggest that the metabolism of YM992 in human liver microsomes is mainly catalysed by CYP1A2, and that YM992 might increase plasma concentration of concomitant drugs metabolized by CYP1A2 due to competitive inhibition.

  16. Mechanism of chloroform-induced renal toxicity: Non-involvement of hepatic cytochrome P450-dependent metabolism

    SciTech Connect

    Fang Cheng; Behr, Melissa; Xie Fang; Lu Shijun; Doret, Meghan; Luo Hongxiu; Yang Weizhu; Aldous, Kenneth; Ding Xinxin; Gu Jun

    2008-02-15

    Chloroform causes hepatic and renal toxicity in a number of species. In vitro studies have indicated that chloroform can be metabolized by P450 enzymes in the kidney to nephrotoxic intermediate, although direct in vivo evidence for the role of renal P450 in the nephrotoxicity has not been reported. This study was to determine whether chloroform renal toxicity persists in a mouse model with a liver-specific deletion of the P450 reductase (Cpr) gene (liver-Cpr-null). Chloroform-induced renal toxicity and chloroform tissue levels were compared between the liver-Cpr-null and wild-type mice at 24 h following differing doses of chloroform. At a chloroform dose of 150 mg/kg, the levels of blood urea nitrogen (BUN) were five times higher in the exposed group than in the vehicle-treated one for the liver-Cpr-null mice, but they were only slightly higher in the exposed group than in the vehicle-treated group for the wild-type mice. Severe lesions were found in the kidney of the liver-Cpr-null mice, while only mild lesions were found in the wild-type mice. At a chloroform dose of 300 mg/kg, severe kidney lesions were observed in both strains, yet the BUN levels were still higher in the liver-Cpr-null than in the wild-type mice. Higher chloroform levels were found in the tissues of the liver-Cpr-null mice. These findings indicated that loss of hepatic P450-dependent chloroform metabolism does not protect against chloroform-induced renal toxicity, suggesting that renal P450 enzymes play an essential role in chloroform renal toxicity.

  17. Effects of restricted feeding on daily fluctuations of hepatic functions including p450 monooxygenase activities in rats.

    PubMed

    Hirao, Jun; Arakawa, Shingo; Watanabe, Kyoko; Ito, Kazumi; Furukawa, Tadashi

    2006-02-10

    Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.

  18. Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures.

    PubMed

    Lemley, C O; Wilson, M E

    2010-10-01

    Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and

  19. Thalidomide increases human hepatic cytochrome P450 3A enzymes by direct activation of the pregnane X receptor.

    PubMed

    Murayama, Norie; van Beuningen, Rinie; Suemizu, Hiroshi; Guguen-Guillouzo, Christiane; Shibata, Norio; Yajima, Kanako; Utoh, Masahiro; Shimizu, Makiko; Chesné, Christophe; Nakamura, Masato; Guengerich, F Peter; Houtman, René; Yamazaki, Hiroshi

    2014-02-17

    Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ( ( 2013 ) Chem. Res. Toxicol. 26 , 486 - 489 ) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.

  20. Suppressive effect of accumulated aluminum trichloride on the hepatic microsomal cytochrome P450 enzyme system in rats.

    PubMed

    Zhu, Yanzhu; Han, Yanfei; Zhao, Hansong; Li, Jing; Hu, Chongwei; Li, Yanfei; Zhang, Zhigang

    2013-01-01

    Aluminum (Al) is a low toxicological metal and can accumulate in the liver. The hepatic microsomal cytochrome P450 enzyme system (CYPS) plays important role in the transformation of the toxic materials. It is not clear if the CYPS is affected by Al exposure. Thus, the aim of this study is to investigate the effects of aluminum trichloride (AlCl(3)) on CYPS in rats. Forty male Wistar rats (5weeks old) weighing 110-120g were randomly allocated and orally exposed to 0, 64.18, 128.36 and 256.72mg/kg body weight (BW) AlCl(3) in drinking water for 120days. The body weight (BW) of rats, hepatosomatic index (HSI), hepatic Al content, the concentrations of cytochrome P450 (CYP450), cytochrome B5 (B5), microsomal protein and the activities of NADPH-cytochrome c reductase (CR), aminopyrin N-demethylase (AND), erythromycin N-demethylase (ERND) and aniline-4-hydeoxylase (AH) were assessed at the end of the experiment. The results showed that the increase in Al concentration decreased BW, HIS, concentrations of CYP450, B5, microsomal protein and the activity of CR, AND, ERND and AH in hepatic microsomes. The results revealed that exposure to AlCl(3) inhibited the microsomal CYP450 dependent enzyme system of liver. Our findings suggest that long term daily exposure of AlCl(3) exerts the suppressive effects and thus may cause dysfunction of hepatic CYP450 dependent enzyme system of rat.

  1. Comparative use of isolated hepatocytes and hepatic microsomes for cytochrome P450 inhibition studies: transporter-enzyme interplay.

    PubMed

    Brown, Hayley S; Wilby, Alison J; Alder, Jane; Houston, J Brian

    2010-12-01

    Accurate assignment of the concentration of victim drug/inhibitor available at the enzyme active site, both in vivo and within an in vitro incubation, is an essential requirement in rationalizing and predicting drug-drug interactions. Inhibitor accumulation within the liver, whether as a result of active transport processes or intracellular binding, may best be accounted for using hepatocytes rather than hepatic microsomes to estimate in vitro inhibitory potency. The aims of this study were to compare K(i) values determined in rat liver microsomes and freshly isolated rat hepatocytes of four cytochrome P450 (P450) inhibitors (clarithromycin, enoxacin, nelfinavir, and saquinavir) with known hepatic transporter involvement and a range of uptake (cell/medium concentration ratios 20-3000) and clearance (10-1200 μl/min/10(6) cells) properties. Inhibition studies were performed using two well established P450 probe substrates (theophylline and midazolam). Comparison of unbound K(i) values showed marked differences between the two in vitro systems for inhibition of metabolism. In two cases (clarithromycin and enoxacin, both low-clearance drugs), inhibitory potency in hepatocytes markedly exceeded that in microsomes (10- to 20-fold), and this result was consistent with their high cell/medium concentration ratios. For nelfinavir and saquinavir (high-clearance, extensively metabolized drugs), the opposite trend was seen in the K(i) values: despite very high cell/medium concentration ratios, stronger inhibition was evident within microsomal preparations. Hence, the consequences of hepatic accumulation resulting from uptake transporters vary according to the clearance of the inhibitor. This study demonstrates that transporter-enzyme interplay can result in differences in inhibitory potency between microsomes and hepatocytes and hence drug-drug interaction predictions that are not always intuitive.

  2. Characterization of the effects of musk ketone on mouse hepatic cytochrome P450 enzymes.

    PubMed

    Stuard, S B; Caudill, D; Lehman-McKeeman, L D

    1997-12-01

    Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/ kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later

  3. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection.

    PubMed

    Kawakami, Hirotaka; Ohtsuki, Sumio; Kamiie, Junichi; Suzuki, Takashi; Abe, Takaaki; Terasaki, Tetsuya

    2011-01-01

    Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10  fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism. PMID:20564338

  4. 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

  5. Genistein decreases basal hepatic cytochrome P450 1A1 protein expression and activity in Swiss Webster mice.

    PubMed

    Froyen, Erik B; Steinberg, Francene M

    2016-05-01

    Soy consumption has been associated with risk reduction for chronic diseases such as cancer. One proposed mechanism for cancer prevention by soy is through decreasing cytochrome P450 1A1 (Cyp1a1) activity. However, it is not known with certainty which soy components modulate Cyp1a1, or the characteristics or mechanisms involved in the responses after short-term (<20 days) dietary treatment without concomitant carcinogen-mediated induction. Therefore, the objective was to test the hypothesis that physiologic concentrations of dietary genistein and/or daidzein will decrease basal hepatic Cyp1a1 protein expression and activity in male and female Swiss Webster mice via inhibiting the bindings of aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (ARNT) and estrogen receptor-α to the Cyp1a1 promoter region xenobiotic response element. The mice were fed the AIN-93G diet supplemented with 1500 mg/kg of genistein or daidzein for up to 1 week. Genistein, but not daidzein, significantly decreased basal hepatic microsomal Cyp1a1 protein expression and activity. AhR protein expression was not altered. Molecular mechanisms were investigated in Hepa-1c1c7 cells treated with 5 μmol/L purified aglycones genistein, daidzein, or equol. Cells treated with genistein exhibited inhibitions in ARNT and estrogen receptor-α bindings to the Cyp1a1 promoter region. This study demonstrated that genistein consumption reduced constitutive hepatic Cyp1a1 protein expression and activity, thereby contributing to the understanding of how soy isoflavone aglycones modulate cytochrome P450 biotransformation enzymes.

  6. Comparative hepatic cytochrome P450 activities and contaminant concentrations in caged carp and juvenile ducks

    SciTech Connect

    O`Keefe, P.; Gierthy, J.; Connor, S.; Bush, B.; Hong, C.S.; Wood, L.; Clayton, W.; Storm, R.

    1995-12-31

    Juvenile carp (Cyprinius carpio) weighing approx. 60 g were placed in cages located on the surface of sediments near an aluminum plant and an automobile parts plant in the Massena area of the St. Lawrence River. Fish were removed at weekly intervals over a 35 day exposure period and composited samples of liver tissue, cranial lipid, and fillet tissue were prepared for analysis of polynuclear aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs). Liver tissue was also stored at {minus}80 C for determination of microsomal Cytochrome P450 activity using the aryl hydrocarbon hydroxylase (AHH) assay. A control exposure was carried out upstream at an uncontaminated site. Juvenile pre-flight ducks (mallards, gadwalls, wood ducks and common mergansers) were collected in the contaminated areas on the St. Lawrence and on the Hudson River two to three months after hatching. Control pre-flight mallards, wood ducks and common mergansers were collected from remote lakes in the Addirondack State Park. Samples of subcutaneous fat and liver tissue were removed for analysis as described above for the carp. There was a three fold increase in AHH activity in the carp liver tissue at the end of the 35 day exposure period and there was a similar increase it activity for the mallards, common mergansers and wood ducks compared to controls. For each species the enzyme activity increases will be compared to the contaminant concentrations.

  7. 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

  8. Avian Cytochrome P450 (CYP) 1-3 Family Genes: Isoforms, Evolutionary Relationships, and mRNA Expression in Chicken Liver

    PubMed Central

    Ikenaka, Yoshinori; Kawata, Minami; Ikushiro, Shin-Ichi; Sakaki, Toshiyuki; Ishizuka, Mayumi

    2013-01-01

    Cytochrome P450 (CYP) of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR) activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene. PMID:24098714

  9. Metabolic activation of acetylenic substituents to derivatives in the rat causing the loss of hepatic cytochrome P-450 and haem

    PubMed Central

    White, Ian N. H.

    1978-01-01

    1. A number of acetylenic-substituted steroidal and non-steroidal compounds, including 2,2-dipropargylacetamide, pregna-2,4-dien-20-yno[2,3-d]isoxazol-17-ol (Danazol) and acetylene gas, when administered to rats in vivo brought about a decrease in the concentrations of hepatic microsomal cytochrome P-450 and haem. Abnormal haem-breakdown products, `green pigments', and porphyrins accumulated in the livers of these animals. 2. For loss of microsomal cytochrome P-450 to occur in vitro, metabolic activation of the acetylenic substituent was necessary. The enzyme system responsible required NADPH and air, and was induced by pretreatment of rats with phenobarbitone; these are characteristics typical of the microsomal mixed-function oxidases. 3. When rats were dosed with 17α-ethynyl-17β-hydroxyandrost-4-en-3-one (ethynyltestosterone, 1mmol/kg) the pattern of green pigments extracted from the liver 4h after dosing and separated by t.l.c. was quite different from that in rats given 17β-hydroxy-17α-vinylandrost-4-en-3-one (vinyltestosterone), suggesting that reduction of the unsaturated triple bond to a double bond is not normally part of the metabolic activation pathway of the acetylenic substituent. 4. The green pigments extracted from the livers of rats 4h after the administration of the acetylenic-substituted compounds (1mmol/kg) when separated by silica-gel t.l.c. had variable RF values. The number and distribution of green pigments was characteristic for each compound examined. There was little correlation between the total loss of hepatic microsomal haem and the apparent intensity of the green pigments seen on the thin-layer chromatograms. 5. After incubation of [14C]acetylene in vitro with microsomal preparations from phenobarbitone-pretreated rats and a NADPH-generating system, no significant covalent binding to microsomal protein was detected over a 30min incubation period, although under similar conditions there was a significant loss of cytochrome P-450

  10. Cytochrome P450-2E1 promotes aging-related hepatic steatosis, apoptosis and fibrosis through increased nitroxidative stress.

    PubMed

    Abdelmegeed, Mohamed A; Choi, Youngshim; Ha, Seung-Kwon; Song, Byoung-Joon

    2016-02-01

    The role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in promoting aging-dependent hepatic disease is unknown and thus was investigated in this study. Young (7 weeks) and aged female (16 months old) wild-type (WT) and Cyp2e1-null mice were used in this study to evaluate age-dependent changes in liver histology, steatosis, apoptosis, fibrosis and many nitroxidative stress parameters. Liver histology showed that aged WT mice exhibited markedly elevated hepatocyte vacuolation, ballooning degeneration, and inflammatory cell infiltration compared to all other groups. These changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT mice although serum ALT and insulin resistance were not significantly altered. Aged WT mice showed the highest rates of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of mitochondrial nitroxidative stress and alteration of mitochondrial complex III and IV proteins in aged WT mice, although hepatic ATP levels seems to be unchanged. In contrast, the aging-related nitroxidative changes were very low in aged Cyp2e1-null mice. These results suggest that CYP2E1 is important in causing aging-dependent hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 could be a potential target for translational research in preventing aging-related liver disease. PMID:26703967

  11. Feed-drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens.

    PubMed

    Mátis, G; Kulcsár, A; Petrilla, J; Hermándy-Berencz, K; Neogrády, Zs

    2016-08-01

    The expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition-derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed-drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP-inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non-specific CYP-inducer, simultaneously with two different doses of intra-ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB-triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP-inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP-inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition.

  12. Hepatic and Renal Cytochrome P450 Gene Regulation During Citrobacter rodentium Infection in Wildtype and Toll-like Receptor 4 Mutant Mice

    PubMed Central

    Richardson, Terrilyn A.; Sherman, Melanie; Antonovic, Leposava; Kardar, Sean S.; Strobel, Henry W.; Kalman, Daniel; Morgan, Edward T.

    2005-01-01

    C. rodentium is the rodent equivalent of human enteropathogenic E. coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice (which lack functional toll-like receptor 4 [TLR4]) were infected with C. rodentium by oral gavage, and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16–55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4-dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5 and 3A13. Hepatic levels of IL-1β, IL-6, and TNFα mRNAs were significantly increased in infected HeOu mice, whereas only TNFα mRNA was significantly increased in HeJ mice. Hepatic α1-acid glycoprotein was induced in both groups, whereas α-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent, and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated. PMID:16339354

  13. Mapping of genes for cytochromes P-450b, P-450e, P-450g and P-450h in the rat

    SciTech Connect

    Rampersaud, A.; Walz, F.G. Jr.

    1987-05-01

    Inbred ACI, WF and RCS rats having characteristic markers for albino (c), hemoglobin ..beta..-chain (Hbb) and pink-eyed dilution (p) loci on chromosome l and expressing electrophoretic variants for hepatic cytochromes P-450b, P-450e and P-450h and a likely Cis-acting regulatory variant of P-450g were used in genetic mapping studies of these hemoproteins. Phenotypes for these microsomal cytochromes P-450 were analyzed using 2-D electrophoresis and the results of WF x (ACI x WF)fl and RCS x (WF x RCS)fl backcrosses revealed two gene clusters designated the P450-b,e and P450-g,h loci. The interval separating P450-b and P450-e was <1 centiMorgan (cM) and that separating P450-g from P450-h was, 3.7 cM at a 90% confidence level. P450-g,h is not linked with P450-b,e and the other markers tested on chromosome 1. The linkage map P450-b,e--p--c--Hbb on rat chromosome 1 was demonstrated and found to be congruent with Coh(P450-b,e)--p--c--Hbb on mouse chromosome 7. It appears that close genetic linkage, rather than common functional/regulatory properties, typify members of cytochrome P-450 subfamilies.

  14. Hepatic P450 enzyme activity, tissue morphology and histology of mink (Mustela vison) exposed to polychlorinated dibenzofurans.

    PubMed

    Moore, Jeremy N; Newsted, John L; Hecker, Markus; Zwiernik, Matthew J; Fitzgerald, Scott D; Kay, Denise P; Zhang, Xiaowei; Higley, Eric B; Aylward, Lesa L; Beckett, Kerrie J; Budinsky, Robert A; Bursian, Steven J; Giesy, John P

    2009-08-01

    Dose- and time-dependent effects of environmentally relevant concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) of 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of these two congeners on hepatic P450 enzyme activity and tissue morphology, including jaw histology, of adult ranch mink were determined under controlled conditions. Adult female ranch mink were fed either TCDF (0.98, 3.8, or 20 ng TEQ(TCDF)/kg bw/day) or PeCDF (0.62, 2.2, or 9.5 ng TEQ(PeCDF)/kg bw/day), or a mixture of TCDF and PeCDF (4.1 ng TEQ(TCDF)/kg bw/day and 2.8 ng TEQ(PeCDF)/kg bw/day, respectively) for 180 days. Doses used in this study were approximately eight times greater than those reported in a parallel field study. Activities of the cytochrome P450 1A enzymes, ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) were significantly greater in livers of mink exposed to TCDF, PeCDF, and a mixture of the two congeners; however, there were no significant histological or morphological effects observed. It was determined that EROD and MROD activity can be used as sensitive biomarkers of exposure to PeCDF and TCDF in adult female mink; however, under the conditions of this study, the response of EROD/MROD induction occurred at doses that were less than those required to cause histological or morphological changes. PMID:19458992

  15. Implication of cytochrome P-450 1A isoforms and the AH receptor in the genotoxicity of coal-tar fume condensate and bitumen fume condensates.

    PubMed

    Genevois, C; Pfohl-Leszkowicz, A; Boillot, K; Brandt, H; Castegnaro, M

    1998-06-01

    During the hot application of bitumen- or coal-tar-containing materials, fumes are emitted that contain polycyclic aromatic compounds. Although workers' exposure to these fumes is low, it might lead to health problems. No study has reported the metabolic pathways involved in the genotoxicity of coal tar or bitumen fume condensates (CTFC, BFCs). We have therefore studied the DNA adducts formed by incubation of CTFC or BFCs with liver microsomes from several type of mice and with yeast microsomes expressing individual human CYP enzymes. Our results demonstrates that: (1) the aryl hydrocarbon receptor (AHR) plays an important role in the biotransformation of BFCs and to a lesser extent of CTFC; (2) for CTFC, both cytochrome P450 (CYP) 1A isoforms are involved in the formation of genotoxic compounds, and the reactive metabolites formed via CYP 1A1, are substrates for epoxide hydrolase (mEH); (3) for BFCs, the genotoxicity is partially dependent upon CYP 1A1 and the reactive metabolites are not substrates for mEH; (4) CYP 1A isoforms are not exclusively responsible for the genotoxicity of the CTFC and BFCs as other CYPs and also enzymes of the [AH] gene battery, may play an important role. PMID:21781875

  16. 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. PMID:23232241

  17. Implication of cytochrome P-450 1A isoforms and the AH receptor in the genotoxicity of coal-tar fume condensate and bitumen fume condensates.

    PubMed

    Genevois, C; Pfohl-Leszkowicz, A; Boillot, K; Brandt, H; Castegnaro, M

    1998-06-01

    During the hot application of bitumen- or coal-tar-containing materials, fumes are emitted that contain polycyclic aromatic compounds. Although workers' exposure to these fumes is low, it might lead to health problems. No study has reported the metabolic pathways involved in the genotoxicity of coal tar or bitumen fume condensates (CTFC, BFCs). We have therefore studied the DNA adducts formed by incubation of CTFC or BFCs with liver microsomes from several type of mice and with yeast microsomes expressing individual human CYP enzymes. Our results demonstrates that: (1) the aryl hydrocarbon receptor (AHR) plays an important role in the biotransformation of BFCs and to a lesser extent of CTFC; (2) for CTFC, both cytochrome P450 (CYP) 1A isoforms are involved in the formation of genotoxic compounds, and the reactive metabolites formed via CYP 1A1, are substrates for epoxide hydrolase (mEH); (3) for BFCs, the genotoxicity is partially dependent upon CYP 1A1 and the reactive metabolites are not substrates for mEH; (4) CYP 1A isoforms are not exclusively responsible for the genotoxicity of the CTFC and BFCs as other CYPs and also enzymes of the [AH] gene battery, may play an important role.

  18. Developmental changes in the expression and function of cytochrome P450 3A isoforms: evidence from in vitro and in vivo investigations.

    PubMed

    Ince, Ibrahim; Knibbe, Catherijne A J; Danhof, Meindert; de Wildt, Saskia N

    2013-05-01

    The aim of this review is to discuss our current understanding of the developmental changes of the drug-metabolizing enzyme cytochrome P450 (CYP) 3A and its impact on drug therapy. In the last 10 years, several methods have been used to study the ontogeny of specific CYP3A isoforms in vitro and in vivo. Although most studies confirm previous findings that CYP3A4/5 activity is low at birth and reaches adult values in the first years of life, there are still important gaps in our knowledge of the exact developmental patterns of individual CYP3A isoforms, especially in this age range. Moreover, most in vivo clinical studies have also failed to cover the whole pediatric age range. To date, this information gap still hampers the design of age-specific dosing guidelines of CYP3A substrate drugs, especially in neonates and infants. Innovative study methods, including opportunistic sampling and sensitive analytical assays used in combination with physiologically based pharmacokinetics, and population pharmacokinetic model concepts may help to improve our understanding of the ontogeny of CYP3A and aid the application of this knowledge in clinical practice.

  19. 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.

  20. Imprinting of cerebral and hepatic cytochrome p450s in rat offsprings exposed prenatally to low doses of cypermethrin.

    PubMed

    Singh, Anshuman; Yadav, Sanjay; Srivastava, Vikas; Kumar, Rakesh; Singh, Dhirendra; Sethumadhavan, Rao; Parmar, Devendra

    2013-08-01

    Oral administration of low doses (1.25 or 2.5 or 5 mg/kg) corresponding to 1/200th or 1/100th or 1/50th of LD50 of cypermethrin, a synthetic type II pyrethroid, to pregnant Wistar rats from gestation day 5 to 21 produced a dose-dependent increase in the expression of xenobiotic metabolizing cytochrome P450 (CYP) 1A-, 2B- and 2E1 in the brain and liver of offsprings postnatally at 3 weeks that persisted up to 12 weeks. This persistent increase in CYPs was associated with alterations in circulating concentrations of testosterone, luteinizing hormone and follicle stimulating hormone, spontaneous locomotor activity and accumulation of cypermethrin in the brain of exposed offsprings. Rechallenge of exposed offsprings at adulthood (12 weeks old) with cypermethrin (p.o., 10 mg/kg × 6 days) led to a much higher increase in the expression of CYPs in the exposed offsprings when compared to the control offsprings treated with cypermethrin. Further, bioinformatic analysis demonstrating absence of specific short interspersed elements in CYPs suggests that persistence in the increase in CYPs in exposed offsprings could be attributed to the imprinting of the cerebral and hepatic CYPs following prenatal exposure to low doses of cypermethrin. This imprinting could be of toxicological relevance as it may modify the response of drugs or environmental exposures in exposed offsprings particularly for those chemicals which require CYP-mediated metabolism to produce their beneficial or toxic effects. PMID:23447098

  1. Comparative 1-substituted imidazole inhibition of cytochrome p450 isozyme-selective activities in human and mouse hepatic microsomes.

    PubMed

    Franklin, Michael R; Constance, Jonathan E

    2007-01-01

    Inhibition of cytochrome P450(CYP)-selective reactions in a single human and a single mouse hepatic microsome preparation by fourteen 1-substituted imidazoles provides a simultaneous ranking of reaction susceptibility to a specific imidazole and the relative inhibitory potency of the imidazoles for a given reaction. CYP3A4/5 activity was inhibited (IC(50) <5 microM) by the greatest number of imidazoles, followed closely by CYP2C9. Seven imidazoles exhibited IC(50) values for CYP3A4/5 <0.3 microM (none for CYP2C9) and were exclusively above 300 MW. Nafimidone (MW, 236) exhibited an IC(50) value <0.3 microM towards CYP2D6 and CYP1A2 reactions. CYP2E1 and CYP2A6 were exclusively inhibited (IC(50) <5 microM) by imidazoles with MWs below approximately 200. In general, mouse activities exhibited lower IC(50) values than in human microsomes. PMID:17786623

  2. Molecular Mimicry of Human Cytochrome P450 by Hepatitis C Virus at the Level of Cytotoxic T Cell Recognition

    PubMed Central

    Kammer, Andreas R.; van der Burg, Sjoerd H.; Grabscheid, Benno; Hunziker, Isabelle P.; Kwappenberg, Kitty M.C.; Reichen, Jürg; Melief, Cornelis J.M.; Cerny, Andreas

    1999-01-01

    Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8+ cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201–restricted HCV-derived epitope, i.e., HCV core 178–187, which shows sequence homology with human CYP2A6 and CYP2A7 8–17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8+ CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity. PMID:10432280

  3. Role of cytochromes P450 1A1/2 in detoxication and activation of carcinogenic aristolochic acid I: studies with the hepatic NADPH:cytochrome P450 reductase null (HRN) mouse model.

    PubMed

    Levová, Katerina; Moserová, Michaela; Kotrbová, Vera; Sulc, Miroslav; Henderson, Colin J; Wolf, C Roland; Phillips, David H; Frei, Eva; Schmeiser, Heinz H; Mares, Jaroslav; Arlt, Volker M; Stiborová, Marie

    2011-05-01

    Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

  4. Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity

    PubMed Central

    Ma, Lei-lei; Wu, Zhi-tao; Wang, Le; Zhang, Xue-feng; Wang, Jing; Chen, Chen; Ni, Xuan; Lin, Yun-fei; Cao, Yi-yi; Luan, Yang; Pan, Guo-yu

    2016-01-01

    Aim: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. Methods: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg·kg−1·d−1, ig) for 4 weeks, their blood samples were analyzed. Results: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 μmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 μmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg−1·d−1) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg−1·d−1, all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. Conclusion: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major

  5. Oxidative metabolism of spironolactone: Evidence for the involvement of electrophilic thiosteroid species in drug-mediated destruction of rat hepatic cytochrome P450

    SciTech Connect

    Decker, C.J.; Rashed, M.S.; Baillie, T.A.; Maltby, D.; Correia, M.A. )

    1989-06-13

    In a preliminary paper, the authors have shown that the antimineralocorticoid spironolactone (SPL) preferentially inactivates dexamethasone (DEX) inducible rat hepatic cytochrome P450p isozymes in a suicidal manner. These findings are now confirmed, and the kinetic characteristics of such a process are detailed. In an effort to elucidate the mechanism of SPL-mediated inactivation of cytochrome P450, they have examined the metabolism of SPL in vitro. Incubation of ({sup 14}C)SPL and NADPH with liver microsomes prepared from DEX-pretreated rats results in the formation of several polar metabolites separable by HPLC with UV detection. This process is found to be dependent on NADPH, O{sub 2}, SPL, and enzyme concentration, as well as temperature. Furthermore, metabolite formation was significantly attenuated by P450 inhibitors CO and n-octylamine. Mass metabolites indicated that these compounds had molecular weights that corresponded to the sulfinic and sulfonic acid derivatives of deacetyl-SPL (SPL-SH). These finding document the formation of previously unreported polar metabolites of SPL by rat liver microsomes enriched in cytochrome P450p and implicate a role for this isozyme in the oxidation of the thiol moiety of deacetyl-SPL. The detection of such metabolites also implicates a catalytic trajectory that includes the thiyl radical and/or sulfenic acid species as a plausible protagonist in drug-mediated inactivation of cytochrome P450p.

  6. Relationship between Differential Hepatic microRNA Expression and Decreased Hepatic Cytochrome P450 3A Activity in Cirrhosis

    PubMed Central

    Goswami, Chirayu Pankaj; Nalamasu, Rohit; Li, Lang; Jones, David; Wei, Rongrong; Liu, Wanqing; Sarasani, Vishal; Janga, Sarath Chandra; Chalasani, Naga

    2013-01-01

    Background and Aim Liver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A) activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA) are associated with decreased hepatic CYP3A activity in cirrhosis. Methods Hepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28) and normal (n=12) liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity. Results Hepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min-1*mg protein-1 (mean ± SEM), P=0.02). Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500) had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05). Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08) and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017). The relative expression (2-ΔΔCt mean ± SEM) of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07) but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08). Conclusion The strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity. PMID:24058572

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

    PubMed

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

    1997-03-01

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

  8. 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.

  9. Cytochrome P450 CYP6DA2 regulated by cap 'n'collar isoform C (CncC) is associated with gossypol tolerance in Aphis gossypii Glover.

    PubMed

    Peng, T; Pan, Y; Gao, X; Xi, J; Zhang, L; Yang, C; Bi, R; Yang, S; Xin, X; Shang, Q

    2016-08-01

    Cotton plants accumulate phytotoxins, such as gossypol and related sesquiterpene aldehydes, to resist insect herbivores. The survival of insects exposed to toxic secondary metabolites depends on the detoxification metabolism mediated by limited groups of cytochrome P450. Gossypol has an antibiotic effect on Aphis gossypii, and as the concentrations of gossypol were increased in the present study, the mortality of cotton aphids increased from 4 to 28%. The fecundity of the cotton aphids exposed to gossypol was also significantly reduced compared with the control. The transcriptional levels of CYP6DA2 in cotton aphids were significantly induced when exposed to gossypol, and knockdown of the CYP6DA2 transcripts by RNA interference (RNAi) significantly increased the toxicity of gossypol to cotton aphids. To further understand the gossypol regulatory cascade, the 5'-flanking promoter sequences of CYP6DA2 were isolated with a genome walker, and the promoter was very active and was inducible by gossypol. Co-transfection of the cap 'n' collar isoform C (CncC) and CYP6DA2 promoters dramatically increased the expression of CYP6DA2, and suppression of the CncC transcripts by RNAi significantly decreased the expression levels of CYP6DA2, and significantly increased the toxicity of gossypol to cotton aphids. Thus, the transcriptional regulation of CYP6DA2 involved the transcriptional factor CncC. PMID:27005728

  10. Hepatic microsomal metabolism of the anthelmintic benzimidazole fenbendazole: enhanced inhibition of cytochrome P450 reactions by oxidized metabolites of the drug.

    PubMed

    Murray, M; Hudson, A M; Yassa, V

    1992-01-01

    Potentiation of the anthelmintic action of benzimidazole carbamates, such as fenbendazole [methyl 5(6)-(phenylthio)-1H-benzimidazol-2-ylcarbamate], has been noted during concurrent administration of benzimidazoles that possess no intrinsic anthelmintic activity. This study investigated the possibility that inhibition of P450 enzymes by fenbendazole and its metabolites could play a role in the potentiation phenomenon. Fenbendazole underwent P450-mediated oxidation in microsomes from untreated rat liver to the sulfoxide and (4'-hydroxyphenyl)thio metabolites [2.92 and 2.87 nmol/(mg of protein.h)]. Pretreatment of rats with phenobarbital or dexamethasone enhanced sulfoxidation by 1.9- and 2.9-fold, respectively. 4'-Hydroxylation was increased slightly (by 28%) by phenobarbital and decreased slightly (by 41%) by dexamethasone. Induction also promoted further metabolism of the sulfoxide to fenbendazole sulfone. Immunoinhibition and chemical inhibition studies suggested that P450 3A proteins and the flavin-containing monooxygenase are involved in sulfoxide and sulfone formation whereas 4'-hydroxylation involved the P450s 2C11, 2C6, and 2B1, depending on the type of induction. In untreated rat liver, the sulfoxide and (4'-hydroxyphenyl)thio metabolites of fenbendazole were relatively potent inhibitors of P450-mediated androstenedione 16 alpha-, 16 beta-, and 6 beta-hydroxylation (IC50 values of 42, 36, and 74 microM, respectively); 7 alpha-hydroxylase activity was uninhibited. In contrast, fenbendazole and its sulfone metabolite were not inhibitors of these reactions. Mixed-function oxidase activities in phenobarbital-induced rat hepatic microsomes were refractory to inhibition by most compounds, but P450 1A1 mediated activities in microsomes from beta-naphthoflavone-induced rat liver were quite susceptible to inhibition by fenbendazole sulfoxide. Studies with two analogous sulfoxides yielded similar findings.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. 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...

  12. 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

  13. Effect of penicillin-based antibiotics, amoxicillin, ampicillin, and piperacillin, on drug-metabolizing activities of human hepatic cytochromes P450.

    PubMed

    Niwa, Toshiro; Morimoto, Mari; Hirai, Takako; Hata, Tomomi; Hayashi, Misato; Imagawa, Yurie

    2016-02-01

    The effects of three kinds of penicillin-based antibiotics, amoxicillin, ampicillin, and piperacillin, on drug-metabolizing activity of human hepatic cytochrome P450 (P450 or CYP) were investigated. Metabolic activities of P450s expressed in recombinant Escherichia coli at substrate concentrations around the Michaelis constant were compared in the presence or absence of the antibiotics. Amoxicillin, ampicillin, and piperacillin at 0.5 or 1 mM concentrations neither inhibited nor stimulated CYP2C9-mediated tolbutamide methylhydroxylation, CYP2D6-mediated dopamine formation from p-tyramine, or CYP3A4- or CYP3A5-mediated testosterone 6β-hydroxylation. However, amoxicillin and piperacillin inhibited CYP2C8-mediated aminopyrine N-demethylation at 50% inhibitory concentration of 0.83 and 1.14 mM, respectively. These results suggest that piperacillin might inhibit CYP2C8 clinically, although the interactions between these three penicillin-based antibiotics and other drugs that are metabolized by P450s investigated would not be clinically significant.

  14. Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

    PubMed

    Wu, Jing-Jing; Ge, Guang-Bo; He, Yu-Qi; Wang, Ping; Dai, Zi-Ru; Ning, Jing; Hu, Liang-Hai; Yang, Ling

    2016-01-01

    Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.

  15. 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.

  16. Study of the upregulation of the activity of cytochrome P450 3A isoforms by Astragalus injection and Astragalus granules in rats and in cells.

    PubMed

    Zhang, Yongli; Huang, Ling; Bi, Huichang; Cui, Yuqiang; Li, Jingqing; Wang, Xiangsheng; Qin, Xiaoling; Chen, Jiangying; Huang, Min

    2013-06-01

    Astragalus injection (AI) and Astragalus granules (AG) are the two representative clinical preparations from Astragali Radix. In order to investigate the regulation of metabolism, AI and AG were tested for their ability to affect the major enzyme cytochrome P450 3A isoforms in vivo and in vitro. In the study of CYP3A1 enzyme activity, male rats were pretreated with AI and AG. The "cocktail" approach-based LC-MS/MS results showed that AI pretreatment at 0.16, 0.8 and 4 g kg(-1) day(-1) significantly increased the rat liver microsome CYP3A1 activity by 1.62-, 1.68- and 2.00-fold, and AG pretreatment at 32, 160 and 800 mg kg(-1) day(-1) significantly increased the rat CYP3A1 activity by 1.86-, 2.16- and 1.76-fold. The effects of AI and AG on liver microsome CYP3A1 mRNA expression in rats were analyzed using real-time PCR technique. The results showed that AI and AG pretreatments significantly increased the CYP3A1 mRNA expression. The induction of CYP3A4 enzyme activity by AI and AG in vitro was measured using a CYP3A4 luciferase reporter gene assay in transiently transfected human intestinal LS174T cells. Compared to the control group, AI at 62.5-1,000 mg/ml could significantly induce CYP3A4 reporter gene luciferase activity of 1.36- to 1.88-fold for 48-h incubated PXR-transfected LS174T cells, and AG at 62.5-1,000 μg/ml significantly transactivated CYP3A4 reporter gene luciferase activity of 1.36- to 2.05-fold. However, the CYP3A4 reporter gene construct was not significantly transactivated by the AI and AG in CAR-transfected LS174T cells. These CYP3A isoforms upregulation results can help us to use AI and AG rationally in the clinic.

  17. 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

  18. Norcocaine and N-hydroxynorcocaine formation in human liver microsomes: role of cytochrome P-450 3A4.

    PubMed

    LeDuc, B W; Sinclair, P R; Shuster, L; Sinclair, J F; Evans, J E; Greenblatt, D J

    1993-05-01

    Cocaine was metabolized to norcocaine by microsomes prepared from lymphoblastoid cells expressing transfected human P-450 3A4. The specific activities of norcocaine formation by microsomes prepared from three human liver samples correlated with the amount of P-450 3A immunoreactive protein detected by immunoblot. Triacetyloleandomycin, a specific inhibitor of P-450 3A isoforms, inhibited formation of norcocaine from cocaine, but not formation of N-hydroxynorcocaine from norcocaine. The chemical identity of the norcocaine and N-hydroxynorcocaine produced by human liver microsomes was established by combination of gas chromatography and mass spectrometry. Thus, human P-450 3A4 is a cocaine demethylase, and P-450 isoforms of the 3A family are responsible for the majority of norcocaine production by human hepatic microsomes.

  19. Adaptive hepatic and intestinal alterations in mice after deletion of NADPH-cytochrome P450 Oxidoreductase (Cpr) in hepatocytes.

    PubMed

    Cheng, Xingguo; Gu, Jun; Klaassen, Curtis D

    2014-11-01

    Cytochrome P450 enzymes (P450) play an important role in first-pass metabolism in both the intestine and liver. NADPH-cytochrome P450 oxidoreductase (Cpr) is an essential electron transfer protein required for microsomal P450 activity. Mice with conditional knockout of Cpr in hepatocytes develop normally and survive even with complete loss of liver microsomal P450 activity. Our current studies were performed to determine whether alternative drug-metabolizing pathways increase in an attempt to maintain whole-body homeostasis. In addition to the liver, Cpr is mainly expressed in tissues such as lung, kidney, and gastrointestinal tract. In livers of H-Cpr-null mice, there is a marked increase in mRNA expression of phase I enzymes (Aldh1a1, 1a7, 3a2; Ces1b2, 2a6, and 2a12), antioxidant enzymes (Ho-1, Nqo1, and epoxide hydrolase), phase II enzymes (Ugt1a9; Gsta1/2, m3, m4, m6, t1, and t3; and Sult1a1 and 1d1), and drug transporters (Oatp1a4, Oct3, Mate1, Mdr1a, and Mrp3 and 4). In addition, glucuronide-conjugated bilirubin concentrations are doubled in serum of H-Cpr-null mice. Both constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein in nuclei are higher in the livers of H-Cpr-null mice, indicating that CAR and Nrf2 are activated. In the small intestine of H-Cpr-null mice, mRNA expression of Cyp3a11 and Mdr1a, two genes critical for intestinal first-pass metabolism, are markedly up-regulated. In addition, nutrient (Pept1) and cholesterol (Npc1l1) transporters are induced in the small intestine of H-Cpr-null mice. In conclusion, in H-Cpr-null mice, adaptive regulation of alternative detoxification genes in liver and small intestine appear to partially compensate for the loss of microsomal P450 function in liver.

  20. Subacute nicotine co-exposure has no effect on 2,2',3,5',6- pentachlorobiphenyl disposition but alters hepatic cytochrome P450 expression in the male rat.

    PubMed

    Stamou, Marianna; Uwimana, Eric; Flannery, Brenna M; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2015-12-01

    Polychlorinated biphenyls (PCBs) are metabolized by cytochrome P450 2B enzymes (CYP2B) and nicotine is reported to alter CYP2B activity in the brain and liver. To test the hypothesis that nicotine influences PCB disposition, 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and its metabolites were quantified in tissues of adult male Wistar rats exposed to PCB 95 (6mg/kg/d, p.o.) in the absence or presence of nicotine (1.0mg/kg/d of the tartrate salt, s.c.) for 7 consecutive days. PCB 95 was enantioselectively metabolized to hydroxylated (OH-) PCB metabolites, resulting in a pronounced enrichment of E1-PCB 95 in all tissues investigated. OH-PCBs were detected in blood and liver tissue, but were below the detection limit in adipose, brain and muscle tissues. Co-exposure to nicotine did not change PCB 95 disposition. CYP2B1 mRNA and CYP2B protein were not detected in brain tissues but were detected in liver. Co-exposure to nicotine and PCB 95 increased hepatic CYP2B1 mRNA but did not change CYP2B protein levels relative to vehicle control animals. However, hepatic CYP2B protein in animals co-exposed to PCB 95 and nicotine were reduced compared to animals that received only nicotine. Quantification of CYP2B3, CYP3A2 and CYP1A2 mRNA identified significant effects of nicotine and PCB 95 co-exposure on hepatic CYP3A2 and hippocampal CYP1A2 transcripts. Our findings suggest that nicotine co-exposure does not significantly influence PCB 95 disposition in the rat. However, these studies suggest a novel influence of PCB 95 and nicotine co-exposure on hepatic cytochrome P450 (P450) expression that may warrant further attention due to the increasing use of e-cigarettes and related products.

  1. Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases.

    PubMed

    Hazai, Eszter; Gagne, Peter V; Kupfer, David

    2004-07-01

    Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of

  2. 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.

  3. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 in Japanese patients with chronic hepatitis.

    PubMed

    Nishioka, M; Morshed, S A; Parveen, S; Kono, K; Matsuoka, H; Manns, M P

    1997-12-01

    Auto-antibodies specific to various antigens in chronic hepatitis (CH) have been detected but their specificities and implications were uncertain. The aims of the present study were to investigate the frequency and the significance of seropositivity of antibodies to P450IID6 or liver/kidney microsome 1 (LKM1), soluble liver antigen (SLA), pyruvate dehydrogenase (PDH) and branched-chain keto acid dehydrogenase (BCKD) in 188 Japanese patients with different forms of CH by western blot or enzyme immunoassay (EIA). Anti-LKM1 was also measured by indirect immunofluorescent test. Anti-P450IID6 was found in 6/188 (3.2%) CH patients including 5/104 (4.8%) with hepatitis C virus (C) infection and 1/12 (8.3%) CH-C patients with antibodies to nuclear and smooth muscle antigens and hypergammaglobulinaemia (> 2.5 g/dL). This patient was the only one diagnosed with autoimmune hepatitis (AIH). All CH patients with hepatitis B (B), hepatitis non-B non-C (NBNC) and AIH were seronegative for anti-LKM1. Antibodies to soluble liver antigen were found in two of 188 (1%) patients, one with AIH and one with CH-B. Anti-BCKD-E2 but not anti-PDH-E2 was found in four patients (2.5%), one with AIH, two with CH-C, and one with NBNC. There was no obvious difference in age, sex ratio and laboratory findings in patients with or without anti-SLA and anti-BCKD-E2. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 are uncommon in adult CH-C, CH-B, CH-NBNC and AIH patients in Japan. Some of these patients positive for auto-antibodies appear to have autoimmune features and might require a careful follow up. The heterogeneity of these antibodies in CH preclude further justification for subtyping of AIH by the presence of the distinct auto-antibodies.

  4. Hepatic cytochrome P450s play a major role in monocrotaline-induced renal toxicity in mice

    PubMed Central

    Yao, Jun; Li, Cheng-gang; Gong, Li-kun; Feng, Chen-chen; Li, Chun-zhu; Gao, Man; Luan, Yang; Qi, Xin-ming; Ren, Jin

    2014-01-01

    Aim: Monocrotaline (MCT) in plants of the genus Crotalaria induces significant toxicity in multiple organs including the liver, lung and kidney. Metabolic activation of MCT is required for MCT-induced toxicity. In this study, we attempted to determine whether the toxicity of MCT in kidney was a consequence of the metabolic activation of MCT in the liver. Methods: Liver-specific cytochrome P450 reductase-null (Null) mice, wild-type (WT) mice and CYP3A inhibitor ketoconazole-pretreated WT (KET-WT) mice were examined. The mice were injected with MCT (300, 400, or 500 mg/kg, ip), and hepatotoxicity and nephrotoxicity were examined 24 h after MCT treatment. The levels of MCT and its metabolites in the blood, liver, lung, kidney and bile were determined using LC-MS analysis. Results: Treatment of WT mice with MCT increased the serum levels of alanine aminotransferase, hyaluronic acid, urea nitrogen and creatinine in a dose-dependent manner. Histological examination revealed that MCT (500 mg/kg) caused severe liver injury and moderate kidney injury. In contrast, these pathological abnormalities were absent in Null and KET-WT mice. After injection of MCT (400 and 500 mg/kg), the plasma, liver, kidney and lung of WT mice had significantly lower MCT levels and much higher N-oxide metabolites contents in compared with those of Null and KET-WT mice. Furthermore, WT mice had considerably higher levels of tissue-bound pyrroles and bile GSH-conjugated MCT metabolites compared with Null and KET-WT mice. Conclusion: Cytochrome P450s in mouse liver play a major role in the metabolic activation of MCT and thus contribute to MCT-induced renal toxicity. PMID:24362331

  5. Cytochrome P450 responsible for the stereoselective S-oxidation of flosequinan in hepatic microsomes from rats and humans.

    PubMed

    Kashiyama, E; Yokoi, T; Odomi, M; Funae, Y; Inoue, K; Kamataki, T

    1997-06-01

    The forms of cytochrome P450 involved in the stereoselective S-oxidation of flosequinan [(+/-)-7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone] were investigated in vitro using liver microsomes from rats and humans. Rat liver microsomes supplemented with NADPH catalyzed the four different S-oxidations, which were from flosequinan sulfide (FS; 7-fluoro-1-methyl-3-methylthio-4-quinolone) to R(+)- and S(-)-flosequinan (R-FSO and S-FSO, respectively) and from R-FSO and S-FSO to flosequinan sulfone (FSO2; 7-fluoro-1-methyl-3-methylsulfonyl-4-quinolone). The activities of all the S-oxidases in liver microsomes from male rats were higher than those from female rats. The activities of the S-oxidases measured at a high substrate concentration (1 mM) were induced by treatment of rats with phenobarbital and dexamethasone. Treatment of rats with 3-methylcholanthrene also induced the activities, but only at a low substrate concentration (50 microM), except for the S-oxidase catalyzing the reaction from FS to R-FSO. Enzymes induced by clofibrate and ethanol were not involved in the oxidations at a low substrate concentration. The activities of S-oxidases were correlated with the contents of cytochrome P450 (CYP)3A enzymes. Anti-CYP3A2 antisera inhibited the activities of the S-oxidases catalyzing the reactions from FS to R-FSO (40%) and to S-FSO (60%) at the high substrate concentration and inhibited the activities of the S-oxidases, thus catalyzing reactions from R-FSO and S-FSO to FSO2 (70%) at both high and low substrate concentrations. These results suggest that CYP3A is the major enzyme involved in all S-oxidation pathways in flosequinan metabolism in rats. On the other hand, except for the S-oxidation of FS to R-FSO, the rates of the other three S-oxidations by liver microsomes from 30 individual humans correlated highly with each other, suggesting that the same enzyme would be involved in the three S-oxidations. Anti-CYP3A2 antisera inhibited the activities of all the S

  6. 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.

  7. 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

  8. 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

  9. Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats.

    PubMed

    Kawase, Atsushi; Ito, Ayami; Yamada, Ayano; Iwaki, Masahiro

    2015-06-01

    Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats.

  10. Mechanism of decrease in levels of hepatic P450 isozymes induced by intracerebral endotoxin: independence from sympathetic nervous and adrenocortical systems.

    PubMed

    Shimamoto, Y; Kitamura, H; Iwai, M; Saito, M; Kazusaka, A; Fujita, S

    1999-02-01

    We previously reported that lipoplysaccharide (LPS) injected intracerebroventricularly (i.c.v.) at an ineffective dose (0.1 microgram/rat) decreased the drug metabolizing activities and related cytochrome P450 (CYP) isozymes in rat liver microsomes when injected intraperitoneally (i.p.). The dose study (doses < 0.1 microgram intracerebrally and > 0.1 microgram i.p.), which was carried out to examine how much more effective is i.c.v.-LPS than i.p.-LPS, showed that the pattern of alteration of expression of CYP isozymes induced by i.c.v.-LPS was different from that caused by i.p.-LPS at an effective dose (10 micrograms/rat). These results indicate that the decrease in hepatic CYP isozymes caused by i.c.v.-LPS could not be explained by the LPS leaked from the brain, suggesting that the decrease in hepatic CYP isozymes by i.c.v.-LPS may be caused by a central action of LPS. In this study, the possible involvement of sympathetic nervous and adrenocortical systems in the down-regulation of CYP isozymes by i.c.v.-LPS was investigated using surgical or chemical sympathetecomized or adrenalectomized rats. The norepinephrine (NE) content in the liver in rats with surgical hepatic sympathetectomy was reduced by 88% compared with that of sham-operated rats that received i.c.v.-saline and 85% compared with that of sham-operated rats that received i.c.v.-LPS, indicating that hepatic denervation was successful. The NE content in the liver in rats chemically sympathetectomized by two i.p. injections of 6-hydroxydopamine (40 mg/kg each time) 1 and 2 days before i.c.v. injection was reduced by 82% in i.c.v.-saline-treated and by 74% in i.c.v.-LPS-treated groups compared with that in rats pretreated with i.p.-saline. These results indicate that sympathetic NE terminals were effectively removed. Intracerebroventricular LPS decreased the total P450 content and the activities of CYP dependent drug metabolizing enzymes, ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O

  11. Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice.

    PubMed

    Xu, S F; Wu, Q; Zhang, B B; Li, H; Xu, Y S; Du, Y Z; Wei, L X; Liu, J

    2016-09-01

    Zuotai (mainly β-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl2) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl2 and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (β-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHg (3.1mg/kg) for 7days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1-4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl2. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl2 and MeHg, but not by Zuotai and HgS. HgCl2 decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl2 and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl2 and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes. PMID:27473830

  12. Dietary tomato powder inhibits alcohol-induced hepatic injury by suppressing cytochrome p450 2E1 induction in rodent models.

    PubMed

    Stice, Camilla P; Liu, Chun; Aizawa, Koichi; Greenberg, Andrew S; Ausman, Lynne M; Wang, Xiang-Dong

    2015-04-15

    Chronic and excessive alcohol consumption leads to the development of alcoholic liver disease (ALD) and greatly increases the risk of liver cancer. Induction of the cytochrome p450 2E1 (CYP2E1) enzyme by chronic and excessive alcohol intake is known to play a role in the pathogenesis of ALD. High intake of tomatoes, rich in the carotenoid lycopene, is associated with a decreased risk of chronic disease. We investigated the effects of whole tomato (tomato powder, TP), partial tomato (tomato extract, TE), and purified lycopene (LYC) against ALD development in rats. Of the three supplements, only TP reduced the severity of alcohol-induced steatosis, hepatic inflammatory foci, and CYP2E1 protein levels. TE had no effect on these outcomes and LYC greatly increased inflammatory foci in alcohol-fed rats. To further support the protective effect of TP against ALD, TP was supplemented in a carcinogen (diethylnitrosamine, DEN)-initiated alcohol-promoted mouse model. In addition to reduced steatosis and inflammatory foci, TP abolished the presence of preneoplastic foci of altered hepatocytes in DEN-injected mice fed alcohol. These reductions were associated with decreased hepatic CYP2E1 protein levels, restored levels of peroxisome proliferator-activated receptor-α and downstream gene expression, decreased inflammatory gene expression, and reduced endoplasmic reticulum stress markers. These data provide strong evidence for TP as an effective whole food prevention strategy against ALD.

  13. Dietary tomato powder inhibits alcohol-induced hepatic injury by suppressing cytochrome p450 2E1 induction in rodent models

    PubMed Central

    Stice, Camilla P.; Liu, Chun; Aizawa, Koichi; Greenberg, Andrew S.; Ausman, Lynne M.; Wang, Xiang-Dong

    2015-01-01

    Chronic and excessive alcohol consumption leads to the development of alcoholic liver disease (ALD) and greatly increases the risk of liver cancer. Induction of the cytochrome p450 2E1 (CYP2E1) enzyme by chronic and excessive alcohol intake is known to play a role in the pathogenesis of ALD. High intake of tomatoes, rich in the carotenoid lycopene, is associated with a decreased risk of chronic disease. We investigated the effects of whole tomato (tomato powder, TP), partial tomato (tomato extract, TE), and purified lycopene (LYC) against ALD development in rats. Of the three supplements, only TP reduced the severity of alcohol-induced steatosis, hepatic inflammatory foci, and CYP2E1 protein levels. TE had no effect on these outcomes and LYC greatly increased inflammatory foci in alcohol-fed rats. To further support the protective effect of TP against ALD, TP was supplemented in a carcinogen (diethylnitrosamine, DEN)-initiated alcohol-promoted mouse model. In addition to reduced steatosis and inflammatory foci, TP abolished the presence of preneoplastic foci of altered hepatocytes in DEN-injected mice fed alcohol. These reductions were associated with decreased hepatic CYP2E1 protein levels, restored levels of peroxisome proliferator-activated receptor-α and downstream gene expression, decreased inflammatory gene expression, and reduced endoplasmic reticulum stress markers. These data provide strong evidence for TP as an effective whole food prevention strategy against ALD. PMID:25592162

  14. Dietary tomato powder inhibits alcohol-induced hepatic injury by suppressing cytochrome p450 2E1 induction in rodent models.

    PubMed

    Stice, Camilla P; Liu, Chun; Aizawa, Koichi; Greenberg, Andrew S; Ausman, Lynne M; Wang, Xiang-Dong

    2015-04-15

    Chronic and excessive alcohol consumption leads to the development of alcoholic liver disease (ALD) and greatly increases the risk of liver cancer. Induction of the cytochrome p450 2E1 (CYP2E1) enzyme by chronic and excessive alcohol intake is known to play a role in the pathogenesis of ALD. High intake of tomatoes, rich in the carotenoid lycopene, is associated with a decreased risk of chronic disease. We investigated the effects of whole tomato (tomato powder, TP), partial tomato (tomato extract, TE), and purified lycopene (LYC) against ALD development in rats. Of the three supplements, only TP reduced the severity of alcohol-induced steatosis, hepatic inflammatory foci, and CYP2E1 protein levels. TE had no effect on these outcomes and LYC greatly increased inflammatory foci in alcohol-fed rats. To further support the protective effect of TP against ALD, TP was supplemented in a carcinogen (diethylnitrosamine, DEN)-initiated alcohol-promoted mouse model. In addition to reduced steatosis and inflammatory foci, TP abolished the presence of preneoplastic foci of altered hepatocytes in DEN-injected mice fed alcohol. These reductions were associated with decreased hepatic CYP2E1 protein levels, restored levels of peroxisome proliferator-activated receptor-α and downstream gene expression, decreased inflammatory gene expression, and reduced endoplasmic reticulum stress markers. These data provide strong evidence for TP as an effective whole food prevention strategy against ALD. PMID:25592162

  15. 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.

  16. Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses.

    PubMed Central

    Yuan, P M; Ryan, D E; Levin, W; Shively, J E

    1983-01-01

    Two isozymes of rat liver microsomal cytochrome P-450--P-450b and P-450e--were compared by micro sequence analyses of their NH2 termini and tryptic fragments. These two phenobarbital-inducible hemoproteins, which are immunochemically indistinguishable with antibody against cytochrome P-450b, have extensive sequence homology. Automated Edman degradation of the native proteins revealed identical amino acids for the first 35 residues. Sequence determinations of the tryptic peptides, which constitute approximately 75% of each protein molecule, have thus far shown 10 amino acid differences between the two isozymes. Results of our amino acid sequence analyses established that two of the cDNAs, pcP-450pb1 and pcP-450pb4, reported by Fujii-Kuriyama et al. [Fujii-Kuriyama, Y., Mizukami, Y., Kamajiri, K., Sogawa, K. & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. USA 79, 2793-2797] encode cytochrome P-450b whereas pcP-450pb2, a third cDNA whose nucleotide sequence differed slightly from that of the other two (six amino acid substitutions), encodes cytochrome P-450e. In addition to establishing the identity of these cloned cDNAs we provide direct evidence for seven additional amino acid differences between cytochromes P-450b and P-450e that occur beyond the region (Arg358) encoded by the cloned cDNA for cytochrome P-450e. Together, the amino acid sequences determined by micro sequence analysis and recombinant DNA techniques reveal 13 amino acid differences between these two isozymes. This report highlights the complementary nature of two different molecular approaches to elucidation of the amino acid sequences of isozymes with extensive structural homology. PMID:6572377

  17. Effect of myricetin on cytochrome P450 isoforms CYP1A2, CYP2C9 and CYP3A4 in rats.

    PubMed

    Guo, Yu-Jin; Zheng, Shuang-Li

    2014-04-01

    Myricetin is one of the main ingredients of Chinese bayberry, which is used as a traditional medicine. The purpose of this study was to find out whether myricetin influences the rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9 and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated for 14 days with myricetin. 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 corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Our study showed that treatment with multiple doses of myricetin had no effects on rat CYP1A2. However, CYP2C9 and CYP3A4 enzyme activities were inhibited after multiple doses of myricetin. Therefore, caution is needed when myricetin is co-administered with CYP2C9 or CYP3A4 substrates, which may result in herb-drug interactions.

  18. Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4.

    PubMed

    Zhu, Hui-dan; Gu, Ni; Wang, Meng; Kong, Hong-ru; Zhou, Meng-tao

    2015-01-01

    Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (15 mg/kg), omeprazole (15 mg/kg), and midazolam (10 mg/kg), was given orally to rats treated for 7 d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug-CAP interactions.

  19. Potential inhibitory effect of herbal medicines on rat hepatic cytochrome P450 2D gene expression and metabolic activity.

    PubMed

    Al-Jenoobi, F I; Korashy, H M; Ahad, A; Raish, M; Al-Mohizea, A M; Alam, M A; Al-Suwayeh, S A; Alkharfy, K M

    2014-11-01

    The aim of current study was to investigate the effect of some commonly used medicinal herbs on the regulation of rat CYP2D gene expression and its metabolic activity. Wistar albino rats were treated for seven consecutive days with selected doses of five commonly used herbs (Trigonella foenum-graecum, Ferula asafoetida, Nigella sativa, Commiphora myrrha and Lepidium sativum). Thereafter, rat livers were harvested and CYP2D mRNA levels were determined by RT-PCR. The metabolic activity of CYP2D was performed on rat hepatic microsomes using dextromethorphan as specific substrate. All investigated herbs produced inhibition of CYP2D mRNA expression and metabolic activity. The inhibitory potential of investigated herbs on rat CYP2D mRNA was in the following order: Commiphora myrrha > Nigella sativa > Lepidium sativum > Trigonella foenum-graecum > Ferula asafoetida. Whereas, the inhibitory potential of investigated herbs on CYP2D mediated enzyme metabolic activity was found in following order: Nigella sativa > Lepidium sativum > Trigonella foenum-graecum > Commiphora myrrha > Ferula asafoetida. The current study shows that only used herbs reduce CYP2D activity in rat liver microsomes at the transcriptional levels. Such effects could lead to undesirable pharmacological effects of clinically used low therapeutic index CYP2D substrate drugs.

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

    PubMed

    Nesnow, Stephen; Grindstaff, Rachel D; Lambert, Guy; Padgett, William T; Bruno, Maribel; Ge, Yue; Chen, Pei-Jen; Wood, Charles E; Murphy, Lynea

    2011-10-15

    Propiconazole induces hepatocellular carcinomas and hepatocellular adenomas in mice and promotes liver tumors in rats. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicated that propiconazole induced oxidative stress. Here we sought to identify the source of the reactive oxygen species (ROS) induced by propiconazole using both AML12 immortalized mouse hepatocytes in culture and liver tissues from mice. We also sought to further characterize the nature and effects of ROS formation induced by propiconazole treatment in mouse liver. ROS was induced in AML12 cells by propiconazole as measured by fluorescence detection and its formation was ameliorated by N-acetylcysteine. Propiconazole induced glutathione-S-transferase (GSTα) protein levels and increased the levels of thiobarbituric acid reactive substances (TBARS) in AML12 cells. The TBARS levels were decreased by diphenylene iodonium chloride (DPIC), a cytochrome P450 (CYP) reductase inhibitor revealing the role of CYPs in ROS generation. It has been previously reported that Cyp2b and Cyp3a proteins were induced in mouse liver by propiconazole and that Cyp2b and Cyp3a proteins undergo uncoupling of their CYP catalytic cycle releasing ROS. Therefore, salicylic acid hydroxylation was used as probe for ROS formation using microsomes from mice treated with propiconazole. These studies showed that levels of 2,3-dihydroxybenzoic acid (an ROS derived metabolite) were decreased by ketoconazole, melatonin and DPIC. In vivo, propiconazole increased hepatic malondialdehyde levels and GSTα protein levels and had no effect on hepatic catalase or superoxide dismutase activities. Based on these observations we conclude that propiconazole induces ROS in mouse liver by increasing CYP protein levels leading to increased ROS levels. Our data also suggest that propiconazole induces the hydroxyl radical as a major

  1. Carbon monoxide-bound red blood cell resuscitation ameliorates hepatic injury induced by massive hemorrhage and red blood cell resuscitation via hepatic cytochrome P450 protection in hemorrhagic shock rats.

    PubMed

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

    2014-07-01

    Red blood cell (RBC) transfusions are the gold standard in cases of massive hemorrhage, but induce hepatic ischemia-reperfusion injury, a serious complication associated with hemorrhage and RBC resuscitation. Thus, the development of a novel resuscitable fluid that is not associated with hepatic ischemia-reperfusion injury would be desirable. It was reported that exogenous carbon monoxide (CO) treatment ameliorated hepatic ischemia-reperfusion injury accompanying liver transplantation. This suggests that transfusions with CO-bound RBC (CO-RBC) might protect against hepatic ischemia-reperfusion injury following massive hemorrhage and resuscitation compared with RBC resuscitation. To investigate this, we created a hemorrhagic shock model rat, followed by resuscitation with RBC and CO-RBC. Hepatic ischemia-reperfusion injury and the destruction of hepatic cytochrome P450 (CYP) were significantly ameliorated in the CO-RBC resuscitation group compared with the RBC resuscitation group. The free heme derived from the destruction of hepatic CYP was correlated with hepatic oxidation and injury, suggesting that CO-RBC was a major factor in the amelioration of hepatic ischemia-reperfusion injury induced by hemorrhage and resuscitation via hepatic CYP protection. These results indicate that CO-RBC has potential for use as a resuscitative fluid in blood transfusion and does not suffer from the limitations associated with the RBC transfusions that are currently in use.

  2. Pyrethroid insecticides: isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor.

    PubMed

    Yang, Dongfang; Wang, Xiliang; Chen, Yi-Tzai; Deng, Ruitang; Yan, Bingfang

    2009-05-15

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

  3. Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

    SciTech Connect

    Yang Dongfang; Wang Xiliang; Chen Yitzai; Deng Ruitang; Yan Bingfang

    2009-05-15

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

  4. 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.

  5. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    PubMed Central

    Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  6. Short communication: Hepatic progesterone-metabolizing enzymes cytochrome P450 2C and 3A in lactating cows during thermoneutral and heat stress conditions.

    PubMed

    McCracken, V L; Xie, G; Deaver, S E; Baumgard, L H; Rhoads, R P; Rhoads, M L

    2015-05-01

    Two experiments were performed to determine the effects of heat stress (HS) and insulin on hepatic mRNA abundance of enzymes responsible for metabolizing progesterone [cytochrome P450 2C and 3A (CYP2C and CYP3A)]. To distinguish the direct effects of HS from decreased dry matter intake, cohorts were pair fed (PF) in thermoneutral conditions to match the intake of the HS cows during both experiments. In the first experiment, multiparous late-lactation Holstein cows (n=12, 305±33 d in milk) housed in climate-controlled chambers were subjected to 2 experimental periods: (1) thermoneutral (TN) conditions (18°C, 20% humidity) with ad libitum intake (TN and well fed) for 9 d; and (2) either HS conditions (cyclical temperature 31-40°C, 20% humidity) fed for ad libitum intake (n=6), or TN conditions and PF to match the HS animal (n=6) for 9 d. To evaluate hepatic gene expression during experiment 1, biopsies were obtained at the end of each period. In the second experiment, multiparous mid-lactation Holstein cows (n=12, 136±8 DIM) were housed and fed in conditions similar to those described for the first experiment. Liver biopsies were obtained immediately before and after an insulin tolerance test administered on d 6 of each period. No effects of exogenous insulin were observed on any of the tested variables, nor were there interactions between environment (TN/HS or well fed/PF) and insulin administration. Heat stress decreased hepatic CYP2C expression during both experiments. The relative abundance of CYP3A was not affected by environmental conditions in the late-lactation cows (first experiment), but was reduced by HS in the mid-lactation cows (second experiment). Interestingly, during experiment 2, hepatic CYP3A expression also decreased during PF. These results suggest that HS reduces the capacity of the liver to metabolize progesterone through distinct effects on CYP2C and CYP3A, and that the effects appear to vary based upon stage of lactation. Ultimately, HS

  7. Short communication: Hepatic progesterone-metabolizing enzymes cytochrome P450 2C and 3A in lactating cows during thermoneutral and heat stress conditions.

    PubMed

    McCracken, V L; Xie, G; Deaver, S E; Baumgard, L H; Rhoads, R P; Rhoads, M L

    2015-05-01

    Two experiments were performed to determine the effects of heat stress (HS) and insulin on hepatic mRNA abundance of enzymes responsible for metabolizing progesterone [cytochrome P450 2C and 3A (CYP2C and CYP3A)]. To distinguish the direct effects of HS from decreased dry matter intake, cohorts were pair fed (PF) in thermoneutral conditions to match the intake of the HS cows during both experiments. In the first experiment, multiparous late-lactation Holstein cows (n=12, 305±33 d in milk) housed in climate-controlled chambers were subjected to 2 experimental periods: (1) thermoneutral (TN) conditions (18°C, 20% humidity) with ad libitum intake (TN and well fed) for 9 d; and (2) either HS conditions (cyclical temperature 31-40°C, 20% humidity) fed for ad libitum intake (n=6), or TN conditions and PF to match the HS animal (n=6) for 9 d. To evaluate hepatic gene expression during experiment 1, biopsies were obtained at the end of each period. In the second experiment, multiparous mid-lactation Holstein cows (n=12, 136±8 DIM) were housed and fed in conditions similar to those described for the first experiment. Liver biopsies were obtained immediately before and after an insulin tolerance test administered on d 6 of each period. No effects of exogenous insulin were observed on any of the tested variables, nor were there interactions between environment (TN/HS or well fed/PF) and insulin administration. Heat stress decreased hepatic CYP2C expression during both experiments. The relative abundance of CYP3A was not affected by environmental conditions in the late-lactation cows (first experiment), but was reduced by HS in the mid-lactation cows (second experiment). Interestingly, during experiment 2, hepatic CYP3A expression also decreased during PF. These results suggest that HS reduces the capacity of the liver to metabolize progesterone through distinct effects on CYP2C and CYP3A, and that the effects appear to vary based upon stage of lactation. Ultimately, HS

  8. The Anticancer Drug Ellipticine Activated with Cytochrome P450 Mediates DNA Damage Determining Its Pharmacological Efficiencies: Studies with Rats, Hepatic Cytochrome P450 Reductase Null (HRN™) Mice and Pure Enzymes

    PubMed Central

    Stiborová, Marie; Černá, Věra; Moserová, Michaela; Mrízová, Iveta; Arlt, Volker M.; Frei, Eva

    2014-01-01

    Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models. PMID:25547492

  9. Effects of Hypoxia Exposure on Hepatic Cytochrome P450 1A (CYP1A) Expression in Atlantic Croaker: Molecular Mechanisms of CYP1A Down-Regulation

    PubMed Central

    Rahman, Md. Saydur; Thomas, Peter

    2012-01-01

    Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress. PMID:22815834

  10. 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...

  11. 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...

  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. Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19.

    PubMed

    Kolukisaoglu, Üner; Wendler, Christian; Goerdes, Dirk; Diener, Annette; Thurow, Kerstin

    2010-12-01

    Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug-drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.

  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. 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

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

    PubMed Central

    2014-01-01

    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

  17. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    SciTech Connect

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V.

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

  18. Tissue-specific expression of rat mRNAs homologous to cytochromes P-450b and P-450e.

    PubMed Central

    Omiecinski, C J

    1986-01-01

    The tissue-specific expression of cytochrome P-450b and P-450e mRNAs was examined with synthetic 18-mer oligomer probes in the liver, lung, kidney, and testis of control and inducer pretreated adult rats. RNAs homologous to the P-450e probe were detected in trace amounts in control and 3-methylcholanthrene (MC) induced livers and at high levels in livers from phenobarbital (PB) induced animals. P-450e mRNA levels were below detection limits in the other tissues examined, regardless of pretreatment. In contrast, mRNAs homologous to the P-450b oligomer were detected at low levels in control and inducer pretreated lung and testis, and at high levels in PB induced liver. No P-450b mRNAs were detected in these assays in RNA isolates from the kidney or from control or MC pretreated liver. Solution hybridization data indicated that the rat lung contained 9-12%, and the testis, 6-9%, respectively, of the levels of P-450b mRNA measured in the PB induced liver. Results from oligo(dT)-cellulose and poly(U)-affinity experiments indicated that the hepatic mRNAs for P-450b and P-450e were present predominantly in the bound, polyadenylated fraction, whereas the homologous lung and testes P-450b mRNAs predominated in the flow-thru fractions. Images PMID:3754047

  19. Evaluation of the HC-04 Cell Line as an In Vitro Model for Mechanistic Assessment of Changes in Hepatic Cytochrome P450 3A during Adenovirus Infection

    PubMed Central

    Wonganan, Piyanuch; Jonsson-Schmunk, Kristina; Callahan, Shellie M.; Choi, Jin Huk

    2014-01-01

    HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 μM) and phenobarbital (500 μM) increased activity by 230 and 124%, whereas ketoconazole (10 μM) and lipopolysaccharide (LPS) (10 μg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic. PMID:24764148

  20. 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.

  1. Origins of P450 diversity.

    PubMed

    Sezutsu, Hideki; Le Goff, Gaëlle; Feyereisen, René

    2013-02-19

    The P450 enzymes maintain a conserved P450 fold despite a considerable variation in sequence. The P450 family even includes proteins that lack the single conserved cysteine and are therefore no longer haem-thiolate proteins. The mechanisms of successive gene duplications leading to large families in plants and animals are well established. Comparisons of P450 CYP gene clusters in related species illustrate the rapid changes in CYPome sizes. Examples of CYP copy number variation with effects on fitness are emerging, and these provide an opportunity to study the proximal causes of duplication or pseudogenization. Birth and death models can explain the proliferation of CYP genes that is amply illustrated by the sequence of every new genome. Thus, the distribution of P450 diversity within the CYPome of plants and animals, a few families with many genes (P450 blooms) and many families with few genes, follows similar power laws in both groups. A closer look at some families with few genes shows that these, often single member families, are not stable during evolution. The enzymatic prowess of P450 may predispose them to switch back and forth between metabolism of critical structural or signal molecules and metabolism dedicated to environmental response. PMID:23297351

  2. Origins of P450 diversity

    PubMed Central

    Sezutsu, Hideki; Le Goff, Gaëlle; Feyereisen, René

    2013-01-01

    The P450 enzymes maintain a conserved P450 fold despite a considerable variation in sequence. The P450 family even includes proteins that lack the single conserved cysteine and are therefore no longer haem-thiolate proteins. The mechanisms of successive gene duplications leading to large families in plants and animals are well established. Comparisons of P450 CYP gene clusters in related species illustrate the rapid changes in CYPome sizes. Examples of CYP copy number variation with effects on fitness are emerging, and these provide an opportunity to study the proximal causes of duplication or pseudogenization. Birth and death models can explain the proliferation of CYP genes that is amply illustrated by the sequence of every new genome. Thus, the distribution of P450 diversity within the CYPome of plants and animals, a few families with many genes (P450 blooms) and many families with few genes, follows similar power laws in both groups. A closer look at some families with few genes shows that these, often single member families, are not stable during evolution. The enzymatic prowess of P450 may predispose them to switch back and forth between metabolism of critical structural or signal molecules and metabolism dedicated to environmental response. PMID:23297351

  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. 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.

  5. Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

    PubMed

    Sagami, I; Ohmachi, T; Fujii, H; Kikuchi, H; Watanabe, M

    1991-10-01

    Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.

  6. Purification and characterization of the dog hepatic cytochrome P-450 isozyme(s) responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl

    SciTech Connect

    Duignan, D.B.; Sipes, I.G.; Leonard, T.B.; Halpert, J.R.

    1986-03-01

    Pretreatment of dogs and rats with phenobarbital (PB) enhances the in vitro metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB); however, in control dog microsomes there is an approximately 5-fold greater rate of metabolism than that observed in PB-induced rat microsomes. At least two PB-induced isozymes of cytochrome P-450 are detected in dog microsomes, and by use of Octylamino-Sepharose. Hydroxylapatite, and DEAE-Sephacel column chromatography, one of these isozymes (which the authors call PBD-2) has been purified to >95%, as determined by SDS-PAGE. In a reconstituted system, PBD-2 can metabolize 245-HCB at a rate similar to that seen for PB-B, the major PB-induced isozyme in the rat. In addition, antibodies raised against PB-B cross-react with PBD-2, and the NH/sub 2/-terminal amino acid sequence of PBD-2 is nearly 70% homologous to that of PB-B. These results suggest that a cytochrome P-450 isozyme capable of metabolizing 245-HCB in the dog (PBD-2) is similar to PB-B. The importance of PBD-2 will be further elucidated by conducting inhibition studies with anti-PBD-2 antibodies in control and PB-induced dog microsomes.

  7. 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.

  8. Hepatic zonation of the induction of cytochrome P450 IVA, peroxisomal lipid beta-oxidation enzymes and peroxisome proliferation in rats treated with dehydroepiandrosterone (DHEA). Evidence of distinct zonal and sex-specific differences.

    PubMed

    Beier, K; Völkl, A; Metzger, C; Mayer, D; Bannasch, P; Fahimi, H D

    1997-08-01

    Dehydroepiandrosterone (DHEA) is an intermediate product in the synthesis of male and female sex hormones in the adrenal cortex of man. In livers of rats and mice DHEA increases the levels of cytochrome P450 IVA and peroxisomal beta-oxidation enzymes associated with peroxisome proliferation. Prolonged treatment of rats with DHEA induces liver tumors that are more frequent in females arising mainly in the periportal regions of the liver lobule (Metzger et al., Toxicol. Pathol. 23, 591-605, 1995). Because of paucity of information on hepatic zonation of peroxisomal response to DHEA and controversial reports on gender-specific differences of its effects the present study was undertaken using qualitative immunohistochemical and quantitative immunoelectron microscopical techniques in addition to Western blotting. Rats were treated for 24 weeks with 0.6% DHEA supplied with diet. Immunoblot analysis revealed marked induction of peroxisomal beta-oxidation enzymes, which by quantitative analysis was equally strong in male and female animals, whilst catalase and urate-oxidase were not increased. Cytochrome P450 IVA, in contrast, was induced significantly stronger in male than in female rats. Immunohistochemistry confirmed the induction of cytochrome P450 IVA showing a marked lobular gradient in female animals with strong induction in pericentral and almost no induction in periportal regions of the liver lobule. In male animals cytochrome P450 IVA was expressed more uniformly across the liver lobule. A similar sex specific zone-dependent response was observed for peroxisomes. DHEA induced in females a significant zonal gradient with marked peroxisome proliferation and a strong induction of peroxisomal hydratase/dehydrogenase in pericentral hepatocytes and a much smaller response in periportal regions. Livers of male animals, in contrast, showed a uniform peroxisomal proliferation to DHEA with only slight zonal differences. The striking homologies of the induction patterns of

  9. 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.

  10. 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

  11. 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...

  12. In Vitro and in Silico Analyses for Predicting Hepatic Cytochrome P450-Dependent Metabolic Potencies of Polychlorinated Biphenyls in the Baikal Seal.

    PubMed

    Yoo, Jean; Hirano, Masashi; Mizukawa, Hazuki; Nomiyama, Kei; Agusa, Tetsuro; Kim, Eun-Young; Tanabe, Shinsuke; Iwata, Hisato

    2015-12-15

    The aim of this study was to understand the cytochrome P450 (CYP)-dependent metabolic pathway and potency of polychlorinated biphenyls (PCBs) in the Baikal seal (Pusa sibirica). In vitro metabolism of 62 PCB congener mixtures was investigated by using liver microsomes of this species. A decreased ratio of over 20% was observed for CB3, CB4, CB8, CB15, CB19, CB22, CB37, CB54, CB77, and CB105, suggesting the preferential metabolism of low-chlorinated PCBs by CYPs. The highly activated metabolic pathways in Baikal seals that were predicted from the decreased PCBs and detected hydroxylated PCBs (OH-PCBs) were CB22 to 4'OH-CB20 and CB77 to 4'OH-CB79. The total amount of OH-PCBs detected as identified and unidentified congeners accounted for only a 3.8 ± 1.7 mol % of loaded PCBs, indicating many unknown PCB metabolic pathways. To explore factors involved in CYP-dependent PCB metabolism, we examined the relationships among the structural and physicochemical properties of PCBs, the in silico PCB-CYP docking parameters, and the in vitro PCB decreased ratios by principal component analysis. Statistical analysis showed that the decreased PCB ratio was at least partly accounted for by the substituted chlorine number of PCBs and the distance from the Cl-unsubstituted carbon of docked PCBs to the heme Fe in CYP2A and 2B. PMID:26579933

  13. In Vitro and in Silico Analyses for Predicting Hepatic Cytochrome P450-Dependent Metabolic Potencies of Polychlorinated Biphenyls in the Baikal Seal.

    PubMed

    Yoo, Jean; Hirano, Masashi; Mizukawa, Hazuki; Nomiyama, Kei; Agusa, Tetsuro; Kim, Eun-Young; Tanabe, Shinsuke; Iwata, Hisato

    2015-12-15

    The aim of this study was to understand the cytochrome P450 (CYP)-dependent metabolic pathway and potency of polychlorinated biphenyls (PCBs) in the Baikal seal (Pusa sibirica). In vitro metabolism of 62 PCB congener mixtures was investigated by using liver microsomes of this species. A decreased ratio of over 20% was observed for CB3, CB4, CB8, CB15, CB19, CB22, CB37, CB54, CB77, and CB105, suggesting the preferential metabolism of low-chlorinated PCBs by CYPs. The highly activated metabolic pathways in Baikal seals that were predicted from the decreased PCBs and detected hydroxylated PCBs (OH-PCBs) were CB22 to 4'OH-CB20 and CB77 to 4'OH-CB79. The total amount of OH-PCBs detected as identified and unidentified congeners accounted for only a 3.8 ± 1.7 mol % of loaded PCBs, indicating many unknown PCB metabolic pathways. To explore factors involved in CYP-dependent PCB metabolism, we examined the relationships among the structural and physicochemical properties of PCBs, the in silico PCB-CYP docking parameters, and the in vitro PCB decreased ratios by principal component analysis. Statistical analysis showed that the decreased PCB ratio was at least partly accounted for by the substituted chlorine number of PCBs and the distance from the Cl-unsubstituted carbon of docked PCBs to the heme Fe in CYP2A and 2B.

  14. 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 ...

  15. 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. PMID:25036135

  16. 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.

  17. 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. PMID:16828826

  18. 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.

  19. Multiple cytochrome P-450 genes are concomitantly regulated by vitamin A under steady-state conditions and by retinoic acid during hepatic first-pass metabolism.

    PubMed

    Ross, A Catharine; Cifelli, Christopher J; Zolfaghari, Reza; Li, Nan-Qian

    2011-01-01

    Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improvement of dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate, and supplemented) and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P-450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P < 0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding retinoic acid receptor (RAR)β, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log 2(6) in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naive rats, evidenced by increased CYP26A1 gene expression and increased conversion of [(3)H]RA to polar metabolites. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling retinol-binding protein (RBP)4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change.

  20. Antioxidant activity of new aramide nanoparticles containing redox-active N-phthaloyl valine moieties in the hepatic cytochrome P450 system in male rats.

    PubMed

    Hassan, Hammed H A M; El-Banna, Sabah G; Elhusseiny, Amel F; Mansour, El-Sayed M E

    2012-07-10

    We report the synthesis of aramide nanoparticles containing a chiral N-phthaloyl valine moiety and their antioxidant activities on hepatic contents of cytochrome P₄₅₀, amidopyrene N-demethylase, aniline-4-hyroxylase and induced the hepatic content of cytochrome b5 and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C-reductase. Polymers were obtained as well-separated spherical nanoparticles while highly aggregated particles via H-bonding organization of the aramide-containing pyridine led to a thin layer formation. The effects of the nanoparticles and CCl₄ on enzyme activities and thiobarbituric acid reactive substances (TBARS) levels of male rat liver were studied. Pretreatments of rats with the polyamides prior to the administration of CCl₄ decreased the hepatic content of the tested enzymes. Doses reduced the toxic effects exerted by (•CCl₃) upon the liver through inhibition of the cytochrome P₄₅₀ system. Inhibition of such metabolizing enzymes could reduce the carcinogenic effects of chemical carcinogens.

  1. 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.

  2. In vitro and in vivo association of porcine hepatic cytochrome P450 3A and 2C activities with testicular steroids.

    PubMed

    Zamaratskaia, G; Zlabek, V; Ropstad, E; Andresen, Ø

    2012-12-01

    The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17β-estradiol, 17α-estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism-based inhibitions were examined. 7-benzyloxyresorufin (BR) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7-benzyloxyresorufin O-dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism-based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17β-oestradiol. Mechanism-based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α-oestradiol through the mechanism-based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in

  3. 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.

  4. Canine cytochrome P-450 pharmacogenetics.

    PubMed

    Court, Michael H

    2013-09-01

    The cytochrome P-450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences.

  5. Faster clearance of omeprazole in rats with acute renal failure induced by uranyl nitrate: contribution of increased expression of hepatic cytochrome P450 (CYP) 3A1 and intestinal CYP1A and 3A subfamilies.

    PubMed

    Lee, Dae Y; Jung, Young S; Shin, Hyun S; Lee, Inchul; Kim, Young C; Lee, Myung G

    2008-07-01

    It has been reported that omeprazole is mainly metabolized via hepatic cytochrome P450 (CYP) 1A1/2, CYP2D1 and CYP3A1/2 in male Sprague-Dawley rats, and the expression of hepatic CYP3A1 is increased in male Sprague-Dawley rats with acute renal failure induced by uranyl nitrate (U-ARF rats). Thus, the metabolism of omeprazole would be expected to increase in U-ARF rats. After intravenous administration of omeprazole (20 mgkg(-1)) to U-ARF rats, the area under the plasma concentration-time curve from time zero to infinity (AUC) was significantly reduced (371 vs 494 microg min mL(-1)), possibly due to the significantly faster non-renal clearance (56.6 vs 41.2 mL min(-1) kg(-1)) compared with control rats. This could have been due to increased expression of hepatic CYP3A1 in U-ARF rats. After oral administration of omeprazole (40 mgkg(-1)) to U-ARF rats, the AUC was also significantly reduced (89.3 vs 235 microg min mL(-1)) compared with control rats. The AUC difference after oral administration (62.0% decrease) was greater than that after intravenous administration (24.9% decrease). This may have been primarily due to increased intestinal metabolism of omeprazole caused by increased expression of intestinal CYP1A and 3A subfamilies in U-ARF rats, in addition to increased hepatic metabolism.

  6. Luminogenic cytochrome P450 assays.

    PubMed

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

    2006-08-01

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

  7. Intact glucosinolates modulate hepatic cytochrome P450 and phase II conjugation activities and may contribute directly to the chemopreventive activity of cruciferous vegetables.

    PubMed

    Abdull Razis, Ahmad F; Bagatta, Manuela; De Nicola, Gina R; Iori, Renato; Ioannides, Costas

    2010-11-01

    The currently accepted view is that the chemopreventive activity of glucosinolates is exclusively mediated by their degradation products, such as isothiocyanates. In the present study, evidence is presented for the first time that intact glucosinolates can modulate carcinogen-metabolising enzyme systems. The glucosinolates glucoraphanin and glucoerucin were isolated from cruciferous vegetables and incubated with precision-cut rat liver slices. Both glucosinolates elevated the O-dealkylations of methoxy- and ethoxyresorufin, markers for CYP1 activity; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished these effects. Moreover, both glucoerucin and glucoraphanin increased the apoprotein levels of microsomal CYP1A1, CYP1A2 and CYP1B1. At higher concentrations, both glucosinolates enhanced quinone reductase activity, whereas glucoraphanin also elevated glutathione S-transferase; in this instance, however, supplementation of the incubation medium with myrosinase exacerbated the inductive effect. Finally, both glucosinolates increased modestly cytosolic quinone reductase, GSTα and GSTμ protein levels, which became more pronounced when myrosinase was added to the incubations with the glucosinolate. It may be inferred that intact glucosinolates can modulate the activity of hepatic carcinogen-metabolising enzyme systems and this is likely to impact on the chemopreventive activity linked to cruciferous vegetable consumption.

  8. Characterization and profiling of hepatic cytochromes P450 and phase II xenobiotic-metabolizing enzymes in beluga whales (Delphinapterus leucas) from the St. Lawrence River Estuary and the Canadian Arctic.

    PubMed

    McKinney, Melissa A; Arukwe, Augustine; De Guise, Sylvain; Martineau, Daniel; Béland, Pierre; Dallaire, André; Lair, Stéphane; Lebeuf, Michel; Letcher, Robert J

    2004-07-30

    Cytochromes P450 (CYP, phase I) and conjugating (phase II) enzymes can be induced by and influence the toxicokinetics (metabolism) and toxicity of xenobiotic contaminants in exposed organisms. Beluga whale (Delphinapterus leucas) from the endangered St. Lawrence (SL) River Estuary population exhibit deleterious health effects and various severe pathologies that have been associated with contaminant exposure. In contrast, such effects (e.g. reproductive and immunological impairment) are generally less frequent in less exposed populations in the Canadian Arctic (CA). In the present study, opportunistic sampling resulted in the collection immediately after death of liver tissue from a single female neonate SL beluga (SL6) and male and female CA beluga (n=10) from the Arviat region of western Hudson Bay, in addition to sampling of stranded carcasses of male and female SL beluga (n=5) at least 12 h postmortem. We immunologically characterized cross-reactive proteins of hepatic microsomal CYP1A, CYP2B, CYP3A, CYP2E, epoxide hydrolase (EH) and uridine diphosphoglucuronosyl transferase (UDPGT) isozymes. Cross-reactive proteins were found in all SL and CA beluga using anti-rat CYP1A1, anti-rainbow trout CYP3A, anti-human CYP2E1, anti-rabbit EH and anti-human UDPGT1A1 polyclonal antibodies (Abs), whereas faintly cross-reactive CYP2B proteins were only found in SL6 and the CA samples using an anti-rabbit CYP2B1 Ab. In corresponding catalytic activity assessments, only SL6 and all CA beluga microsomal samples exhibited CYP1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activity (51-260 pmol/mg/min), CYP3A-mediated activity (113-899 pmol/mg/min) based on the formation of 6beta-hydroxytestosterone using a testosterone hydroxylase assay, and UDPGT activity (830-4956 pmol/mg/min) based on 1-naphthylglucuronide formation. The marginal cross-reactivity with the anti-CYP2B1 Ab and lack of catalytically measurable hydroxytestosterone isomers associated with CYP2B-type activity in

  9. 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

  10. Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

    PubMed

    Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

    2004-03-01

    The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

  11. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

  12. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  13. Cytochrome P450 monooxygenase system in echinoderms.

    PubMed

    den Besten, P J

    1998-11-01

    The results of a limited number of studies on echinoderms provide evidence for the presence of a cytochrome P450 monooxygenase system in representatives of three classes of the phylum Echinodermata: the asteroids (sea stars), holothuroids (sea cucumbers) and echinoids (sea urchins). The monooxygenase system has been demonstrated to be involved in the metabolism of xenobiotic compounds, but is assumed to have its primary function in the metabolism of endogenous substrates, such as steroids. Available data on P450 cofactor requirement, P450-dependent metabolism of benzo[a]pyrene, studies with classical inhibitors of P450, specificity of P450 induction by planar compounds, and the changes in the benzo[a]pyrene metabolite profile in induced animals suggest similarities with the MO system present in vertebrates. However, the relatively high capacity of the monooxygenase system in sea stars to catalyse reactions with organic hydroperoxide as donor for activated oxygen, and the low induceability during exposure to xenobiotics indicate also important differences between the echinoderm cytochrome P450 monooxygenase system and that of vertebrates. Some evidence was found for the existence of different forms of cytochrome P450 in sea stars. Catalytic functions of the cytochrome P450 monooxygenase system of sea stars in the metabolism of steroids may be suppressed as a result of the induction of cytochrome P450 by xenobiotics. PMID:9972455

  14. Structural characterization of a monoclonal antibody immunopurified pulmonary cytochrome P-450 from 3-methylcholanthrenetreated rats

    SciTech Connect

    Robinson, R.C.; Cheng, K.C.; Park, S.S.; Gelboin, H.V.; Friedman, F.K.

    1986-05-01

    Extrahepatic cytochromes P-450 have not been as extensively studied as the hepatic forms, owing to the low concentrations of these enzymes in extrahepatic tissues. A cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to the major MC-inducible form of rat liver cytochrome P-450. The lung cytochrome P-450 is related to this liver form by at least two common epitopes, recognized by monoclonal antibodies 1-7-1 and 1-31-2. The isolated pulmonary cytochrome P-450 is MC-inducible and has an apparent molecular weight of 57 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight as well as the NH/sub 2/-terminal sequence of the pulmonary cytochrome P-450 is identical to that of the major MC-inducible form of rat liver cytochrome P-450. In addition, limited proteolytic digestion of both cytochromes P-450 generates the same peptide patterns on SDS-PAGE. By several criteria, treatment of rats with MC thus induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme.

  15. Role of Cytochrome P450s in Inflammation.

    PubMed

    Christmas, Peter

    2015-01-01

    Cytochrome P450 epoxygenases and hydroxylases play a regulatory role in the activation and suppression of inflammation by generating or metabolizing bioactive mediators. CYP2C and CYP2J epoxygenases convert arachidonic acid to anti-inflammatory epoxyeicosatrienoic acids, which have protective effects in a variety of disorders including cardiovascular disease and metabolic syndrome. CYP4A and CYP4F hydroxylases have the ability to metabolize multiple substrates related to the regulation of inflammation and lipid homeostasis, and it is a challenge to determine which substrates are physiologically relevant for each enzyme; the best-characterized activities include generation of 20-hydroxyeicosatetraenoic acid and inactivation of leukotriene B4. The expression of hepatic drug-metabolizing cytochrome P450s is modulated by cytokines during inflammation, resulting in changes to the pharmacokinetics of prescribed medications. Cytochrome P450s are therefore the focus of intersecting challenges in the pharmacology of inflammation: not only do they represent targets for development of new anti-inflammatory drugs but they also contribute to variability in drug efficacy or toxicity in inflammatory disease. Animal models and primary hepatocytes have been used extensively to study the effects of cytokines on cytochrome P450 expression and activity. However, it is difficult to predict changes in drug exposure in patients because the response to inflammation varies depending on the disease state, its time course, and the cytochrome P450 involved. In these circumstances, the development of endogenous markers of cytochrome P450 metabolism might provide a useful tool to reevaluate drug dosage and choice of therapy.

  16. 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

  17. SMARTCyp: A 2D Method for Prediction of Cytochrome P450-Mediated Drug Metabolism.

    PubMed

    Rydberg, Patrik; Gloriam, David E; Zaretzki, Jed; Breneman, Curt; Olsen, Lars

    2010-06-10

    SMARTCyp is an in silico method that predicts the sites of cytochrome P450-mediated metabolism of druglike molecules. The method is foremost a reactivity model, and as such, it shows a preference for predicting sites that are metabolized by the cytochrome P450 3A4 isoform. SMARTCyp predicts the site of metabolism directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. This is a major advantage, because it makes SMARTCyp very fast. Other advantages are that experimental data are not a prerequisite to create the model, and it can easily be integrated with other methods to create models for other cytochrome P450 isoforms. Benchmarking tests on a database of 394 3A4 substrates show that SMARTCyp successfully identifies at least one metabolic site in the top two ranked positions 76% of the time. SMARTCyp is available for download at http://www.farma.ku.dk/p450.

  18. A world of cytochrome P450s.

    PubMed

    Nelson, David R

    2013-02-19

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

  19. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

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

  20. 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.

  1. Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics.

    PubMed

    Lewis, D F; Watson, E; Lake, B G

    1998-06-01

    The evolution of the cytochrome P450 (CYP) superfamily is described, with particular reference to major events in the development of biological forms during geological time. It is noted that the currently accepted timescale for the elaboration of the P450 phylogenetic tree exhibits close parallels with the evolution of terrestrial biota. Indeed, the present human P450 complement of xenobiotic-metabolizing enzymes may have originated from coevolutionary 'warfare' between plants and animals during the Devonian period about 400 million years ago. A number of key correspondences between the evolution of P450 system and the course of biological development over time, point to a mechanistic molecular biology of evolution which is consistent with a steady increase in atmospheric oxygenation beginning over 2000 million years ago, whereas dietary changes during more recent geological time may provide one possible explanation for certain species differences in metabolism. Alignment between P450 protein sequences within the same family or subfamily, together with across-family comparisons, aid the rationalization of drug metabolism specificities for different P450 isoforms, and can assist in an understanding of genetic polymorphisms in P450-mediated oxidations at the molecular level. Moreover, the variation in P450 regulatory mechanisms and inducibilities between different mammalian species are likely to have important implications for current procedures of chemical safety evaluation, which rely on pure genetic strains of laboratory bred rodents for the testing of compounds destined for human exposure.

  2. Effect of cytochrome P450 inducers on cocaine-mediated hepatotoxicity.

    PubMed

    Bornheim, L M

    1998-05-01

    The effect of several cytochrome P450 (P450) inducers on cocaine metabolism were examined in order to characterize the metabolic events contributing to cocaine-induced hepatotoxicity. Phenobarbital (PB)-pretreatment of mice induced P450s 3A and 2B and markedly increased serum alanine aminotransferase (ALT) activity after cocaine or norcocaine administration. Although dexamethasone (Dex) induced P450s 3A and 2B at least to the same extent as PB, no increase in serum ALT activity was observed after cocaine or norcocaine administration. Phencyclidine (PCP) pretreatment did not increase either P450s 3A or 2B, yet it markedly enhanced cocaine- or norcocaine-induced serum ALT activity. In contrast to the marked induction of P450s 3A and 2B, P450 2C was increased only 2.5-fold by PB and to an even lesser extent by Dex or PCP. Cannabidiol (CBD), which inactivates P450s 3A and 2C in mice, completely protected mice against cocaine- or norcocaine-induced hepatotoxicity irrespective of whether they were induced or not with PB or PCP. Both PB and Dex pretreatment increased the in vitro hepatic microsomal formation of the first two sequential oxidative metabolites of cocaine (norcocaine and N-hydroxynorcocaine), whereas PCP pretreatment did not. Hepatic esterase activity was also determined after pretreatment with P450 inducers, since this is the major detoxification pathway in cocaine metabolism. Dex pretreatment markedly increased (> 11-fold) total hepatic esterase activity, whereas PB pretreatment increased it more modestly (less than fourfold) and PCP pretreatment had little effect. This marked effect of Dex pretreatment may decrease liver cocaine concentrations and thus protect mice against cocaine-induced hepatotoxicity, despite their increased P450 2B and 3A contents.

  3. The effects of azole-based heme oxygenase inhibitors on rat cytochromes P450 2E1 and 3A1/2 and human cytochromes P450 3A4 and 2D6.

    PubMed

    Hum, Maaike; McLaughlin, Brian E; Roman, Gheorghe; Vlahakis, Jason Z; Szarek, Walter A; Nakatsu, Kanji

    2010-09-01

    Heme oxygenases (HOs) catalyze the degradation of heme to biliverdin, carbon monoxide (CO), and free iron. The two major isoforms, HO-1 (inducible) and HO-2 (constitutive), are involved in a variety of physiological functions, including inflammation, apoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (P450s), soluble guanylyl cyclase (sGC), and nitric-oxide synthase (NOS). Our laboratory has successfully synthesized a number of nonporphyrin azole-based HO inhibitors (QC-xx) that had little or no effect on sGC and NOS activity. However, their effects on various P450 isoforms have yet to be fully elucidated. To determine the effects of the QC-xx inhibitors on P450 enzyme activity, microsomal preparations of two rat P450 isoforms (2E1 and 3A1/3A2) and two human P450 supersome isoforms (3A4 and 2D6) were incubated with varying concentrations of HO inhibitor, and the activity was determined by spectrophotometric or fluorometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of the P450s, whereas others did inhibit these P450 isoforms. Four structural regions of QC-xx were analyzed, leading to the identification of structures that confer a decreased effect on both rat and human P450 isoforms studied while maintaining an inhibitory effect on the HOs.

  4. Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver.

    PubMed

    Sun, Guobin; Thai, Sheau-Fung; Tully, Douglas B; Lambert, Guy R; Goetz, Amber K; Wolf, Douglas C; Dix, David J; Nesnow, Stephen

    2005-02-15

    Propiconazole is a N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. In the present study, propiconazole was examined for its effects on the expression of hepatic cytochrome P450 genes and on the activities of P450 enzymes in male Sprague-Dawley rats and male CD-1 mice. Rats and mice were administered propiconazole by gavage daily for 14 days at doses of 10, 75, and 150 mg/kg body weight/day. Quantitative real time RT-PCR assays of rat hepatic RNA samples from animals treated at the 150 mg/kg body weight/day dose revealed significant mRNA overexpression of the following genes compared to control: CYP1A2 (1.62-fold), CYP2B1 (10.8-fold), CYP3A1/CYP3A23 (2.78-fold), and CYP3A2 (1.84-fold). In mouse liver, propiconazole produced mRNA overexpression of Cyp2b10 (2.39-fold) and Cyp3a11 (5.19-fold). mRNA expression of CYP1A1 was not detected in liver tissues from treated or controls animals from either species. Propiconazole significantly induced both pentoxyresorufin O-dealkylation (PROD) and methoxyresorufin O-dealkylation (MROD) activities in both rat and mouse liver at the 150 mg/kg body weight/day and 75 mg/kg body weight/day doses. In summary, these results indicated that propiconazole induced CYP1A2 in rat liver and CYP2B and CYP3A families of isoforms in rat and mouse liver.

  5. 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

  6. Citrulline-malate effect on microsome phospholipids and cytochrome P450 in Euglena grown with ethanol.

    PubMed

    Thuillier-Bruston, F; Julistiono, H; Briand, J

    1991-04-01

    This study indicates for the first time the presence of cytochrome P450 in the microsomes of Euglena grown in lactate medium and substantiates the use of Euglena as a hepatic cell model. Similar effects of ethanol on Euglena and on rat hepatic microsomes were demonstrated: (i) decrements in the quantities of FA per milligram of proteins; (ii) increases in the proportions of PE; (iii) decreases in the proportions of PC; and (iv) production of cytochrome P450, degraded in P420. The citrulline-malate reestablishes in the microsomes the phospholipid environment and the cytochrome P450 concentration. These findings illustrate that the complex acts on the lipid peroxidation via the changes in cytochrome P450 activity. PMID:1909150

  7. Genomic and bioinformatic analysis of NADPH-cytochrome P450 reductase in Anopheles stephensi (Diptera: Culicidae).

    PubMed

    Suwanchaichinda, C; Brattsten, L B

    2014-01-01

    The cytochrome P450 monooxygenase (P450) enzyme system is a major mechanism of xenobiotic biotransformation. The nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is required for transfer of electrons from NADPH to P450. One CPR gene was identified in the genome of the malaria-transmitting mosquito Anopheles stephensi Liston (Diptera: Culicidae). The gene encodes a polypeptide containing highly conserved flavin mononucleotide-, flavin adenine dinucleotide-, and NADPH-binding domains, a unique characteristic of the reductase. Phylogenetic analysis revealed that the A. stephensi and other known mosquito CPRs belong to a monophyletic group distinctly separated from other insects in the same order, Diptera. Amino acid residues of CPRs involved in binding of P450 and cytochrome c are conserved between A. stephensi and the Norway rat Rattus norvegicus Berkenhout (Rodentia: Muridae). However, gene structure particularly within the coding region is evidently different between the two organisms. Such difference might arise during the evolution process as also seen in the difference of P450 families and isoforms found in these organisms. CPR in the mosquito A. stephensi is expected to be active and serve as an essential component of the P450 system.

  8. 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

  9. The P450 gene superfamily: recommended nomenclature.

    PubMed

    Nebert, D W; Adesnik, M; Coon, M J; Estabrook, R W; Gonzalez, F J; Guengerich, F P; Gunsalus, I C; Johnson, E F; Kemper, B; Levin, W

    1987-02-01

    A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge. PMID:3829886

  10. Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3.

    PubMed

    Sowden, Rebecca J; Yasmin, Samina; Rees, Nicholas H; Bell, Stephen G; Wong, Luet-Lok

    2005-01-01

    The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3. PMID:15602599

  11. 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.

  12. Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes.

    PubMed Central

    Kolars, J C; Schmiedlin-Ren, P; Schuetz, J D; Fang, C; Watkins, P B

    1992-01-01

    Enzymes within the P450IIIA (CYP3A) subfamily appear to account for significant "first pass" metabolism of some drugs in the intestine. To identify which of the known P450IIIA genes are expressed in intestine, enterocyte RNA was hybridized on Northern blots with synthetic oligonucleotides complementary to hypervariable regions of hepatic P450IIIA4, P450IIIA5, and P450IIIA7 cDNAs. Hybridization was detected only with the P450IIIA4-specific oligonucleotide. The identity of the hybridizing mRNA was confirmed to be P450IIIA4 by direct sequencing of a DNA fragment amplified from enterocyte cDNA by the polymerase chain reaction. To determine if enterocyte P450IIIA4 is inducible, biopsies of small bowel mucosa were obtained from five volunteers before and after they received 7d of treatment with rifampin, a known inducer of P450IIIA4 in liver. Rifampin treatment resulted in a five- or eightfold mean increase (P < 0.05) in the biopsy concentration of P450IIIA4 mRNA when normalized for content of sucrase isomaltase or intestinal fatty acid binding protein mRNAs, respectively. Rifampin also induced P450IIIA immunoreactive protein in enterocytes in each of the subjects, as judged by immunohistochemistry, and resulted in a 10-fold increase in P450IIIA4-specific catalytic activity (erythromycin N-demethylation) in the one patient studied. Our identification of inducible P450IIIA4 in enterocytes may in part account for drug interactions characteristic of P450IIIA4 substrates and suggests a strategy for controlling entry into the body of a major class of xenobiotics. Images PMID:1430211

  13. 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.

  14. Impacts of diversification of cytochrome P450 on plant metabolism.

    PubMed

    Mizutani, Masaharu

    2012-01-01

    Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.

  15. Interactions of phospholipase D and cytochrome P450 protein stability

    SciTech Connect

    Zangar, Richard C.; Fan, Yang-Yi; Chapkin, Robert S.

    2004-08-01

    Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

  16. Cytochrome P-450 epitope typing in animals and humans with monoclonal antibodies to ethanol induced rat liver microsomal cytochrome P-450 (P-450et)

    SciTech Connect

    Park, S.S.; Ko, I.Y.; Yang, C.; Guengerich, F.G.; Schenkman, J.B.; Coon, M.J.; Gelboin, H.V.

    1986-05-01

    Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice that had been immunized with P-450et. The monoclonal antibody (MAb)-producing hybridomas were screened by RIA. Thirty one independent hybrid clones were isolated with each producing an MAb of a single immunoglobulin subclass. All of these MAbs had high affinities for P-450et but only one MAb had a strong inhibitory effect on aniline rho-hydroxylase and N-nitrosodimethylamine demethylase. Western blots and RIAs based on ten MAbs (C1-C10) were used to determine the epitope homology of purified cytochromes P-450 from rats, rabbits, and humans. All ten MAbs had high affinity for both P-450et and a rat P-450 which is induced by acetone (P-450ac). Classes of these MAbs were identified which crossreacted toward different forms of rat P-450. In addition, several MAbs (C3, C6, C9) recognized a P-450 form of human liver, while other MAbs (C7, C9) recognized P-450/sub LM2/ of rabbits. Three MAbs (C4, C5, C8) were specific for only P-450et and P-450ac. These results demonstrate the different degrees of epitope relatedness among the multiple forms of cytochrome P-450.

  17. 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

  18. Pharmacogenomics of human P450 oxidoreductase

    PubMed Central

    Pandey, Amit V.; Sproll, Patrick

    2014-01-01

    Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley–Bixler syndrome (ABS) but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations, with Japanese (R457H), Caucasian (A287P), and Turkish (399–401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V is found in about 30% of all alleles but there are some differences across different population groups. PMID:24847272

  19. 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.

  20. The anticarcinogen 3,3'-diindolylmethane is an inhibitor of cytochrome P-450.

    PubMed

    Stresser, D M; Bjeldanes, L F; Bailey, G S; Williams, D E

    1995-08-01

    Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now report a similar potent inhibition by 3,3'-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3'-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we found 3,3'-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3'-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans.

  1. 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

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

    PubMed

    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

  3. 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

  4. 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.

  5. Aldehyde Reduction by Cytochrome P450

    PubMed Central

    Amunom, Immaculate; Srivastava, Sanjay; Prough, Russell A.

    2011-01-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  6. Interaction of fluoroethane chlorofluorocarbon (CFC) substitutes with microsomal cytochrome P450. Stimulation of P450 activity and chlorodifluoroethene metabolism.

    PubMed

    Wang, Y; Olson, M J; Baker, M T

    1993-07-01

    The abilities of halothane and the fluoroethane chlorofluorocarbon (CFC) substitutes, FC-123, FC-133a, FC-124, FC-134a and FC-125, to stimulate cytochrome P450 activities and 2-chloro-1,1-difluoroethene (CDE) defluorination in hepatic microsomes from phenobarbital-treated rabbits were compared. At 1% (v/v) each, halothane and FC-123 similarly increased the consumption of NADPH and O2 by 300 and 100%, respectively, over that in microsomes without substrate. FC-133a and FC-124 were less effective, increasing NADPH and O2 consumption by 150-200 and 70%. FC-134a and FC-125 were the least effective, increasing NADPH and O2 consumption by only 70 and 50%, respectively. No metabolism of any fluoroethane could be detected under the incubation conditions used. Halothane and FC-123 were most effective in stimulating CDE metabolism with increases of CDE defluorination ranging from 1.5- to 2-fold. FC-133a and FC-124 enhanced CDE oxidation 89 and 74%, respectively, and FC-134a and FC-125 had no effect. While CDE metabolism was enhanced in the presence of the fluoroethanes, no additional NADPH or O2 was consumed when halothane or FC-124 was incubated with CDE compared with incubations containing only halothane or FC-124. Log-log plots of NADPH consumption and CDE metabolism with the olive oil/gas partition coefficients of each fluoroethane showed linear relationships. These data demonstrate that the activity of the fluoroethanes in stimulating P450 activity and CDE metabolism is a function of their lipid solubility, and fluoroethane-enhanced CDE metabolism is related to the ability of these compounds to increase uncoupled P450 activity.

  7. 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.

  8. UNDERSTANDING THE MECHANISM OF CYTOCHROME P450 3A4: RECENT ADVANCES AND REMAINING PROBLEMS

    PubMed Central

    Sevrioukova, Irina F.; Poulos, Thomas L.

    2013-01-01

    Cytochromes P450 (CYPs) represent a diverse group of heme-thiolate proteins found in almost all organisms. CYPs share a common protein fold but differ in substrate selectivity and catalyze a wide variety of monooxygenation reactions via activation of molecular oxygen. Among 57 human P450s, the 3A4 isoform (CYP3A4) is the most abundant and the most important because it metabolizes the majority of the administered drugs. A remarkable feature of CYP3A4 is its extreme promiscuity in substrate specificity and cooperative substrate binding, which often leads to undesirable drug-drug interactions and toxic side effects. Owing to its importance in drug development and therapy, CYP3A4 has been the most extensively studied mammalian P450. In this review we provide an overview on recent progress and remaining problems in the CYP3A4 research. PMID:23018626

  9. Recent Structural Insights into Cytochrome P450 Function.

    PubMed

    Guengerich, F Peter; Waterman, Michael R; Egli, Martin

    2016-08-01

    Cytochrome P450 (P450) enzymes are important in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides, and many other types of chemicals. Their catalytic activities are important issues in areas such as drug-drug interactions and endocrine function. During the past 30 years, structures of P450s have been very helpful in understanding function, particularly the mammalian P450 structures available in the past 15 years. We review recent activity in this area, focusing on the past 2 years (2014-2015). Structural work with microbial P450s includes studies related to the biosynthesis of natural products and the use of parasitic and fungal P450 structures as targets for drug discovery. Studies on mammalian P450s include the utilization of information about 'drug-metabolizing' P450s to improve drug development and also to understand the molecular bases of endocrine dysfunction. PMID:27267697

  10. Genetics Home Reference: cytochrome P450 oxidoreductase deficiency

    MedlinePlus

    ... P450 oxidoreductase deficiency is a disorder of hormone production. This condition specifically affects steroid hormones, which are ... activity of cytochrome P450 oxidoreductase, which disrupts the production of steroid hormones. Changes in sex hormones such ...

  11. 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.

  12. 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

  13. 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.

  14. Biological diversity of cytochrome P450 redox partner systems.

    PubMed

    McLean, Kirsty J; Luciakova, Dominika; Belcher, James; Tee, Kang Lan; Munro, Andrew W

    2015-01-01

    Cytochrome P450 enzymes (P450s or CYPs) catalyze an enormous variety of oxidative reactions in organisms from all major domains of life. Their monooxygenase activity relies on the reductive scission of molecular oxygen (O2) bound to P450 heme iron, and thus on the delivery of two electrons to the heme iron at discrete points in the catalytic cycle. Early studies suggested that P450 redox partner machinery fell into only two major classes: either the eukaryotic diflavin enzyme NADPH-cytochrome P450 oxidoreductase, or bacterial/mitochondrial NAD(P)H-ferredoxin reductase and ferredoxin partners. However, more recent studies, aided by genome sequence data, reveal a much more complex scenario. Several new types of P450 redox partner systems have now been characterized, including P450s naturally linked to their redox partners, or to a component protein of their P450 electron delivery system. Other P450s have evolved to bypass requirements for redox partners, and instead react directly with hydrogen peroxide or NAD(P)H to facilitate oxidative or reductive catalysis. Further P450s are fused to non-redox partner enzymes and can catalyse consecutive reactions in a common pathway. This chapter describes the biochemistry and the enormous natural diversity of P450 redox systems, including descriptions of novel P450s fused to non-redox partner proteins.

  15. 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...

  16. 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...

  17. 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

  18. 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

  19. Mammalian cytochromes P-450: Volume I and Volume II

    SciTech Connect

    Guengerich, F.P.

    1987-01-01

    This two volume set summarizes the current knowledge of mammalian cytochromes. Ten chapters cover the current understanding of the enzymology of rat, rabbit, and human liver cytochromes P-450, extrahepatic cytochromes P-450, the diversity of substrates for the individual cytochromes P0-450 proteins, the metabolism of pro-toxicants and -carcinogens by cytochrome P-450, the degradation of cytochrome P-450 proteins, and the regulation of cytochrome P-450 activities in vitro and in vivo. The individual chapters outline the historical development of each area, the approaches which are applied, the current state of knowledge, and future directions towards unresolved questions; and index.

  20. Functional coupling of ATP-binding cassette transporter Abcb6 to cytochrome P450 expression and activity in liver.

    PubMed

    Chavan, Hemantkumar; Li, Feng; Tessman, Robert; Mickey, Kristen; Dorko, Kenneth; Schmitt, Timothy; Kumer, Sean; Gunewardena, Sumedha; Gaikwad, Nilesh; Krishnamurthy, Partha

    2015-03-20

    Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s. PMID:25623066

  1. Crystal structure of cindoxin, the P450cin redox partner.

    PubMed

    Madrona, Yarrow; Hollingsworth, Scott A; Tripathi, Sarvind; Fields, James B; Rwigema, Jean-Christophe N; Tobias, Douglas J; Poulos, Thomas L

    2014-03-11

    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

  2. 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 ...

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

    PubMed Central

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

    1998-01-01

    , 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. PMID:9667645

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Characterization of Drosophila melanogaster cytochrome P450 genes

    PubMed Central

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

    2009-01-01

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

  9. 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.

  10. Deletion of 30 murine cytochrome p450 genes results in viable mice with compromised drug metabolism.

    PubMed

    Scheer, Nico; McLaughlin, Lesley A; Rode, Anja; Macleod, A Kenneth; Henderson, Colin J; Wolf, C Roland

    2014-06-01

    In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity. PMID:24671958

  11. Gadolinium chloride reduces cytochrome P450: relevance to chemical-induced hepatotoxicity.

    PubMed

    Badger, D A; Kuester, R K; Sauer, J M; Sipes, I G

    1997-08-15

    The Kupffer cell inhibitor, gadolinium chloride (GdCl3), protects the liver from a number of toxicants that require biotransformation to elicit toxicity (i.e. 1,2-dichlorobenzene and CCl4), as well as compounds that do not (i.e. cadmium chloride and beryllium sulfate). The mechanism of this protection is thought to result from reduced secretion of inflammatory and cytotoxic products from Kupffer cells (KC). However, since other lanthanides have been shown to decrease cytochrome P450 (P450) activity, the following studies were designed to determine if GdCl3 pretreatment alters hepatic P450 levels or activity. The toxicological relevance of GdCl3-mediated alterations in P450 activity was also estimated by determining the effect of GdCl3 pretreatment on the susceptibility of primary cultured hepatocytes to CCl4 and cadmium chloride (CdCl2). Male and female Sprague-Dawley rats were given GdCl3 (i.v., 10 mg/kg). Twenty-four hours later, livers were either processed for preparation of microsomes or for primary cultures of hepatocytes. Gadolinium chloride treatment reduced total hepatic microsomal P450 as well as aniline hydroxylase activity by approximately 30% in males and 20% in females. In hepatocytes isolated from rats pretreated with GdCl3, the toxicity caused by CCl4, but not CdCl2 was reduced. Interestingly, when GdCl3 was administered in vitro to microsomes, there was no effect on either the microsomal P450 difference spectra or p-hydroxylation of aniline. However, when GdCl3 was incubated with isolated hepatocytes, the cytotoxicity of CCl4 (but not CdCl2) was partially attenuated. These results suggest that, in addition to its inhibitory effects on KC, GdCl3 produces other effects which may alter the susceptibility of hepatocytes to toxicity caused by certain chemicals.

  12. Influence of recipient gender on intrasplenic fetal liver tissue transplants in rats: cytochrome P450-mediated monooxygenase functions.

    PubMed

    Lupp, Amelie; Hugenschmidt, Sabine; Rost, Michael; Müller, Dieter

    2004-05-01

    Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern with consecutive differences in P450-mediated monooxygenase activities, which have been shown to be due to a differential profile of growth hormone (GH) secretion. Parallel to previous investigations on P450 isoforms expression, the aim of the present study was to elucidate the influence of recipient gender on P450-mediated monooxygenase activities in intrasplenic liver tissue transplants in comparison to orthotopic liver. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant-recipients and age-matched controls were treated with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the vehicles and sacrificed 24 or 48 h thereafter. P450-dependent monooxygenase activities were assessed by a series of model reactions for different P450 subtypes in liver and spleen 9000 g supernatants. In spleens of male and female control rats only very low monooxygenase activities were detectable, whereas with most model reactions distinct activities were observed in transplant-containing organs. Livers and transplant-containing spleens from male rats displayed higher basal ethoxycoumarin O-deethylase and testosterone 2alpha-, 2beta-, 6beta-, 14alpha-, 15alpha-, 15beta-, 16alpha-, 16beta- and 17-hydroxylase activities than those from females. On the other hand, like the respective livers, spleens from female transplant-recipients demonstrated more pronounced p-nitrophenol- and testosterone 6alpha- and 7alpha-hydroxylase activities than those from male hosts. With nearly all model reactions gender-specific differences in inducibility by BNF, PB or DEX could be demonstrated in livers as well as in transplant-containing spleens. These results further confirm that the P450 system of intrasplenic liver tissue transplants and the respective orthotopic livers is similarly influenced

  13. Insect P450 inhibitors and insecticides: challenges and opportunities.

    PubMed

    Feyereisen, René

    2015-06-01

    P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing.

  14. [Cytochrome P450 enzymes and microbial drug development - A review].

    PubMed

    Li, Zhong; Zhang, Wei; Li, Shengying

    2016-03-01

    Cytochrome P450 enzymes broadly exist in animals, plants and microorganisms. This superfamily of monooxygenases holds the greatest diversity of substrate structures and catalytic reaction types among all enzymes. P450 enzymes play important roles in natural product biosynthesis. In particular, P450 enzymes are capable of catalyzing the regio- and stereospecific oxidation of non-activated C-H bonds in complex organic compounds under mild conditions, which overrides many chemical catalysts. This advantage thus warrants their great potential in microbial drug development. In this review, we introduce a variety of P450 enzymes involved in natural product biosynthesis; provide a brief overview on protein engineering, biotransformation and practical application of P450 enzymes; and discuss the limits, challenges and prospects of industrial application of P450 enzymes.

  15. [Cytochrome P450 enzymes and microbial drug development - A review].

    PubMed

    Li, Zhong; Zhang, Wei; Li, Shengying

    2016-03-01

    Cytochrome P450 enzymes broadly exist in animals, plants and microorganisms. This superfamily of monooxygenases holds the greatest diversity of substrate structures and catalytic reaction types among all enzymes. P450 enzymes play important roles in natural product biosynthesis. In particular, P450 enzymes are capable of catalyzing the regio- and stereospecific oxidation of non-activated C-H bonds in complex organic compounds under mild conditions, which overrides many chemical catalysts. This advantage thus warrants their great potential in microbial drug development. In this review, we introduce a variety of P450 enzymes involved in natural product biosynthesis; provide a brief overview on protein engineering, biotransformation and practical application of P450 enzymes; and discuss the limits, challenges and prospects of industrial application of P450 enzymes. PMID:27382792

  16. 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.

  17. Role of inducer binding in cytochrome P-450 IA2-mediated uroporphyrinogen oxidation.

    PubMed

    Jacobs, J M; Sinclair, P R; Lambrecht, R W; Sinclair, J F; Jacobs, N J

    1990-01-01

    The oxidation of uroporphyrinogen, an intermediate of the heme biosynthetic pathway, by methylcholanthrene-inducible isozymes(s) of cytochrome P-450 has been proposed to play a role in the development of chemically induced uroporphyria. Prior work from this laboratory indicated that although addition of 3,4,3',4'-tetrachlorobiphenyl is required for uroporphyrinogen oxidation by methylcholanthrene-induced chick embryo liver microsomes, this biphenyl is not required for the oxidation catalyzed by hepatic microsomes from methylcholanthrene-induced rodents. Here we investigated whether rodent microsomes catalyze uroporphyrinogen oxidation without addition of 3,4,3',4'-tetrachlorobiphenyl because the chemical used as an inducer remains bound to cytochrome P-450. Hepatic microsomes containing almost no residual inducer were isolated from rats treated with a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These microsomes oxidized uroporphyrinogen at high rates without addition of 3,4,3',4'-tetrachlorobiphenyl. Inducer-free microsomal cytochrome P-450 was also obtained by inducing cytochrome P-450 in rats and mice with isosafrole, which was then removed from the isolated microsomes by butanol treatment. This procedure resulted in microsomes with high activity for uroporphyrinogen oxidation. Furthermore, addition of chlorobiphenyl to these inducer-free microsomes was inhibitory. Hepatic microsomes from isosafrole-induced C57BL/6 and DBA mice, rendered inducer-free by butanol treatment, oxidized uroporphyrinogen at the same rate even though these two strains differ markedly in their susceptibility to chemically induced uroporphyria. We conclude that uroporphyrinogen oxidation is catalyzed by cytochrome P-450 that is free of inducer.

  18. 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.

  19. Myeloid expression of cytochrome P450 4F3 is determined by a lineage-specific alternative promoter.

    PubMed

    Christmas, Peter; Carlesso, Nadia; Shang, Haibo; Cheng, Shing-Ming; Weber, Brittany M; Preffer, Frederic I; Scadden, David T; Soberman, Roy J

    2003-07-01

    The cytochrome P450 4F3 (CYP4F3) gene encodes two functionally distinct enzymes that differ only by the selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B). CYP4F3A inactivates leukotriene B4, a reaction that has significance for controlling inflammation. CYP4F3B converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a potent activator of protein kinase C. We have previously shown that mRNAs coding for CYP4F3A and CYP4F3B are generated from distinct transcription start sites in neutrophils and liver. We therefore investigated mechanisms that regulate the cell-specific expression of these two isoforms. Initially, we analyzed the distribution of CYP4F3 in human leukocytes and determined a lineage-specific pattern of isoform expression. CYP4F3A is expressed in myeloid cells and is coordinate with myeloid differentiation markers such as CD11b and myeloperoxidase during development in the bone marrow. In contrast, CYP4F3B expression is restricted to a small population of CD3+ T lymphocytes. We identified distinct transcriptional features in myeloid, lymphoid, and hepatic cells that indicate the presence of multiple promoters in the CYP4F3 gene. The hepatic promoter depends on a cluster of hepatocyte nuclear factor sites 123-155 bp upstream of the initiator ATG codon. The myeloid promoter spans 400 bp in a region 468-872 bp upstream of the ATG codon; it is associated with clusters of CACCT sites and can be activated by ZEB-2, a factor primarily characterized as a transcriptional repressor in cells that include lymphocytes. ZEB-2 interacts with C-terminal binding protein and Smads, and this would provide opportunities for integrating environmental signals in myelopoiesis and inflammation.

  20. FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

    PubMed

    Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane

    2007-03-01

    Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

  1. 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.

  2. 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. PMID:24814259

  3. Effect of swimming exercise and ethanol on rat liver P450-dependent monooxygenases.

    PubMed

    Ardies, C M; Zachman, E K; Koehn, B J

    1994-12-01

    The interactive effects of 6 wk of repeated swimming exercise and chronic ethanol consumption (36% of total calories) on the hepatic cytochrome P450-dependent monooxygenase system were studied utilizing four groups of male rats in a 2 x 2 factorial design. The sedentary-control (S/C), sedentary-ethanol (S/E), and swim-control (SW/C) groups received the same amount of food that the swim-ethanol (SW/E) group consumed. The swimming groups were trained to swim for 2 h.d-1, 5 d.wk-1. Significant main effects due to ethanol (P < 0.002) and exercise (P < 0.02) were observed for the enhanced cytochrome P450 content and cytochrome P450 reductase activity, respectively. In addition, significant main effects for ethanol (P < 0.001), exercise (P < 0.0001), and significant interaction effects (P < 0.005) on aniline p-hydroxylase activity and significant main effects for ethanol (P < 0.01), exercise (P < 0.01), and interaction effects (P < 0.04) on 7-ethoxycoumarin o-deethylase activity were observed. Because the SW/C treatment had no effect on any of the measured cytochrome P450 activities and the SW/E treatment enhanced P450 activities much more than the S/E treatment, the main effects observed for exercise are accounted for by the alterations produced by combining swimming with the ethanol treatment. Based on these results, repeated exercise combined with ethanol consumption produces a synergistic increase in ethanol-inducible cytochrome P450-dependent activities. PMID:7869878

  4. A targeted proteomics approach for profiling murine cytochrome P450 expression.

    PubMed

    Hersman, Elisabeth M; Bumpus, Namandjé N

    2014-05-01

    The cytochrome P450 (P450) superfamily of enzymes plays a prominent role in drug metabolism. Although mice are a widely used preclinical model in pharmacology, the expression of murine P450 enzymes at the protein level has yet to be fully defined. Twenty-seven proteins belonging to P450 subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 2F, 2J, 2U, 3A, 4A, 4B, 4F, and 4V were readily detectable in Balb/c mouse tissue using a global mass spectrometry-based proteomics approach. Subsequently, a targeted mass spectrometry-based assay was developed to simultaneously quantify these enzymes in ranges of femtomoles of P450 per microgram of total protein concentration range. This screen was applied to mouse liver microsomes and tissue lysates of kidney, lung, intestine, heart, and brain isolated from mixed-sex fetuses; male and female mice that were 3-4 weeks, 9-10 weeks, and 8-10 months of age; and pregnant mice. CYP1A2 was consistently more abundant in male mouse liver microsomes compared with age-matched females. Hepatic expression of CYP2B9 was more abundant in 3- to 4-week-old male and female mice than in mice of other ages; in addition, CYP2B9 was the only enzyme that was detectable at higher levels in pregnant mouse liver microsomes compared with age-matched females. Interestingly, sexually dimorphic expression of CYP2B9, 2D26, 2E1, and 4B1 was observed in kidney only. The targeted proteomics assay described here can be broadly used as a tool for investigating the expression patterns of P450 enzymes in mice.

  5. 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

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

    PubMed Central

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

    2013-01-01

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

  7. 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

  8. Effect of natamycin on cytochrome P450 enzymes in rats.

    PubMed

    Martínez, María Aránzazu; Martínez-Larrañaga, María Rosa; Castellano, Victor; Martínez, Marta; Ares, Irma; Romero, Alejandro; Anadón, Arturo

    2013-12-01

    Natamycin is a polyene macrolide antibiotic widely used in the food industry as a feed additive to prevent mold contamination of foods. There are many contradictory results on the genotoxic effects of macrolides which could suggest a potential risk for humans. In the present study, the effects of natamycin on the activities of some drug metabolizing enzymes in rat liver microsomes were determined in vivo. Rats were treated orally with natamycin at doses of 0.3, 1, 3 and 10 mg/kg body weight (bw)/day for 6 days. Determinations of cytochrome P450 (CYP) enzyme activities were carried out in hepatic microsomes isolated from rats treated. The activities of CYP2E1, CYP1A1/2 CYP2B1/2 and CYP4A1/2 enzymes significantly decreased after treatment with 1, 3 and 10 mg/kg bw/day, in a dose-dependent manner as compared to control. This effect was not observed after natamycin treatment at dose of 0.3 mg/kg bw/day. Our results suggest that natamycin may not potentiate the toxicity of many xenobiotics via metabolic activation and/or accumulation of reactive metabolites but also might affect the clearance of other xenobiotics detoxified by the studied CYP enzymes.

  9. 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...

  10. 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. PMID:26507217

  11. 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.

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

    PubMed

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

    2013-02-19

    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.

  13. Cytochrome P450: taming a wild type enzyme

    PubMed Central

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

    2011-01-01

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

  14. 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

  15. Alterations in cytochrome P-450 levels in adult rats following neonatal exposure to xenobiotics

    SciTech Connect

    Zangar, R.C. Pacific Northwest Laboratories, Richland, WA ); Springer, D.L. ); Buhler, D.R. )

    1993-01-01

    Neonatal exposure to certain xenobiotics has been shown to alter hepatic metabolism in adult rats in a manner that indicates long-term changes in enzyme regulation. Previously, the authors have observed changes in adult testosterone metabolism and in cytochrome P-450 (P-450) mRNA levels in animals neonatally exposed to phenobarbital (PB) or diethylstilbestrol (DES). In order to test for other enzyme alterations, they used Western blot procedures for specific P-450s to analyze hepatic microsomes from adult rats (24 wk old) that had been exposed neonatally to DES, PB, 7,12-dimethylbenz[a]anthracene (DMBA), or pregnenolone 16[alpha]-carbonitrile (PCN). The most striking effects were observed in the DES-treated males: P-4502C6 and an immunologically similar protein were increased 60 and 90%, respectively, relative to control values, but P-4503A2 was decreased by 44%. No changes were observed in the DES-treated males in levels of P-4502E1, P-4502B, or the male-specific P-4502C13. Adult males neonatally treated with PB had 150% increase in levels of anti-P4502B-reactive protein without significant changes in the other enzymes. The DES- and DMBA-treated females had increased levels of the female-specific P-4502C12 of 38 and 48%, respectively, but no other observed alterations. The results confirm that neonatal exposure to DES or PB can cause alterations in adult hepatic cytochrome P-450 levels but show that these chemicals act on different enzymes. Neonatal DMBA resulted in changes in adult females similar to those produced by the synthetic estrogen DES, but did so at about two-thirds lower dose. 37 refs., 5 figs.

  16. 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.

  17. 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.

  18. Induction of cytochrome P450 1A1 and monooxygenase activity in Tilapia by sediment extract

    SciTech Connect

    Ueng, Y.F.; Ueng, T.H.; Liu, T.Y.

    1995-01-01

    Cytochrome P450 (P450)-dependent monooxygenases of fishes are inducible by a variety of environmental pollutants including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Induction of fish monoxygenases may serve as a biological monitor for PAH- and PCB-types of environmental chemicals. Many studies have demonstrated environmental induction of fish monooxygenases using various experimental approaches. However, relatively few studies have been conducted using fish treated with contaminated river sediment extracts. Damsui River is the largest river in the north of Taiwan. The lower section of the river in the Taipei Metropolitan area is heavily polluted by industrial and municipal wastes. Tilapia (Oreochromis mossambicus) is one of the few species of fish that occur in the polluted river. Previous field studies showed that the levels of P450 1A1, benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase activities in tilapia collected at Fu-Ho Bridge, a polluted section of Damsui River, were higher than respective levels in fish collected from an unpolluted section. These results suggested that tilapia caught at the polluted site were exposed to substances similar in action to PAHs and PCBs, because these chemical pollutants are potent inducers of P450 1A1. PAHs and PCBs are persistent compounds that can accumulate in sediment. Tilapia are occasionally associated with the bottom and could ingest chemically contaminated sediment. In the present study, we determined the induction properties of monooxygenases using tilapia treated with extract of sediment collected from a polluted section of Damsui River. The present study demonstrates that Damsui River sediment extract has the ability to induce hepatic P450 1A1 and dependent monooxygenase activities in tilapia. 17 refs., 2 figs., 2 tabs.

  19. Cytochrome P450 dysregulations in thioacetamide-induced liver cirrhosis in rats and the counteracting effects of hepatoprotective agents.

    PubMed

    Xie, Yuan; Wang, Guangji; Wang, Hong; Yao, Xilin; Jiang, Shan; Kang, An; Zhou, Fang; Xie, Tong; Hao, Haiping

    2012-04-01

    Dysregulations of cytochromes P450 (P450s) under liver injury have been extensively studied. However, little is known about the possible reversing effects of hepatoprotective agents, the understanding of which is of great importance in guiding clinical dosage adjustment for patients with liver injury. This study aims to investigate the dysregulation patterns of major P450s in thioacetamide (TAA)-induced liver cirrhosis in rats and the potential counteracting effects of hepatoprotective agents schisandra lignans extract (SLE) and dimethyl diphenyl bicarboxylate (DDB). TAA intoxications for 6 weeks induced apparent liver injury and dramatically reduced the hepatic protein expressions of CYP1A2, CYP2C6, CYP2E1, and CYP3A2 to 18, 71, 30, and 21% of that in the normal control, respectively. Both SLE and DDB treatments could significantly reverse the TAA-induced loss of P450 protein levels, which may be ascribed to their hepatoprotective effects and direct P450-inducing effects that have been confirmed in healthy rats. However, the recovery of enzyme activities of most P450s by SLE and DDB treatment was less evident than that for the protein expression levels. TAA exhibited NADPH-, time-, and concentration-dependent inactivating effects on all of the four major P450 isozymes; both DDB and GSH showed little effects on counteracting such an inactivation efficacy. These findings provided a good explanation on the disproportional effects of hepatoprotective agents in recovering the protein levels and enzyme activities of TAA-induced dysregulated P450s.

  20. 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.

  1. 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

  2. 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

  3. Unusual properties of the cytochrome P450 superfamily

    PubMed Central

    Lamb, David C.; Waterman, Michael R.

    2013-01-01

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

  4. N-demethylation of cocaine to norcocaine. Evidence for participation by cytochrome P-450 and FAD-containing monooxygenase.

    PubMed

    Kloss, M W; Rosen, G M; Rauckman, E J

    1983-03-01

    Experiments were conducted to determine which microsomal enzymes are involved in the in vitro hepatic oxidative N-demethylation of cocaine to norcocaine, the first step in the biotransformation of cocaine to its ultimate hepatotoxic metabolite. Cocaine was found to undergo conversion to norcocaine by two alternate pathways, one involving only cytochrome P-450 and the other requiring both cytochrome P-450 and FAD-containing monooxygenase. In the first pathway, cocaine was directly N-demethylated to norcocaine by cytochrome P-450; this reaction was enhanced by phenobarbital induction and was inhibited by both n-octylamine and metyrapone. The second route was found to be a two-step reaction involving cocaine N-oxide as an intermediate. In this pathway, cocaine is first oxidized to cocaine N-oxide by FAD-containing monooxygenase, followed by a cytochrome P-450-catalyzed N-demethylation to norcocaine. This latter step was enhanced by phenobarbital treatment and inhibited by n-octylamine. Cocaine N-oxide also exhibited a Type I binding spectrum with mouse hepatic microsomes. In addition, a model system consisting of ferrous sulfate was found to catalyze the N-demethylation of cocaine N-oxide. On the basis of these experiments, it is concluded that cytochrome P-450 and FAD-containing monooxygenase participate in the initial oxidation of cocaine to norcocaine. We also propose a mechanism to account for the conversion of cocaine N-oxide to norcocaine.

  5. 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

  6. Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

    PubMed

    Hrycay, E G; Bandiera, S M

    2009-12-01

    The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

  7. Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450.

    PubMed

    Stevenson, P M; Ruettinger, R T; Fulco, A J

    1983-05-16

    Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450. PMID:6405752

  8. Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

    PubMed

    Zhang, W; Parentau, H; Greenly, R L; Metz, C A; Aggarwal, S; Wainer, I W; Tracy, T S

    1999-01-01

    Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.

  9. 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

  10. 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.

  11. Electrochemical investigations on the oxygen activation by cytochrome P-450.

    PubMed

    Scheller, F; Renneberg, R; Schwarze, W; Strnad, G; Pommerening, K; Prümke, H J; Mohr, P

    1979-01-01

    The application of cytochrome P-450 in substrate conversion is complicated both due to the limited stability and the cofactor regeneration problems. To overcome the disadvantages of NADPH consumption the transfer of the reduction equivalents from an electrode into the cytochrome P-450-system was studied: 1. NADPH was cathodically reduced at a mercury pool electrode. By immobilization of NADP on dialdehyde Sephadex the reductive recycling was possible. 2. Different forms of reduced oxygen were produced by the cathode: a) The reaction of O2- with deoxycorticosterone yields a carboxylic acid derivative. In contrast the cytochrome P-450 catalyzed NADPH-dependent reaction with the same substrate gives corticosterone, O2- represents only an intermediate in the activation of oxygen and is not the "activated oxygen" species. b) Molecular oxygen was reduced to HO2- and H2O2, respectively. The interaction of adsorbed cytochrome P-450 on the electrode surface with the reduced oxygen species in the absence of NADPH was studied. The electrochemically generated peroxide seems to be more active than added H2O2. 3. In a model of electro-enzyme-reactor several substrates were hydroxylated by microsomal cytochrome P-450 with cathodically reduced oxygen which substitutes NADPH.

  12. Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

    PubMed

    Cook, D J; Finnigan, J D; Cook, K; Black, G W; Charnock, S J

    2016-01-01

    Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s. PMID:27567486

  13. 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.

  14. Inducing effect of oxfendazole on cytochrome P450IA2 in rabbit liver. Consequences on cytochrome P450 dependent monooxygenases.

    PubMed

    Gleizes, C; Eeckhoutte, C; Pineau, T; Alvinerie, M; Galtier, P

    1991-06-15

    Male New Zealand rabbits were dosed with either 0.9, 4.5 or 22.5 mg/kg/day of oxfendazole by gastric intubation for 10 days. Oxfendazole administered at the therapeutic dose (4.5 mg/kg) and at the highest dose (22.5 mg/kg) increased 1.54- and 2.36-fold the total liver microsomal cytochrome P450 and more particularly the isoenzyme P450IA2 (95 and 184% increases) as demonstrated by western blotting. Increases in ethoxyresorufin O-deethylation and hydroxylations of benzopyrene and acetanilide occurred in livers of the same animals without any change in N-demethylation of aminopyrine, benzphetamine or erythromycin. Because of the unchanged level of mRNA specific to cytochrome P450IA2, as shown by northern blot analysis of poly mRNA, an enzyme stabilization rather than a transcriptional activation of IA2 genes should be involved in the P450IA2 regulation mechanisms. Oxfendazole bound strongly to cytochrome P450, giving rise to a type II spectrum, and inhibited noncompetitively the ethoxyresorufin O-deethylase and acetanilide hydroxylase activities, this confirmed that oxfendazole interacts only with the P450IA2 family. On the basis of a comparison of the enzymatic activities induced by various imidazole drugs, it was concluded that oxfendazole, like omeprazole and albendazole, behaved as a 3-methylcholanthrene-type inducer. These three benzimidazoles did not all belong to the same category of cytochrome P450 inducers as the antifungal drugs miconazole, clotrimazole and ketoconazole.

  15. Pivotal Role of P450-P450 Interactions in CYP3A4 Allostery: the Case of α-Naphthoflavone

    PubMed Central

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

    2014-01-01

    SYNOPSIS We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to α-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) ratio ≥ 750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization we probed P450-P450 interactions with a new technique based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. Addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore, the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions. PMID:23651100

  16. Activation of Oxygen by Cytochrome P-450 and Other Haemoproteins

    NASA Astrophysics Data System (ADS)

    Metelitsa, D. I.

    1982-11-01

    Data on the activation of molecular oxygen by the full microsomal hydroxylating system containing cytochrome P-450 as the terminal oxygenase are examined. The nature of the hydroxylating agent, which is the oxenoid Fe3+O, is analysed. The autoxidation reactions of cytochrome P-450 from various sources, haemoglobin, myoglobin, and peroxidases are compared and the role of the axial ligands of the haem iron and the structure of the active centres of the haemoproteins in this process is demonstrated. The possible mechanisms of the oxidation of organic compounds by peroxides with participation of cytochrome P-450, cytochrome c, haemoglobin, and catalase are examined critically. Haemoproteins have been divided into three groups in terms of the type of peroxide oxidation reactions. The relative contributions of the radical and two-electron reactions in the oxidation of compounds by peroxides with participation of different haemoproteins are analysed. The bibliography includes 184 references.

  17. Inhibition of cytochrome p450 enzymes by quinones and anthraquinones.

    PubMed

    Sridhar, Jayalakshmi; Liu, Jiawang; Foroozesh, Maryam; Klein Stevens, Cheryl L

    2012-02-20

    In silico docking studies and quantitative structure-activity relationship analysis of a number of in-house cytochrome P450 inhibitors have revealed important structural characteristics that are required for a molecule to function as a good inhibitor of P450 enzymes 1A1, 1A2, 2B1, and/or 2A6. These insights were incorporated into the design of pharmacophores used for a 2D search of the Chinese medicine database. Emodin, a natural anthraquinone isolated from Rheum emodi and known to be metabolized by cytochrome P450 enzymes, was one of the hits and was used as the lead compound. Emodin was found to inhibit P450s 1A1, 1A2, and 2B1 with IC(50) values of 12.25, 3.73, and 14.89 μM, respectively. On the basis of the emodin molecular structure, further similarity searches of the PubChem and ZINC chemical databases were conducted resulting in the identification of 12 emodin analogues for testing against P450s 1A1-, 1A2-, 2B1-, and 2A6-dependent activities. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) showed the best inhibition potency for P450 1A1 with an IC(50) value of 0.40 μM. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) and 1-amino-4-hydroxyanthracene-9,10-dione (compound 2) both inhibited P450 1A2 with the same IC(50) value of 0.53 μM. In addition, compound 1 acted as a mechanism-based inhibitor of cytochrome P450s 1A1 and 1A2 with K(I) and K(inactivation) values of 5.38 μM and 1.57 min(-1) for P450 1A1 and 0.50 μM and 0.08 min(-1) for P450 1A2. 2,6-Di-tert-butyl-5-hydroxynaphthalene-1,4-dione (compound 8) directly inhibited P450 2B1 with good selectivity and inhibition potency (IC(50) = 5.66 μM). Docking studies using the 3D structures of the enzymes were carried out on all of the compounds. The binding modes of these compounds revealed the structural characteristics responsible for their potency and selectivity. Compound 1, which is structurally similar to compound 2 with the presence of an amino group at position 1, showed a

  18. 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

  19. Reactive intermediates produced from the metabolism of the vanilloid ring of capsaicinoids by p450 enzymes.

    PubMed

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

    2013-01-18

    This study characterized electrophilic and radical products derived from the metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from the 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 the bimolecular 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

  20. 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

  1. 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.

  2. Cytochrome P-450 3A enzymes are responsible for biotransformation of FK506 and rapamycin in man and rat.

    PubMed

    Sattler, M; Guengerich, F P; Yun, C H; Christians, U; Sewing, K F

    1992-01-01

    The hepatic cytochrome P-450 responsible for metabolism of the structurally related macrolides FK506 and rapamycin in humans was identified using in vitro studies. FK506 and rapamycin metabolism was significantly correlated with nifedipine oxidation in human liver microsomes of eight different individuals. Immunoinhibition with anti-P450 3A4 abolished almost all FK506 and rapamycin metabolite formation. Inactivation of P450 3A4 by incubation of human liver microsomes with triacetyl oleandomycin (50 microM) or gestodene (10 microM) inhibited metabolism of FK506 and rapamycin. In liver microsomes from dexamethasone-treated rats FK506 and rapamycin metabolism was increased compared to liver microsomes from uninduced, phenobarbital-, or 3-methylcholanthrene-induced rats. FK506 and rapamycin were metabolized by reconstituted recombinant human liver P450 3A4. It is concluded that in human and rat liver FK506 and rapamycin are metabolized primarily by cytochrome P-450 3A4. PMID:1385058

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

    PubMed

    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 generate

  4. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    PubMed Central

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

  5. 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

  6. The effect of 17β-estradiol on sex-dimorphic cytochrome P450 expression patterns induced by hyperoxia in the liver of male CBA/H mice.

    PubMed

    Šafranko, Željka Mačak; Balog, Tihomir; Musa, Marina; Bujak, Ivana Tartaro; Sobočanec, Sandra

    2016-10-01

    The aim of this study was to determine whether treatment of male CBA/H mice with 17β-estradiol (E2) had protective effect on survival and hepatic oxidative damage of lipids and proteins against hyperoxia. Furthermore, we wanted to explore the effect of E2 treatment on the expression of sex-specific cytochrome P450 isoforms, and their possible involvement in E2-induced resistance to hyperoxia. Lipid peroxidation and protein carbonylation were analysed spectrophotometrically and were used as a measure of lipid and protein oxidative damage. Real-time PCR and western blot analysis were used to measure both gene and protein expression levels of Cyp2E1, Cyp7B1 and Cyp2A4, respectively. We found that treatment of male CBA/H mice with E2 increased survival upon hyperoxia exposure, and provided protection against hepatic lipid and protein oxidative damage. Hyperoxia had feminizing effect on the expression of sex-specific CYPs, which resembled the lifespan-promoting conditions. E2 administration had the opposite effect on the expression pattern of these CYPs in hyperoxic versus normoxic conditions. Results of this research proposed possible male strategy in adaptive response to oxidative stress, which may finally result in their longer lifespan. PMID:27576492

  7. 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.

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

    PubMed

    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

  9. 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

  10. Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates, astemizole and terfenadine.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Okamoto, Eriko; Sasaki, Erika; Yamazaki, Hiroshi

    2016-11-01

    1. Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes. 2. Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice. 3. Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver. 4. Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes. 5. These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans.

  11. Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates, astemizole and terfenadine.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Okamoto, Eriko; Sasaki, Erika; Yamazaki, Hiroshi

    2016-11-01

    1. Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes. 2. Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice. 3. Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver. 4. Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes. 5. These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans. PMID:26899760

  12. 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...

  13. Spectroscopic features of cytochrome P450 reaction intermediates

    PubMed Central

    Luthra, Abhinav; Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Preface Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2-3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV-Vis absorption spectra of the reduced CO-saturated state [4-5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle. PMID:21167809

  14. Rational redesign of the biodegradative enzyme cytochrome P450 cam:

    SciTech Connect

    Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

    1991-03-01

    Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs.

  15. 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. PMID:25225740

  16. Covalent linkage of prosthetic heme to CYP4 family P450 enzymes.

    PubMed

    Henne, K R; Kunze, K L; Zheng, Y M; Christmas, P; Soberman, R J; Rettie, A E

    2001-10-30

    An extensive body of research on the structural properties of cytochrome P450 enzymes has established that these proteins possess a b-type heme prosthetic group which is noncovalently bound at the active site. Coordinate, electrostatic, and hydrogen bond interactions between the protein backbone and heme functional groups are readily overcome upon mild acid treatment of the enzyme, which releases free heme from the protein. In the present study, we have used a combination of HPLC, LC/ESI-MS, and SDS-PAGE techniques to demonstrate that members of the mammalian CYP4B, CYP4F, and CYP4A subfamilies bind their heme in an unusually tight manner. HPLC chromatography of CYP4B1 on a POROS R2 column under mild acidic conditions caused dissociation of less than one-third of the heme from the protein. Moreover, heme was not substantially removed from CYP4B1 under electrospray or electrophoresis conditions that readily release the prosthetic group from other non-CYP4 P450 isoforms. This was evidenced by an intact protein mass value of 59,217 +/- 3 amu for CYP4B1 (i.e., apoprotein plus heme) and extensive staining of this approximately 60 kDa protein with tetramethylbenzidine/H(2)O(2) following SDS-PAGE. In addition, treatment of CYP4B1, CYP4F3, and CYP4A5/7 with strong base generated a new, chromatographically distinct, polar heme species with a mass of 632.3 amu rather than 616.2 amu. This mass shift is indicative of the incorporation of an oxygen atom into the heme nucleus and is consistent with the presence of a novel covalent ester linkage between the protein backbone of the CYP4 family of mammalian P450s and their heme catalytic center.

  17. 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.

  18. In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries.

    PubMed

    Utkarsh, Doshi; Loretz, Carol; Li, Albert P

    2016-08-01

    A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the

  19. Quantum mechanically derived AMBER-compatible heme parameters for various states of the cytochrome P450 catalytic cycle

    PubMed Central

    Shahrokh, Kumars; Orendt, Anita; Yost, Garold; Cheatham, Tom

    2011-01-01

    Molecular mechanics (MM) methods are computationally affordable tools for screening chemical libraries of novel compounds for sites of P450 metabolism. One challenge for MM methods has been the absence of a consistent and transferable set of parameters for the heme within the P450 active-site. Experimental data indicates that mammalian P450 enzymes vary greatly in the size, architecture, and plasticity of their active sites. Thus, obtaining x-ray based geometries for the development of accurate MM parameters for the major classes of hepatic P450 remains a daunting task. Our previous work with preliminary gas-phase quantum mechanics (QM) derived atomic partial charges, greatly improved the accuracy of docking studies of raloxifene to CYP3A4. We have therefore developed and tested a consistent set of transferable MM parameters based on gas-phase QM calculations of two model systems of the heme—a truncated (T-HM) and a full (F-HM) for four states of the P450 catalytic cycle. Our results indicate that the use of the atomic partial charges from the F-HM model further improves the accuracy of docked predictions for raloxifene to CYP3A4. Different patterns for substrate docking are also observed depending on the choice of heme model and state. Newly parameterized heme models are tested in implicit and explicitly solvated MD simulations in the absence and presence of enzyme structures, for CYP3A4, and appear to be stable on the nanosecond simulation timescale. The new force field for the various heme states may aid the community for simulations of P450 enzymes and other heme containing enzymes. PMID:21997754

  20. 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.

    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).

  1. 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

  2. Computer-aided design of aptamers for cytochrome p450.

    PubMed

    Shcherbinin, Dmitrii S; Gnedenko, Oksana V; Khmeleva, Svetlana A; Usanov, Sergey A; Gilep, Andrei A; Yantsevich, Aliaksei V; Shkel, Tatsiana V; Yushkevich, Ivan V; Radko, Sergey P; Ivanov, Alexis S; Veselovsky, Alexander V; Archakov, Alexander I

    2015-08-01

    Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M. PMID:26166326

  3. Reversible inhibition of three important human liver cytochrome p450 enzymes by tiliroside.

    PubMed

    Sun, Dong-Xue; Lu, Jin-Cai; Fang, Zhong-Ze; Zhang, Yan-Yan; Cao, Yun-Feng; Mao, Yu-Xi; Zhu, Liang-Liang; Yin, Jun; Yang, Ling

    2010-11-01

    Tiliroside, an active flavonoid extensively found in many medicinal plants including Helichrysum italicum, Geranium mexicanum and Helianthemum glomeratum, has been demonstrated to exert multiple biological effects including antiinflammatory, antimicrobial, antioxidant and antitumor activities. Cytochrome P450 (CYP) enzymes play an important role in the Phase I oxidation metabolism of a wide range of xenobiotics and inhibition of CYP isoforms might influence the elimination of drugs and induce serious adverse drug response. The inhibition of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2D6, CYP2C9, CYP2C8 and CYP2E1) by tiliroside was investigated using in vitro human liver microsomal incubation assays. The results showed that tiliroside strongly inhibited the activity of CYP3A4 (IC(50) = 9.0 ± 1.7 μm), CYP2C8 (IC(50) = 12.1 ± 0.9 μm) and CYP2C9 (IC(50) = 10.2 ± 0.9 μm) with other CYP isoforms negligibly influenced. Further kinetic analysis showed that inhibition of these three CYP isoforms by tiliroside is best fit to a competitive way. The K(i) value was calculated to be 5.5 μm, 3.3 μm, 9.4 μm for CYP3A4, CYP2C9 and CYP2C8, respectively. The relatively low K(i) values suggested that tiliroside might induce drug-drug interactions with many clinically used drugs which are mainly metabolized by these three CYP isoforms. Therefore, attention should be given to the probable drug-drug interaction between tiliroside-containing herbs and substrates of CYP3A4, CYP2C9 and CYP2C8.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Differential Oxidation of Two Thiophene-Containing Regioisomers to Reactive Metabolites by Cytochrome P450 2C9

    PubMed Central

    Rademacher, Peter M; Woods, Caleb M; Huang, Qingbiao; Szklarz, Grazyna D; Nelson, Sidney D

    2012-01-01

    The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this manuscript was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated 18O from 18O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product, but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver

  10. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

    PubMed

    Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

    2009-12-01

    Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

  11. 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.

  12. Structure, genetic mapping, and function of the cytochrome P450 3A37 gene in the turkey (Meleagris gallopavo).

    PubMed

    Rawal, S; Mendoza, K M; Reed, K M; Coulombe, R A

    2009-01-01

    Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB1 is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB1-8,9-epoxide (AFBO). Using 3'-5'-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB(1). Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB(1), this study will contribute to the identifying allelic variants of this important gene in poultry.

  13. Crystallization and preliminary x-ray diffraction analysis of P450terp and the hemoprotein domain of P450BM-3, enzymes belonging to two distinct classes of the cytochrome P450 superfamily.

    PubMed Central

    Boddupalli, S S; Hasemann, C A; Ravichandran, K G; Lu, J Y; Goldsmith, E J; Deisenhofer, J; Peterson, J A

    1992-01-01

    Cytochromes P450 are members of a superfamily of hemoproteins that are involved in the metabolism of various physiologic and xenobiotic organic compounds. This superfamily of proteins can be divided into two classes based on the electron donor proximal to the P450: an iron-sulfur protein for class I P450s or a flavoprotein for class II. The only known tertiary structure of any of the cytochromes P450 is that of P450cam, a class I soluble enzyme isolated from Pseudomonas putida (product of the CYP101 gene). To understand the details of the structure-function relationships within and between the two classes, structural studies on additional cytochromes P450 are crucial. We report here characterization of the crystal forms of two soluble, bacterial enzymes: cytochrome P450terp [class I enzyme from a Pseudomonas species (product of CYP108 gene)] and the hemoprotein domain of cytochrome P450BM-3 [class II enzyme from Bacillus megaterium (product of the CYP102 gene)]. The crystals of cytochrome P450terp are hexagonal and belong to the space group P6(1)22 (or its enantiomorph, P6(5)22) with unit cell dimensions a = b = 68.9 A and c = 458.7 A. The crystals of the hemoprotein domain of cytochrome P450BM-3 are monoclinic and belong to the space group P2(1) with unit cell dimensions a = 59.4 A, b = 154.0 A, c = 62.2 A, and beta = 94.7 degrees. Diffraction data for the crystals of these two proteins were obtained to a resolution better than 2.2 A. Assuming the presence of two molecules in the asymmetric unit for the hemoprotein domain of P450BM-3 and one molecule for P450terp, the calculated values of Vm are 2.6 and 3.3 A3/Da, respectively. Images PMID:1608967

  14. The effects of milk thistle (Silybum marianum) on human cytochrome P450 activity.

    PubMed

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

    2014-10-01

    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.

  15. Transcriptional Regulation of the Human P450 Oxidoreductase Gene: Hormonal Regulation and Influence of Promoter Polymorphisms

    PubMed Central

    Tee, Meng Kian; Huang, Ningwu; Damm, Izabella

    2011-01-01

    P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at −325/−1 bp from the untranslated exon. Common human POR polymorphisms at −208 and −173 had little influence on transcription, but the polymorphism at −152 reduced transcription significantly in both cell lines. EMSA and supershift assays identified binding of Smad3/Smad4 between −249 and −261 and binding of thyroid hormone receptor-β (TRβ) at −240/−245. Chromatin immunoprecipitation showed that Smad3, Smad4, TRα, TRβ, and estrogen receptor-α were bound between −374 and −149. Cotransfection of vectors for these transcription factors and POR promoter-reporter constructs into both cell types followed by hormonal treatment showed that T3 exerts major tropic effects via TRβ, with TRα, estrogen receptor-α, Smad3, and Smad4 exerting lesser, modulatory effects. T3 also increased POR mRNA in both cell lines. Thyroid hormone also is essential for rat liver POR expression but acts via different transcription factor complexes. These are the first data on human POR gene transcription, establishing roles for TRβ and Smad3/4 in its expression and indicating that the common polymorphism at −152 may play a role in genetic variation in steroid biosynthesis and drug metabolism. PMID:21393444

  16. 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

  17. Regulation of cytochrome P-450Ia1 gene expression

    SciTech Connect

    Kamps, C.A.

    1989-01-01

    The mechanism by which cytochrome P-450IA1 gene expression is induced by polycyclic aromatic hydrocarbons and various polychlorinated dibenzo-p-dioxins involves an intracellular protein known as the Ah receptor. Within the past few years, a second protein has been identified which binds to certain polycyclic aromatic hydrocarbons (PAHs) but not to the receptor ligand, 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD). The protein, named the 4S PAH binding protein, has been reported to bind to a site on the DNA in the 5{prime} regulatory region for the cytochrome P-450IA1 gene. This finding led to the hypothesis that the 4S PAH binding protein may be involved in the trans-regulation of this gene. The work presented in this manuscript addressed this hypothesis by (1) screening animals and cell lines for the presence or absence of the Ah receptor and 4S PAH binding protein, (2) screening polycyclic aromatic hydrocarbons (PAHs) to identify ligands which specifically bind only the 4S protein, (3) determining dose-response curves for TCDD and 4S protein specific ligands in mammalian cell lines, (4) co-administering a 4S binding protein ligand and TCDD in mammalian cell lines to determine the effects of the 4S protein-ligand complex on TCDD-induced cytochrome P-450IA1 expression, and (5) co-administering TCDD and 6-methyl 1,3,8-trichlorodibenzofuran (MCDF), a compound reported to be an antagonist of TCDD-induced benzo(a)pyrene-3-hydroxylase (AHH) activity, to determine whether antagonism occurs at the transcriptional level. The results of gradient assays show that the Ah receptor and the 4S binding protein were expressed in the rat strains which were studied. In the cell lines, H4IIE cells (rat hepatoma expressed only the receptor whereas Hepa1c1c7 cells mouse hepatoma) expressed both proteins.

  18. 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.

  19. 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.

  20. Interactions between nitric oxide and cytochrome P-450 in the liver.

    PubMed

    Khatsenko, O

    1998-07-01

    Bacterial lipopolysaccharide and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing the activity of hepatic cytochrome P-450 mixed function oxidase system. Although this effect of immunostimulants was first described almost 40 years ago, the mechanism is obscure. Immunostimulants are now known to cause nitric oxide overproduction by cells via induction of nitric oxide synthase. The highly reactive NO radical binds to prosthetic groups such as heme or iron-sulfur clusters leading to either activation or (more often) inhibition of iron-containing enzymes. It has been known for years that NO also binds to the heme moiety of cytochrome P-450 (CYP) with high affinity. However it was only recently demonstrated that binding of NO to CYPs also inhibits their enzymatic activity. This applies to both exogenously derived as well as endogenously synthesized NO. Suppression of CYP-dependent metabolism, which is a major problem of inflammatory liver diseases, can be significantly reversed by inhibition of NO synthesis in vivo under experimental conditions. The present paper reviews the findings implicating NO as a major factor mediating the suppression of CYP expression caused by endotoxins and immunostimulants in general. NO-mediated suppression of the metabolism of endogenous and exogenous substances under inflammatory conditions may contribute to the clinical manifestations and may be an important consideration for rational drug therapy in these conditions. PMID:9721336

  1. Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

    SciTech Connect

    Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

    1985-02-01

    The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H/sub 2/O/sub 2/ such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization.

  2. Structure of a bovine gene for P-450c21 (steroid 21-hydroxylase) defines a novel cytochrome P-450 gene family.

    PubMed Central

    Chung, B C; Matteson, K J; Miller, W L

    1986-01-01

    P-450c21, a cytochrome P-450 enzyme [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], mediates the 21-hydroxylation of glucocorticoid and mineralocorticoid hormones in the adrenal gland. The complete sequence of a bovine P-450c21 gene shows it is 3447 base pairs long and contains 10 exons. The intron/exon organization and encoded amino acid sequence indicate that P-450c21 represents a unique family of genes in the P-450 gene superfamily. Primer extension and S1 nuclease protection experiments identified several cap sites for initiation of transcription; the principal cap site produces mRNA with a 5' untranslated region only 11 bases long. S1 nuclease protection experiments confirm that P-450c21 is actively expressed in the adrenal and the testis, an organ not known to secrete 21-hydroxylated steroids. Images PMID:3487086

  3. Musk xylene is a novel specific inducer of cytochrome P-450IA2.

    PubMed

    Iwata, N; Minegishi, K; Suzuki, K; Ohno, Y; Kawanishi, T; Takahashi, A

    1992-04-15

    The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.

  4. Kupffer cell stimulation with Corynebacterium parvum reduces some cytochrome P450-dependent activities and diminishes acetaminophen and carbon tetrachloride-induced liver injury in the rat.

    PubMed

    Raiford, D S; Thigpen, M C

    1994-11-01

    Chemical activation of Kupffer cells in vivo by vitamin A or latex beads is associated with a worsening of hepatic injury induced by the P450-dependent hepatotoxins acetaminophen (ACET) and carbon tetrachloride (CCl4) and by the P450-independent toxin galactosamine (GLN). Immunostimulants such as Corynebacterium parvum (CP) also activate Kupffer cells, but do so while prompting release of soluble mediators which depress microsomal oxidative activities in cultured hepatocytes. Therefore, we sought to characterize the effects of CP on hepatic injury in vivo due to ACET and CCl4 while employing GLN as a control. Hepatic microsomal oxidative activity and glutathione (GSH) disposition were examined since each influences susceptibility to injury from ACET or CCl4. Rats were given CP 28 mg/kg i.v. 5 days before challenge with hepatotoxicant. Hepatic injury was assessed 24 hr after hepatotoxicant administration by measurement of serum alanine aminotransferase (ALT) activity and review of histological sections. Livers from parallel groups of rats were used to prepare microsomal and cytosolic fractions, to measure tissue GSH, or for perfusion to assess GSH efflux. Significant reductions in injury due to ACET or CCl4 were observed while injury due to GLN was potentiated. Serum ALT levels after ACET were 3000 +/- 620 in controls vs 170 +/- 45 IU/liter in the CP-treated group and ALT levels after CCl4 were 3100 +/- 500 in controls vs 1700 + 450 IU/liter in the CP-treated group. In contrast, serum ALT levels after GLN were 920 +/- 230 in controls vs 1700 +/- 370 in the CP-treated group. Patterns of hepatic injury observed on histological sections were those characteristic for each toxin and the severity of injury correlated well with alterations in serum ALT levels for each agent. Hepatic microsomal fractions from rats pretreated with CP showed significantly diminished total cytochrome P450 content as well as reduced activity for two P450IIE1 substrates, p-nitrophenol and 7

  5. Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.

    PubMed

    Nakayama, S M M; Ikenaka, Y; Hayami, A; Mizukawa, H; Darwish, W S; Watanabe, K P; Kawai, Y K; Ishizuka, M

    2016-10-01

    Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.

  6. Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

    PubMed Central

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-01-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. PMID:25804257

  7. 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.

  8. 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.

  9. Induction of cytochrome P-450 1A1 in human hepatoma HepG2 and lung carcinoma NCI-H322 cells by motorcycle exhaust particulate.

    PubMed

    Ueng, T H; Hu, S H; Chen, R M; Wang, H W; Kuo, M L

    2000-05-26

    The effects of motorcycle exhaust particulate (MEP) on human cytochrome P-450 (P-450)-dependent monooxygenases were determined using human hepatoma cell line HepG2 and lung carcinoma cell line NCI-H322 treated with organic extracts of MEP from a two-stroke engine. Gas chromatography and mass spectrometry analysis of MEP extract revealed the presence of carcinogens benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, chrysene, and indeno[1,2,3-c,d]pyrene in the chemical mixture. Treatment with MEP extract produced concentration- and time-dependent increases of monooxygenase activity in HepG2 cells. Treatment of the cells with 100 microg/ ml MEP extract for 24 h markedly increased benzo[a]pyrene hydroxylation, 7-ethoxycoumarin, and 7-ethoxyresorufin O-deethylation activities in microsomes. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 against P-450 1A1 revealed that MEP extract induced a P-450-immunorelated protein in the hepatoma cells. RNA blot analysis of cellular total RNA using a human P-450 1A1 3'-end cDNA probe showed that MEP extract increased the level of a hybridizable P-450 mRNA. These P-450 1A1 inductive effects of MEP extract were similar to those from treatment with 10 microM benzo[a]pyrene or 3-methylcholanthrene (3-MC) in HepG2 cells. Treatment of lung carcinoma NCI-H322 cells with 100 microg/ml MEP extract, 10 microM benzo[a]pyrene, or 3-MC resulted in induction of monooxygenase activity, protein, and mRNA of P-450 1A1, similar to the induction observed with the hepatoma cells. The present study demonstrates that MEP extract has the ability to induce human hepatic and pulmonary P-450 1A1 in the liver- and lung-derived cell lines, and the induction involves a pretranslational mechanism. Induction of the human hepatic and pulmonary P-450 1A1 in vitro may provide important information in the assessment of MEP metabolism and toxicity in humans.

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

    PubMed Central

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

    2014-01-01

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

  11. Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues.

    PubMed

    Liu, L; Massey, T E

    1992-04-01

    In the present investigation, we have examined the role of lipoxygenases in the bioactivation of aflatoxin B1 (AFB1) in hepatic and extrahepatic tissues. The enzyme activities were evaluated by determining [3H]AFB1-DNA adduct formation. The results demonstrated that both purified soybean lipoxygenase and guinea-pig tissue cytosolic lipoxygenases were able to activate AFB1 to form [3H]AFB1-DNA adduct(s). The reaction was completely inhibited by nordihydroguaiaretic acid (NDGA, 0.1 mM), a lipoxygenase inhibitor and an antioxidant, but not by indomethacin (0.1 mM), an inhibitor of prostaglandin H synthase (PHS), indicating that this reaction is associated with lipoxygenase activity, and/or is involved in a peroxyl radical process. While purified lipoxygenase showed arachidonic acid (AA)-dependent properties, the omission of AA did not diminish guinea-pig tissue cytosolic [3H]AFB1-DNA adduct formation, possibly because AA was released from lipid particles by AFB1. Within the range of hemoglobin (Hb) concentrations found in lung, kidney and liver cytosols (1.4-11.1 microM) and microsomes (0-0.5 microM), neither pure Hb, nor Hb of cytosols or microsomes from whole blood caused detectable AA-dependent AFB1-DNA binding. This indicates that Hb, as a contaminant with quasi-lipoxygenase activity, did not contribute to AFB1 activation attributed to guinea-pig tissue lipoxygenases. [3H]AFB1 concentrations at half-maximal DNA binding rate of pulmonary cytochrome P450 monooxygenases (P450) and lipoxygenases were similar, though P450 had a much higher maximum DNA binding rate. Pulmonary microsomal PHS activity for AFB1 activation was too low for its half-maximal binding concentrations of [3H]AFB1 and maximum rate to be accurately determined. In kidney, maximum rates for lipoxygenase, PHS and P450 were similar, whereas half-maximal binding concentrations for reactions by lipoxygenase and P450 were lower compared to that of PHS. The half-maximal binding concentration of hepatic

  12. Quantum mechanics/molecular mechanics modeling of regioselectivity of drug metabolism in cytochrome P450 2C9.

    PubMed

    Lonsdale, Richard; Houghton, Kerensa T; Żurek, Jolanta; Bathelt, Christine M; Foloppe, Nicolas; de Groot, Marcel J; Harvey, Jeremy N; Mulholland, Adrian J

    2013-05-29

    Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation. Here we present a comprehensive study of the oxidation of three widely used pharmaceutical compounds (S-ibuprofen, diclofenac, and S-warfarin) by one of the major drug-metabolizing P450 isoforms, CYP2C9. The reaction barriers to substrate oxidation by the iron-oxo species (Compound I) have been calculated at the B3LYP-D/CHARMM27 level for different possible metabolism sites for each drug, on multiple pathways. In the cases of ibuprofen and warfarin, the process with the lowest activation energy is consistent with the experimentally preferred metabolite. For diclofenac, the pathway leading to the experimentally observed metabolite is not the one with the lowest activation energy. This apparent inconsistency with experiment might be explained by the two very different binding modes involved in oxidation at the two competing positions. The carboxylate of diclofenac interacts strongly with the CYP2C9 Arg108 side chain in the transition state for formation of the observed metabolite-but not in that for the competing pathway. We compare reaction barriers calculated both in the presence and in the absence of the protein and observe a marked improvement in selectivity prediction ability upon inclusion of the protein for all of the substrates studied. The barriers calculated with the protein are generally higher than those calculated in the gas phase. This suggests that active-site residues surrounding the substrate play an important role in controlling selectivity in CYP2C9. The results show that inclusion of sampling (particularly) and dispersion effects is important in making accurate predictions of drug

  13. Drug Oxidation by Cytochrome P450BM3 : Metabolite Synthesis and Discovering New P450 Reaction Types.

    PubMed

    Ren, Xinkun; Yorke, Jake A; Taylor, Emily; Zhang, Ting; Zhou, Weihong; Wong, Luet Lok

    2015-10-12

    There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.

  14. Cytochrome P450 polymorphism--molecular, metabolic and pharmacogenetic aspects. I. Mechanisms of activity of cytochrome P450 monooxygenases.

    PubMed

    Pachecka, Jan; Tomaszewski, Piotr; Kubiak-Tomaszewska, Grazyna

    2008-01-01

    Cytochrome P450, initially perceived as a type of cell pigment, was soon identified as a hemoprotein with an enzymatic activity characteristic for monooxygenases with an affinity for differentiated endo- or exogenous substrates, including drugs. So far in the human organism 58 CYP isoenzymes belonging to 18 families have been described. Most from the CYP monooxygenases superfamily turned out to be integral elements of hepatocytic reticular monooxygenase complexes which also contain NADPH-dependent cytochrome P450 reductase (CPR). Later investigations indicated the possibility of the participation in electron transport for reticular CYP isoenzymes, alternative NADH-dependent reticular system composed of cytochrome b5 reductase (CBR) and cytochrome b5. The demonstration of the activity of some CYP superfamily isoenzymes not only in hepatocytes but also in many other cells of the human organism, numerous plant and animal tissues and even in cells of fungi, protists and prokaryotes has contributed to the significantly increased understanding of the role of CYP in biological systems. In addition, some CYP isoenzymes were found to be characteristic for the inner mitochondrial membrane monooxygenase complexes which contain NADPH-dependent adrenodoxin reductase (AR) and adrenodoxin (Ad), which is identical with ferredoxin-1 (Fd-1) and hepatoredoxin (Hd).

  15. Ethynyl and Propynylpyrene Inhibitors of Cytochrome P450

    PubMed Central

    Zhu, Naijue; Lightsey, Danielle; Liu, Jiawang; Foroozesh, Maryam; Morgan, Kathleen M.; Stevens, Edwin D.

    2010-01-01

    The single-crystal X-ray structures and in vivo activities of three aryl acetylenic inhibitors of cytochromes P450 1A1, 1A2, 2A6, and 2B1 have been determined and are reported herein. These are 1-ethynylpyrene, 1-propy-nylpyrene, and 4-propynylpyrene. To investigate electronic influences on the mechanism of enzyme inhibition, the experimental electron density distribution of 1-ethynylpy-rene has been determined using low-temperature X-ray diffraction measurements, and the resulting net atomic charges compared with various theoretical calculations. A total of 82,390 reflections were measured with Mo Kα radiation to a (sinθ/λ)max = 0.985 Å−1. Averaging symmetry equivalent reflections yielded 8,889 unique reflections. A least squares refinement procedure was used in which multipole parameters were added to describe the distortions of the atomic electron distributions from spherical symmetry. A map of the model electron density distribution of 1-ethynylpyrene was obtained. Net atomic charges calculated from refined monopole population parameters yielded charges that showed that the terminal acetylenic carbon atom (C18) is more negative than the internal carbon (C17). Net atomic charges calculated by ab initio, density functional theory, and semi-empirical methods are consistent with this trend suggesting that the terminal acetylenic carbon atom is more likely to be the site of oxidation. This is consistent with the inhibition mechanism pathway that results in the formation of a reactive ketene intermediate. This is also consistent with assay results that determined that 1-ethynylpyrene acts as a mechanism-based inhibitor of P450s 1A1 and 1A2 and as a reversible inhibitor of P450 2B1. Crystallographic data: 1-ethynylpyrene, C18H10, P21/c, a = 14.571(2) Å, b = 3.9094(5) Å, c = 20.242(3) Å, β = 105.042(2)°, V = 1,113.5(2) Å3; 1-propynylpyrene, C19H12, P21/n, a = 8.970(2) Å, b = 10.136(1) Å, c = 14.080(3) Å, β = 99.77(2)°, V = 1,261.5(4) Å3; 4

  16. 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.

  17. Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements

    PubMed Central

    Liu, Jin; Tawa, Gregory J.; Wallqvist, Anders

    2013-01-01

    Cytochrome P450 (CYP) enzymes play key roles in drug metabolism and adverse drug-drug interactions. Despite tremendous efforts in the past decades, essential questions regarding the function and activity of CYPs remain unanswered. Here, we used a combination of sequence-based co-evolutionary analysis and structure-based anisotropic thermal diffusion (ATD) molecular dynamics simulations to detect allosteric networks of amino acid residues and characterize their biological and molecular functions. We investigated four CYP subfamilies (CYP1A, CYP2D, CYP2C, and CYP3A) that are involved in 90% of all metabolic drug transformations and identified four amino acid interaction networks associated with specific CYP functionalities, i.e., membrane binding, heme binding, catalytic activity, and dimerization. Interestingly, we did not detect any co-evolved substrate-binding network, suggesting that substrate recognition is specific for each subfamily. Analysis of the membrane binding networks revealed that different CYP proteins adopt different membrane-bound orientations, consistent with the differing substrate preference for each isoform. The catalytic networks were associated with conservation of catalytic function among CYP isoforms, whereas the dimerization network was specific to different CYP isoforms. We further applied low-temperature ATD simulations to verify proposed allosteric sites associated with the heme-binding network and their role in regulating metabolic fate. Our approach allowed for a broad characterization of CYP properties, such as membrane interactions, catalytic mechanisms, dimerization, and linking these to groups of residues that can serve as allosteric regulators. The presented combined co-evolutionary analysis and ATD simulation approach is also generally applicable to other biological systems where allostery plays a role. PMID:24312617

  18. Potential of decursin to inhibit the human cytochrome P450 2J2 isoform.

    PubMed

    Lee, Boram; Wu, Zhexue; Sung, Sang Hyun; Lee, Taeho; Song, Kyung-Sik; Lee, Min Young; Liu, Kwang-Hyeon

    2014-08-01

    CYP2J2 enzyme is highly expressed in human tumors and carcinoma cell lines, and epoxyeicosatrienoic acids, CYP2J2-mediated metabolites, have been implicated in the pathologic development of human cancers. To identify a CYP2J2 inhibitor, 50 natural products obtained from plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes. Of these, decursin noncompetitively inhibited CYP2J2-mediated astemizole O-demethylation and terfenadine hydroxylation activities with Ki values of 8.34 and 15.8μM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner while it did not show cytotoxicity against mouse hepatocytes. The present data suggest that decursin is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities. Studies are currently underway to test decursin as a potential therapeutic agent for cancer. PMID:24788058

  19. Novel Bioactivation Pathway of Benzbromarone Mediated by Cytochrome P450.

    PubMed

    Kitagawara, Yumina; Ohe, Tomoyuki; Tachibana, Kumiko; Takahashi, Kyoko; Nakamura, Shigeo; Mashino, Tadahiko

    2015-09-01

    Benzbromarone (BBR) is a hepatotoxic drug, but the detailed mechanism of its toxicity remains unknown. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (2-ethyl-3-(3-bromo-4,5-dihydroxybenzoyl)benzofuran; CAT) as novel metabolites of BBR in rat and human liver microsomal systems by comparison with chemically synthesized authentic compounds, and we also elucidated that DBH is formed by cytochrome P450 2C9 and that CAT is formed mainly by CYP1A1, 2D6, 2E1, and 3A4. Furthermore, CAT, DBH, and the oxidized form of DBH are highly cytotoxic in HepG2 compared with BBR. Taken together, our data demonstrate that DBH, a novel reactive metabolite, may be relevant to BBR-induced hepatotoxicity. PMID:26106235

  20. Cytochrome P450 epoxygenase pathway of polyunsaturated fatty acid metabolism

    PubMed Central

    Spector, Arthur A.; Kim, Hee-Yong

    2014-01-01

    Polyunsaturated fatty acids (PUFA) are oxidized by cytochrome P450 epoxygenases to PUFA epoxides which function as potent lipid mediators. The major metabolic pathways of PUFA epoxides are incorporation into phospholipids and hydrolysis to the corresponding PUFA diols by soluble epoxide hydrolase. Inhibitors of soluble epoxide hydrolase stabilize PUFA epoxides and potentiate their functional effects. The epoxyeicosatrienoic acids (EETs) synthesized from arachidonic acid produce vasodilation, stimulate angiogenesis, have anti-inflammatory actions, and protect the heart against ischemia-reperfusion injury. EETs produce these functional effects by activating receptor-mediated signaling pathways and ion channels. The epoxyeicosatetraenoic acids synthesized from eicosapentaenoic acid and epoxydocosapentaenoic acids synthesized from docosahexaenoic acid are potent inhibitors of cardiac arrhythmias. Epoxydocosapentaenoic acids also inhibit angiogenesis, decrease inflammatory and neuropathic pain, and reduce tumor metastasis. These findings indicate that a number of the beneficial functions of PUFA may be due to their conversion to PUFA epoxides. PMID:25093613

  1. Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis.

    PubMed

    Saruwatari, Takayoshi; Yagishita, Fumitoshi; Mino, Takashi; Noguchi, Hiroshi; Hotta, Kinya; Watanabe, Kenji

    2014-03-21

    As dimeric natural products frequently exhibit useful biological activities, identifying and understanding their mechanisms of dimerization is of great interest. One such compound is (−)-ditryptophenaline, isolated from Aspergillus flavus, which inhibits substance P receptor for potential analgesic and anti-inflammatory activity. Through targeted gene knockout in A. flavus and heterologous yeast gene expression, we determined for the first time the gene cluster and pathway for the biosynthesis of a dimeric diketopiperazine alkaloid. We also determined that a single cytochrome P450, DtpC, is responsible not only for pyrroloindole ring formation but also for concurrent dimerization of N-methylphenylalanyltryptophanyl diketopiperazine monomers into a homodimeric product. Furthermore, DtpC exhibits relaxed substrate specificity, allowing the formation of two new dimeric compounds from a non-native monomeric precursor, brevianamide F. A radical-mediated mechanism of dimerization is proposed.

  2. Identification of Small-Molecule Scaffolds for P450 Inhibitors

    PubMed Central

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

    2015-01-01

    Mycobacterium tuberculosis cytochrome P450 enzymes (P450, CYP) attract ongoing interest for their pharmacological development potential, as evidenced by the activity of antifungal azole drugs that inhibit sterol 14α-demethylase CYP51 in fungi, tightly bind M. tuberculosis CYP enzymes, and display inhibitory potential against latent and multi drug resistant forms of tuberculosis both in vitro and in tuberculosis-infected mice. Although “piggy-backing” onto existing antifungal drug development programs would have obvious practical and economic benefits, the substantial differences between fungal CYP51 and potential CYP targets in M. tuberculosis are driving direct screening efforts against CYP enzymes 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. Newly discovered compounds can be evaluated in in vitro assays or in vivo disease models for inhibitory/therapeutic effects. The best inhibitors in complex with the target protein can be further characterized by x-ray crystallography. In conjunction with knowledge about compound inhibition potential, detailed structural characterization of the protein-inhibitor binding mode can guide lead optimization strategies to assist drug design. This unit includes protocols for compound library screening, analysis of inhibitory potential of the screen hits, and co-crystallization of top hits with the target CYP. Support protocols are provided for expression and purification of soluble CYP enzymes. PMID:20131225

  3. 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

  4. 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.

  5. A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes.

    PubMed

    De Mot, René; Parret, Annabel H A

    2002-11-01

    The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications. PMID:12419614

  6. Electrochemistry of cytochromes p450: analysis of current-voltage characteristics of electrodes with immobilized cytochromes p450 for the screening of substrates and inhibitors.

    PubMed

    Shumyantseva, V V; Bulko, T V; Kuznetsova, G P; Samenkova, N F; Archakov, A I

    2009-04-01

    In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14alpha-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450.

  7. 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

  8. 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.

  9. Progesterone receptor membrane component 1 inhibits the activity of drug-metabolizing cytochromes P450 and binds to cytochrome P450 reductase.

    PubMed

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2011-03-01

    Progesterone receptor membrane component 1 (PGRMC1) has been shown to interact with several cytochromes P450 (P450s) and to activate enzymatic activity of P450s involved in sterol biosynthesis. We analyzed the interactions of PGRMC1 with the drug-metabolizing P450s, CYP2C2, CYP2C8, and CYP3A4, in transfected cells. Based on coimmunoprecipitation assays, PGRMC1 bound efficiently to all three P450s, and binding to the catalytic cytoplasmic domain of CYP2C2 was much more efficient than to a chimera containing only the N-terminal transmembrane domain. Down-regulation of PGRMC1 expression levels in human embryonic kidney 293 and HepG2 cell lines stably expressing PGRMC1-specific small interfering RNA had no effect on the endoplasmic reticulum localization and expression levels of P450s, whereas enzymatic activities of CYP2C2, CYP2C8, and CYP3A4 were slightly higher in PGRMC1-deficient cells. Cotransfection of cells with P450s and PGRMC1 resulted in PGRMC1 concentration-dependent inhibition of the P450 activities, and this inhibition was partially reversed by increased expression of the P450 reductase (CPR). In contrast, CYP51 activity was decreased by down-regulation of PGRMC1 and expression of PGRMC1 in the PGRMC1-deficient cells increased CYP51 activity. In cells cotransfected with CPR and PGRMC1, strong binding of CPR to PGRMC1 was observed; however, in the presence of CYP2C2, interaction of PGRMC1 with CPR was significantly reduced, suggesting that CYP2C2 competes with CPR for binding to PGRMC1. These data show that in contrast to sterol synthesizing P450, PGRMC1 is not required for the activities of several drug-metabolizing P450s, and its overexpression inhibits those P450 activities. Furthermore, PGRMC1 binds to CPR, which may influence P450 activity.

  10. N-methylcarbazole metabolism by a purified isozyme of rat liver microsomal cytochrome P-450: membrane effects and autocatalytic inactivation

    SciTech Connect

    Gurka, D.P.

    1985-01-01

    The catalytic properties of purified cytochrome P-450b, the major isozyme found in hepatic microsomes from phenobarbital-pretreated rats, for the metabolism of N-methylcarbazole (NMC) were analyzed in two membranous systems in order to investigate membrane bilayer-dependent parameters. Incorporation of the enzymes of the monoxygenase system into bilayer liposomal vesicles composed of total microsomal lipid had no effect on the rates or regioselectivity of the mono-hydroxylation of the substrate when compared to the same enzymes reconstituted in a soluble system with small amounts of dilauroylphosphatidylcholine (DLPC). The membranous system exhibited K/sub m/ values of NMC which were 4-5 times higher than the non-vesicular system. The rapid decline in the rates of NMC metabolism by cytochrome P-450b during catalysis suggested enzyme inactivation during substrate processing. Radiolabel from (methyl-/sup 14/C)NMC was incorporated into the P-450b apoprotein as a result of enzyme turnover co-incident with the loss of enzyme activity. NMC-stimulated NADPH oxidation by the reconstituted enzyme system containing DLPC was biophasic, with a slow rate preceding a two-fold higher rate. Carbazole and some N-substituted derivatives were unique in stimulating this unusual oxidation.

  11. 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.

  12. Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

    PubMed

    Liu, Nannan; Li, Ming; Gong, Youhui; Liu, Feng; Li, Ting

    2015-05-01

    P450s are known to be critical for the detoxification and/or activation of xenobiotics such as drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In insects, P450s play an important role in detoxifying exogenous compounds such as insecticides and plant toxins and their overexpression can result in increased levels of P450 proteins and P450 activities. This has been associated with enhanced metabolic detoxification of insecticides and has been implicated in the development of insecticide resistance in insects. Multiple P450 genes have been found to be co-overexpressed in individual insect species via several constitutive overexpression and induction mechanisms, which in turn are co-responsible for high levels of insecticide resistance. Many studies have also demonstrated that the transcriptional overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of regulatory genes/factors and resistance genes. This chapter reviews our current understanding of how the molecular mechanisms of P450 interaction/gene regulation govern the development of insecticide resistance in insects and our progress along the road to a comprehensive characterization of P450 detoxification-mediated insecticide resistance.

  13. 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

  14. 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.

  15. Temporal induction of cytochrome P450 1A in the mirror carp (Cyprinus carpio) after administration of several polycyclic aromatic hydrocarbons

    SciTech Connect

    Weiden, M.E.J. Van Der; Hanegraaf, F.H.M.; Seinen, W.; Berg, M. Van Den . Research Inst. of Toxicology); Eggens, M.L. . Dept. of Ecotoxicology); Celander, M. . Dept. of Zoophysiology)

    1994-05-01

    High inductions of 7-ethoxyresorufin O-deethylation (EROD) activities, cytochrome P450 1A protein levels (P4501A), and total cytochrome P450 content (P450) were observed 1, 2, 5, and 14 d after a single i.p. injection of 99.5 [mu]mol/kg benzo[a]pyrene (BaP) and chrysene (Chrys). The highest inductions in EROD activities and P450 1A protein levels were seen 2 d after treatment, approximately 35- and 11-fold relative to the controls. Less pronounced increase of EROD activities and P4501A protein levels, approximately 3- to 10-fold and 6-fold, respectively, were observed after treatments with equimolar concentrations of fluoranthenes (Fluor) and pyrene (Pyr). Benzo[ghi]perylene (BghiP) and the carrier dimethyl sulfoxide (DMSO) did not affect these parameters. The highest concentrations of PAHs in the liver, equivalent to 2 to 25% of the dose, were found 1 or 2 d after treatment. The highest percentage of the dose in the liver was observed for Chrys, followed by BghiP, BaP, Fluor, and Pyr. Poor correlations were found between hepatic concentrations of PAHs and P450 1A increases, which might be attributed to the rapid metabolism of these compounds.

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

    PubMed Central

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

    2010-01-01

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

  17. Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man.

    PubMed Central

    Sesardic, D; Boobis, A R; Murray, B P; Murray, S; Segura, J; de la Torre, R; Davies, D S

    1990-01-01

    1. Furafylline (1,8-dimethyl-3-(2'-furfuryl)methylxanthine) is a methylxanthine derivative that was introduced as a long-acting replacement for theophylline in the treatment of asthma. Administration of furafylline was associated with an elevation in plasma levels of caffeine, due to inhibition of caffeine oxidation, a reaction catalysed by one or more hydrocarbon-inducible isoenzymes of P450. We have now investigated the selectivity of inhibition of human monooxygenase activities by furafylline. 2. Furafylline was a potent, non-competitive inhibitor of high affinity phenacetin O-deethylase activity of microsomal fractions of human liver, a reaction catalysed by P450IA2, with an IC50 value of 0.07 microM. 3. Furafylline had either very little or no effect on human monooxygenase activities catalysed by other isoenzymes of P450, including P450IID1, P450IIC, P450IIA. Of particular interest, furafylline did not inhibit P450IA1, assessed from aryl hydrocarbon hydroxylase activity of placental samples from women who smoked cigarettes. 4. It is concluded that furafylline is a highly selective inhibitor of P450IA2 in man. 5. Furafylline was a potent inhibitor of the N3-demethylation of caffeine and of a component of the N1- and N7-demethylation. This confirms earlier suggestions that caffeine is a selective substrate of a hydrocarbon-inducible isoenzyme of P450 in man, and identifies this as P450IA2. Thus, caffeine N3-demethylation should provide a good measure of the activity of P450IA in vivo in man. 6. Although furafylline selectively inhibited P450IA2, relative to P450IA1, in the rat, this was at 1000-times the concentration required to inhibit the human isoenzyme, suggesting a major difference in the active site geometry between the human and the rat orthologues of P50IA2. PMID:2378786

  18. Human cytochrome p450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes.

    PubMed

    Jeurissen, Suzanne M F; Punt, Ans; Boersma, Marelle G; Bogaards, Jan J P; Fiamegos, Yiannis C; Schilter, Benoit; van Bladeren, Peter J; Cnubben, Nicole H P; Rietjens, Ivonne M C M

    2007-05-01

    Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1'-hydroxyestragole were identified and compared to the enzymes of importance for 1'-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that all enzymes tested, except P450 2C8, are intrinsically able to 1'-hydroxylate estragole. Experiments with Gentest microsomes, expressing P450 enzymes to roughly average liver levels, indicated that P450 1A2, 2A6, 2C19, 2D6, and 2E1 might contribute to estragole 1'-hydroxylation in the human liver. Especially P450 1A2 is an important enzyme based on the correlation between P450 1A2 activity and estragole 1'-hydroxylation in human liver microsomal samples and inhibition of estragole 1'-hydroxylation by the P450 1A2 inhibitor alpha-naphthoflavone. Kinetic studies revealed that, at physiologically relevant concentrations of estragole, P450 1A2 and 2A6 are the most important enzymes for bioactivation in the human liver showing enzyme efficiencies (kcat/Km) of, respectively, 59 and 341 min-1 mM-1. Only at relatively high estragole concentrations, P450 2C19, 2D6, and 2E1 might contribute to some extent. Comparison to results from similar studies for safrole and methyleugenol revealed that competitive interactions between estragole and methyleugenol 1'-hydroxylation and between estragole and safrole 1'-hydroxylation are to be expected because of the involvement of, respectively, P450 1A2 and P450 2A6 in the bioactivation of these compounds. Furthermore, poor metabolizer phenotypes in P450 2A6 might diminish the chances on bioactivation of estragole and safrole, whereas lifestyle factors increasing P450 1A2 activities such as cigarette smoking and consumption of charbroiled food might increase those chances for estragole and methyleugenol.

  19. Classification and characterization of putative cytochrome P450 genes from Panax ginseng C. A. Meyer.

    PubMed

    Devi, Balusamy Sri Renuka; Kim, Yu-Jin; Sathiyamoorthy, Subramaniyum; Khorolragchaa, Altanzul; Gayathri, Sathiyaraj; Parvin, Shohana; Yang, Dong-Uk; Selvi, Senthil Kalai; Lee, Ok Ran; Lee, Sungyoung; Yang, Deok-Chun

    2011-12-01

    In plants heme containing cytochrome P450 (P450) is a superfamily of monooxygenases that catalyze the addition of one oxygen atom from O2 into a substrate, with a substantial reduction of the other atom to water. The function of P450 families is attributed to chemical defense mechanism under terrestrial environmental conditions; several are involved in secondary and hormone metabolism. However, the evolutionary relationships of P450 genes in Panax ginseng remain largely unknown. In the present study, data mining methods were implemented and 116 novel putative P450 genes were identified from Expressed Sequence Tags (ESTs) of a ginseng database. These genes were classified into four clans and 22 families by sequence similarity conducted at amino acid level. The representative putative P450 sequences of P. ginseng and known P450 family from other plants were used to construct a phylogenetic tree. By comparing with other genomes, we found that most of the P450 genes from P. ginseng can be found in other dicot species. Depending on P450 family functions, seven P450 genes were selected, and for that organ specific expression, abiotic, and biotic studies were performed by quantitative reverse transcriptase-polymerase chain reaction. Different genes were found to be expressed differently in different organs. Biotic stress and abiotic stress transcript level was regulated diversely, and upregulation of P450 genes indicated the involvement of certain genes under stress conditions. The upregulation of the P450 genes under methyl jasmonate and fungal stress justifies the involvement of specific genes in secondary metabolite biosynthesis. Our results provide a foundation for further elucidating the actual function and role of P450 involved in various biochemical pathways in P. ginseng.

  20. Clinical Pharmacogenetics Implementation Consortium Guidelines for Cytochrome P450 2D6 Genotype and Codeine Therapy: 2014 Update

    PubMed Central

    Crews, K R; Gaedigk, A; Dunnenberger, H M; Leeder, J S; Klein, T E; Caudle, K E; Haidar, C E; Shen, D D; Callaghan, J T; Sadhasivam, S; Prows, C A; Kharasch, E D; Skaar, T C

    2014-01-01

    Codeine is bioactivated to morphine, a strong opioid agonist, by the hepatic cytochrome P450 2D6 (CYP2D6); hence, the efficacy and safety of codeine are governed by CYP2D6 activity. Polymorphisms are a major cause of CYP2D6 variability. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for codeine based on CYP2D6 genotype. This document is an update to the 2012 Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for CYP2D6 genotype and codeine therapy. PMID:24458010

  1. Menthol reduces the anticoagulant effect of warfarin by inducing cytochrome P450 2C expression.

    PubMed

    Hoshino, Motohiro; Ikarashi, Nobutomo; Tsukui, Makoto; Kurokawa, Asako; Naito, Rina; Suzuki, Midori; Yokobori, Kohsuke; Ochiai, Takumi; Ishii, Makoto; Kusunoki, Yoshiki; Kon, Risako; Ochiai, Wataru; Wakui, Nobuyuki; Machida, Yoshiaki; Sugiyama, Kiyoshi

    2014-06-01

    Recently, it was reported that the anticoagulant effect of warfarin was reduced when patients receiving warfarin also took menthol. The purpose of this study is to reveal the mechanism of this reduced anticoagulant effect of warfarin from the pharmacokinetic point of view. Warfarin was orally administered to mice 24h after the administration of menthol for 2 days, and the plasma warfarin concentration was measured. In the menthol administration group, the area under the blood concentration time curve of warfarin was decreased by approximately 25%, while total clearance was increased to 1.3-fold compared to the control group. The hepatic cytochrome P450 (CYP) 2C protein expression level in the menthol administration group was significantly increased compared to that in the control group. An increase in the nuclear translocation of constitutive androstane receptor (CAR) was also observed. The addition of menthol to human hepatic cells, HepaRG cells, caused an increase in the mRNA expression level of CYP2C9. The results of this study revealed that menthol causes an increase in CYP2C expression levels in the liver, which leads to an enhancement of warfarin metabolism, resulting in a decreased anticoagulant effect of warfarin. It was also suggested that menthol acted directly on the liver and increased the expression level of CYP2C by enhancing the nuclear translocation of CAR. PMID:24594507

  2. 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

  3. Effects of icaritin on cytochrome P450 enzymes in rats.

    PubMed

    Liang, Dong-Lou; Zheng, Shuang-Li

    2014-04-01

    The purpose of this study was to find out whether icaritin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated with multiple doses of icaritin. 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 corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Treatment with multiple doses of icaritin had inhibitive effects on rat CYP1A2, CYP2C9 and CYP3A4 enzyme activities. However, icaritin has no inductive or inhibitory effect on the activity of CYP2E1. Therefore, caution is needed when icaritin is co-administered with some CYP1A2, CYP2C9 or CYP3A4 substrates, which may result in treatment failure and herb-drug interactions.

  4. Interaction of rocuronium with human liver cytochromes P450.

    PubMed

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed.

  5. Three-dimensional model of cytochrome P450 human aromatase.

    PubMed

    Loge, Cedric; Le Borgne, Marc; Marchand, Pascal; Robert, Jean-Michel; Le Baut, Guillaume; Palzer, Martina; Hartmann, Rolf W

    2005-12-01

    A three-dimensional (3-D) structure of human aromatase (CYP 19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys 119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles ofAsp309 and His480 in the aromatization of the steroid A ring. PMID:16408794

  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. Pharmacogenetic biomarkers: cytochrome P450 3A5.

    PubMed

    MacPhee, Iain A M

    2012-09-01

    The immunosuppressive drugs used for solid organ transplantation all have a narrow therapeutic index with wide variation between individuals in the blood concentration achieved by a given dose. Therapeutic drug monitoring is employed routinely but may not allow optimisation of drug exposure during the critical period two to three days following transplantation. A key factor in the inter-individual variability for tacrolimus, and probably sirolimus, is whether an individual is genetically predicted to express the drug metabolising enzyme cytochrome P450 3A5 (CYP3A5). Individuals predicted to express CYP3A5 by possession of at least one wild-type CYP3A5*1 allele require 1.5-2 times higher doses of tacrolimus to achieve target blood concentrations than individuals homozygous for the CYP3A5*3 allele who are functional non-expressers of CYP3A5. Planning the initial tacrolimus dose based on the CYP3A5 genotype has been shown to allow more rapid achievement of target blood concentrations after transplantation than a standard dose given to all patients. However, it remains to be demonstrated that use of this approach as an adjunct to therapeutic drug monitoring can reduce either efficacy failure (transplant rejection) or toxicity. Use of a pharmacogenetic approach to dosing sirolimus awaits testing and it is unlikely to be useful for ciclosporin or everolimus.

  8. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    PubMed Central

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

    2015-01-01

    High prevalence of cigarette smoking in HIV patients is associated with increased HIV pathogenesis and disease progression. While the effect of smoking on the occurrence of lung cancer has been studied extensively, the association between smoking and HIV pathogenesis is poorly studied. We have recently shown the possible role of cytochrome P450 (CYP) in smoking/nicotine-mediated viral replication. In this review, we focus on the potential role of CYP pathway in polycyclic aromatic hydrocarbons (PAH), important constituents of cigarette smoke, mediated HIV pathogenesis. More specifically, we will discuss the role of CYP1A1 and CYP1B1, which are the major PAH-activating CYP enzymes. Our results have shown that treatment with cigarette smoke condensate (CSC) increases viral replication in HIV-infected macrophages. CSC contains PAH, which are known to be activated by CYP1A1 and CYP1B1 into procarcinogens/toxic metabolites. The expression of these CYPs is regulated by aryl hydrocarbon receptors (AHR), the cellular target of PAH, and an important player in various diseases including cancer. We propose that PAH/AHR-mediated CYP pathway is a novel target to develop new interventions for HIV positive smokers. PMID:26082767

  9. Versatile biocatalysis of fungal cytochrome P450 monooxygenases.

    PubMed

    Durairaj, Pradeepraj; Hur, Jae-Seoun; Yun, Hyungdon

    2016-01-01

    Cytochrome P450 (CYP) monooxygenases, the nature's most versatile biological catalysts have unique ability to catalyse regio-, chemo-, and stereospecific oxidation of a wide range of substrates under mild reaction conditions, thereby addressing a significant challenge in chemocatalysis. Though CYP enzymes are ubiquitous in all biological kingdoms, the divergence of CYPs in fungal kingdom is manifold. The CYP enzymes play pivotal roles in various fungal metabolisms starting from housekeeping biochemical reactions, detoxification of chemicals, and adaptation to hostile surroundings. Considering the versatile catalytic potentials, fungal CYPs has gained wide range of attraction among researchers and various remarkable strategies have been accomplished to enhance their biocatalytic properties. Numerous fungal CYPs with multispecialty features have been identified and the number of characterized fungal CYPs is constantly increasing. Literature reveals ample reviews on mammalian, plant and bacterial CYPs, however, modest reports on fungal CYPs urges a comprehensive review highlighting their novel catalytic potentials and functional significances. In this review, we focus on the diversification and functional diversity of fungal CYPs and recapitulate their unique and versatile biocatalytic properties. As such, this review emphasizes the crucial issues of fungal CYP systems, and the factors influencing efficient biocatalysis. PMID:27431996

  10. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application.

    PubMed

    Urlacher, Vlada B; Girhard, Marco

    2012-01-01

    Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospecific oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.

  11. Therapeutic doses of SkQ1 do not induce cytochromes P450 in rat liver.

    PubMed

    Myasoedova, K N; Silachev, D N

    2014-10-01

    The effect of SkQ1 (a mitochondria-targeted antioxidant) on the level of cytochromes P450 in rat liver was studied. It was found that administration of therapeutic dose of SkQ1 with drinking water for 5 days (250 nmol/kg of body weight per day) did not alter the level of cytochromes P450. Under the same conditions, the standard dose of phenobarbital used for the induction of cytochromes P450 caused the 2.7-fold increase in the content of these cytochromes. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in rats.

  12. 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.

  13. Cytochrome P450IA mRNA expression in feral Hudson River tomcod.

    PubMed

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

    1991-06-01

    We sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, we found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of beta-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers. PMID:1855491

  14. 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. PMID:16711749

  15. How similar are P450s and what can their differences teach us?

    PubMed

    Graham, S E; Peterson, J A

    1999-09-01

    Cytochromes P450 form a very large superfamily of proteins which metabolize substrates from steroids to fatty acids to drugs and are found in organisms from protists to mammals. P450s all appear to take on a similar structural fold, yet frequently having less than 20% sequence identity and having vastly different substrates. Within the structural fold there appears to be a highly conserved core, as determined from the comparison of the structures of the six crystallized, soluble P450s. There are also variable regions which by and large appear to be associated with substrate recognition, substrate binding, and redox partner binding. Molecular dynamics simulations of motion in P450cam and P450BM-3 indicate that substrate binding and product release require substantial motion around the "substrate access channel." Additionally, at the 11th International Conference on Cytochrome P450 Biochemistry, Biophysics, and Molecular Biology and briefly here, the first structure of a microsomal eukaryotic P450 will be presented and compared to the already determined structures by Drs. Johnson and McRee. Finally, with a better understanding of the structure/function relationship of P450s, one will be better able to modify P450s to metabolize the substrates of choice or produce needed valuable chemicals.

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

    SciTech Connect

    Prasad, Swati . E-mail: swati@scripps.edu; Murugan, Rajamanickam; Mitra, Samaresh

    2005-09-23

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

  17. 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.

  18. Suppression of male-specific cytochrome P450 2c and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl in rat liver is not causally related to changes in serum testosterone.

    PubMed

    Yeowell, H N; Waxman, D J; LeBlanc, G A; Linko, P; Goldstein, J A

    1989-06-01

    Rat cytochrome P450 2c (P450 gene IIC11) is a constitutive, male-specific hepatic enzyme which is suppressed greater than 90% by treatment with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) [H. N. Yeowell et al. (1987) Mol. Pharmacol. 32, 340-347]. HCB also decreases serum testosterone levels in adult male rats (greater than 98% loss). The present study assesses whether the suppression of P450 2c by HCB is a direct result of its effects on serum testosterone levels. Further, the site along the hypothalamic-pituitary-testicular axis at which HCB acts to depress testosterone secretion was examined. Administration of the synthetic androgen methyltrienolone to HCB-treated rats failed to prevent the suppression of P450 2c mRNA and its associated microsomal steroid 16 alpha-hydroxylase activity under conditions where it effectively reversed the large decrease in P450 2c mRNA and steroid 16 alpha-hydroxylase activity produced by castration. Hepatic steroid 6 beta-hydroxylase activity, which is catalyzed primarily by P450 2a (P450 gene IIIA2), was also suppressed by HCB and was not protected by methyltrienolone. Administration of either human chorionic gonadotropin, an analog of pituitary-derived luteinizing hormone, or the hypothalamic luteinizing hormone releasing hormone elevated serum testosterone levels to a much smaller extent in HCB-treated rats than in control rats. These results indicate that the effects of HCB on serum testosterone levels reflect its effects on testicular function rather than the pituitary or hypothalamus. However, the present study demonstrates that the consequential reduction in serum testosterone levels in HCB-treated rats is not causally related to the reduction in hepatic P450 2c levels. Thus, HCB must also act on some other regulatory mechanism involved in the expression of this protein.

  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 Central

    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. PMID:25671424

  1. 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. PMID:25671424

  2. 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.

  3. 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

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

    PubMed

    Nicoli, Elena-Raluca; Al Eisa, Nada; Cluzeau, Celine V M; 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

  5. 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

  6. Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by inducible and constitutive cytochrome P450 enzymes in rats.

    PubMed

    Guo, Z; Smith, T J; Thomas, P E; Yang, C S

    1992-10-01

    The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces tumor formation in the liver, lung, nasal cavity, and pancreas of rats. Metabolic activation is required for the tumorigenicity of this compound. The involvement of cytochrome P450 enzymes in NNK bioactivation was investigated in rats by studies with chemical inducers and antibodies against P450s. Liver microsomal enzymes catalyzed the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) from NNK. When the activity was expressed on a per nanomole P450 basis, treatments of rats with 3-methylcholanthrene (MC), phenobarbital (PB), pregnenolone 16-alpha-carbonitrile (PCN), Aroclor 1254 (AR), safrole (SA), and isosafrole (ISA) increased the keto aldehyde formation in liver microsomes 2.0-, 2.4-, 3.8-, 2.5-, 2.1-, and 1.8-fold, respectively; PB, AR, SA, and ISA increased the keto alcohol formation 1.7-, 1.3-, 2.0-, and 1.3-fold, respectively. The extents of induction were more pronounced when expressed on a per milligram protein basis, due to the higher microsomal P450 contents in the induced microsomes. The formation of NNK-N-oxide was markedly increased by PB and PCN and slightly increased by AR, SA, and ISA. However, the formation of NNAL, the major metabolite due to carbonyl reduction, was not increased by the treatments but was decreased by AR, ISA, and acetone (AC). The kinetic parameters of NNK metabolism by control, MC-, PB-, and PCN-induced liver microsomes were obtained. A panel of monoclonal (anti-1A1, -2B1, -2C11, and -2E1) and polyclonal (anti-1A2, -2A1, and -3A) antibodies were used to assess the involvement of constitutive hepatic P450 enzymes in NNK metabolism. Keto aldehyde formation was inhibited by anti-1A2 and anti-3A (about 15%) but not by others; the formation of keto

  7. Renal drug metabolism in humans: the potential for drug–endobiotic interactions involving cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT)

    PubMed Central

    Knights, Kathleen M; Rowland, Andrew; Miners, John O

    2013-01-01

    Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug–endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. PMID:23362865

  8. Renal drug metabolism in humans: the potential for drug-endobiotic interactions involving cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT).

    PubMed

    Knights, Kathleen M; Rowland, Andrew; Miners, John O

    2013-10-01

    Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug-endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. PMID:23362865

  9. Brain isoform glycogen phosphorylase as a novel hepatic progenitor cell marker.

    PubMed

    Huang, Yu-Wen; Chiu, Chien-Chang; Liang, Ja-Der; Chiou, Ling-Ling; Huang, Guan-Tarn; Yu, Ming-Jiun; Lee, Hsuan-Shu

    2015-01-01

    An appropriate liver-specific progenitor cell marker is a stepping stone in liver regenerative medicine. Here, we report brain isoform glycogen phosphorylase (GPBB) as a novel liver progenitor cell marker. GPBB was identified in a protein complex precipitated by a monoclonal antibody Ligab generated from a rat liver progenitor cell line Lig-8. Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The levels of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, consistent with roles of GPBB as a liver progenitor cell marker. Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. We conclude that GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under low glucose conditions and cell differentiation. PMID:25826279

  10. 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

  11. 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

  12. 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

  13. 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 ...

  14. 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

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

    PubMed

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

    2014-01-21

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

  16. 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

  17. 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...

  18. Measurement of Cytochrome P450 Enzyme Induction and Inhibition in Human Hepatoma Cells.

    PubMed

    Rodrigues, Robim M; De Kock, Joery; Doktorova, Tatyana Y; Rogiers, Vera; Vanhaecke, Tamara

    2015-01-01

    Cytochrome P450 enzymes are a diverse group of catalytic enzymes in the liver that are mainly responsible for the biotransformation of organic substances. Cytochrome P450 activity as well as both its induction and inhibition are key factors in drug biotransformation and can be involved in deactivation, activation, detoxification and toxification processes. Thus, the modulation of cytochrome P450 activity is an important parameter when evaluating the potential toxicity of chemical compounds using an in vitro system. The cytochrome P450 3A subfamily proteins are among the most important drug-metabolizing enzymes in human liver and are responsible for about half of all cytochrome P450-dependent drug oxidations. In vitro, these enzymes are active not only in primary human hepatocyte cultures, but also in differentiated human hepatoma HepaRG cells. The present protocol describes the culture of cryopreserved differentiated HepaRG cells and the evaluation of its cytochrome P450 activity upon exposure to a chemical compound using a commercially available luminogenic cytochrome P450 assay. This in vitro model can be used to monitor the induction and inhibition of cytochrome P450 3A following exposure to a particular test compound.

  19. Expression of a Ripening-Related Avocado (Persea americana) Cytochrome P450 in Yeast.

    PubMed

    Bozak, K R; O'keefe, D P; Christoffersen, R E

    1992-12-01

    One of the mRNAs that accumulates during the ripening of avocado (Persea americana Mill. cv Hass) has been previously identified as a cytochrome P450 (P450) monooxygenase and the corresponding gene designated CYP71A1. In this report we demonstrate that during ripening the accumulation of antigenically detected CYP71A1 gene product (CYP71A1) correlates with increases in total P450 and two P450-dependent enzyme activities: para-chloro-N-methylaniline demethylase, and trans-cinnamic acid hydroxylase (tCAH). To determine whether both of these activities are derived from CYP71A1, we have expressed this protein in yeast (Saccharomyces cerevisiae) using a galactose-inducible yeast promoter. Following induction, the microsomal fraction of transformed yeast cells undergoes a large increase in P450 level, attributable almost exclusively to the plant CYP71A1 protein. These membranes exhibit NADPH-dependent para-chloro-N-methylaniline demethylase activity at a rate comparable to that in avocado microsomes but have no detectable tCAH. These results demonstrate both that the CYP71A1 protein is not a tCAH and that a plant P450 is fully functional upon heterologous expression in yeast. These findings also indicate that the heterologous P450 protein can interact with the yeast NADPH:P450 reductase to produce a functional complex.

  20. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1.

    PubMed

    Chun, Young-Jin; Kim, Donghak

    2016-04-01

    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

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

    PubMed

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

    1993-12-01

    1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation

  2. Georges Brohee Prize 1996. Major cytochrome P-450 families: implications in health and liver diseases.

    PubMed

    Horsmans, Y

    1997-01-01

    Cytochromes P-450 are a superfamily of hemoproteins which represent the main pathway for drug and chemical oxidation. This superfamily is divided into families, subfamilies and/or single enzymes. The majority of P-450s involved in drug metabolism appear to belong to three distinct families termed CYP1, CYP2 and CYP3. Numerous invasive and non-invasive methodologies have been developed to study these enzymes. Their activities are modulated by genetic and nongenetic factors as well as pathological conditions. In this work, the significance of genetic and nongenetic control of P-450s activities in normal subjects is described. Thereafter, the impact of P-450s on the apparition of liver diseases and the effects of liver disease on P-450s activities is emphasized. In conclusion, future perspectives on this field are presented.

  3. 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.

  4. Ethylbenzene induces microsomal oxygen free radical generation: antibody-directed characterization of the responsible cytochrome P450 enzymes.

    PubMed

    Serron, S C; Dwivedi, N; Backes, W L

    2000-05-01

    Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to its fluorescent product 2',7'-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H(2)O(2)) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to

  5. 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.

  6. Role of rat cytochromes P450 in the oxidation of 17α-ethinylestradiol.

    PubMed

    Bořek-Dohalská, Lucie; Valášková, Petra; Černá, Věra; Stiborová, Marie

    2014-11-01

    17α-Ethinylestradiol (EE2) is an endocrine disruptor (ED) used as an ingredient of oral contraceptives. Rat hepatic microsomes metabolize EE2 to three products; two of them are hydroxylated EE2 derivatives. Of the hydroxylation reactions, 2-hydroxylation, is the major reaction. Cytochrome P450 (CYP) plays a major role in EE2 hydroxylation. To resolve which rat CYPs are responsible for EE2 oxidation, three approaches were used: induction of specific CYPs, selective inhibition of CYPs, and recombinant rat CYPs. The results demonstrate that EE2 is hydroxylated by several rat CYPs, among them CYP2C6 and 2C11 are most efficient in 2-hydroxy-EE2 formation, while CYP2A and 3A catalyze EE2 hydroxylation to the second product. EE2 is also an inhibitor of CYP2C- and CYP3A-catalyzed hydroxylation of endogenous EDs progesterone and testosterone. EE2 acts as a reversible inhibitor of CYP3A-mediated progesterone 6β-hydroxylation and inactivates CYP3A- and CYP2C-catalyzed testosterone 6β-hydroxylation and progesterone 21- or 16α-hydroxylation, respectively, in a mechanism-based manner.

  7. 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.

  8. [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.

  9. Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States.

    PubMed

    Liou, Shu-Hao; Mahomed, Mavish; Lee, Young-Tae; Goodin, David B

    2016-08-17

    In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states. PMID:27452076

  10. 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.

  11. Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States.

    PubMed

    Liou, Shu-Hao; Mahomed, Mavish; Lee, Young-Tae; Goodin, David B

    2016-08-17

    In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states.

  12. Computer modeling of 3D structures of cytochrome P450s.

    PubMed

    Chang, Y T; Stiffelman, O B; Loew, G H

    1996-01-01

    The understanding of structure-function relationship of enzymes requires detailed information of their three-dimensional structure. Protein structure determination by X-ray and NMR methods, the two most frequently used experimental procedures, are often difficult and time-consuming. Thus computer modeling of protein structures has become an increasingly active and attractive option for obtaining predictive models of three-dimensional protein structures. Specifically, for the ubiquitous metabolizing heme proteins, the cytochrome P450s, the X-ray structures of four isozymes of bacterial origin, P450cam, P450terp, P450BM-3 and P450eryF have now been determined. However, attempts to obtain the structure of mammalian forms by experimental means have thus far not been successful. Thus, there have been numerous attempts to construct models of mammalian P450s using homology modeling methods in which the known structures have been used to various extents and in various strategies to build models of P450 isozymes. In this paper, we review these efforts and then describe a strategy for structure building and assessment of 3D models of P450s recently developed in our laboratory that corrects many of the weaknesses in the previous procedures. The results are 3D models that for the first time are stable to unconstrained molecular dynamics simulations. The use of this method is demonstrated by the construction and validation of a 3D model for rabbit liver microsomal P450 isozyme 2B4, responsible for the oxidative metabolism of diverse xenobiotics including widely used inhalation anesthetics. Using this 2B4 model, the substrate access channel, substrate binding site and plausible surface regions for binding with P450 redox partners were identified. PMID:9010606

  13. Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole. Isolation of an NN-bridged benzyne-protoporphyrin IX adduct.

    PubMed

    Ortiz de Montellano, P R; Mathews, J M

    1981-06-01

    Destruction of hepatic cytochrome P-450 during catalytic processing of 1-amino-benzotriazole is accompanied by an equal loss of microsomal haem but not by loss of cytochrome b5, or stimulation of lipid peroxidation. An abnormal porphyrin, tentatively identified as an NN-bridged benzyne-protoporphyrin IX adduct, appears to be formed by the addition of catalytically generated benzyne to prosthetic haem.

  14. Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency

    PubMed Central

    Jin, Yi; Chen, Mo; Penning, Trevor M.; Miller, Walter L.

    2015-01-01

    Cytochrome P450 oxidoreductase (POR) is a 2-flavin protein that transfers electrons from NADPH via its FAD and FMN moieties to all microsomal cytochrome P450 enzymes, including steroidogenic and drug-metabolizing P450s. Defects in the POR gene can cause POR deficiency (PORD), manifested clinically by disordered steroidogenesis, genital anomalies and skeletal malformations. We examined the POR mutant A287P, which is the most frequent cause of PORD in patients of European ancestry and partially disrupts most P450 activities in vitro. Flavin content analysis showed that A287P is deficient in FAD and FMN binding, although the mutation site is distant from the binding sites of both flavins. Externally added flavin partially restored the cytochrome c reductase activity of A287P, suggesting that flavin therapy may be useful for this frequent form of PORD. Transient kinetic dissection of the reaction of POR with NADPH and the reduction in cytochrome c by POR using stopped-flow techniques revealed defects in individual electron transfer steps mediated by A287P. A287P had impaired ability to accept electrons from NADPH, but was capable of a fast FMN ➔ cytochrome c electron donation reaction. Thus the reduced rates of P450 activities with A287P may be due to deficient flavin and impaired electron transfer from NADPH. PMID:25728647

  15. Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

    SciTech Connect

    Hamdane, Djemel; Xia, Chuanwu; Im, Sang-Choul; Zhang, Haoming; Kim, Jung-Ja P.; Waskell, Lucy

    2010-01-20

    NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.

  16. Purification of a sheep liver cytochrome P-450 from the P450IIIA gene subfamily. Its contribution to the N-dealkylation of veterinary drugs.

    PubMed

    Pineau, T; Galtier, P; Bonfils, C; Derancourt, J; Maurel, P

    1990-03-01

    Oral administration of troleandomycin at a dose of 100 mg/kg/day for 6 days to three adult male Lacaune sheep produced a 1.6-fold increase in specific content of liver microsomal cytochrome P-450. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, microsomal preparations from treated animals exhibited a strong band in the zone of electrophoretic mobility of cytochromes P-450. This band corresponded to a cytochrome P-450 which cross-reacted with rabbit P450IIIA6 antibodies, as demonstrated by immunoblotting. The ovine isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, CM cellulose and hydroxylapatite chromatographic separations. This hemoprotein had an apparent molecular weight of 52 kD as determined by calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was characterized in terms of spectral data, NH2-terminal amino acid sequence, immunologic and catalytic properties. This study revealed some interspecies differences with the orthologous rabbit isozyme. The contribution of this form to the N-demethylation of erythromycin and of three veterinary drugs: chlorpromazine, chlorpheniramine and bromhexine was demonstrated from inhibition by TAO, from immunoinhibition studies, using polyclonal antibodies raised in rabbit and from the existence of significant correlations between its microsomal level and these N-demethylase activities. In contrast, the results suggest that ovine P450IIIA could not be predominantly involved in the N-dealkylation of benzphetamine, ephedrine, ivermectine or spiramycin. PMID:2310415

  17. Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium.

    PubMed

    He, J S; Ruettinger, R T; Liu, H M; Fulco, A J

    1989-12-22

    The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439. The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987-10995). PMID:2597681

  18. 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.

  19. Mechanism of induction of cytochrome p450 enzymes by the proestrogenic endocrine disruptor pesticide-methoxychlor: interactions of methoxychlor metabolites with the constitutive androstane receptor system.

    PubMed

    Blizard, D; Sueyoshi, T; Negishi, M; Dehal, S S; Kupfer, D

    2001-06-01

    Methoxychlor, a structural analog of the DDT pesticide, was previously shown to induce rat hepatic CYP2B and -3A mRNAs and the corresponding proteins [J Biochem Mol Toxicol 1998;12:315-323], Additionally, methoxychlor was found to activate the constitutive androstane receptor (CAR) system and induce CYP2B6 (J Biol Chem 1999;274:6043-6046), suggesting a mechanism for methoxychlor-mediated cytochrome P450 (P450) 2B induction. However, it has not been established whether CAR activation and P450 induction was due to methoxychlor per se and/or due to its metabolites. Also, a possible link between the estrogenic potency of methoxychlor metabolites and CAR activation or P450 induction was not investigated. The current study explores the ability of methoxychlor and its metabolites to activate CAR and whether their potency of CAR activation correlates with their respective estrogenicity. Methoxychlor and its metabolites [mono-OH-M [1,1,1-trichloro-2 (4-hydroxyphenyl)-2'-(4-methoxyphenyl)ethane]; bis-OH-M [1,1,1-trichloro-2,2'-bis(4-hydroxyphenyl)ethane]; ring-OH-M [1,1,1-trichloro-2(4-methoxyphenyl)-2'-(3-hydroxy-4-methoxyphenyl)ethane]; and tris-OH-M [1,1,1-trichloro-2(4-hydroxyphenyl)-2'-(3,4-dihydroxyphenyl)ethane

  20. Antioxidant-mediated attenuation of the induction of cytochrome P450BM-3(CYP102) by ibuprofen in Bacillus megaterium ATCC 14581.

    PubMed

    English, N T; Rankin, L C

    1997-08-15

    Bacillus megaterium contains a soluble cytochrome P450 termed BM-3, which is highly inducible by barbiturates, peroxisome proliferators, and nonsteroidal antiinflammatory drugs. In rats and mice, the chronic administration of peroxisome proliferators induces a sustained oxidative stress in hepatic tissue and may be responsible for the nongenotoxic carcinogenesis observed with prolonged treatment. Here it is shown that ibuprofen induces a variety of enzymes associated with the oxidative stress response in Bacillus, including catalase, glucose-6-phosphate-dehydrogenase, and aldehyde reductase in a dose-related manner. Furthermore, evidence is presented to show that the expression of cytochrome P450 in Bacillus is associated with a marked depletion in cellular glutathione levels and that it renders these cells considerably more sensitive to oxidant insult. Finally, this work reports that a variety of structurally diverse antioxidants such as ascorbic acid, reduced glutathione, alpha-tocopherol acetate and the artificial antioxidant, butylated hydroxyanisole, all dramatically attenuate the expression of the cytochrome P450BM-3 gene and its repressor, Bm3R1, following ibuprofen treatment. These observations provide the first evidence that the expression of cytochrome P450 genes can lead to increased oxidant sensitivity but can be strongly modulated by dietary and artificial antioxidants, as well as antioxidant enzymes. The important implications of this phenomenon are also discussed. PMID:9313770

  1. A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A.

    PubMed

    Dai, Zi-Ru; Ge, Guang-Bo; Feng, Lei; Ning, Jing; Hu, Liang-Hai; Jin, Qiang; Wang, Dan-Dan; Lv, Xia; Dou, Tong-Yi; Cui, Jing-Nan; Yang, Ling

    2015-11-18

    Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

  2. Inhibition of human cytochrome P450 enzymes by licochalcone A, a naturally occurring constituent of licorice.

    PubMed

    He, Wei; Wu, Jing-Jing; Ning, Jing; Hou, Jie; Xin, Hong; He, Yu-Qi; Ge, Guang-Bo; Xu, Wei

    2015-10-01

    Licochalcone A (LCA) is a major bioactive compound in traditional Chinese herbal liquorice that possesses multiple pharmacological activities. However, the effects of the potential herb-drug interactions (HDIs) between LCA and therapeutic drugs on the inhibition of human cytochrome P450 (CYP) enzymes remain unclear. In the present study, the inhibitory effects of LCA on seven major human CYP isoforms, including CYP1A2, 2D6, 2E1, 2C19, 2C8, 2C9 and 3A4, were investigated in human liver microsomes (HLMs). The results demonstrated that LCA significantly inhibited the activities of CYP1A2, 2C19, 2C8, 2C9 and 3A4 and exhibited weak inhibitory effects on CYP2E1 and CYP2D6. Dixon and Lineweaver-Burk plots revealed that the inhibition types of LCA against CYP1A2, 2C9, 2C19 and 2C8 were best fit as mixed-type inhibitions, while LCA was a competitive inhibitor towards CYP3A4. The inhibition kinetic parameters (K(i)) were calculated to be 1.02 μM, 0.17 μM, 3.89 μM 0.89 μM, and 2.29 μM, for CYP1A2, 2C9, 2C19, 2C8, and 3A4, respectively. Furthermore, the areas under the plasma concentration-time curves (AUCs) of several drugs that are primarily metabolized by CYPs were estimated to increase by 2-398% in the presence of LCA, which suggested that LCA exhibited high HDI potentials via CYP inhibition. These data are significant for the clinical applications of LCA-containing herbs.

  3. Preparation and characterization of monoclonal antibodies recognizing unique epitopes on sexually differentiated rat liver cytochrome P-450 isozymes.

    PubMed

    Morgan, E T; Rönnholm, M; Gustafsson, J A

    1987-07-14

    Cytochrome P-450 isozymes P-450(16 alpha), P-450(15 beta), and P-450DEa are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to P-450(15 beta) and one antibody to P-450(16 alpha) that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among P-450(16 alpha), P-450(15 beta), and P-450DEa. All of the P-450(15 beta-specific antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay. Western immunoblotting, and antibody-Sepharose immunoadsorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for P-450(15 beta) by these criteria. However, the apparent specificities of antibodies F3 and F20 for P-450(15 beta), and of M16 for P-450(16 alpha), were highly dependent on the analytical technique used. The five anti-P-450(15 beta) antibodies all inhibited the catalytic activity of microsomal P-450(15 beta), by a maximum of 70%. However, they also produced a similar inhibition of microsomal P-450(16 alpha-specific antibody M16 and F23 have a low-affinity interaction with an epitope on P-450(16 alpha). The P-450(16 alpha)-specific antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Metabolism of styrene to styrene oxide and vinylphenols in cytochrome P450 2F2- and P450 2E1-knockout mouse liver and lung microsomes

    PubMed Central

    Shen, Shuijie; Li, Lei; Ding, Xinxin; Zheng, Jiang

    2014-01-01

    Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 (CYP) enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e. styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. Dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes, relative to that in the wild-type mouse lung microsomes. However, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knock–out and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed similar susceptibility to lung toxicity of styrene as the wild-type animals. However, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene. PMID:24320693

  5. P450cam visits an open conformation in the absence of substrate.

    PubMed

    Lee, Young-Tae; Wilson, Richard F; Rupniewski, Igor; Goodin, David B

    2010-04-27

    P450cam from Pseudomonas putida is the best characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K(+)]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B' helix, and loss of the K(+) binding site. Crystallization in the presence of K(+) alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Redissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K(+). These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand. PMID:20297780

  6. 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 P