Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11. To clarify the influences of P450 enzymes on the catalytic activity of mEH, the hydrolyzing activity for styrene oxide and benzo(a)pyrene-7,8-oxide [B(a)P-oxide] was analyzed in the presence or absence of P450s. Styrene oxide hydrolysis was activated by all P450s including the CYP1A, 2B, 2C, and 3A subfamilies. In agreement with the association affinity determined by resonant mirror, CYP2C11 tends to have enhanced activity for styrene oxide hydrolysis. On the other hand, B(a)P-oxide hydrolysis was enhanced by only CYP2C11 while CYP1A1 and CYP2B1 had no effect. These results suggest that (1) many P450 enzymes associate nonspecifically with mEH, (2) the CYP2C11 plays a greater role in the association/activation of mEH and (3) the P450-mediated activation of mEH depends upon the substrate of mEH.     

Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP-glucuronosyltransferase

Protein-protein interactions between cytochrome P450 (P450) and other drug-metabolizing enzymes were studied by affinity chromatography using CYP1A1-, glycine-, and bovine serum albumin (BSA)-conjugated Sepharose 4B columns. Sodium cholate-solubilized microsomes from phenobarbital-treated rat liver were applied to the columns and the material eluted with buffer containing NaCl was analyzed by immunoblotting. Microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferases (UGTs), as well as NADPH-P450 reductase, were efficiently trapped by the CYP1A1 column. Glycine and BSA columns exhibited no ability to retain these proteins. Protein disulfide isomerase and calnexin, non-drug-metabolizing enzymes expressed in the endoplasmic reticulum, were unable to associate with the CYP1A1 column. These results suggest that CYP1A1 interacts with mEH and UGT to facilitate a series of multistep drug metabolic conversions.     

Understanding the role of xenobiotic-metabolism in chemical carcinogenesis using gene knockout mice

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutations. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and microsomal epoxide hydrolase, and various phase II transferase enzymes. It is extremely important to determine the role P450s play in the carcinogenesis and to establish if they are the rate limiting and critical interface between the chemical and its biological activities. The latter is essential in order to validate the use of rodent models to test safety of chemicals in humans. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disrupted genes encoding the P450s CYP1A2, CYP2E1, and CYP1B1 were developed. Mice lacking expression of microsomal epoxide hydrolase (mEH) and NADPH-quinone oxidoreductase (NQO1) were also made. All of these mice exhibit no gross abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in xenobiotic-metabolizing enzymes among humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying the human carcinogens and validate the molecular epidemiological studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.     

温肾咳喘片主要成分对HepG2细胞中CYP相关基因表达的影响

观察温肾咳喘片组方中5种主要单体成分甘草酸、厚朴酚、和厚朴酚、蛇床子素和 欧前胡素对细胞色素P450(cytochrome P450, CYP) 1A2,2D6,2E1和3A4基因表达的影 响. 采用实时荧光定量PCR技术检测HepG2细胞中药物处理后各CYP mRNA的表达.厚朴酚 、和厚朴酚、蛇床子素和欧前胡素在不同浓度均能明显的诱导CYP2E1和CYP3A4,同时欧 前胡素也能诱导CYP1A2的表达,而甘草酸、厚朴酚、和厚朴酚、蛇床子素和欧前胡素在 不同浓度对CYP2D6的表达均具有较弱的抑制作用.甘草酸、厚朴酚、和厚朴酚、蛇床子 素和欧前胡素能明显影响CYP1A2、2D6、2E1或3A4的表达.此研究为中西药物代谢性相互 作用及毒理学的研究提供实验依据.     

Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons

Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix.Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested.In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways.     

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.     

Identification of novel enzyme-prodrug combinations for use in cytochrome P450-based gene therapy for cancer

Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene-CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated approximately 250-fold by liver microsomes from dexamethasone-induced rats (IC(50) (MMDX) approximately 0.1nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug-CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.     

3′-Hydroxypterostilbene Inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Mouse Skin Carcinogenesis

BackgroundA natural pterostilbene analogue isolated from the herb Sphaerophysa salsula, 3′-hydroxypterostilbene (HPSB), exhibits antiproliferative activity in several cancer cell lines; however, the inhibitory effects of HPSB on skin carcinogenesis remains unclear.PurposeThe aim of this study was to evaluate the inhibitory effects of HPSB on two-stage skin carcinogenesis in mice and its potential mechanism.Study Design and MethodsThis study investigated the anti-inflammatory and anti-tumor effects of HPSB in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated acute skin inflammation and 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced two-stage skin carcinogenesis model. In addition, the effects of HPSB on the modulation of the phase I and phase II metabolizing enzymes in the DMBA-induced HaCaT cell model were investigated.ResultsThe results provide evidence that topical treatment with HPSB significantly inhibits TPA-induced epidermal hyperplasia and leukocyte infiltration through the down-regulation of cyclooxygenase-2 (COX-2), matrix metalloprotein-9 (MMP-9), and ornithine decarboxylase (ODC) protein expression in mouse skin. Furthermore, HPSB suppresses DMBA/TPA-induced skin tumor incidence and multiplicity via the inhibition of proliferating cell nuclear antigen (PCNA), Cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression in the two-stage skin carcinogenesis model. In addition, pretreatment with HPSB markedly reduces DMBA-induced cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) gene expression in human keratinocytes; however, HPSB does not significantly affect the gene expression of the phase II enzymes.ConclusionThis is the first study to show that topical treatment with HPSB prevents mouse skin tumorigenesis. Overall, our study suggests that natural HPSB may serve as a novel chemopreventive agent capable of preventing carcinogen activation and inflammation-associated tumorigenesis.     

Induction of cytochrome P450 3A by Shexiang Baoxin Pill and its main components

The expression of cytochrome P450 is regulated by both endogenous factors and xenobiotics including chemical drugs and natural medicines. Induction on cytochrome P450 can reduce the therapeutic efficacy from drugs inactivated by this enzyme system, but may increase the efficacy or lead to intoxication for prodrugs. Shexiang Baoxin Pill (SBP) is a traditional Chinese medicine widely used for the treatment of angina pectoris and myocardial infarction in China and other oriental countries. To assess the potential of SBP to alter the activity and expression of cytochrome P450 3A (CYP3A) extensively involved in drug metabolism, we investigated the enzyme-inducing effects of SBP in HepG2 cells and in rats. The results showed that treatment with SBP increased the enzyme activity, mRNA levels and protein expression of CYP3A4 in a concentration-dependent manner in HepG2 cells. Moreover, treatment with SBP enhanced the activities and mRNA expressions of CYP3A1 and CYP3A2 ex vivo in rats. Furthermore, we utilized HepG2 cell line to identify individual components in SBP as potential inducers of CYP3A4. It was found that bufalin, cinobufagin, and resibufogenin were novel CYP3A4 inducers. Among them, bufalin and cinobufagin significantly promoted the CYP3A4 enzyme activity, mRNA and protein levels, with the maximal induction challenging or exceeding that of the induction by rifampicin, indicating that they might play a critical role in CYP3A4 enzyme-inducing effects of SBP. In addition, the metabolic studies with specific inhibitors of CYP isoforms suggested that the three CYP3A4 inducers in SBP are also the substrates for the enzyme. Overall, our results show that SBP contains constituents that can potently induce CYP3A and suggest that this traditional Chinese medicine should be examined clinically for potential drug metabolic interactions.     

Allelic variation in the Depressaria pastinacella CYP6AB3 protein enhances metabolism of plant allelochemicals by altering a proximal surface residue and potential interactions with cytochrome P450 reductase

CYP6AB3v1, a cytochrome P450 monooxygenase in Depressaria pastinacella (parsnip webworm), is highly specialized for metabolizing imperatorin, a toxic furanocoumarin in the apiaceous host plants of this insect. Cloning and heterologous expression of CYP6AB3v2, an allelic variant identified in D. pastinacella, reveals that it metabolizes imperatorin at a rate (V(max) of 10.02 pmol/min/pmol of cytochrome P450 monooxygenase (P450)) significantly higher than CYP6AB3v1 (V(max) of 2.41 pmol/min/pmol) when supplemented with even low levels of cytochrome P450 reductase. Comparisons of the NADPH consumption rates for these variants indicate that CYP6AB3v2 utilizes this electron source at a faster rate than does CYP6AB3v1. Molecular modeling of the five amino acid differences between these variants and their potential interactions with P450 reductase suggests that replacement of Val(92) on the proximal face of CYP6AB3v1 with Ala(92) in CYP6AB3v2 affects interactions with P450 reductase so as to enhance its catalytic activity. Allelic variation at this locus potentially allows D. pastinacella to adapt to both intraspecific and interspecific variation in imperatorin concentrations in its host plants.     

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

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

CYP2D6 and CYP1A1 mutations in the Turkish population

Drugs and carcinogens are substrates of a group of metabolic enzymes including cytochrome p450 enzymes and gluthatione S-transferases. Many of the genes encoding these enzymes exhibit functional polymorphisms that contribute individual cancer susceptibility and drug response. Molecular studies based on these polymorphic enzymes also explain the aetiology of cancer and therapeutic management in clinics. We analysed the cytochrome p4501A1 (CYP1A1) and 2D6 (CYP2D6) variant genotype and allele frequencies by PCR-RFLP in Turkish individuals (n=140). The frequency of the CYP1A1*2A mutant allele was found to be 15.4%, and the CYP2D6*3 and *4 mutant allele (poor metabolizer) frequencies were 2.5% and 13.9%, respectively. This study presents the first results of CYP1A1 and CYP2D6 mutant allele distributions in the Turkish population and these data provide an understanding of epidemiological studies that correlate therapeutic approaches and aetiology of several types of malignancy in Turkish patients.     

Feeding of Ginkgo biloba extract (GBE) enhances gene expression of hepatic cytochrome P-450 and attenuates the hypotensive effect of nicardipine in rats

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany and is ingested widely as a herbal medicine in some countries. However, accurate information about its safety as an herbal medicine has not been sufficiently established. To address this issue, we examined the effect of GBE on hepatic drug metabolizing enzymes and their influence on hypotensive drug in rats. Male rats were fed either a control diet or diet containing GBE (0.5% w/w) for 4 weeks. The feeding of a GBE diet did not change the serum transaminase activity, but increased the liver weight and the phospholipid concentration in the liver. In addition, the GBE diet markedly increased the content of cytochrome P-450 (CYP), and the activity of glutathione S-transferase in the liver. Furthermore, the GBE diet markedly induced levels of CYP2B1/2, CYP3A1 and CYP3A2 mRNA in the liver. The levels of CYP1A1, CYP1A2, CYP2E1, CYP2C11 and CYP4A1 were unchanged. The feeding of GBE for 4 weeks significantly reduced the hypotensive effect of nicardipine that was reported to be metabolized by CYP3A2 in rats. These findings suggest that GBE reduces the therapeutic potency of the Ca2+ channel blocker, nicardipine, via enhancement of cytochrome P-450 expression.     

Toxicogenomics of resveratrol in rat liver

Resveratrol, a polyphenolic compound found in grape skin and peanuts has been shown to prevent many diseases including cardiovascular diseases and cancer. To better understand resveratrol's potential in vivo toxicity, we studied the dose response using cDNA stress arrays coupled with drug metabolizing enzymatic (DME) assays to investigate the expression of stress-responsive genes and Phase I and II detoxifying enzymes in rat livers. Male and female CD rats were treated with high doses of resveratrol (0.3, 1.0 and 3.0 gm/kg/day) for a period of 28 days. Total RNA from rat liver was reverse-transcribed using gene-specific primers and hybridized to stress-related cDNA arrays. Among female rats, Phase I DME genes were repressed at 0.3 and 1.0 gm/kg/day doses, while genes such as manganese superoxide dismutase, cytochrome P450 reductase, quinone oxidoreductase and thiosulfate sulfurtransferase demonstrated a dose-dependent increase in gene expression. The modulation of these liver genes may implicate the potential toxicity as observed among the rats at the highest dose level of resveratrol. Real-Time PCR was conducted on some of the Phase II DME genes and anti-oxidant genes to validate the cDNA array data. The gene expression from real-time PCR demonstrated good correlation with the cDNA array data. UGT1A genes were amongst the most robustly induced especially at the high doses of resveratrol. We next performed Phase I and Phase II enzymatic assays on cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Induction of Phase II detoxifying enzymes was most pronounced at the highest dose of resveratrol. CYP1A1 activity demonstrated a decreasing trend among the 3 dose groups and CYP2E1 activity increased marginally among female rats over controls. In summary, at lower doses of resveratrol there are few significant changes in gene expression whereas the modulation of liver genes at the high dose of resveratrol may implicate the potential toxicity observed.     

Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes

Estrogen exposure and metabolism may play an important role in the development of estrogen-sensitive cancers in postmenopausal women. In this study we investigated whether past oral contraceptive (OC) administration or current dietary isoflavonoids (IF) affected expression and/or activity of steroid hormone-metabolizing cytochrome P450 (CYP) enzymes using complementary primate and cell culture models. One-hundred-eighty-one female cynomolgus macaques were randomized to receive OC or nothing for 26 months premenopausally, then ovariectomized and randomized to one of three diets for 36 months: an IF-depleted soy protein isolate (Soy−) diet, a Soy diet with IF (Soy+), or a Soy− diet supplemented with conjugated equine estrogens (CEE). Prior OC-treatment significantly reduced CYP gene expression in the mammary gland (≤60% of OC-). Dietary IFs had no effect on CYP expression, while CEE-treatment decreased CYP1A1 and increased CYP3A4 mRNA in a tissue-specific manner. For in vitro studies, we measured effects of the isoflavonoids genistein, daidzein and equol on CYP activity using intact V79 cells stably transfected to express CYP1A1, CYP1B1, or CYP3A4. All three IFs significantly altered CYP activity in a dose-dependent and isoform-specific manner (20–95% inhibition versus controls). These results suggest potential mechanisms for prior OC and dietary IF effects on cancer risk in estrogen-responsive tissues.     

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

The Ginkgo biloba extract EGb761 was tested for its ability to inhibit the major human cytochrome P450 enzymes (CYPs). The full extract was found to strongly inhibit CYP2C9 (Ki = 14+/- 4 microg/mL), and to a lesser extent, CYP1A2 (Ki = 106 +/- 24 microg/mL), CYP2E1 (Ki = 127 +/- 42 microg/mL), and CYP3A4 (Ki = 155 +/- 43 microg/mL). The terpenoidic and flavonoidic fractions of the extract were tested separately against the same P450s to identify the source of inhibition by EGb761. The terpenoidic fraction inhibited only CYP2C9 (Ki = 15 +/-6 microg/mL) whereas the flavonoidic fraction of EGb761 showed high inhibition of CYP2C9, CYP1A2, CYP2E1, and CYP3A4 (Ki's between 4.9 and 55 microg/mL). The flavonoidic fraction was further fractionated using extraction and chromatography. Inhibition studies indicated that the majority of these fractions inhibited P450s at a significant level (IC50 < 40 microg/mL).     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

Indole-3-carbinol modulation of hepatic monooxygenases CYP1A1, CYP1A2 and FMO1 in guinea pig, mouse and rabbit

Indole-3-carbinol (I3C), a major component of cruciferous vegetables, has been shown to be chemoprotective against cancer in a number of animal models and is being evaluated as a potential agent to prevent breast cancer in healthy women. Some concern has been raised related to the long-term use of I3C, as in some models chronic dietary post-initiation exposures promote cancers. I3C administration to rats marked induces several cytochrome P450s (CYPs), especially CYP1A1 (approx. 25-fold), while at the same time inhibiting the expression of FMO1. The consequence is a marked shift in the metabolic profile of drugs such as nicotine and tamoxifen, that are substrates for both monooxygenases. Such an effect could lead to adverse drug reactions in humans. In order to determine if the effect of I3C was manifest in species other than the rat, we fed 2000-ppm I3C to male guinea pigs, mice and rabbits for a period of 4 weeks. In each species, induction of CYP1A1/1A2 expression was observed in the liver but little or no effect on FMO1 was evident, with the possible exception of the rabbit. These data demonstrate that the ability of I3C to both induce CYP1A1 and inhibit FMO1, as observed in the rat, may not be common to other mammals for which FMO1 is the major isoform in the liver.