Suicide inhibitors of cytochrome P450 1A1 and P450 2B1.

The inhibition of the P450 1A1 dependent de-ethylation of 7-ethoxyphenoxazone (7EPO) and the P450 2B1 dependent de-pentylation of 7-pentoxyphenoxazone (7PPO) by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 1-ethynylanthracene (1EA), 2-ethynylanthracene (2EA), 9-ethynylanthracene (9EA), 2-ethynylphenathrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-ethynylphenanthrene (9EPh), 1-ethynylpyrene (1EP) and 2-ethynylpyrene (2EP) was studied in hepatic microsomal preparations from rats. Although all of the polycyclic aromatic acetylenes studied inhibited the dealkylation of 7EPO or 7PPO, only some of the acetylenes produced a mechanism-based irreversible inactivation (suicide inhibition) of the P450 dependent dealkylation of 7EPO or 7PPO. Of the molecules tested, only 1EP, 1EN, 2EN, 2EPh and 3EPh were effective suicide inhibitors of the P450 1A1 dependent de-ethylation of 7EPO and only 1EN, 2EN, 1EA and 9EPh were effective suicide inhibitors of the P450 2B1 dependent de-pentylation of 7PPO. In addition to the size and shape of the polycyclic aromatic ring system, placement of the carbon--carbon triple bond on the ring system was critical for suicide inhibition. In contrast to 1EP, 2EP was not a mechanism-based inhibitor of P450 1A1; 9EPh, but not 2EPh or 3EPh, was a suicide inhibitor of P450 2B1. None of the aryl acetylenes tested produced heme destruction under assay conditions that produced the suicide inhibition of the P450 dependent 7EPO or 7PPO dealkylation activities. Because a precise orientation of the terminal acetylene is required to produce suicide inhibition without heme destruction, acetylenic suicide inhibitors can potentially be used to differentiate between P450 isozymes and to establish some distinguishing geometric features of the active site of these isozymes.     

On the substrate specificity of cytochrome P450IIIA1

The instability of the solubilized/purified form, the lack of catalytic activity of the stabilized, macrolide-complexed form, and the compromised catalytic activity of the decomplexed form of steroid-inducible cytochrome P450IIIA1 motivated further investigations of the substrate specificity of this isozyme. A major complementary goal was to identify reactions utilizable as sensitive, specific diagnostic probes for the detection and partial characterization of this isozyme in tissues for which isolation and purification are not practical (e.g., extrahepatic, embryonic tissues, etc.). The approach utilized a combination of a specific, purified inducer, specific inhibitors including triacetyloleandomycin and inhibitory antibodies, and diagnostic probe substrates including the phenoxazone ethers, testosterone, warfarin, 2-acetylaminofluorene, estradiol-17 beta and benzo[a]pyrene. The results obtained indicated that steroid-inducible, rat hepatic P450IIIA1 would catalyze minimal or no O-dealkylation of methoxy-, ethoxy- or pentoxyphenoxazone but catalyzed rapid O-debenzylation of benzyloxyphenoxazone. Hydroxylation of testosterone was specific for the beta face of the molecule at the 2-, 6-, 15- and 16-positions with no detectable conversion to androstenedione and minimal hydroxylation on the alpha face. Both the R- and S-enantiomers of warfarin were attacked at positions 9 and 10, and these reactions appeared to be specific to isozymes of the IIIA family. Aromatic hydroxylation of estradiol-17 beta was efficiently catalyzed, particularly at the 2-position. Hydroxylations of 2-acetylaminofluorene at positions 5 and 7 were catalyzed at relatively rapid rates, but N-hydroxylation of the same substrate was not catalyzed effectively. Hydroxylation of benzo[a]pyrene occurred preferentially at carbon 3 with much lesser activity at carbon 9 and little or no detectable attack at positions 7 or 1. The results indicated that the 2 beta- and 15 beta-hydroxylation of testosterone and the 10-hydroxylation of warfarin would serve as the most useful probes thus far available for detection of the presence of functional P450IIIA1 isozymes in tissues for which isolation and purification are impractical. The results also indicated a very broad, yet selective substrate specificity for the steroid-inducible P450IIIA1.     

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

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

The mechanism of halothane binding to microsomal cytochrome P450

Summary The unusual difference spectrum obtained with halothane and reduced rat liver microsomal cytochrome P450 can be simulated by addition of trifluoro diazoethane to dithionite reduced microsomes. Chemical evidence and model reactions suggest that in both cases a trifluoromethyl carbene complex is formed with the reduced hemoprotein. The spectral dissociation constants of the two species are similar as are their competitive reactions with carbon monoxide. The partial destruction of the carbenoid-cytochrome P450 complex characterizes the ligand as a highly reactive species. It is assumed that under anaerobic conditions this complex is formed by a two electron reduction of halothane and that covalent binding to microsomal proteins proceeds by this carbenoid species. A possible relationship to the hepatotoxicity of polyhalogenated compounds and anesthetics is discussed.     

Characterization of the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 using different in vitro systems

An important step in the drug development process is identification of enzymes responsible for metabolism of drug candidates and determination of enzyme kinetic parameters. These data are used to increase understanding of the pharmacokinetics and possible metabolic-based drug interactions of drug candidates. The aim of the present study was to characterize the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 [1-(3-{2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2(R)-methylpiperazine], a potent and selective 5HT2c-receptor agonist. The enzyme kinetic parameters were determined for formation of three main metabolites of BVT.2938 using human liver microsomes and expressed cytochrome P450 (CYP) isoforms. The major metabolite was formed by hydroxylation of the pyridine ring (CL_int = 27 μl/mg min), and was catalysed by both CYP2D6*1 and CYP1A1, with K_m values corresponding to 1.4 and 2.7 μM, respectively. The results from enzyme kinetic studies were confirmed by incubation of BVT.2938 in the presence of the chemical inhibitor of CYP2D6*1, quinidine. Quinidine inhibited the formation of the major metabolite by approximately 90%. Additionally, studies with recombinant expressed CYP isoforms from rat indicated that formation of the major metabolite of BVT.2938 was catalysed by CYP2D2. This result was further confirmed by experiments with liver slices from different rat strains, where the formation of the metabolite correlated with phenotype of CYP2D2 isoform (Sprague–Dawley male, extensive; Dark Agouti male, intermediate; Dark Agouti female, poor metabolizer). The present study showed that the major metabolite of BVT.2938 is formed by hydroxylation of the pyridine ring and catalysed by CYP2D6*1. CYP1A1 is also involved in this reaction and its role in extra-hepatic metabolism of BVT.2938 might be significant.     

Characterization of mouse small intestinal cytochrome P450 expression.

The expression of biotransformation enzymes in mouse small intestine is poorly characterized, which limits the utility of transgenic or knockout mouse models for first-pass drug metabolism studies. In response, we have systematically examined the composition and inducibility of cytochrome P450 (P450) protein and mRNA in mouse small intestinal epithelial cells (enterocytes). RNA-PCR was conducted to confirm the expression and identity of CYP1A1, 1B1, 2B10, 2B19, 2B20, 2C29, 2C38, 2C40, 2E1, 3A11, 3A13, 3A16, 3A25, and 3A44 in the enterocytes of untreated mice, but CYP1A2, 2A4/5, 2A12, 2C37, 2C39, and 2F2 were not detected. The inducibility of CYP2B, 2C, and 3A subfamily forms was determined by real-time quantitative RNA-PCR. All five CYP3A forms were induced, in a range from 1.7- to 4.5-fold, by dexamethasone (DEX). Phenobarbital (PB) induced CYP2B9, CYP2B10, and CYP2B20 mRNAs and suppressed CYP2B19 mRNA levels. PB also induced CYP2C29 and CYP2C40, but not CYP2C38 mRNA. At the protein level, CYP1A1, CYP1B1, CYP2B, CYP2C, CYP2E1, and CYP3A were detected in enterocytes from untreated mice by immunoblot analysis. CYP1A1 was inducible by beta-naphthoflavone (BNF), CYP2B and CYP2C by PB, and CYP3A by DEX. CYP2B, 2C, and 3A proteins were all expressed at high levels proximally, and decreased distally. The inducibility of CYP1A1 followed a similar pattern. Intestinal P450 expression was compared between C57BL/6 (B6) and 129/sv (129) mice, strains commonly used in the preparation of transgenic and knockout mouse models. There was no significant strain difference in constitutive levels or induction patterns for CYP2B, 2C, and 3A protein. However, CYP1A1 was induced to a high level by BNF in B6 mice, but was not induced in the 129 mice.     

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.     

Tissue- and cell type-specific expression of cytochrome P450 1A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization.

We used in situ hybridization to examine organ- and cell type-specific constitutive and 3-methylcholanthrene (3MC)-inducible cytochrome P450 (CYP)1A1 and CYP1A2 mRNA expression in various tissues of the C57BL/6N mouse. In situ hybridization was carried out 10 hr after the mice had received intraperitoneal 3MC, or vehicle alone. We detected levels of 3MC-induced CYP1A1 mRNA in: liver (centrilobular, more so than periportal, regions); lung (Clara Type II cells much more than Type I epithelial cells); brain, especially endothelial cells lining the vascular surface of the choroid plexus; the digestive tract (duodenum > jejunum > ileum > colon > esophagus > stomach--in particular, the villous epithelium, plus cells surrounding glands in the lamina propria); renal corpuscles of the kidney; the ovary (medulla more so than cortex); and the endothelial cells of blood vessels throughout the animal. Constitutive CYP1A1 mRNA was not detectable by in situ hybridization in any of these tissues. In contrast, constitutive CYP1A2 mRNA was measurable in liver, and 3MC-inducible CYP1A2 mRNA was observed only in liver, lung, and duodenum (having cell-type locations similar to those of CYP1A1); the other above-mentioned tissues were negative for CYP1A2 mRNA. These data demonstrate the striking differences in tissue- and cell type-specific expression between the two members of the mouse Cypla subfamily. Because of the ubiquitous nature of 3MC-inducible CYP1A1 throughout the animal rather than just "portals of entry," these results support our hypothesis that CYP1A1, induced by particular endogenous signals in various tissues and cell types, might participate in one or more critical life processes--in addition to its well-established role of metabolism of polycyclic hydrocarbons, certain drugs, and other environmental pollutants.     

Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.

CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.     

Nefiracetam metabolism by human liver microsomes: role of cytochrome P450 3A4 and cytochrome P450 1A2 in 5-hydroxynefiracetam formation.

An in-vitro study was conducted to investigate the metabolism of nefiracetam in human liver microsomes and to identify the enzymes responsible for the metabolism. Nefiracetam was hydroxylated by human liver microsomes to 5-hydroxynefiracetam (5-OHN). Eadie-Hofstee plots for the formation of 5-OHN suggested substrate activation. The kinetic parameters, apparent Km, Vmax, and Hill coefficient, for the formation of 5-OHN by pooled human liver microsomes were 4012 microM, 2.66 nmol min(-1) (mg protein)(-1), and 1.65, respectively. The formation of 5-OHN was significantly correlated with cytochrome P450 (CYP)3A4-mediated testosterone 6beta-hydroxylase activity and dextromethorphan N-demethylase activity. The 5-OHN formation was inhibited (94%) by antibody to human CYP3A4/5. The 5-OHN formation was also inhibited by the CYP3A4 inhibitors ketoconazole and troleandomycin, but not significantly inhibited by several other P450 inhibitors. The microsomes containing cDNA-expressed CYP3A4 formed 5-OHN with sigmoidal kinetics. CYP3A5-containing microsomes did not form 5-OHN. These results indicated that CYP3A, most likely CYP3A4, was the major isozyme responsible for the formation of 5-OHN in human liver microsomes. CYP1A2 and CYP2C19 microsomes were also capable of forming 5-OHN. However, the contribution of CYP1A2 was considered to be relatively minor compared with that of CYP3A4, and the contribution of CYP2C19 was assumed to be negligible, based on the result of the immunoinhibition study and taking into account both the turnover rate by each isozyme and the relative abundance of each isozyme in human liver. We conclude that on average the formation of 5-OHN, the major metabolite of nefiracetam, is principally mediated by CYP3A4 with a relatively minor contribution by CYP1A2.     

Measurement of Michaelis constants for cytochrome P450-mediated biotransformation reactions using a substrate depletion approach.

The Michaelis constant (KM) for cytochrome P450-mediated drug biotransformation reactions can be an important parameter in understanding the potential for a drug to exhibit saturable metabolism in vivo and nonlinear dose-exposure relationships. KM values were measured for several drug biotransformation reactions using recombinant heterologously expressed human enzymes. These determinations were made using an approach of monitoring substrate loss ("in vitro t1/2" method) at multiple substrate concentrations, with the objective of comparing KM values determined by this approach with KM values determined using the conventional approach of measuring product formation rates at several substrate concentrations. The reactions examined were CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation and thioridazine S-oxidation, CYP2C19-catalyzed imipramine N-demethylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, and CYP1A2-catalyzed tacrine 1-hydroxylation. KM values spanned an 80-fold range from 0.12 microM (CYP2D6-catalyzed thioridazine S-oxidation) to 9.8 microM (CYP2C19-catalyzed imipramine N-demethylation). On average, KM values determined by the substrate depletion approach were within 1.54-fold of those determined by measuring product formation. Thus, KM values can be determined for drug metabolism reactions without requiring knowledge of metabolite structures or requiring authentic standards of metabolites for use in construction of standard curves for quantitative bioanalysis. The in vitro t1/2 approach of determining KM values should be useful in early drug discovery efforts to identify those compounds with low KM values and, hence, a greater probability of exhibiting supraproportional dose-exposure relationships.     

Comparison of kinetic parameters for drug oxidation rates and substrate inhibition potential mediated by cytochrome P450 3A4 and 3A5

Cytochrome P450 (P450 or CYP) 3A is one of the most important P450 subfamilies in terms of its broad substrate specificity and relatively high abundance in humans. The substrate specificities of CYP3A4 and CYP3A5 are generally overlapped, but sometimes could differ from each other. It is still important to understand drug interactions more precisely in individual subjects. However, there are few review articles regarding comparative drug oxidation rates catalyzed by CYP3A4 and CYP3A5 and/or substrate inhibition potential towards CYP3A4 and CYP3A5. In this article, we summarize 1) Michaelis-Menten constants (Km), maximal velocities (Vmax), and intrinsic clearance (Vmax/Km) values for 63 substrates (94 reactions) mediated by CYP3A4 and/or CYP3A5, 2) inhibition constants (Ki) and 50% inhibitory concentrations (IC50) of 18 substrates, and 3) maximum inactivation rate constants (kinact) of 14 inhibitors from the literature. The relative contribution of polymorphic CYP3A5 compared with inducible CYP3A4 varies with the substrates and the reaction positions of the substrates. Inhibitory effects of azole antifungal agents and macrolide antibiotics, with low Ki and/or IC50 values for CYP3A4, are likely to be determinant factors for predominant drug interactions in humans, although Asian subjects with relatively high frequency of genetic CYP3A5 expressers should be carefully treated with CYP3A substrates. The collective findings in our present survey provide fundamental and useful information for drug oxidations catalyzed by CYP3A4 and CYP3A5, in spite of some contradictive kinetic parameters for the same reactions reported from many laboratories in different conditions. To understand causal factor(s) and mechanism(s) for such different reports summarized here is still one of the hot research topics to be solved in current drug metabolism.     

Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism.

The primary metabolite of nicotine in smokers is cotinine. Cotinine is further metabolized to trans-3'-hydroxycotinine, the major urinary metabolite of nicotine in tobacco users. It was recently reported that cytochrome P450 2A6 catalyzes the conversion of cotinine to trans-3'-hydroxycotinine. In this work, we report that P450 2A6 metabolizes cotinine not only to trans-3'-hydroxycotinine but also to 5'-hydroxycotinine, norcotinine, and a fourth as yet unidentified metabolite. The products of baculovirus-expressed P450 2A6 [methyl-(3)H]cotinine metabolism were analyzed by radioflow HPLC. Three (3)H-labeled metabolites were detected and were present in approximately equal amounts. The identities of two of the metabolites were confirmed to be 5'-hydroxycotinine and trans-3'-hydroxycotinine by LC/MS/MS and LC/MS analysis and comparison to standards. The third product was not identified. A fourth product of P450 2A6-catalyzed cotinine metabolism was detected by LC/MS. It was identified by cochromatography with a standard and MS and MS/MS data to be norcotinine. An attempt was made to further characterize the unidentified (3)H-labeled metabolite by comparison to the cotinine metabolites generated by hamster liver microsomes. Hamster liver microsomes contain a P450, 2A8, which is closely related to P450 2A6, and have previously been shown to metabolize cotinine to three hydroxylated products, trans-3'-hydroxycotinine, 5'-hydroxycotinine, and N-(hydroxymethyl)norcotinine. We were unable to confirm that N-(hydroxymethyl)norcotinine was the unidentified cotinine metabolite generated by P450 2A6.     

Involvement of cytochrome P450 1A in sanguinarine detoxication

Sanguinarine (SA), a member of the benzo[c]phenanthridine alkaloids, is a potent anti-microbial agent with anti-inflammatory and anti-neoplastic properties. However, toxicity of the alkaloid severely limits its medical applications. Recent report by Williams et al. implicated rat hepatic cytochrome P450 (CYP) 1A2 as a likely modulator of SA toxicity. Indeed, the in vitro toxicity of SA in primary culture of rat hepatocytes and human hepatic cell line HepG2, demonstrated as lactate dehydrogenase leakage and metabolic capability (MTT assay), was diminished following induction of CYP1A by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphtoflavone. Using microsomes containing recombinant CYP1A1 or CYP1A2 we show that SA causes non-competitive inhibition of the former and competitive inhibition of the latter as assessed by ethoxyresorufin de-ethylation (EROD). In human hepatic microsomes SA exhibits competitive inhibition of EROD activity with apparent K(i) of 2 microM, a value identical to that observed for CYP1A2 inhibition in recombinant system. Pre-incubation of SA with human liver microsomes resulted in time-dependent, but not dose-dependent decline in EROD activity suggesting CYP1A2 inhibition is not mechanism based. SA also inhibits activity of NADPH:CYP reductase, an enzyme required for CYP activity, with IC(50) very similar to that observed for EROD inhibition. Tentative mechanism for CYP1A involvement in decreased in vitro SA toxicity is discussed.     

Characterization of transgenic mouse strains using six human hepatic cytochrome P450 probe substrates

1. Transgenic mice were evaluated with six human cytochrome P450 (CYP) selective probe substrates, as little is known about their metabolism in the mouse. Mouse strains characterized include C57BL/SJL, FVB/N, mdr 1a/1b (-/-), ob/ob and ACCA. 2. Human CYP probe substrates used for characterization of mouse CYP activities included bufuralol, testosterone, dextromethorphan, phenacetin, diclofenac and S-mephenytoin. Activities were compared with those obtained in human liver microsomes and in human recombinant enzyme preparations. All transgenic mouse strains showed similar apparent K_m with bufuralol, testosterone and dextromethorphan which compared favourably with those observed in human liver microsomes. 3. K_m for phenacetin O-deethylase and S-mephenytoin 4'-hydroxylation were more variable across strains and in some cases demonstrated biphasic kinetics. Phenacetin O-deethylase activity was low in all mouse strains except FVB/N and mdr 1a/1b (-/-). Diclofenac 4-hydroxylation did not occur to any significant extent in the five strains of mouse evaluated here. 4. The findings suggest the validity of using five of the probes for transgenic mouse hepatic CYP characterization and gross comparison with data generated with human CYP.     

Characterization of transgenic mouse strains using six human hepatic cytochrome P450 probe substrates

1. Transgenic mice were evaluated with six human cytochrome P450 (CYP) selective probe substrates, as little is known about their metabolism in the mouse. Mouse strains characterized include C57BL/SJL, FVB/N, mdr 1a/1b (-/-), ob/ob and ACCA. 2. Human CYP probe substrates used for characterization of mouse CYP activities included bufuralol, testosterone, dextromethorphan, phenacetin, diclofenac and S-mephenytoin. Activities were compared with those obtained in human liver microsomes and in human recombinant enzyme preparations. All transgenic mouse strains showed similar apparent K(m) with bufuralol, testosterone and dextromethorphan which compared favourably with those observed in human liver microsomes. 3. K(m) for phenacetin O-deethylase and S-mephenytoin 4'-hydroxylation were more variable across strains and in some cases demonstrated biphasic kinetics. Phenacetin O-deethylase activity was low in all mouse strains except FVB/N and mdr 1a/1b (-/-). Diclofenac 4-hydroxylation did not occur to any significant extent in the five strains of mouse evaluated here. 4. The findings suggest the validity of using five of the probes for transgenic mouse hepatic CYP characterization and gross comparison with data generated with human CYP.     

Heteroactivation of cytochrome P450 1A1 by teas and tea polyphenols

We studied 7-ethoxyresorufin deethylase as an index of cytochrome P4501A1 (CYP1A1) activity in liver microsomes from rats pretreated with 3-methylcholanthrene. The enzyme had complex kinetics compatible with a multisite model. At 1 microM substrate, brewed black, green and white teas had complex effects on enzyme activity consisting of activation at low concentrations and inhibition at higher concentrations. Data fit well to a two-site model that allowed us to determine maximal activation (% increase above control), pEC(50) for activation (g ml(-1)) and pIC(50) for inhibition (g ml(-1)). These parameters were 190+/-40, 5.9+/-0.1 and 4.51+/-0.09 for green tea, 350+/-40, 5.43+/-0.05 and 5.43+/-0.05 for black tea and 230+/-80, 5.3+/-0.3 and 4.7+/-0.2 for white tea, respectively. The effects of the brewed teas were mimicked to different degrees by the green tea polyphenols. Maximal activation, pEC(50) (M) and pIC(50) (M) were: (-)-epicatechin, 55+/-9, 5.4+/-0.3, 2+/-1; (-)-epicatechin gallate, 160+/-60, 6.2+/-0.3, 5.28+/-0.06; (-)-epigallocatechin 30+/-10, 6.5+/-0.5, 3.37+/-0.08; and (-)-epigallocatechin gallate 130+/-40, 6.7+/-0.3, 5.0+/-0.1. A crude extract of black tea polyphenols inhibited 7-ethoxyresorufin deethylase, but did not cause enzyme activation consistently. Enzyme activation was dependent upon substrate concentration. Heteroactivation of CYP1A1 may partially explain the lack of agreement between biological and epidemiological evidence of a role for tea in cancer prevention.