P450s 3A4 Research Articles

Rate-limiting steps in cytochrome P450 catalysis.

Cytochrome P450 (P450) reactions are of interest because of their relevance to the oxidative metabolism of drugs, steroids, carcinogens, and other chemicals. One of the considerations about functional characterization is which steps of the catalytic cycle are rate-limiting. Detailed analysis indicates that several different steps can be rate-limiting with individual P450 reactions. N-Dealkylation of para-substituted N,N-dimethylanilines is a function of the electron withdrawing/donating properties of the substituent and the oxidation-reduction potential of the substrate, supporting a role in rate-limiting electron transfer from substrate to the high valent P450. In the oxidations of ethanol and acetaldehyde by human P450 2E1, a step following product formation must be the slow step (but not product release per se). Several oxidations catalyzed by human P450s 1A2 and 2D6 show slow C-H bond breaking, and apparent high-valent iron complexes accumulate in the reaction steady-state. Kinetic simulations were used to test the suitability of potential schemes and to probe the effects of changes in individual reaction steps.

Design, synthesis, and discovery of novel trans-stilbene analogues as potent and selective human cytochrome P450 1B1 inhibitors.

A series of trans-stilbene derivatives containing a 3,5-dimethoxyphenyl moiety were prepared through a new efficient solution phase synthetic pathway, and their inhibitory activities were evaluated on human cytochrome P450s (CYP) 1A1, 1A2, and 1B1 to find a potent and selective CYP1B1 inhibitor. We found that a substituent at the 2-position of the stilbene skeleton plays a very important role in discriminating between CYP1As and CYP1B1. Among the compounds tested, the most selective and potent CYP1B1 inhibitor was 2,3',4,5'-tetramethoxystilbene. Compound 7j, 2-[2-(3,5-dimethoxy-phenyl)vinyl]thiophene, showed greater inhibitory activities but had a lower selectivity toward all of the CYP1s tested.

Differential N-Demethylation of l-α-Acetylmethadol (LAAM) and norLAAM by Cytochrome P450s 2B6, 2C18, and 3A4

Incubation of l-α-acetylmethadol (LAAM) or norLAAM with cDNA-expressed P450s 3A4, 2B6, and 2C18 produced significant N-demethylation products. P450s 2C19, 2C8, 3A5, 2C9, 3A7, 1A1, and 2D6 (norLAAM only), also produced detectable product. Coexpression of cytochrome b5 enhanced LAAM N-demethylation, most dramatically for 3A4, but had marginal effects on norLAAM N-demethylation. Modeling total liver metabolism using immunoquantification and relative activity factors of P450s suggests contributions of P450 3A4 > 2B6 > 2C18, with the importance of 2B6 to 2C isozymes enhanced by relative activity factors. The ratio of dinorLAAM to norLAAM plus dinorLAAM formed from LAAM did not exceed 20%, and was isozyme and cytochrome b5 coexpression dependent. This ratio decreased with concentration with 3A4, but was relatively constant for 2B6 and 2C18. The human liver microsomes substrate-concentration response was similar to cDNA-expressed 3A4, but the ratio was higher. Changes in the environment of cDNA-expressed 3A4 also effected the magnitude of the ratio, but not the concentration-dependent decrease. These studies show that the N-demethylation of LAAM and norLAAM is not restricted to P450 3A4, particularly P450s 2B6 and 2C18, and suggest that the mechanism of sequential metabolism for 3A4 differs from that of 2B6 and 2C18.

Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells.

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.

Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites.

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5, 6-epoxy-RA formation (30-min incubation, 10 microM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V(max)/K(m) of 127.7, followed by 3A5 (V(max)/K(m) = 25.6), 2C8 (V(max)/K(m) = 24.5), 2C18 (V(max)/K(m) = 15.8), 3A4 (V(max)/K(m) = 5.7), 1A1 (V(max)/K(m) = 5.0), and 4A11 (V(max)/K(m) = 1.9); for 4-oxo-RA, 3A7 showed a V(max)/K(m) of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V(max)/K(m) = 1.2); and for 18-OH-RA, 3A7 showed a V(max)/K(m) of 10.5 compared with a V(max)/K(m) of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 microM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.

Oxidation of indole by cytochrome P450 enzymes.

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.

Cytochrome P450 Isoform Expression in Human Vascular Endothelial Cells

P29 Epoxy derivatives of arachidonic acid may act as important autocrine and paracrine mediators of endothelial function including regulation of vascular tone and control of inflammation. To identify potential candidates for catalyzing the synthesis of these and further arachidonic acid metabolites, we studied human vascular endothelial cells for the expression of individual cytochrome P450 isoforms belonging to the CYP families 1, 2, 3 and 4. An RT-PCR screening performed with subfamily- and isoform-specific primer pairs revealed mRNAs for the P450 forms 1A1, 1B1, 2C8, 2E1, 2J2, 3A7, 4A11 and 4F2. The identity of the RT-PCR products was confirmed by DNA sequencing. In addition, P450 1A2 mRNA was detected after induction with β-naphthoflavone which also enhanced the expresion of P450s 1A1 and 1B1. P450s 2B6 and 3A4 were not detectable. Similar P450 isoform patterns were obtained analyzing primary human endothelial cells originating from aorta, coronary arteries, dermal microvessels and umbilical veins, as well as an immortalized human endothelial cell line (HMEC-1). Further studies with HMEC-1 cells showed the expression of all human members of the P450 2C subfamily (2C8, 2C9, 2C18 and 2C19). We next used gaschromatography-mass spectrometry to identify the regioisomeric epoxeicosatrienoic acids produced by HMEC-1 cells. Among the P450 forms detected by the RT-PCR screening, P450 2C8 and 2J2 are the leading candidates for producing vasoactive epoxyeicosatrienoic acids. Using recombinant human P450 1A1, we then found that this P450 form catalyzes the formation of various regioisomeric hydroxy derivatives of arachidonic acid. We conclude that P450 1A1 known primarily for its role in polycyclic aromatic hydrocarbon metabolism, may interfere with endothelial arachidonic acid metabolism, particularly after its induction by drugs and xenobiotics. Furthermore, P450s 4A11 and 4F2 probably contribute to the degradation of lipid mediators of inflammation.

Poor metabolizers at the cytochrome P450 2D6 and 2C19 loci are at increased risk of developing adult acute leukaemia.

We have genotyped over 550 cases of acute leukaemia and 950 matched controls from a population-based case-control study, to investigate the impact cytochrome P450s 2D6, 2C19 and 1A1 have on susceptibility to adult acute leukaemia. Analysis included potential associations between polymorphic status and acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), plus the FAB and cytogenetic subtypes therein. A significant increased risk was found for CYP2D6 poor metabolizer phenotype and acute leukaemia [odds ratio (OR) = 1.69, 95% confidence interval (CI) 1.17-2.43], a risk also found in AML and ALL. No interaction was found with smoking. However, a significant age-related association between CYP2D6 polymorphism and acute myeloid leukaemia implied that the excess risk was confined to persons aged 40 years and over (OR 2.38, 95% CI 1.53-3.71). Amongst AML cases, increased odds ratios were observed in both de-novo (OR 1.54, 95% CI 1.02-2.32) and secondary leukaemia (OR 2.83, 95% CI 0.91-8.77), and among patients with a chromosomal abnormality (OR 2.00, 95% CI 1.11-3.61). An increased risk was found for the CYP2C19 poor metabolizer phenotype (OR 1.68, 95% CI 0.97-2.92) which was also present in AML and ALL. For this CYP450 locus, an increased risk was suggested in secondary leukaemia (OR 2.67, 95% CI 0.44-16.3) and amongst AML cases with a chromosomal abnormality (OR 6.72, 95% CI 2.22-20.4). No difference in CYP1A1 genotype distribution was found for acute leukaemia, AML, ALL or any other diagnostic classification group used. No significant interactions between CYP2D6, CYP2C19 or CYP1A1 were found.

Xenobiotic-metabolizing Cytochromes P450 Convert Prostaglandin Endoperoxide to Hydroxyheptadecatrienoic Acid and the Mutagen, Malondialdehyde

Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC(50) values of 1 and 0.4 microM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 microM substrate, the catalytic activities of purified human P450s varied from 10 +/- 1 to 0.62 +/- 0.02 min(-1), 3A4 >> 2E1 > 1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with K(s) values of 240 +/- 20 and 130 +/- 10 microM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.

Inhibition of human cytochrome P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its derivatives, and sulforaphane.

Recent studies have demonstrated that two chemoprotective agents, oltipraz (OPZ), a synthetic derivative of the natural compound 1, 2-dithiole-3-thione (D3T), and sulforaphane (SF), an isothiocyanate, are not only inducers of glutathione S-transferases but also inhibitors of some major cytochrome P450 enzymes (P450s) involved in xenobiotic metabolism. We examined P450 inhibition by the two compounds and compared two OPZ metabolites (OPZ M(3) and M(8)) and D3T using human P450s expressed in Escherichia coli membranes. OPZ was a more potent inhibitor than D3T or SF, in the following order of inhibition: P450 1A2 > 3A4 > 1A1 approximately 1B1 > 2E1. OPZ M(3) also inhibited P450s 1A2, 1A1, 1B1, and 3A4 but not more effectively than OPZ. OPZ M(8) was not inhibitory. OPZ behaved as a competitive inhibitor of P450 1A2, with a K(i) of 1.5 microM. Incubation of P450 1A2 with OPZ and NADPH led to a first-order loss of the P450 spectrum, and the loss was not blocked by glutathione. No such time-dependent loss of P450 was seen with P450 1A2 and D3T, P450 1A2 and OPZ M(3), P450 1A2 and SF, P450 3A4 and OPZ, P450 3A4 and D3T, P450 2E1 and OPZ, or P450 2E1 and D3T. The time- and concentration-dependent loss of P450 1A2 activity in the presence of OPZ was characterized with a K(i) of 9 microM and a k(inactivation) of 0.19 min(-)(1). The activation of 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline (MeIQ) in an E. coli lac-based mutagenicity tester system containing functional human P450 1A2 was inhibited by OPZ (IC(50) < 1 microM) but not appreciably by 40 microM D3T. Our results indicate that OPZ is a competitive and mechanism-based inhibitor of P450 1A2, and the extent of this inhibition is significantly greater than that of other chemoprotective chemicals with P450 1A2 or other human P450s.

A quantitative comparison of dibenzo[a,l]pyrene‐DNA adduct formation by recombinant human cytochrome P450 microsomes

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11,12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using 32P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74–82, 1999. Published 1999 Wiley-Liss, Inc.

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11, 12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using (32)P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a, l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74-82, 1999. Published 1999 Wiley-Liss, Inc.

Mechanism-based inactivation of cytochrome P450s 1A2 and 3A4 by dihydralazine in human liver microsomes.

Dihydralazine is known to induce immunoallergic hepatitis. Since anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that dihydralazine is biotransformed into a reactive metabolite, which covalently binds to cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing P450 enzymes were preincubated with dihydralazine in the presence of NADPH, followed by an assay of several monooxygenase activities. Preincubation of human liver microsomes with dihydralazine in the presence of NADPH resulted in decreases in phenacetin O-deethylase activity (an indicator of P450 1A2 activity) and testosterone 6beta-hydroxylase activity (P450 3A4), but not in diclofenac 4'-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-first-order kinetics and was saturable with increasing dihydralazine concentrations. Similar time-dependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the enzyme themselves, suggesting that dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM antibodies in dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the autoantibodies.

Inhibition of Drug-Metabolizing Enzymes in Mouse Liver by a Water Soluble Fullerene C60

The microsomal cytochrome P450-dependent monooxygenase system is responsible for the metabolism a variety of xenobiotics including drugs and carcinogens. Fullerenol-1 is a water soluble polyhydroxylated C60 derivative synthesized by a sequence of reactions involving the electrophilic attack of nitronium tetrafluoroborate on fullerenes in the presence of organocarboxylic acids. The present study has evaluated the acute toxicity of fullerenol-1 and determined the effects of the C60 compound on the P450-dependent drug-metabolzing activities. Pretreatments with 0.5 and 1.0g/kg fullerenol-1 decreased P450 and cytochrome b5 contents, and NADPH-cytochrome P450 reductase, as well as drug metabolizing activities toward test substrates benzo[a]pyrene, 7-ethoxycoumarin, aniline, and erythromycin in liver microsomes. Pretreatments with 0.01 and 0.1 g/kg fullerenol-1 had no effect on these monooxygenases. Immunoblotting anlysis of the microsomal proteins revealed that pretreatments with fullerenol-1 did not alter the protein levels of P450s 1A, 2E and 3A. Additions of fullerenol-1 to mouse liver microsomes suppressed monooxygenases activities toward benzo[a]pyrene, 7-ethoxycoumarin, aniline, and erythromycin with IC50 values of 42, 94, 102 and 349 μM, respectively. These results demonstrate that fullerenol-1 can decrease the activities of microsomal drug-metabolizing enzymes in vivo and in vitro. Proper adjustment of the functionalized fullerene compound may facilitate the biomedical application of the new material.

Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening.

In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M. J. De Groot et al. [(1997) Chem. Res. Toxicol. 10, 41-48]. The substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes. Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 microM with only P450 1A2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 microM. K(m) and v(max) values of MAMC for P450 2D6 were found to be 26.2 +/- 2.8 microM and 2.9 +/- 0.07 min(-)(1), respectively. For P450 1A2, MAMC was found to have a K(m) value of 29.7 +/- 6.2 microM and a v(max) of 0.57 +/- 0.07 min(-)(1). Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 microM selective for P450 2D6, and furafylline, at a concentration of 30 microM selective for P450 1A2. In conclusion, O-demethylation of 7-methoxy-4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes.

Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase.

Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.

Metabolism of genistein by rat and human cytochrome P450s.

The metabolism of genistein (4',5,7-trihydroxyisoflavone), a phytoestrogen derived from soy products, was investigated using rat and human liver microsomes and recombinant human cytochrome P450 enzymes. Metabolism of genistein by microsomes obtained from rats treated with pyridine, phenobarbital, beta-naphthoflavone, isosafrole, pregnenolone-16alpha-carbonitrile, or 3-methylcholanthrene resulted in very different product profiles consisting of five different NADPH- and time-dependent metabolites as observed by HPLC reverse-phase analysis at 260 nm. The metabolism of genistein was also investigated with recombinant human cytochrome P450 1A1, 1A2, 1B1, 2B6, 2C8, 2E1, or 3A4. P450s 1A1, 1A2, 1B1, and 2E1 metabolized genistein to form predominantly one product (peak 3) with smaller amounts of peaks 1 and 2. P450 3A4 produced two different products (peaks 4 and 5). Product peaks 1-3 eluted off the HPLC column prior to the parent compound genistein, and the UV/vis spectra, GC/MS, and ESI/MS/MS analyses support the conclusion that these products result from hydroxylation of genistein. The product peak 3 has been identified by tandem mass spectrometry as 3',4',5, 7-tetrahydroxyisoflavone, also known as orobol, and peaks 1 and 2 appear to be hydroxylated at position 6 or 8.

The role of metabolism in determining susceptibility to parathion toxicity in man

Human liver microsomes ( n=16) activated parathion ( O, O, diethyl O- p-nitrophenyl phosphorothioate, 20 and 200 μM) to paraoxon at a rate of 23.3–199.3 and 18.7–310.3 pmol/min per mg protein, respectively. p-Nitrophenol, was also formed, at 321.1–769.2 and 406.2–778.3 pmol/min per mg protein. This represented a 16-fold and 2-fold range in capacity to activate and detoxify parathion, respectively. Parathion was activated with an apparent K m of 9–16 μM ( n=3). The activation of parathion (200 μM) was positively correlated with nifedipine oxidation, indicating the involvement of CYP3A. Correlations were not significant with ethoxyresorufin- O-dealkylation (CYP1A1/2), pentoxyresorufin- O-dealkylation (CYP2B6), p-nitrophenol hydroxylation (CYP2E1), paraoxon hydrolysis (A-esterase) or phenylvalerate hydrolysis (B-esterase). Paraoxon formation from parathion was markedly reduced by CYP3A inhibitors. Experiments with EDTA indicated that A-esterase was not functionally important at low levels of paraoxon. Human P450s 3A4 and 3A5 expressed microsomes were the most efficient at biotransforming parathion to paraoxon, although P450s 1A1, 2B6 and 2C8 also catalysed the reaction. This study has determined wide interindividual variations in capacity to metabolise parathion, mainly by CYP3A, which may influence its manifest toxicity.

Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil.

The present study was conducted to evaluate metabolism of the enantiomers of verapamil and norverapamil using a broad range of cytochrome P450 isoforms and measure the kinetic parameters of these processes. Cytochrome P450 cDNA-expressed cells and microsomes from a P450-expressed lymphoblastoid cell line were incubated with 40 microm concentrations of R- or S-verapamil and R- or S-norverapamil and metabolite formation measured by h.p.l.c. as an initial screening. Those isoforms exhibiting substantial activity were then studied over a range of substrate concentrations (2.5-450 microm ) to estimate the kinetic parameters for metabolite formation. P450s 3A4, 3A5, 2C8 and to a minor extent 2E1 were involved in the metabolism of the enantiomers of verapamil. Estimated Km values for the production of D-617 and norverapamil by P450 s 3A4 and 3A5 were similar (range=60-127 microm ) regardless of the enantiomer of verapamil studied while the Vmax estimates were also similar (range=4-8 pmol min-1 pmol-1 P450). Only nominal production of D-620 by these isoforms was noted. Interestingly, P450 2C8 readily metabolized both S- and R-verapamil to D-617, norverapamil and PR-22 with only slightly higher Km values than noted for P450s 3A4 and 3A5. However, the Vmax estimates for P450 2C8 metabolism of S- and R-verapamil were in general greater (range=8-15 pmol min-1 pmol-1 P450) than those noted for P450 s 3A4 and 3A5 with preference noted for metabolism of the S-enantiomer. Similarly, P450 s 3A4, 3A5 and 2C8 also mediated the metabolism of the enantiomers of norverapamil with minor contributions by P450 s 2D6 and 2E1. P450s 3A4 and 3A5 readily formed the D-620 metabolite with generally a lower Km and higher Vmax for S-norverapamil than for the R-enantiomer. In contrast, P450 2C8 produced both the D-620 and PR-22 metabolites from the enantiomers of norverapamil, again with stereoselective preference seen for the S-enantiomer. These results confirm that P450s 3A4, 3A5 and 2C8 play a major role in verapamil metabolism and demonstrate that norverapamil can also be further metabolized by the P450s.

Cytochrome P450 3A Degradation in Isolated Rat Hepatocytes: 26S Proteasome Inhibitors as Probes

Mechanism-based inactivation of liver microsomal cytochromes P450 3A (CYP 3A, P450s 3A)in vivoand/orin vitro,via heme modification of the protein, results in accelerated proteolytic degradation of the enzyme that is preceded by the ubiquitination of the protein, thereby implicating the ubiquitin-ATP-dependent 26S proteasomal system. In this study, this involvement is confirmed with the use of the proteasomal inhibitors aclarubicin and MG-132 as probes, in isolated rat hepatocytes treated with the P450 3A mechanism-based inactivator, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP). In addition, the findings reveal that during the course of this proteolysis, the endoplasmic reticulum (ER)-anchored DDEP-inactivated P450 3A is translocated from the ER to the cytosol in a brefeldin A-insensitive manner.