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1. Nine forms of recombinant cytochrome P450 (P450 or CYP) enzymes were used to study roles of individual P450 enzymes in the oxidation of flavone and some other flavonoids, 4´-hydroxyflavone and 4´-, 3´-, and 2´-methoxyflavones, by human liver microsomes using LC-MS/MS analysis. 2. As has been reported previously (Nagayoshi et al., Xenobiotica 50, 1158-1169, 2020), 4´-, 3´-, and 2´-methoxyflavones were preferentially O-demethylated by human liver P450 enzymes to form 4´-, 3´-, and 2´-hydroxylated flavones and also 3´,4´-dihydroxyflavone from the former two substrates. 3. In comparisons of product formation by oxidation of these methoxylated flavones, CYP2A6 was found to be a major enzyme catalyzing flavone 4´- and 3´-hydroxylations by human liver microsomes but did not play significant roles in 2´-hydroxylation of flavone, O-demethylations of three methoxylated flavones, and the oxidation of 4´-hydroxyflavone to 3´,4´-dihydroxyflavone. 4. The effects of anti-CYP2A6 IgG and chemical P450 inhibitors suggested that different P450 enzymes, as well as CYP2A6, catalyzed oxidation of these flavonoids at different positions by liver microsomes. 5. These studies suggest that CYP2A6 catalyzes flavone 4´- and 3´-hydroxylations in human liver microsomes and that other P450 enzymes have different roles in oxidizing these flavonoids.

Background/Aim: We examined the mechanism of nitric oxide (NO) production in a tissue-factor (TF)-induced disseminated intravascular coagulation (DIC) model in rats, using inducible nitric oxide synthase (iNOS) inhibitor (L-NIL), endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME), Factor Xa inhibitor (DX-9065a), and thrombin inhibitor argatroban. Materials and Methods: Experimental DIC was induced by sustained infusion of 3.75 U/kg TF for 4 h via the tail vein. We then investigated the effect of these four agents on TF-induced DIC. Results: Administration of L-NIL or L-NAME during induction of TF-induced DIC did not affect hemostatic markers, whereas elevated plasma levels of NO metabolites (NOX) were significantly suppressed by co-administration of L-NAME. A significant increase in eNOS-mRNA expression was observed in the TF-induced DIC model. Argatroban almost completely suppressed eNOS-mRNA expression. Conclusion: eNOS plays an important role in the NO production in the TF-induced DIC, and thrombin is a key stimulant of eNOS-mRNA expression in this model.

2′-Hydroxyflavanone (2′OHFva), 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry.Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3′OHFva and 4′OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring.Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4′OHFva (m/z 241) was used as a substrate, formation of 4′OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4′OHFvo by P450s.Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes. 2′-Hydroxyflavanone (2′OHFva), 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3′OHFva and 4′OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4′OHFva (m/z 241) was used as a substrate, formation of 4′OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4′OHFvo by P450s. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

2′-, 3′-, and 4′-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products form...

1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2′- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min−1 and 1.3 min−1, respectively. Other flavanone metabolites were 4′-, 3′- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min−1 that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min−1). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.

A variety of xenobiotic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), aryl- and heterocyclic amines and tobacco related nitrosamines, are ubiquitous environmental carcinogens and are required to be activated to chemically reactive metabolites by xenobiotic-metabolizing enzymes, including cytochrome P450 (P450 or CYP), in order to initiate cell transformation. Of various human P450 enzymes determined to date, CYP1A1, 1A2, 1B1, 2A13, 2A6, 2E1, and 3A4 are reported to play critical roles in the bioactivation of these carcinogenic chemicals. In vivo studies have shown that disruption of Cyp1b1 and Cyp2a5 genes in mice resulted in suppression of tumor formation caused by 7,12-dimethylbenz[a]anthracene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, respectively. In addition, specific inhibitors for CYP1 and 2A enzymes are able to suppress tumor formation caused by several carcinogens in experimental animals in vivo, when these inhibitors are applied before or just after the administration of carcinogens. In this review, we describe recent progress, including our own studies done during past decade, on the nature of inhibitors of human CYP1 and CYP2A enzymes that have been shown to activate carcinogenic PAHs and tobacco-related nitrosamines, respectively, in humans. The inhibitors considered here include a variety of carcinogenic and/or non-carcinogenic PAHs and acethylenic PAHs, many flavonoid derivatives, derivatives of naphthalene, phenanthrene, biphenyl, and pyrene and chemopreventive organoselenium compounds, such as benzyl selenocyanate and benzyl selenocyanate; o-XSC, 1,2-, 1,3-, and 1,4-phenylenebis( methylene)selenocyanate.

1. 1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2. CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3. Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower kcat value than other CYP2A13.1 variants. 4. CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5. Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2 A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.

1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans.

2,5,2′,5′-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with K s values of 9.4 and 0.51 µ M, respectively. However, CYP2A6 oxidized 2,5,2′,5′-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute −1 ) than CYP2A13 (∼0.02 minute −1 ) based on analysis by liquid chromatography–tandem mass spectrometry. Formation of 4-hydroxy-2,5,2′,5′-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2′,5′-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2′,5′-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2′,5′-TCB at a higher rate (∼0.3 minute −1 ) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute −1 ) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute −1 ). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3′,4′-, 3,4,3′,4′-, and 3,5,3′,5′-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2′,5′-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies ( U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2′,5′-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences.

One of the mediators of osteoclast differentiation is receptor activator of nuclear factor κB ligand (RANKL), which is produced by osteoblasts. Binding of RANKL to its receptor, RANK, activates several signaling pathways, including those involving mitogen-activated protein kinases (MAPKs), nuclear factor κB (NF-κB), nuclear factor of activated T cells c1 (NFATc1) and Ca(2+)-calcineurin. In the present study, we found that tetrandrine, a bisbenzylisoquinoline alkaloid extracted from the root of Stephania tetrandra S. MOORE, significantly ameliorated the decrease of bone mass in sciatic-neurectomized osteoporosis model mice. It appears that tetrandrine acts directly on osteoclast precursors, since tetrandrine inhibited osteoclast differentiation not only in mouse bone marrow cells, but also in monocultures of murine macrophage RAW 264.7 cells without osteoblasts. Tetrandrine suppressed RANKL-induced amplification of NFATc1, a master regulator of osteoclast differentiation. However, it did not affect other signaling molecules such as MAPKs and NF-κB. These results suggest that tetrandrine is a candidate for the treatment of bone-destructive diseases, or at least a suitable lead compound for further development.

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.

金沢大学医学部附属病院薬剤部, This study was designed to investigate the induction of CYP2E1 in obese Zucker rats and its effect on the disposition kinetics of chlorzoxazone (CZX). CZX 20 mg/kg was administered to three groups of rats: normal Zucker rats fed a normal diet (ND), normal Zucker rats fed a high-fat diet (HF), and genetically obese Zucker rats fed a normal diet (OB). The values of the area under the plasma concentration–time curve from 0 to 1 (AUC1) of CZX were in the order of ND > HF > OB rats. The AUC1 values of total 6-hydroxychlorzoxazone (6OHCZX-T), which is considered to be a CYP2E1 metabolic marker, were in the opposite order. The values of the AUC1 ratio (6OHCZX–T/CZX) in ND, HF and OB rats were approximately 0.2, 0.3 and 0.4, respectively. The CZX concentration in fat was much higher than the concentrations in plasma, liver and kidney in all groups. Induction of CYP2E1 protein was greater in both liver and fat of OB rats than in those of HF rats. Microsomal activity of CYP2E1 in liver and fat was also in the order of OB > HF > NMrats. These results suggest that CYP2E1 may be induced in liver and fat of obese patients, thereby potentially altering the disposition kinetics of not only CZX, but also other lipophilic drugs metabolized by CYP2E1.©2006 Published by Elsevier Inc.

Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3162–3169, 2015

Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k(cat), 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (approximately 0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process.

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

We examined the influence of basic drugs and protein variants on the binding disposition of ropivacaine to α1-acid glycoprotein (AGP). On doing this, we found the values of the competitive inhibition constant (Ki) for dipyridamole, verapamil, lidocaine and disopyramide with respect to the binding of ropivacaine to commercial AGP (70 mg/dL) to be 2.1, 5.2, 6.0 and 11.0μM, respectively. Also, there was a strong correlation between the fu value and the AGP concentration when ropivacaine was added to plasma samples from ten healthy volunteers (r=0.861). Among the volunteers, eight showed F1S variants and two showed F1 variants without the S variant of AGP. There was no difference in the fu value of ropivacaine between these two groups. However, when ropivacaine was added together with dipyridamole, the fu values of ropivacaine in plasma from volunteers with ES variants were clearly higher than those from volunteers without the S variant. When ropivacaine was added together with disopyramide or lidocaine, however, there was no difference in fu values between these variants.Our results indicate that variability in the effectiveness and/or adverse effects of ropivacaine are caused by changes in fu as a consequence of changes in AGP concentration. They also suggest that in combination therapy, it is also important to consider the AGP variant-dependence of the inhibitory effect of concomitantly administered drugs.

1. To determine the biological effects of 23 polycyclic aromatic hydrocarbons (PAHs) and 3,4,3',4'-tetrachlorobiphenyl, the dose-response studies of the induction of CYP1-dependent xenobiotic oxidation activities by these chemicals in liver microsomes of C57BL/6J mice were studied. 2. In arylhydrocarbon-responsive C57BL/6J mice, the liver microsomal xenobiotic oxidation with substrates of 7-ethoxyresorufin, 7-ethoxycoumarin, (+/-)-benzo[a]pyrene-7,8-diol, dibenzo[a, pyrene-11,12-diol and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline increased by increasing the doses of PAHs to mice, particularly when the PAHs that have been reported to be carcinogenic in experimental animals were used. In arylhydrocarbon receptor-knockout mice, there were no increases in liver microsomal 7-ethoxyresorufin O-deethylation activities nor in liver mRNA levels of CYP1A1, 1A2 and 1B1 by these chemicals. 3. Of the chemicals examined, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[j]-fluoranthene, 3-methylcholanthrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene and 3,4,3',4'-tetrachlorobiphenyl were potent inducers of the induction of liver microsomal 7-ethoxyresorufin O-deethylation in mice. 4. Other PAHs such as 5-methylchrysene, benzo[a]pyrene, dibenzo[a,l]pyrene, dibenz[a,j]acridine, benzo[a]anthracene and 7,12-dimethylbenz[a]anthracene moderately induced 7-ethoxyresorufin O-deethylation activities in mice. PAHs reported to be weak or less carcinogenic in experimental animals did not induce the xenobiotic oxidation activities of CYP1A1 and 1B1 in the mice. 5. The results suggest that induction of liver microsomal CYP1-dependent xenobiotic oxidation activities is a good tool in determining the potencies of carcinogenic PAHs in arylhydrocarbon-responsive C57BL/6J mice.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.

Cytochrome P450 (P450) 1B1 is expressed in a number of human tissues in which cancers occur (e.g. prostate, ovary, uterus, mammary gland). P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis. We have examined the activities of several of the major allelic variants of human P450 1B1 in these reactions. Another interest has been the development of chemical inhibitors of P450 1B1. 2-Ethynylpyrene and alpha-naphthoflavone preferentially inhibit human P450 1B1 compared to P450 1A1, which may be present in the same tissue sites. The natural product resveratrol is also an inhibitor of P450 1B1. Further studies with rhapontigenin and synthetic stilbenes led to the discovery of 2,4,3',5'-tetramethoxystilbene, a selective inhibitor of P450 1B1 relative to other P450s. Inhibition is competitive, with a K(i) value of 3nM, and the inhibitor is resistant to metabolism. In addition to blocking 17beta-estradiol 4-hydroxylation, this stilbene also inhibited the activation of heterocyclic amines to mutagens. 2,4,3',5'-Tetramethoxystilbene also suppressed expression of P450 1B1 and growth of human mammary tumor cells. 3,3',4',5,5'-Pentamethoxystilbene was a selective inhibitor of P450 1A1, showing mixed inhibition, and also suppressed P450 1A1 expression in HepG2 cells. Substituted stilbenes may be useful in preventing cancer caused by estrogens and xenobiotics.

Limonene, a monocyclic monoterpene, is present in orange peel and other plants and has been shown to have chemopreventive activities. (+)- and (-)-Limonene enantiomers were incubated with human liver microsomes and the oxidative metabolites thus formed were analyzed using gas chromatography-mass spectrometry. Two kinds of metabolites, (+)- and (-)-trans-carveol (a product by 6-hydroxylation) and (+)- and (-)-perillyl alcohol (a product by 7-hydroxylation), were identified, and the latter metabolites were found to be formed more extensively, the former ones with liver microsomes prepared from different human samples. Sulfaphenazole, flavoxamine, and antibodies raised against purified liver cytochrome P450 (P450) 2C9 that inhibit both CYP2C9- and 2C19-dependent activities, significantly inhibited microsomal oxidations of (+)- and (-)-limonene enantiomers. The limonene oxidation activities correlated well with contents of CYP2C9 and activities of tolbutamide methyl hydroxylation in liver microsomes of 62 human samples, whereas these activities did not correlate with contents of CYP2C19 and activities of S-mephenytoin 4-hydroxylation. Of 11 recombinant human P450 enzymes (expressed in Trichoplusia ni cells) tested, CYP2C8, 2C9, 2C18, 2C19, and CYP3A4 catalyzed oxidations of (+)- and (-)-limonenes to respective carveols and perillyl alcohol. Interestingly, human CYP2B6 did not catalyze limonene oxidations, whereas rat CYP2B1 had high activities in catalyzing limonene oxidations. These results suggest that both (+)- and (-)-limonene enantiomers are oxidized at 6- and 7-positions by CYP2C9 and CYP2C19 in human liver microsomes. CYP2C9 may be more important than CYP2C19 in catalyzing limonene oxidations in human liver microsomes, since levels of the former protein are more abundant than CYP2C19 in these human samples. Species-related differences exist in the oxidations of limonenes in CYP2B subfamily in rats and humans.

(+)-Limonene is shown to cause renal toxicity in male rats, but not in female rats and other species of animals including mice, guinea pigs, rabbits, and dogs. We have previously shown that male-specific rat CYP2C11 (but not female-specific CYP2C12) is able to convert limonenes to carveols and perillyl alcohols (M. Miyazawa, M. Shindo, and T. Shimada: Chem. Res. Toxicol., 15, 15-20, 2002). Here, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Limonene enantiomers were converted to respective carveols, perillyl alcohols, and carvones (oxidative metabolites of carveols) by liver microsomes of dogs, rabbits, and guinea pigs. Mice, rats, monkeys, and humans produced carveols and perilly alcohols, but not carvones. Reconstituted monooxygenase systems containing purified rabbit CYP1A2 and 2B4 and NADPH-P450 reductase were found to catalyze (+)-limonene to (+)-carveol, (+)-carvone, and (+)-perillyl alcohol, being more active with CYP2B4. When (+)-carveol and (+)-carvone were used as substrates, dogs, rabbits, and guinea pigs metabolized them to (+)-carvone and (+)-carveol, respectively. Again humans, monkeys, rats, and mice did not convert (+)-carveol to (+)-carvone, but metabolized (+)-carvone to (+)-carveol, with male rats having the highest rates. CYP2C enzymes were suggested to play major roles in metabolizing (+)-carveol to (+)-carvone and (+)-carvone to (+)-carveol by liver microsomes, since the activities were inhibited significantly by anti-human CYP2C9 antibodies in these animal species. Studies with recombinant P450 enzymes suggested that CYP2C9 and 2C19 in humans and CYP2C11 in untreated male rats were the major enzymes in metabolizing (+)-carvone. These results suggest that there are species-related differences in the metabolism of limonenes by P450 enzymes, particularly in the way from (+)-carveol to (+)-carvone. However, it remains unclear whether these differences in limonene metabolism by these animal species explain species-related differences in limonene-induced renal toxicity.

Salacia reticulata Wight (S. reticulata) is a herbal medicine used for treatment of early diabetes in Ayurvedic medicine. In previous reports, the extract of S. reticulata showed preventive effects on obesity and various metabolic disorders and a suppressive effect on differentiation in premature adipocytes. The aim of this research was to elucidate the therapeutic efficacy of the extract of S. reticulata on obesity and various metabolic disorders in 12-week-old TSOD mice with obesity and metabolic disorders and in mature 3T3-L1 adipocytes. In TSOD mice, S. reticulata therapy produced a reduction in body weight and mesenteric fat accumulation, an improvement in abnormal glucose metabolism, and an increase in adiponectin level in plasma. In addition, the mRNA expressions of hormone-sensitive lipase (HSL) and adiponectin were increased in mesenteric fat. In in vitro experiments, S. reticulata therapy produced suppression of intracellular triacylglycerol accumulation and enhancement of glycerol release into the medium in mature 3T3-L1 cells. The mRNA expressions of lipogenesis factor (peroxisome proliferator-activated receptor γ, lipoprotein lipase, CD36, and fatty acid binding protein 4) were down-regulated, while the expressions of lipolysis factor (adipose tissue triacylglycerol lipase and HSL) and adiponectin were up-regulated. Moreover, the extract of S. reticulata enhanced the expression of total AMP-activated protein kinase α (AMPKα) and phosphorylated AMPKα in mature adipocytes. These findings demonstrate that the extract of S. reticulata has therapeutic effects on obesity and metabolic disorders by enhancing lipogenesis genes and suppressing lipolysis genes through the activation of AMPKα in adipocytes.

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b 5(b 5). Two major explanations are (i) direct electron transfer from b 5 and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by eitherb 5 or b 5 devoid of heme (apo-b 5), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated byb 5 but not by apo-b 5. Recently, a proposal has been made by Guryev et al.(Biochemistry 40, 5018–5031, 2001) that the stimulation by apo-b 5 can be explained only by transfer of heme from P450 preparations to apo-b 5, enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6β-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only ∼5% of the heme can be transferred to apo-b 5, which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally,b 5 supported NADH-b 5reductase/P450 3A4-dependent testosterone 6β-hydroxylation, but apo-b 5 did not. Thus, apo-b 5 can stimulate P450 3A4 reactions as well as b 5 in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b 5 cannot be used to explain the catalytic stimulation.

1. Oxidation of 1,4-cineole, a monoterpene cyclic ether, was studied in rat and human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes expressed in insect cells in which human P450 and NADPH-P450 reductase cDNAs have been introduced. On analysis with gas chromatography/mass spectrometry, 2-exo-hydroxy-1,4-cineole was identified as a principal oxidation product of 1,4-cineole catalysed by rat and human P450 enzymes. 2. CYP3A4 was a major enzyme involved in the 2-hydroxylation of 1,4-cineole by human liver microsomes, based on the following lines of evidence. First, 1,4-cineole 2-hydroxylation activities catalysed by human liver microsomes were inhibited by ketoconazole, a potent inhibitor of CYP3A activities, and an anti-CYP3A4 antibody. Second, there was a good correlation beteeen CYP3A4 contents and 1,4-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples examined. Finally, of 10 recombinant human P450 enzymes examined, CYP3A4 had the highest activity for 1,4-cineole 2-hydroxylation. 3. Liver microsomal 1,4-cineole 2-hydroxylation activities were induced in rat by pregnenolone 16alpha-carbonitrile and dexamethasone and extensively inhibited by ketoconazole, indicative of the possible roles of CYP3A enzymes in this reaction. 4. Kinetic analysis showed that Vmax/Km for 1,4-cineole 2-hydroxylation catalysed by liver microsomes was higher in a human sample HL-104 (4.6 microM(-1) min(-1)) than those of rat treated with pregnenolone 16alpha-carbonitrile (0.49 microM(-1) min(-1)) and dexamethasone (0.36 microM(-1) min(-1)). 5. 1,8-Cineole, a structurally related monoterpene previously shown to be catalysed by CYP3A enzymes, inhibited 1,4-cineole 2-hydroxylation catalysed by human liver microsomes, whereas 1,4-cineole did not inhibit 1,8-cineole 2-hydroxylation activities. Both compounds caused inhibition of testosterone 6beta-hydroxylation by human liver microsomes, the former compound being more inhibitory than the latter. 6. These results suggest that 1,4-cineole and 1,8-cineole, two plant essential oils present in Citrus medica L. var. acida and Eucalyptus polybractea, respectively, are converted to 2-hydroxylated products by CYP3A enzymes in rat and human liver microsomes. It is unknown at present whether the 2-hydroxylation products of these compounds are more active biologically than the parent compound.

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.

Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized. Purified human liver microsomal cytochromes P450 (P450)—P450 1A2 and P450 2E1—were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline. The activity was confirmed using recombinant and mutated human P450s expressed in bacteria. In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1. Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.

Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons; a similar level of expression was found with a bicistronic construct that also expressed human NADPH-P450 reductase, P450 1B1 was purified (from the monocistronic system) to electrophoretic homogeneity and a specific content of 9.2 nmol P450/mg protein using DEAE, CM, and hydroxylapatite chromatography, The absolute spectra showed a considerable fraction of high-spin iron and little cytochrome P420, The catalytic activity of the purified enzyme was considerably enhanced in the presence of cholate. Both reconstituted P450 1B1 and the bacterial membranes prepared from the bicistronic vector system had similar 7-ethoxyresorufin O-deethylation activities; as expected, 17 beta-estradiol was hydroxylated primarily at the 4-position. The ability of human P450 1B1 to activate several heterocyclic amines and polycyclic hydrocarbon dihydrodiols was confirmed with reconstituted P450 1B1 and the P450 1B1 membranes in which NADPH-P450 reductase was coexpressed. (C) 1998 Academic Press.

Human cytochrome P450s 1A1, 1A2, and 1B1 are known to have overlapping substrate specificities. All are regulated in part by the Ah locus; P450 1A2 is expressed essentially only in liver, but P450s 1A1 and 1B1 are both expressed in many extrahepatic tissues. Twenty-five polycyclic hydrocarbons, many containing acetylenic side chains, were examined as inhibitors of the three enzymes using 7-ethoxyresorufin O-deethylation as the enzyme assay in all cases. Several compounds were inhibitory at low nanomolar concentrations. 1-(1-Propynyl)pyrene and 2-(1-propynyl)phenanthrene nearly completely inhibited P450 1A1 at concentrations at which no P450 1B1 inhibition was observed. 2-Ethynylpyrene and alpha-naphthoflavone (7, 8-benzoflavone) nearly completely inhibited P450 1B1 at concentrations at which no P450 1A1 inhibition was noted. All four of the above compounds also inhibited P450 1A2. Several polycyclic hydrocarbons devoid of acetylenic groups were also inhibitory with respect to all three P450s. Some of the acetylenic compounds examined showed enhanced inhibition following preincubation with the P450s in the presence of cofactors NADPH and O2. However, of seven compounds (five acetylenes) tested with P450 1B1, only two [2-ethynylpyrene and 4-(1-propynyl)biphenyl] showed such evidence for mechanism-based inactivation. We conclude that (i) several polycyclic hydrocarbons and their oxidation products are very inhibitory with respect to human P450s 1A1, 1A2, and 1B1; (ii) of these inhibitors only some are mechanism-based inactivators; and (iii) some of the inhibitors are potentially useful for distinguishing between human P450s 1A1 and 1B1.

Aflatoxin B1 (AFB1) must be activated to the electrophilic AFB1 exo-8,9-epoxide to be genotoxic and carcinogenic. A role for epoxide hydrolase in detoxication has been suggested but never directly addressed. In light of recent studies determining the instability of AFB1 exo-8,9-epoxide in H2O, a role for epoxide hydrolase appears dubious. Rat liver or recombinant rat epoxide hydrolase provided an enhancement to the already fast hydrolysis rate of up to 22%. Purified human epoxide hydrolase provided no detectable enhancement to the rate of chemical hydrolysis. Some reduction in the genotoxicity of AFB1 was observed when the ratio of rat epoxide hydrolase to cytochrome P450 was high (approximately 50-fold). An 80-fold excess of human epoxide hydrolase over cytochrome P450 only produced an effect of approximately 25% inhibition. It appears, therefore, that there is little evidence to support a role for epoxide hydrolase in the detoxication of AFB1.

Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.

Levels of cytochrome P450 (P450 or CYP) proteins immunoreactive to antibodies raised against human CYP1A2, 2A6, 2C9, 2E1, and 3A4, monkey CYP2B17, and rat CYP2D1 were determined in liver microsomes of rats, guinea pigs, dogs, monkeys, and humans. We also examined several drug oxidation activities catalyzed by liver microsomes of these animal species using eleven P450 substrates such as phenacetin, coumarin, pentoxyresorufin, phenytoin, S-mephenytoin, bufuralol, aniline, benzphetamine, ethylmorphine, erythromycin, and nifedipine; the activities were compared with the levels of individual P450 enzymes. Monkey liver P450 proteins were found to have relatively similar immunochemical properties by immunoblotting analysis to the human enzymes, which belong to the same P450 gene families. Mean catalytic activities (on basis of mg microsomal protein) of P450-dependent drug oxidations with eleven substrates were higher in liver microsomes of monkeys than of humans, except that humans showed much higher activities for aniline p-hydroxylation than those catalyzed by monkeys. However, when the catalytic activities of liver microsomes of monkeys and humans were compared on the basis of nmol of P450, both species gave relatively similar rates towards the oxidation of phenacetin, coumarin, pentoxyresorufin, phenytoin, mephenytoin, benzphetamine, ethylmorphine, erythromycin, and nifedipine, while the aniline p-hydroxylation was higher and bufuralol 1'-hydroxylation was lower in humans than monkeys. On the other hand, the immunochemical properties of P450 proteins and the activities of P450-dependent drug oxidation reactions in dogs, guinea pigs, and rats were somewhat different from those of monkeys and humans; the differences in these animal species varied with the P450 enzymes examined and the substrates used. The results presented in this study provide useful information towards species-related differences in susceptibilities of various animal species regarding actions and toxicities of drugs and xenobiotic chemicals.

Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6β hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6β hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (

Roxithromycin and erythromycin were incubated with rat and human liver microsomal or reconstituted cytochrome P450 (P450 or CYP) monooxygenase systems in the presence of an NADPH-generating system, and the effects of these chemicals on testosterone 6 beta-hydroxylation and nifedipine oxidation activities were compared with those of typical CYP3A4 inhibitors including ketoconazole, troleandomycin, and gestodene. Roxithromycin and erythromycin were found to be relatively weak inhibitors of testosterone 6 beta-hydroxylation and nifedipine oxidation activities by rat and human liver microsomes or by reconstituted systems containing CYP3A4/5. Formation of an inhibitory P450-metabolite complex was determined spectrally by incubating troleandomycin with human liver microsomes; the extents of the complex formation were lesser in liver microsomes of humans than those of rats treated with dexamethasone. Erythromycin and roxithromycin were also activated slightly by rat liver microsomes to form P450.Fe(II)-metabolite complex, although these chemicals caused very little or undetectable levels, respectively, of spectral changes by human liver microsomes even when a human sample which contained relatively high levels of CYP3A4 was used. These results suggested that roxithromycin and erythromycin were relatively less potent to inhibit CYP3A4-catalytic activities in human liver microsomes, because of their low capabilities to form P450.Fe(II)-metabolite complex.

1. Interethnic differences in cytochrome P4502A6 (CYP2A6) levels and coumarin 7-hydroxylation activities were determined in liver microsomes of 30 Japanese and 30 Caucasians. 2. Although CYP2A6 levels and coumarin 7-hydroxylation activities varied very significantly in the 60 human samples examined, both CYP2A6 levels and coumarin 7hydroxylation activities were found to be higher in Caucasian than Japanese population. 3. Interestingly, eight of the 30 Japanese examined showed very low or undetectable levels of coumarin 7-hydroxylation activities as well as of CYP2A6 in liver microsomes. All of the Caucasians, however, had significant CYP2A6 levels and variable 7-hydroxylation activities. 4. Kinetic analvsis of coumarin 7-hydroxylation activities in liver microsomes of various human samples suggested that although there were 260-fold differences in Vmax's in 10 human samples examined, the Km's were very similar (2.1 + or - 107 mu M); a value consistent with that obtained (1.2 mu M) with purified CYP2A6 in reconstituted system. 5. The results suggest that CYP2A6 is actually involved in the 7-hydroxylation of coumarin in human liver microsomes, and that interethnic differences in coumarin 7-hydroxylation activities in Japanese and Caucasian population may be ascribed to the differences in expression of CYP2A6 protein.

NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-hydroxylation activities in the presence of NADPH-P450 reductase but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH-P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 affinity chromatography) inhibited testosterone 6 beta-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH-P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2 stimulated rates of reduction of b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH-P450 reductase are accelerated by complexation with substrates and b5 is supported in this study.

A new P450 responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) which is a potent procarcinogen was purified from renal microsomes of male mice using an index of umu gene expression. The purified P450 had high bioactivation toward 3-MeO-AAB and also 2-aminofluorene and 2-aminoanthracene. The antibody against this P450 completely inhibited mutagenic activation of 3-MeO-AAB of mouse renal microsomes. With immunoblotting, this form was present abundantly in renal microsomes of male mice but not in those of female mice. This P450 was also present in pulmonary microsomes of male and female mice but not in hepatic microsomes. The NH2-terminal amino acid sequence analysis indicated that this form belonged to the CYP4B subfamily. Thus, mouse kidney cDNA library was screened with rat CYP4B1 probe. The cDNA-deduced amino acid sequence of isolated cDNA consisted of 511 amino acids and bore 90, 86, and 84% similarities to rat, rabbit, and human CYP4B1, respectively. The NH2-terminal amino acid sequence of the purified renal P450 and amino acid sequence of BrCN-digested peptides from the purified P450 agreed with the cDNA-deduced amino acid sequence. These results suggest that CYP4B1 is a major form in renal microsomes of male mice and plays a major role in mutagenic activation of 3-MeO-AAB. In extrahepatic tissue, CYP4B1 may contribute to chemical carcinogenesis.

Recombinant cytochrome P450 (P450) 3A4 was most active in nifedipine and testosterone oxidation in a system including NADPH-P450 reductase, cytochrome b5 (b5), a semisynthetic phospholipid mixture plus cholate, glutathione, and MgCl2. The MgCl2 effect could be seen with high concentrations of Ca2+ or Sr2+ but not readily when these cations were replaced with monovalent cations. The divalent cation effect was also seen in liver microsomes. Part of the basis of this effect appears to be enhanced rates of b5 reduction, as judged from studies on deletions of reconstitution components and analysis of steady-state spectral studies. Rapid reduction of ferric P450 3A4 to ferrous was dependent upon the presence of substrate, either testosterone or ethylmorphine. When testosterone was present, reduction was also highly dependent upon the presence of b5 and Mg2+. In the case of the substrate ethylmorphine, the need to add b5 and Mg2+ to obtain optimal reduction rates was less pronounced. These patterns are consistent with the dramatic dependence of testosterone 6 beta-hydroxylation on b5 and the lack of dependence of ethylmorphine N-demethylation on b5. Our interpretation is that divalent cations stimulate electron transfer from NADPH-P450 reductase to several acceptors and that substrates and b5 can bind to P450 3A4 to influence its rate of reduction by the reductase. P450 3A4 catalyzed testosterone 6 beta-hydroxylation within Escherichia coli cells. The reactions could be supported by E. coli cytosol or by purified E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase also supported catalytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers. P450s immunoreactive with anti-human P450 1A1 and anti-human P450 3A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related to P450 1A1, 2A6, 2C9, 2E 1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P450 1A2 was only the 1A subfamily of P450 found in adults. Drug oxidation activities with several substrates were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers. Activation of procarcinogens by P450 enzymes was also examined and it was found that activities with (+)- and (-)-enantiomers of 7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene were higher in fetal liver microsomes than adult lung or liver microsomes. Anti-human P450 1A1 and 1A2 and a-naphthoflavone, known to inhibit P450 IA-related activities, did not affect these procarcinogen activation in fetal liver microsomes. Fetal liver microsomes catalyzed activation of aflatoxin B1 and sterigmatocystin, although activation of carcinogenic arylamines was much lower in microsomes of fetal livers and adult lungs than of adult livers. These results suggest that in human fetal livers at least two P450 enzymes, namely P450 IA-related enzyme and P450 3A7, are actually expressed and these enzymes are involved in the activation of the (+)- and (-)-enantiomers of 7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and the carcinogenic mycotoxins, respectively. In adult human lungs, several P450 enzymes are expressed, though the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.

Recent studies indicate that cytochrome P450 (P450) 3A4 plays important roles in the activation of procarcinogens such as aflatoxin B1 and sterigmatocystin, as well as in the oxidation of a number of structurally diverse chemicals and endogenous compounds. Since P450 3A5 has been reported to be present at significant levels in liver microsomes in approximately 25% of human adults, we examined and compared the role of P450 3A4 and 3A5 in procarcinogen activation in humans. Immunoblot experiments with liver microsomes from 60 human samples suggested that 4/30 Japanese and 4/30 Caucasians contained considerable levels of P450 3A5, although P450 3A4 could be determined at relatively high levels in all of the human samples examined. Good correlation was observed between P450 3A4, but not P450 3A5, levels versus activation of aflatoxin B1 and stergmatocystin in these human samples. Comparisons of the activation of procarcinogens in reconstituted monooxygenase systems containing modified P450 3A4 and 3A5 enzymes expressed in Escherichia coli were carried out in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strain for genotoxicity assay, and it was found that P450 3A4 had similar activities to or higher rates than P450 3A5 for the 24 procarcinogens tested.

A new P450 responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) which is a potent procarcinogen was purified from renal microsomes of male mice using an index of umu gene expression. The purified P450 had high bioactivation toward 2-aminofluorene (2-AF), 2-aminoanthracene (2-AA), and 3, 3'-dichlorobenzidine (DCB) as well as 3-MeO-AAB. The antibody against this P450 completely inhibited mutagenic activation of 3-MeO-AAB, 2-AF, 2-AA, and DCB by mouse renal microsomes. With immunoblotting, this form was present abundantly in renal microsomes of male mice but not in those of female mice. This P450 was also present in mouse pulmonary and bladder microsomes but not in hepatic microsomes. The NH2-terminal amino acid sequence analysis indicated that this form belonged to the CYP4B subfamily. Thus, mouse kidney cDNA library was screened with rat CYP4B1 probe. The cDNA-deduced amino acid sequence of isolated cDNA consisted of 511 amino acids and the NH2-terminal amino acid sequence of the purified renal P450 agreed with the cDNA-deduced amino acid sequence, indicating that renal P450 purified in this study is mouse CYP4B1. Human pulmonary and renal cDNA library were screened with mouse CYP4B1 cDNA probe. Two full-length cDNAs were isolated and they had two or three amino acid changes to human CYP4B1 reported previously.

Differences in the catalytic roles of rat and human cytochrome P450 2A enzymes in testosterone 7α- and coumarin 7-hydroxylase activities were examined. Liver microsomes from 18 human samples catalyzed coumarin 7-hydroxylation at a mean rate of about 60pmol/min/nmol P450, but did not show any measurable activity for testosterone 7α-hydroxylation. In rats, both activities were found to be developmentally regulated; 3-week-old rats had the highest activities for these two reactions. Anti-P450 2A1 antibodies and methoxsalen, a potent inhibitor of P450 2A-dependent monooxygenase activities in several animal species, inhibited almost completely both testosterone 7α- and coumarin 7-hydroxylations catalyzed by liver microsomes prepared from 3-week-old male rats. Interestingly, although K m values for coumarin 7-hydroxylation activities in liver microsomes from 3-week-old rats were not different from those of adult humans, the V max value in rats was only 1/30 of that obtained in 18 human samples. Thus, the present results support the view that marked differences exist in the catalytic roles of rat and human P450 2A enzymes, which, in turn, may sometimes cause species-related differences in susceptibilities toward drug actions and toxicities.

P450 HFLb purified from human fetal livers has been shown to be constitutively expressed in fetal livers. In the present study, the occurrence of proteins immunochemically related to P450 HFLb in extrahepatic tissues of human fetuses and their contribution to mutagenic activation of promutagens were investigated. The mutagenic activation of aflatoxin B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and benzo[a]pyrene were observed in human fetal extrahepatic tissues, including adrenal glands, kidneys and lungs, at varying rates. Immunoblot analysis of homogenates of extrahepatic tissues with antibodies to P450 HFLb revealed the occurrence of proteins immunochemically related to P450 HFLb in adrenal glands, kidneys and lungs. Immuno-inhibition studies suggested that in fetal adrenal gland and kidney, the proteins cross-reactive with antibodies to P450 HFLb were capable of activating IQ and MeIQ to mutagens.

In order to address the hypothesis that 6-aminochrysene (6-AC) is converted to genotoxic products by cytochrome P450 enzymes via two activation pathways (N-hydroxylation and epoxidation), the activation of 6-AC and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (6-AC-diol) to genotoxic metabolites was examined in rat and human liver microsomal cytochrome P450 enzymes using Salmonella typhimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009) as tester strains. The latter bacteria, an O-acetyltransferase-overexpressing strain, was highly sensitive to metabolites derived from activation of 6-AC, but not those from 6-AC-diol, using liver microsomes from phenobarbital-treated rats or a reconstituted monooxygenase system containing P4502B1 or -2B2, thus suggesting the roles of P450 and acetyltransferase systems in the activation process. 6-AC-diol, on the other hand, was activated very efficiently by liver microsomes prepared from beta-naphthoflavone-treated rats or a reconstituted system containing P4501A1 or -1A2; the activation reaction is considered to proceed through diol-epoxide formation. The contribution of rat P4501A enzymes towards activation of 6-AC-diol was confirmed by the inhibitory effects on the activation process of alpha-naphthoflavone, a specific inhibitor of P4501A-related activities, and antibodies raised against purified P4501A1 and -1A2. In humans, P4501A2 was found to be the major enzyme involved in the activation of 6-AC-diol to genotoxic metabolites while the parent compound 6-AC was activated mainly by P4503A4. Experiments using recombinant P450 proteins expressed in human lymphoblastoid cell lines showed that human P4501A1 could also activate 6-AC-diol to reactive metabolites at almost the same rate measured with P4501A2. In addition, P4502B6 was found to efficiently catalyze the activation of 6-AC to genotoxic metabolites, and P4503A4 was active in the activation of 6-AC-diol as well as 6-AC. Addition of purified rat epoxide hydrolase to the incubation mixture containing purified rat P4501A1 or microsomes expressing human P4501A1 caused inhibition of activation of 6-AC-diol. These results suggest the existence of different enzymatic activation pathways for 6-AC and 6-AC-diol. The former carcinogen may be N-hydroxylated principally by P4502B enzymes in rats and P4503A4 and -2B6 in humans and activation to its ultimate metabolites may proceed through esterification of the N-hydroxy metabolites by an N-acetyltransferase. The 6-AC-diol is metabolized to its ultimate diolepoxide product by P4501A enzymes in rat and human liver microsomes. P4503A4 (humans) and P4503A2 (rats) may also contribute to some extent in the activation of 6-AC-diol, albeit at lower rates than those of P4501A enzymes.

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1,1A2, 2A1, 2A2,2B1,2B2,2C6,2C7, 2C11,2C12,2C13,2E1,3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2,2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

Using an O-acetyltransferase-overexpressing strain Salmonella typhimurium NM2009 we measured the activities for metabolic activation of several carcinogenic arylamines to genotoxic products by rat liver microsomal cytochrome P-450 enzymes, and compared them with the activities obtained in the original tester strain Salmonella typhimurium TA1535/pSK1002 or the O-acetyltransferase-defective strain Salmonella typhimurium NM2000. Since all of the tester strains had introduced the umuC'-'lacZ gene, we could detect the genotoxic activities by measuring bacterial beta-galactosidase activity resulting from the DNA damage. In the O-acetyltransferase-defective strain NM2000 most of the arylamines tested showed weak responses in inducing umu gene expression after metabolic activation by liver microsomes. The strain NM2009, on the other hand, was found to be highly sensitive towards a variety of aromatic amines, and these activities were greater than those seen in the original tester strain S. typhimurium TA1535/pSK1002. The chemicals which marked responses in strain NM2009 include 2-aminoanthracene, 6-aminochrysene, 2-aminofluorene, 2-acetylaminofluorene, 3-methoxy-4-aminoazobenzene, O-aminoazotoluene, Glu-P-1, Trp-P-2, A alpha C, MeA alpha C, MeIQ, MeIQx and IQ. Of these procarcinogens tested MeIQ, MeIQx and IQ also showed strong cytotoxic effects in S. typhimurium NM2009 after metabolic activation by liver microsomes. Only PhIP was the substrate showing similar responses in strains TA1535/pSK1002 and NM2009. The results with the reconstituted monooxygenase system containing purified cytochrome P-450 enzymes support the above findings obtained with the liver microsomal enzyme system. Thus, the usefulness of the newly developed strain NM2009 for the detection of reactive metabolites of several carcinogenic aromatic amines after metabolism by the liver microsomal cytochrome P-450-linked monooxygenase system has been ascertained.

The genotoxic and mutagenic activation of aflatoxin B1 (AFB1) by hepatic, renal, and pulmonary microsomes and purified cytochrome P450s was investigated in Salmonella typhimurium TA1535/pSK1002 cells in which an umu response shows DNA damage. The activity of the hepatic microsomes was greatest. Pulmonary microsomes had moderate activity and renal microsomes had low activity. P450 2C11, 2B1, 3A2, 4A2, 4B1, K-2, and K-4 were assayed in a reconstituted system with dilauroylphosphatidylcholine (DLPC). P450 2C11 (a major hepatic cytochrome P450 in male rats) had high activity. P450 2B1 (a major form as well as P450 4B1 in pulmonary microsomes) and K-2 (a minor form in renal microsomes) had moderate activity. P450 4A2 (a major form in renal microsomes), P450 K-4 (a renal form), and P450 4B1 had low activity. P450 3A2 did not have high activity in these conditions but it had high activity toward AFB1 in a modified reconstituted system with a lipid mixture and sodium cholate instead of DLPC only. The activities of other forms were not enhanced by the modification of reconstituted system. Anti-P450 2C11 or 3A2 antibodies inhibited the bioactivation of AFB1 by hepatic microsomes to 50%. These results suggest that the greater ability of hepatic microsomes as compared with pulmonary and renal microsomes to metabolize AFB1 to mutagenic products is a function of the relative proportions of the highly active cytochrome P450s, P450 2C11 and 3A2, in the liver.

The specific profile of hepatic drug-metabolizing enzymes and isomer-specific residue of PCBs were characterized in the same family of sea gulls such as black-headed gull (Larus ridibundus) and black-tailed gull (Larus crassirostris). The examined enzyme contents and activities were higher in the former species. On the other hand, higher residual level of polychlorinated biphenyls (PCBs) were observed in black-tailed gull. There was no relationship between PCB concentrations and any enzyme activities in both species. A significant correlation between 2,3,7,8-TCDD toxicity equivalents (TEQs) of coplanar PCBs in pectoral muscles and 7-ethoxyresorufin O-deethylase activities in liver microsomes was observed only in black-headed gulls. According to the electrophoretic/immunochemical analysis using antibodies raised against rat P-450 enzymes, it was clear that both birds have the gull P-450 2B protein which is homologous with rat P-450 2B1. However, gull P-450 1A1 and gull P-450 1A2 (these are homologous protein with rat P-450 1A1 and rat P-450 1A2 respectively) were detected only in liver microsomes of black-headed gull. The variation in contents of these P-450 molecular species in these birds are discussed in relation to induction by PCBs.