Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene.

Tissue-specific induction of mRNA of cytochrome P450 (P450 or CYP) 1A1 and 1B1 by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was investigated in wild and arylhydrocarbon receptor (AhR)-deficient C57BL/6J mice. Ratios of mRNA expression of CYP1A1 or CYP1B1 over beta-actin were determined and used to compare levels of expression and induction of these P450s by PAHs and PCBs in various organs. CYP1A1 mRNA was detected in control mice at very low levels in liver, lung, heart, kidney, intestine, thymus, testis, uterus, ovary, and brain and was highly induced in these organs by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. In AhR(+/+) and AhR(-/-) mice, CYP1B1 mRNA was found to be constitutively expressed at significant levels in heart (the ratio of mRNAs of CYP1B1 to beta-actin was approximately 0.6), kidney ( approximately 0.8), intestine ( approximately 0.3), testis ( approximately 0.9), thymus ( approximately 0.4), uterus ( approximately 0.3), ovary ( approximately 1.4), and brain ( approximately 0.4), whereas it was low in liver and lung (the mRNA ratio to beta-actin was <0.2 in these cases). CYP1B1 in the latter two organs was highly induced by PAHs and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. The induction of CYP1B1 by PAHs and PCBs was more extensive in organs in which the constitutive expression of CYP1B1 was low. For example, CYP1B1 was induced 9-fold and 10-fold by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in livers of male and female mice, respectively, whereas in testis and ovary, the fold induction of CYP1B1 by two inducers was only 1.1 and 1.4, respectively. Liver microsomal xenobiotic oxidation activities were induced by these PAHs and PCBs in male and female AhR(+/+) mice. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in their expression in extrahepatic organs of mice and could be induced by PAHs and PCBs with different extents of induction depending on the inducers used and the organs examined in AhR(+/+) mice. The findings of significant levels of constitutive expression of CYP1B1 in AhR(-/-) mice as well as AhR(+/+) mice in several organs including heart, kidney, thymus, testis, ovary, and brain in AhR(-/-) mice as well as AhR(+/+) mice are of importance in understanding the basis of toxicity and carcinogenesis by chemicals that are metabolized by CYP1B1.

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice.

Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y. Shimizu, Y. Nakatsuru, M. Ichinose, Y. Takahashi, H. Kume, J. Mimura, Y. Fujii-Kuriyama and T. Ishikawa (2000) PROC: Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice.

Metabolism of (+)- and (-)-limonenes to respective carveols and perillyl alcohols by CYP2C9 and CYP2C19 in human liver microsomes.

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.

Sex differences in the metabolism of (+)- and (-)-limonene enantiomers to carveol and perillyl alcohol derivatives by cytochrome p450 enzymes in rat liver microsomes.

(+)-Limonene is reported to cause nephropathy in male rats, but not in female rats and other species of animals including mice, rabbits, guinea pigs, and dogs. Male rats contain high levels of alpha2u-globulin in kidneys, and it has been shown that limonene and/or its metabolites are able to bind noncovalently to alpha2u-globulin, resulting in an accumulation of protein droplets in the renal tubules. In this study, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by liver microsomes of male and female rats. (+)- and (-)-limonene enantiomers were found to be oxidized to their respective trans-carveol (6-hydroxylation) and perillyl alcohol (7-hydroxylation) derivatives in greater amounts by liver microsomes of male rats than those of female rats. The limonene hydroxylation activities were not detected in liver microsomes of rat fetuses and were increased developmentally after birth, only in male rats. Treatment of male rats with phenobarbital significantly increased liver microsomal 6-hydroxylation activities with both enantiomers whereas beta-naphthoflavone, isosafrole, and pregnenolone 16alpha-carbonitrile did not cause such effects. Anti-P450 2C9 which cross-reacts with rat P450 2C11 inhibited limonene hydroxylations catalyzed by liver microsomes of untreated male rats, and it was also found that anti-P450 2B1 suppressed the activities catalyzed by liver microsomes of phenobarbital-treated rats. Possible roles of P450 2C11 and P450 2B1 in the limonene hydroxylation activities were supported by the experiments with purified rat liver P450s in reconstitution systems and with recombinant rat P450s in Trichoplusia ni. Our present results showing that there are sex-related differences in the oxidative metabolism of limonene enantiomers by liver microsomes may provide useful information on the basis of limonene-induced toxicities in different animal species.

Biotransformation of (-)-verbenone by human liver microsomes.

The biotransformation of (-)-verbenone was investigated with human liver microsomes by using GC-MS. Regioselective biotransformation was observed when (-)-verbenone was incubated with the liver microsomes. (-)-10-Hydroxyverbenone was formed from (-)-verbenone of kinetic analysis showed that the Km and Vmax values for the hydroxylation of (-)-verbenone by liver microsomes from three human samples, HG-70, HG-56 and HG-23, were 1.1 mM and 4.8 nmol/min/nmol P450, 0.6 mM and 2.1 nmol/min/nmol P450, and 2.8 mM and 4.6 nmol/min/nmol P450, respectively.

Species differences in the metabolism of (+)- and (-)-limonenes and their metabolites, carveols and carvones, by cytochrome P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, 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.