Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas

CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.

Conserved promoter elements in the CYP6B gene family suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification.

Despite the fact that Papilio glaucus and Papilio polyxenes share no single hostplant species, both species feed to varying extents on hostplants that contain furanocoumarins. P. glaucus contains two nearly identical genes, CYP6B4v2 and CYP6B5v1, and P. polyxenes contains two related genes, CYP6B1v3 and CYP6B3v2. Except for CYP6B3v2, the substrate specificity of which has not yet been defined, each of the encoded cytochrome P450 monooxygenases (P450s) metabolizes an array of linear furanocoumarins. All four genes are transcriptionally induced in larvae by exposure to the furanocoumarin xanthotoxin; several are also induced by other furanocoumarins. Comparisons of the organizational structures of these genes indicate that all have the same intron/exon arrangement. Sequences in the promoter regions of the P. glaucus CYP6B4v2/CYP6B5v1 genes and the P. polyxenes CYP6B3v2 gene are similar but not identical to the -146 to -97 region of CYP6B1v3 gene, which contains a xanthotoxin-responsive element (XRE-xan) important for basal and xanthotoxin-inducible transcription of CYP6B1v3. Complements of the xenobiotic-responsive element (XRE-AhR) in the dioxin-inducible human and rat CYP1A1 genes also exist in all four promoters, suggesting that these genes may be regulated by dioxin. Antioxidant-responsive elements (AREs) in mouse and rat glutathione S-transferase genes and the Barbie box element (Bar) in the bacterial CYP102 gene exist in the CYP6B1v3, CYP6B4v2, and CYP6B5v1 promoters. Similarities in the protein sequences, intron positions, and xanthotoxin- and xenobiotic-responsive promoter elements indicate that these insect CYP6B genes are derived from a common ancestral gene. Evolutionary comparisons between these P450 genes are the first available for a group of insect genes transcriptionally regulated by hostplant allelochemicals and provide insights into the process by which insects evolve specialized feeding habits.

Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage.

Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2'-deoxyguanosine (oxo8dG) residues in DNA. Treatment of this cell line with 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua. The essential role of Ah-receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor-defective c4 mutant cells. While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer.

Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism.

Cytochrome P450 1A2 (CYP1A2) is a predominantly hepatic enzyme known to be important in the metabolism of numerous foreign chemicals of pharmacologic, toxicologic, and carcinogenic significance. CYP1A2 substrates include aflatoxin B1, acetaminophen, and a variety of environmental arylamines. To define better the developmental and metabolic functions of this enzyme, we developed a CYP1A2-deficient mouse line by homologous recombination in embryonic stem cells. Mice homozygous for the targeted Cyp1a2 gene, designated Cyp1a2(-/-), are completely viable and fertile; histologic examination of 15-day embryos, newborn pups, and 3-week-old mice revealed no abnormalities. No CYP1A2 mRNA was detected by Northern blot analysis. Moreover, mRNA levels of Cyp1a1, the other gene in the same subfamily, appear unaffected by loss of the Cyp1a2 gene. Because the muscle relaxant zoxazolamine is a known substrate for CYP1A2, we studied the Cyp1a2(-/-) genotype by using the zoxazolamine paralysis test: the Cyp1a2(-/-) mice exhibited dramatically lengthened paralysis times relative to the Cyp1a2(+/+) wild-type animals, and the Cyp1a2(+/-) heterozygotes showed an intermediate effect. Availability of a viable and fertile CYP1A2-deficient mouse line will provide a valuable tool for researchers wishing to define the precise role of CYP1A2 in numerous metabolic and pharmacokinetic processes.