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.Cytochromes P450 (P450) are a superfamily of hemecontaining monooxygenases. A limited number of P450s participate in pathways of steroid hormone synthesis whereas the majority of these enzymes are involved in oxidative metabolism of drugs, other foreign compounds, and endogenous substrates, including steroids (1). These xenobioticmetabolizing P450s mostly fall within the CYP1, CYP2, and CYP3 families and exhibit broad and sometimes overlapping substrate specificity. A limited number of P450s within these families are responsible for the metabolic activation of chemical carcinogens. In the CYP1 family, CYP1A1 and CYP1B1 metabolically activate polycyclic aromatic hydrocarbons and CYP1A2 participates in the metabolic activation of arylamine, heterocylic amines, and aflatoxin B1. CYP2E1 activates a large number of low molecular weight carcinogens including benzene and N-nitrosodimethylamine. These carcinogenmetabolizing P450s are also among the most well conserved of the P450 superfamily and can be found in several mammalian species, including mouse and human (2). CYP1B1 is a conserved member of the P450 superfamily that was first identified and purified from mouse embryonic fibroblasts (EFs) (3) and rat adrenals (4). This form was characterized by its ability to metabolically act...

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.

Inflammatory stimulation of the liver leads to the induction of nitric oxide (NO) biosynthesis. Because NO binds to the catalytic heme moiety ofcytochromes P450 (CYPs), we investigated whether NO interferes with specific CYPdependent metabolic pathways. In a first experimental approach V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYPlAl and -1A2 were used. Incubation with the NO donors sodium nitroprusside and S-nitrosylacetylpenicillamine led to a concentration-dependent inhibition of all four CYP enzymes. CYPlAl was more sensitive to the inhibitory effect of NO than CYP1A2. In the second part of the study, endogenous NO synthesis was induced in rat hepatocytes by incubation with a mixture of cytokines and endotoxin. Concurrently, as NO production in hepatocytes increased within 24 hr, a decrease in CYPlAl-dependent benzo[a]pyrene turnover was observed to almost undetectable levels. The competitive inhibitor of NO synthesis, NGmonomethyl-L-arginine, was able to significantly restore CYPlAl activity in the presence of cytokines and endotoxin. Inhibition of hepatocellular CYP activity by NO was predominantly due to a direct effect on the enzymes. However, NO-dependent inhibition of CYP expression at a transcriptional level was also demonstrated. Our results indicate that inhibition of NO biosynthesis in patients suffering from systemic inflammatory response syndromes may help to restore biotransformation capacity of the liver.Hepatocellular dysfunction is a detrimental consequence of the metabolic response ofthe liver to prolonged inflammatory stimulation. Characteristic features are suppression of synthetic performance and a profound inhibition of xenobiotic biotransformation (1, 2). Therapy is not available because the molecular basis of these phenomena remains to be elucidated. However, a better understanding of the pathophysiology of the inflamed liver may result from the discovery of L-arginine-dependent nitric oxide (NO) production in hepatocytes and nonparenchymal liver cells (3, 4). As in many other cell types, an inducible NO synthase (iNOS) was identified in hepatocytes upon stimulation with cytokines and endotoxin (5, 6). Induction of iNOS was also observed in the course of parasitic infections, such as malaria (7). Recently, the gene encoding iNOS has been cloned from human hepatocytes in full length for heterologous expression (8).NO exerts a plethora of biologic functions (9). Many of these effects are based on modulation of enzyme activity through binding of NO to prosthetic iron complexes. In this context it is interesting that NO was used for years as a spin-label probe to investigate the role of heme groups in the catalytic centers of cytochrome P450 (CYP) enzymes (10). Consequently, it was demonstrated that NO inhibits CYPdependent reactions when microsomal preparations were exposed to NO (11). CYP enzymes, also referred to as microsomal monooxygenases, catalyze oxidative key reactions in the biotransformation of xenobiotics (12). Therefore, in...