The long-term goal of this project is to understand the role that the CYP1A2 (cytochrome P3450) enzyme plays in genotoxicity caused by environmental pollutants in the intact mouse. The CYP1A2 enzyme participates primarily in the oxidative metabolism of arylamines but has also been shown to metabolize polycyclic hydrocarbons. This laboratory has previously found that the murine Cypla-2 gene is constitutively expressed at high levels only in liver, and highly induced only in liver, lung and duodenum following exposure to polycyclic aromatic compounds such as those found in cigarette smoke and other combustion products. To define the involvement of the CYP1A2 enzyme in genotoxicity of environmental chemicals, we propose to develop a transgenic mouse line lacking both alleles of the Cypla-2 gene. The methods used to generate a transgenic mouse having one copy of the inactivated gene in the germ line will include knocking out the Cypla-2 gene in embryonic stem (ES) cells with an homologous recombination construct; we shall also attempt a """"""""double-knockout"""""""" in ES cell cultures. We shall then inject the targeted ES cells into the blastocoele cavity of embryos and transfer surviving blastocysts to a foster mother by uterine implantation. If necessary, two heterozygotes will be bred in order to generate mice homozygous for the disrupted Cypla-2 gene. In the CYP1A2-deficient mouse, we have chosen to examine the genotoxicity of two environmentally important chemicals that are CYP1A2 substrates: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Pharmacokinetics, DNA adduct formation, and mutagenesis will be examined as a function of the route of administration. These two chemicals were chosen because they are environmentally relevant and the CYP1A2 enzyme plays contrasting roles in their metabolism. NNK is one of the major tobacco smoke-specific nitrosamines that require CYP1A2 for metabolic activation. AFB1, a highly carcinogenic mycotoxin contaminant found in a variety of foods, is activated by some P450 enzymes; however, CYP1A2 appears to be involved in a detoxification pathway. Thus, one would expect that, in the CYP1A2-deficient mouse, NNK-induced genotoxicity int he lung would be decreased and AFB1 genotoxicity in the liver would be enhanced. We shall measure the pharmacokinetics of these two chemicals, using commercially available radiolabeled NNK and AFB1. DNA adduct formation in several tissues will be determined by the 32P- postlabeling assay. to measure tissue-specific mutagenesis, we shall breed the CYP1A2-deficient mouse with the Stratagene Big Blue (TM) indicator mouse to establish an inbred line; mutations in the lacI transgene will be quantitated by established methodologies. The studies proposed in this application should help in evaluating the relative risk for adverse health effects in humans exposed to certain environmental pollutants. This is particularly important, since recent studies have demonstrated a trimodal distribution and >60-fold interindividual variation in the constitutive expression of CYP1A2 in human liver.
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