The long-term goal of this project is the elucidation of cytochrome P450 CYP1A2's role in toxicity and cancer caused by environmentally hazardous chemicals. CYP1A2 substrates include environmentally important heterocyclic amines, nitrosamines, aflatoxins, nitrated polycyclic aromatic hydrocarbons, and arylamines such as 4-aminobiphenyl (ABP). The mouse CYP1A2 gene shows high basal activity only in the liver; is inducible in liver, lung, and GI tract following exposure to inducers such as dioxin and chemicals found in cigarette smoke; and is not expressed in blood cells, kidney or urinary bladder. The mouse CYP1A2 and human CYP1A2 are known to exhibit large differences (6- to 10-fold) in activity/substrate specificity. Acutely, ABP causes hemoglobin (Hb) adducts, methemoglobinemia, and liver toxicity. Chronically, ABP causes bladder ABP-DNA adducts and tumor formation as the consequence of hepatic CYP1A2 and N-acetyltransferase (NAT2) levels, as well as urinary pH. Our hypothesis is that ABP toxicity and cancer will be correlated with hepatic CYP1A2 and low NAT2 activities. To define the role of CYP1A2 in toxicity and cancer, we have generated during the first 4.25 years of this grant a viable, fertile CYP1A2(-/-) genotype with other relevant genotypes: the Stratagene Big Blue indicator and marked differences in Ah receptor affinity [Ahr(b1/b1), Ahr(d/d) or Ahr(-/-)] and rapid vs slow N-acetylation [Nat(b/b), Nat(a/a)]. We are now ready to use these mice to answer questions relevant to human environmental diseases. Comparing Cyp1a2(-/-) with Cyp1a2(+/+) wild-type and the above genotypes, we predict the Cyp1a2(+/+), Nat(a/a) mouse will show more ABP acute toxicity and more DNA adducts/mutations/tumors in the bladder, and dioxin-treated Ahr(b1/b1) will show even higher toxicity/cancer. We also believe that human-mouse differences in ABP metabolism by CYP1A2 are very important. Thus, for the next funding period we will: (1) measure acute toxicity (red cell; liver), ABP-DNA adducts, mutagenesis and tumor genesis (liver, bladder, and kidney)--comparing Cyp1a2(-/-) vs (+/+), and Ahr(b1/b1) vs (d/d) or (-/-), and Nat(b/b) vs (a/a)--following topical ABP treatment, as low vs high urine pH, and with or without dioxin pretreatment; (2) generate an hCYP1A2 mouse line by targeted replacement of the mouse Cyp1a2 gene with the human CYP1A2 gene as a single copy under 5' flanking regulatory controls of the mouse Cyp1A2 gene; and (3) examine in the """"""""humanized"""""""" hCYP1A2 mouse line the parameters as listed in Specific Aim 1. The studies proposed in the applications should help in evaluating the relative risk for adverse health effects in humans exposed to a known human bladder carcinogen that is present in cigarette smoke. This is particularly important, since studies have shown >60-fold interindividual variation in the basal levels of human liver CYP1A2, >30-fold variation in AHR affinity, and slow acetylators comprise about 50 percent of the U.S. population.
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