Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants found in certain foods, cigarette smoke and combustion products of oil, coal, and gas. The prototype PAH, benzo[a}pyrene (B[a]P) is one of the most potent chemical carcinogens known in animal tumorigenesis assays. The level of hepatic glucuronidating activity towards benzo[a]pyrene phenols, quinols, and dihydrodiols, appears to represent a key risk factor underlying susceptibility to B[a]P's genotoxic effects. Evidence has been obtained that oltipraz (4-methyl-5-pyrazinyl- 1 ,2-dithiole-3-thione), an investigational cancer preventive drug, may act in part through induction of hepatic UDPglucuronosyltransferases (UGTs) with activity towards B[a]P metabolites. UGTs in the UGT1 family appear to be particularly critical. A previously identified UGT1 isoform, UGT1A7, appears highly active in the glucuronidation of B[a]P metabolites.
Specific Aim 1 will directly compare the activity of recombinant UGT1A7 to other UGTIA isoforms to determine if any other of these forms are potentially relevant either in protection against B[a}P or in the response to oltipraz. The UGTs will be expressed in HEK293 cells and tested for activity in vitro against key metabolites of B[a]P and other carcinogenic PAHs.
Specific Aim 2 will study whether the corresponding human UGT1A isozymes are also active. In addition, variation in human liver microsomal UGT activities towards B[a]P metabolites will be characterized in relation to expression of specific UGTIA subsets (UGTIAI, UGTIA3-.1A5, UGTIA6, and UGT1A7-lO).
Specific Aim 3 will continue studies of the mechanisms controlling the hepatic expression of rat UGT1A6 and UGT1A7 by oltipraz and PAHs. To characterize possible species differences in regulation, a study will be performed to assess the responsiveness of human UGT1As in cultured human hepatocytes.
Specific Aim 4 will develop novel in vivo approaches to assess the functional significance of UGT1A deficiency and replacement of UGTIA7 liver expression for B[a]P excretion and mutagenesis. To examine the influence of UGTIA deficiency and monitor B[a]P induced mutagenesis in a short term in vivo assay, a special strain of rat will be developed by crossing the Gunn rat onto the Big Blue lad transgenic F344 rat background. Replacement of UGT1A7 expression in liver will be achieved through the use of a recombinant adenovirus 5 engineered to express UGT1A7.
