UDP-glucuronosyltransferases (UGT) are phase II xenobiotic metabolizing enzymes that glucuronidate a variety of exogenous and endogenous compounds. This family of enzymes play an important role in the metabolism of many carcinogens such as hydroxylated benzo[a]pyrene, ben[a]anthracene, 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD), aflatoxin B1, acetylaminofluorene, and tobacco-specific nitrosamine such as 4- (methylnitrosamino)-1-(3-pyridyl)-butan-1-ol NNAL). This proposal focuses on the isolation and characterization of the genes encoding for two major types of UDP-glucuronosyltransferases, i.e., bilirubin/phenol (UGT-br/p) and bile acid/steroid (UGT-ba/s) UGT in mice. The underlying hypothesis is that cloning, gene expression and regulation studies will offer unique insights into the biological mechanisms of detoxification and/or toxicity of different carcinogens catalyzed by UGTs in the mouse and will aid in the extrapolation of chemical toxicity, carcinogenicity and mutagenicity studies from mouse to human.
The specific aims of this research proposal are: 1) To clone, sequence and characterize the cDNAs encoding for the mouse bilirubin/phenol (UGTbr/p) and the bile acid/steroid (UGTba/s) UDP-glucuronosyltransferases. This will involve a series of experiments aimed at screening and re-screening of a female mouse liver cDNA library using human and mouse cDNA probes, followed by restriction-mapping and sequencing. 2) To elucidate the catalytic activities and toxicology of mouse UGT via cDNA expression in cultured cells. This will involve the expression of the cloned mouse UGTbr/p and UGTba/s cDNAs in fibroblast cell lines and baculovirus system and assaying for glucuronidation activities towards model compounds as well as known carcinogenic substrates. 3) To characterize the regulation/expression of UGTs in hepatoma cell lines and in whole animals. These experiments will involve administration of known inducers for Phase II drug metabolizing enzymes as well as environmental pollutants such as TCDD and DDT (1,1-bis[p-chlorophenyl]-2,2,2- trichloroethane) to: 2) hepatoma cell lines - Hepa 1c1c7 (mouse), Fao (rat) and Hep G2 (human); and b) different strains and gender of mice - Balb/c, C3H, C57BL/6J, B6C3F1, ICR and DBA/2J mice, and assaying for mRNA levels as well as UGT activities toward model and carcinogenic substrates. 4) To isolate, sequence and characterize the genomic DNA of the mouse UGTbr/p and UGTba/s genes to elucidate the conserved domain(s) at the 5' flanking regions of the genes. This involves screening of a mouse genomic library with their respective cloned mouse UGTbr/p and UGTba/s cDNAs and subsequent restriction-mapping and sequencing two kb 5' flanking regions of the genes. The results of the proposed experiments are likely to significantly enhance our fundamental understanding of the biological mechanisms of UGTs in the glucuronidation and detoxification of carcinogens in mice and may lend insights into human carcinogenesis and toxicity induced by chemical and environmental carcinogens.
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