UC San Diego

Drug metabolism can be divided into Phase I and Phase II reactions, which catalyze the conversion of a lipophilic compound into a more water soluble metabolite that can be readily eliminated from the body. Cytochrome P450s classically represent the Phase I enzymes and detoxify a myriad of substrates. The human CYP1A1 gene is regulated by the aryl hydrocarbon receptor (AhR) and induction of CYP1A1 is known to play an important role in xenobiotic metabolism. Exposure to prevalent environmental contaminants result in the induction of cytochrome P450 1A1 (CYP1A1), and this response is widely used as a biomarker for toxicant exposure. To examine the regulation of human CYP1A1 in vivo, we created a transgenic mouse strain (Tg-CYP1A1GFP) expressing a chimeric gene consisting of the entire human CYP1A1 gene (15 kb) fused with a GFP reporter gene. The treatment of Tg-CYP1A1GFP mice with TCDD led to induction of CYP1A1GFP in both the liver and lung as determined by fluorescence and Western blot analysis. The ability to identify fluorescently labeled CYP1A1 in vivo provides a sensitive measurement of gene response, and links exposure to potential environmental toxicants and activation of the AhR. The process of glucuronidation is catalyzed by UDP- glucuronosyltransferases (UGTs), and is the most important Phase II reaction for the detoxification of xenobiotic compounds. Recent biochemical evidence indicates that the UGT proteins may oligomerize within the ER. To investigate the potential homo/heterodimerization capacity of UGT1A proteins, fluorescence resonance energy transfer was utilized to study dimerization in live cells, in conjunction with co-immunoprecipitation experiments. These complementary techniques demonstrated that all of the UGT1A proteins homodimerize and heterodimerize with UGT1A1. These studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity. The regions facilitating protein interactions among UGTs was also explored using truncation analysis and co-immunoprecipitation experiments, which revealed that multiple dimerization domains may exist. A homology model of UGT1A1 was constructed which gave insight into the potential orientation of the dimers, as well as the pertinent regions that could be involved in dimerization