The long-term goal of this research is to understand the molecular basis for the susceptibility to polycyclic aromatic hydrocarbon (PAH)-induced toxicity. This proposal specifically focuses on a detailed analysis of the mechanisms which regulate the expression of an important enzyme activity, aryl hydrocarbon hydroxylase (AHH). This enzyme is known to participate in the bioactivation of PAHs to toxic metabolites and is believed to play a central role in environmentally-induced cancers.
The specific aims of this proposal are: 1) Determine the role of the 8S and 4S PAH binding proteins in the PAH-induced expression of the rat P45OIAl gene in rat H4-II-E, mouse Hepa-1, human HepG2 and MCF-7 cells using heterologous gene fusion assays with the reporter gene chloramphenicol acetyltransferase; 2) Determine the location and consensus sequence of the PAH-responsive proximal cis-element in the 5'-flanking region of the rat P45OIAl gene using heterologous gene fusion assays; 3) Determine the role of the proximal Spl binding site in the PAH-induced expression of the rat P45OIAl gene in H4-II-E, Hepa-1, and HepG2 cells; 4) Further characterize the binding interactions of the purified rat 4S PAH binding protein with the proximal cis-element from the rat P45OIAl gene by gel mobility shift assays; 5) Further purify the rat 4S PAH binding protein by affinity chromatography utilizing oligonucleotides containing consensus binding sequences coupled to Sepharose and; 6) Prepare AHH deficient rat H4-II-E cell lines analogous to those prepared from mouse Hepa-1 cells. This proposal will initially examine the regulation of expression of the rat cytochrome P45OIA1 gene in mouse Hepa-1, rat H4-II-E, and human HepG2 and MCF-7 cell lines using heterologous gene fusion assays with the reporter gene chloramphenicol acetyltransferase. Competitive antagonists will be utilized to examine the relative role of the two PAH binding proteins in the regulation of the rat P45OIAI gene in these cells. The interaction of the 4S PAH binding protein with the rat P45OIA1 gene will be examined using gel mobility shift assays and purified rat 4S protein. Finally, an AHH deficient rat H4-II-E cell line will be prepared to isolate variants deficient in the 8S and 4S PAH binding proteins. These studies should contribute to our understanding of the complex regulation of this important enzyme.
