Research in this lab and others has provided evidence for an important role for expression of glutathione S-transferases (GSTs) (Phase II detoxification) in normal cellular defenses against reactive electrophilic metabolites of carcinogens, and as part of the biological response induced by chemopreventive agents. We have employed a transgenic cell modeling approach to examine the specific contributions of GSTs expressed individually and in combination with relevant genes, including members of the cytochrome P450 (Phase I activation) and the multidrug resistance protein (Phase III efflux) multigene families. We have found that protection against cytotoxicity and/or genotoxicity (DNA adducts or mutagenesis) can be quite different, depending in some cases primarily on GST characteristics and expression level, or in other cases on the nature of the carcinogen, whether it requires activation, and the P450 activation enzyme co-expressed with the GST. We propose to continue these investigations with our existing single-and dual-transfected V79 cell lines that stably express human GSTP1, GSTM1, or GSTA1, alone and also in combination with human P450-1A1 or-1B1, with addition of -1A2. We will focus on a limited set of polycyclic aromatic hydrocarbon (PAH) substrates for these P450s and GSTs, Benzo[a]Pyrene (and its two 7,8-dihydrodiol enantiomers); the more potent DiBenzo[a,I]Pyrene (and its intermediate 11,12-diol metabolites); and 5-methylchrysene. We will also examine metabolism and toxicities of estradiol, reportedly activated by these CYP isozymes and detoxified by hGSTP1, in these cell lines.
In Aim #1 we will examine the unique metabolic interactions between each P450 and GST combination, with both cytotoxicity/apoptosis, DNA adducts, and mutagenicity as endpoints.
Aim # 2 will model cell-cell interactions, asking whether binary mixtures of these cell lines show simple additivity, or synergy or antagonism in the presence of PAHs (e.g. via exchange of stable intermediates), and if GST expression is more effective in concert with one or the other P450 in the two different cell lines co-cultured.
Aim #3 will determine the mechanisms that underlie the large differences observed in GST protection against the cytotoxic vs. genotoxic effects of certain carcinogens.
In Aim #4 we will determine the mechanism for the intriguing observation that expression of GSTs with high efficiency for 4-nitroquinoline oxide or 4-hydroxynonenal conjugation confer paradoxical sensitivity instead of protection. These studies will enhance our knowledge of the factors that govern chemoprotective functions of GSTs.
