Human malignancies are believed to be caused in a large part by exposure of cells to mutagenic agents, resulting in genetic changes that lead to abnormal growth control. The problem of chemical carcinogenesis has been attacked mainly from the environmental perspective; however, naturally occurring carcinogens are present in the diet, and are also produced endogenously during oxidative metabolism. An alternative approach to cancer prevention is to find ways to enhance natural cellular protective mechanisms that enzymatically detoxify carcinogenic mutagens. Transgenic cell lines and their isogenic (empty vector-transfected) controls will allow measurement of the antimutagenic activity of cloned genes directly, quantitatively, and unambiguously. Several phase II detoxifying enzymes are consistently induced by cancer chemopreventive agents (e.g. oltipraz), including glutathione S-transferases (GST). The GST isozyme family has the ability to detoxify electrophilic mutagens by several mechanisms, including conjugation with the thiol tripeptide glutathione (GSH). The many isozymes of GST have distinct substrate specificities, and can conjugate numerous mutagens and carcinogens in vitro and in vivo. However, the sufficiency of GST expression for protection against DNA damage and mutagenesis has not previously been directly tested. The goal of this project is to utilize transgenic model cell lines for assessing the potential role of glutathione transferases in protection against chemical toxicity and mutagenesis. We have obtained the first direct evidence that GST isozymes can function effectively in situ to prevent or reduce alkylation, DNA fragmentation, and induction of sister- chromatid exchanges (SCEs) by the carcinogen 4-nitroquinoline-1-oxide (NQO) and covalent modification of DNA by benzo[a]pyrene diol-epoxide.
Specific aims : 1) to assess the effect of GST expression on DNA damage by known mutagens, in MCF-7 cells transfected with GST expression vectors; measured indices will include alkylation of macromolecules by [3H]-carcinogens; induction of DNA strand scission as measured by the alkaline elution method; and induction of SCEs; P 2) transfection of these GST expression vectors into the V79 mutagenicity tester cell line, and into V79 clones previously transfected with cytochrome P-450 isozymes (which activate benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1)); induction of selectable mutations by B[a]P or AFB1 in the hprt gene will be quantitated; 3) to conduct in vitro GST enzymology studies with the electrophiles studied; and 4) to assess protection against toxic and mutagenic products of lipid peroxidation in cell lines transfected with GST isozymes, including mGST 5.7. Mutagens studied initially will be: 1) nitroquinoline oxide; 2) aflatoxin B1; 3) benzo[a]pyrene anti -diol- epoxide; and 4) 4-hydroxyalkenals, all of which are GST substrates. Class-specific GST inhibitors will confirm the role of GST. Improved understanding of the roles of specific gene products in prevention of genetic damage will promote the development of strategies for reduction of human cancer risk, e.g. by dietary or pharmacologic modulation of gene expression, and may aid in estimation of individual relative risk of exposure to certain agents.
