Inactivation of the thymidine kinase gene after in vitro modification with benzo(a)pyrene-diol-epoxide and transfer to LTK- cells as a eukaryotic test for carcinogens

Abstract

A recombinant plasmid containing the thymidine kinase (TK) gene (pAGO; 6.36 kilobases) was reacted in vitro with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, an ultimate carcinogenic metabolite of benzo(a)pyrene. The covalent binding of the metabolite to the circular forms of pAGO was visible by a drastic change in their mobility during agarose gel electrophoresis. The 4% modified DNA was only partially restricted by different endonucleases. Modification and limited restriction were correlated to the biological activity by transfer of the plasmid (TK gene), modified and unmodified, to TK-deficient cells. Upon transfection of mouse LTK- cells with modified plasmid or modified TK gene, no or only a few TK-positive cells were obtained, in contrast to the formation of many colonies after transfection with the unmodified plasmid (gene). Benzo(a)-pyrene itself and phenanthrene oxide, a weakly reactive but noncarcinogenic chemical, did not induce this effect. The reactive diol-epoxides of noncarcinogenic benzo(a)acridine and carcinogenic benzo(c)acridine showed a weaker but similar decreasing effect on the formation of TK+ clones. This inhibition of transformation efficiency suggests inactivation of the gene by chemical modification. Our experimental approach challenges the repair capacity of the eukaryotic cell and thus renders the strategy suitable not only as a eukaryotic test for carcinogens but also as a tool for the study of carcinogenesis as aberrant gene expression.