Inhibition by 6-Mercaptopurine of the Binding of a Benzo(a)pyrene Diol-Epoxide to DNA in Chinese Hamster Ovary Cells
Abstract
The finding that 7 r ,8 t -dihydroxy-9,10 t -oxy-7,8,9,10-tetrahydrobenzo( a )pyrene (BPDE-I) is stabilized against hydrolysis by binding to cellular membranes suggested that nucleophilic compounds which would colocalize with BPDE-I in membranes might inhibit the deleterous biological effects of BPDE-I. We have explored the possibility that hydrophobic, sulfhydryl-containing compounds might provide such inhibition using the binding of BPDE-I to DNA in Chinese hamster ovary cells as a biological end point. Of several such compounds tested, 6-mercaptopurine (6-MP) was the most potent, exhibiting 50% inhibition of BPDE-I:DNA binding at about 30 µ M and about 95% inhibition at 500 µ M . 6-MP, at concentrations of 30 µ M or greater, was also effective in preventing the induction of mutations by BPDE-I at the aprt locus. By varying the time of addition of the two compounds, it was shown that the action of 6-MP is intracellular. In vitro , 6-MP readily forms an adduct with BPDE-I, and the same adduct is found as a major metabolite in cells treated with BPDE-I and 6-MP. These findings are consistent with the hypothesis that 6-MP and BPDE-I colocalize in membranes of Chinese hamster ovary cells and form a covalent adduct, thus preventing the BPDE-I from interacting with critical cellular macromolecules such as DNA. Several nontoxic derivatives of 6-MP (9-methyl-6-MP, 2,6-dithiopurine) or analogues of 6-MP (4-mercapto-1 H -pyrazolo3,4- d pyrimidine) were also tested in the Chinese hamster ovary cell system and found to inhibit binding of BPDE-I to DNA with potencies comparable to that of 6-MP. 1 This research was supported by Grant ES-03602 from the National Institute of Environmental and Health Sciences, by Grant SIG-14 from the American Cancer Society, and by a grant from the Kleberg Foundation. 2 To whom requests for reprints should be addressed.