Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts

Abstract

A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B1 dichloride, a model for the reactive 2,3-epoxide of aflatoxin B1. Exponentially growing cells were exposed to agents and assayed for mutations (induction of 6-thioguanine resistance) and cell killing (loss of colony-forming ability). Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. Density-inhibited cultures were exposed to cytotoxic and mutagenic doses. Immediately after treatment, or after various lengths of time in confluence, the cells were harvested and analyzed for the number of lesions remaining in DNA. A portion was plated at lower densities and assayed for mutations and/or for survival. In several instances, the adducts were analyzed by high-pressure liquid chromatography. As an alternative approach, cells were synchronized and treated at various times prior to the onset of DNA synthesis and analyzed for survival and/or the frequency of mutations. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and the frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation (to anchorage independence) of normal and repair-deficient cells showed this also to be true for transformation.