Error-Free Excision of the Cytotoxic, Mutagenic N2-Deoxyguanosine DNA Adduct Formed in Human Fibroblasts by (± )-7β ,8α -dihydroxy-9α ,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

The ability of normal diploid human fibroblasts and excision repair-deficient xeroderma pigmentosum cells (XP12BE, complementation group A) to excise potentially cytotoxic or mutagenic lesions induced in DNA by (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BzaP-diol epoxide) was determined. Large populations of cells were prevented from replicating by being grown to confluence; after 3 days they were exposed to tritiated BzaP-diol epoxide for 2 hr. One set of cultures was immediately released and assayed for the number of residues covalently bound to DNA, percent survival of colony-forming ability, and frequency of induced mutations. After various periods of time in confluence, other sets were similarly released and assayed. The normal cells exhibited a gradual increase in survival with time held in confluence (recovery from potentially cytotoxic lesions) which was directly correlated with a gradual loss of radioactivity from their DNA and a gradual decrease in the frequency of induced mutations. In contrast, no loss of radioactively labeled carcinogen from the DNA of the XP12BE cells could be detected during a 6-day period and their percent survival and frequency of induced mutations did not change. DNA from normal cells harvested immediately after treatment or after 2, 4, or 8 days in confluence was enzymatically hydrolyzed and analyzed by high-pressure liquid chromatograhy. Only a single peak was detected that cochromatographed with a standard prepared from deoxyguanosine treated with BzaP-diol epoxide. The kinetics of decrease of tritium label in this specific peak corresponded to the decrease in radioactivity of the total DNA with time and with the kinetics of recovery of the cells from the potentially cytotoxic and mutagenic effects of BzaP-diol epoxide. These results suggest that the N2-deoxyguanosine adduct is responsible for these biological effects and indicate that excision repair of this lesion by the normal human cells is "error free."