DNA repair in primary human keratinocyte cultures after low level exposureto bis(2-chloroethyl)sulfide

The literature has reported the appearance and disappearance of single-stra nd breaks (SSBs) in the DNA of rat keratinocytes after exposure to low leve ls of bis(2-chloroethyl) sulfide (BCES). Since SSBs are a consequence of de purination or depyrimidination followed by excision of the apurinic or apyr imidinic site and deoxyguanosine (GdR) is the major alkylation site in DNA exposed to BCES, it was hypothesized that repair occurred by a GdR-specific base replacement and not by large section repair. To test this hypothesis, cultures of human keratinocytes (HK) were preincubated with 5-bromo-2'-deo xyuridine (BUdR), a heavy analog of thymidine (TdR) incorporated into repli cating DNA, immediately before exposure to BCES. Cultures were incubated po stexposure with BUdR, radiolabeled GdR, and/or deoxyadenosine (AdR), to mea sure base-specific repair, and/or radiolabeled TdR, to measure DNA replicat ion and large section repair. A CsCl density gradient was used to remove an y BUdR-containing postexposure DNA replication. Each gradient was assayed f or radioactivity (cpm) and DNA content (absorbance at 260 nm). The peak A(2 60) fractions were pooled and rebanded in another CsCl gradient, if DNA rep air had occurred, the specific activity (cpm/A(260)) of the peak A(260) fra ction in the gradient would be greater than control. After exposure of the cultures to BCES, there was a concentration-dependent increase in the speci fic activity for [H-3]GdR but not [C-14]TdR over the concentration range us ed (20-50 mu M BCES). A concentration-dependent increase in specific activi ty was also detected after [C-14]AdR exposure. The literature has also repo rted that the removal of damaged DNA bases after alkylation is via glycosyl ases. In this series of experiments, we have demonstrated that cultures of HK exposed to the alkylating agent BCES repair their damaged DNA by the rep lacement of the damaged base only. In the case of BCES exposure, it is the GdR base and to a lesser extent the AdR base.