Pretreatment of primary rat cutaneous epidermal keratinocyte culture with a low concentration of MNNG: effect on DNA cross-linking measured in situ after challenge with bis-2-chloroethyl sulfide.

Bis-2-chloroethyl sulfide- (BCES-) induced DNA cross-links in confluent, primary cultures of newborn rat cutaneous epidermal keratinocytes were detected using an assay that includes in situ unwinding of the DNA followed by separation of single-stranded DNA and double-stranded DNA (DSDNA) with hydroxylapatite. DNA cross-links in BCES-challenged cultures were inferred from increases in the percentage of DNA that remained double-stranded, compared with control cultures, after a 60-min alkaline unwinding incubation. The amount of DNA cross-linking after 5 or 10 microM BCES was increased when keratinocytes were first pretreated with 0.05 microM MNNG for 1 h at 8 a.m., 2 p.m., and 8 p.m. for two consecutive days and challenged with BCES the following morning. This increase was statistically significant (p less than .05, by ANOVA). For example, after 5 microM BCES challenge, cultures not pretreated with MNNG had 114.14% control DSDNA, whereas MNNG pretreated cultures had 122.78% control DSDNA. The level of BCES-induced cross-linking was maximal immediately after 30-min challenge and decreased during postchallenge incubation. At 24 and 48 h post 5, 10, or 20 microM BCES challenge, the level of DSDNA was actually depressed below unchallenged levels. This postchallenge decrease in the level of DSDNA, indicative of SSB in DNA, suggests repair activity by glycosylases and endonucleases. However, completion of repair (i.e., a return to control levels of DSDNA) was not seen in these experiments. The activity that resulted in decreases in the level of DSDNA during postchallenge incubation response was unaffected by MNNG pretreatment.