Processing of DNA damage induced by hydrogen peroxide and methyl methanesulfonate in human lymphocytes: analysis by alkaline single cell gel electrophoresis and cytogenetic methods

The persistence of induced DNA damage in human lymphocytes after mitogen st imulation and its relationship to subsequent cytogenetic alterations were i nvestigated. The analysis of single-strand breaks and alkali-labile sites b y single cell gel electrophoresis (SCGE) showed the almost complete repair of damage induced in resting lymphocytes by methyl methanesulfonate (MMS, 1 40-210 mu M) and hydrogen peroxide (H2O2, 25-100 mu M) during the first 16 h of culture. On the other hand, DNA damage was shown to persist to a large extent when cells were cultured in the presence of the repair inhibitor cy tosine beta-D-arabinofuranoside (Ara-C) (1 mu g/ml). Although highly effect ive in the induction of DNA lesions detectable by SCGE, both agents failed to significantly increase the rate of micronucleus formation in cytokinesis -blocked cells harvested 66 h after treatment. However, when Ara-C was pres ent during the first 16 h of culture, micronuclei were significantly increa sed at all doses. Conversely, sister chromatid exchange (SCE) rates were in creased by chemical treatments to a higher extent in cultures without Ara-C , Delayed treatments, 16 h after mitogen stimulation, led to a significant induction of micronuclei in the case of MMS but not with H2O2. These result s suggest that only a minor fraction of DNA damage induced in resting lymph ocytes is available for fixation through misreplication, because of its eff ective repair prior to S phase. However, the processing of damage through r ecombination pathways can lead to increased SCE rates in treated cells, The se features of the processing of DNA damage in human lymphocytes should be taken into account when structural cytogenetic alterations in cultured lymp hocytes are used in monitoring human exposure to genotoxic agents.