Mutagenesis, Vol. 14, No. 5, 497-504,
September 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Processing of DNA damage induced by hydrogen peroxide and methyl methanesulfonate in human lymphocytes: analysis by alkaline single cell gel electrophoresis and cytogenetic methods
Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Roma, Italy
The persistence of induced DNA damage in human lymphocytes after mitogen stimulation and its relationship to subsequent cytogenetic alterations were investigated. The analysis of single-strand breaks and alkali-labile sites by single cell gel electrophoresis (SCGE) showed the almost complete repair of damage induced in resting lymphocytes by methyl methanesulfonate (MMS, 140–210 µM) and hydrogen peroxide (H2O2, 25–100 µM) 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 cytosine ß-D-arabinofuranoside (Ara-C) (1 µg/ml). Although highly effective 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 present during the first 16 h of culture, micronuclei were significantly increased at all doses. Conversely, sister chromatid exchange (SCE) rates were increased 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 results suggest that only a minor fraction of DNA damage induced in resting lymphocytes is available for fixation through misreplication, because of its effective repair prior to S phase. However, the processing of damage through recombination pathways can lead to increased SCE rates in treated cells. These features of the processing of DNA damage in human lymphocytes should be taken into account when structural cytogenetic alterations in cultured lymphocytes are used in monitoring human exposure to genotoxic agents.
1 To whom correspondence should be addressed. Tel: +39 06 49902840; Fax: +39 06 49387139; Email: crebelli{at}iss.it
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