REPRODUCIBILITY OF BASAL AND INDUCED DNA SINGLE-STRAND BREAKS DETECTED BY THE SINGLE-CELL GEL-ELECTROPHORESIS ASSAY IN HUMAN PERIPHERAL MONONUCLEAR LEUKOCYTES

The aim of the reported study was to investigate the reproducibility o f the single-cell gel electrophoresis (SCGE) assay in the determinatio n of DNA single-strand breaks (SSBs) and to estimate the statistical r equirements when the SCGE assay is used for the detection of genotoxic ity in humans. In human peripheral mononuclear leukocytes (PMLs), we r epeatedly measured the rate of SSBs after in vitro incubation of cells for 1 h at 4 degrees C in phosphate buffered saline (PBS, basal) or 1 0 mu M or 50 mu M H2O2 (induced). Intra-assay variation was determined from cryopreserved PMLs of a single donor. To assess intrasubject and intersubject variation, PMLs of ten healthy, nonsmoking subjects (age d 19-37 years) were tested 5-9 times. Cryopreserved cells revealed a m ean coefficient of variation of 18% (PBS) and 7%-9% (H2O2). There were statistically significant differences between individuals in the rate of SSBs after incubation in PBS (P < 0.01), 10 mu M H2O2 (P < 0.001), and 50 mu M H2O2 (P < 0.001). The range of interindividual variabilit y was 26% for basal and 12%-13% for induced SSBs, and the coefficient of intra-individual variation was 18%-72% (PBS) and 7%-23% (H2O2). Nei ther basal nor induced rates of DNA damage were related to gender or a ge. Estimates of the minimum detectable effects were based on these ob served sources of variability (power 90%, level of significance 5%, as sumed sample size 50). With two different groups, a difference of 31% in basal SSBs or 12% in induced SSBs would be detectable. Repeated mea surement within one group could detect a difference of 26% in basal an d 9% in induced SSBs. In summary, the SCGE assay appears to be suitabl e for the detection of single-strand breaks, e.g., in biomonitoring or environmental medicine, and the statistical requirements could be der ived from our analysis of the sources of variability.