Reproducibility of basal and induced DNA single-strand breaks detected by the single-cell gel electrophoresis assay in human peripheral mononuclear leukocytes

Abstract

The aim of the reported study was to investigate the reproducibility of the single-cell gel electrophoresis (SCGE) assay in the determination of DNA single-strand breaks (SSBs) and to estimate the statistical requirements when the SCGE assay is used for the detection of genotoxicity in humans. In human peripheral mononuclear leukocytes (PMLs), we repeatedly 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 10 microM or 50 microM 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 (aged 19-37 years) were tested 5-9 times. Cryopreserved cells revealed a mean 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 microM H2O2 (P < 0.001), and 50 microM H2O2 (P < 0.001). The range of interindividual variability was 26% for basal and 12%-13% for induced SSBs, and the coefficient of intraindividual variation was 18%-72% (PBS) and 7%-23% (H2O2). Neither basal nor induced rates of DNA damage were related to gender or age. Estimates of the minimum detectable effects were based on these observed sources of variability (power 90%, level of significance 5%, assumed sample size 50). With two different groups, a difference of 31% in basal SSBs or 12% in induced SSBs would be detectable. Repeated measurement within one group could detect a difference of 26% in basal and 9% in induced SSBs. In summary, the SCGE assay appears to be suitable for the detection of single-strand breaks, e.g., in biomonitoring or environmental medicine, and the statistical requirements could be derived from our analysis of the sources of variability.

Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay

“…Increased DNA damage by older age as detected by the comet assay was also reported in other studies [46][47][48][49][50][51]. In contrast, there are studies that showed no effect of age on the level of DNA damage [52][53][54][55][56][57][58]. As the children in our study were in the similar age range spreading over only ten years it was difficult to determine if there is an actual age-difference in DNA damage among them.…”

Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N‐Nitroso compound formation attributed to red meat consumption

“…The alkaline Comet assay is a sensitive technique that enables the detection of DNA strand breaks and alkalilabile sites in individual cells [Fairbairn et al, 1995;Holz et al, 1995]. Previous studies carried out using this technique showed that the known NOC carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was genotoxic in human colon cells [Pool-Zobel and Leucht, 1997].…”

Answer to comments by A. Lerchl on “Radiofrequency electromagnetic fields (UMTS, 1,950 MHz) induce genotoxic effects in vitro in human fibroblasts but not in lymphocytes” published by C. Schwarz et al. 2008

“…He does not present own comet assay results and has not published any, instead he refers to a CV ''in the order of 30 and 40%'' published by Speit et al (2007), obtained by densitometric evaluation of 50 cells per assay. In contrast, published data from others gave an intra-assay variability of 4.1-21.3% (Holz et al 1995), or 0.5-4% (Hughes et al 1997). In addition, the low CV's published by Schwarz et al are not ''in sharp contrast to previously published results''.…”