The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders

Abstract

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.

Trial registration: ClinicalTrials.gov NCT02231736.

Keywords: Biomarkers; Comet assay; DNA damage; Human biomonitoring; Pooled analysis.

Fig. 1.

Geographical distribution of the 26 countries contributing data to the hCOMET dataset (dark grey).

Fig. 2.

Frequency (median and interquartile distance) of DNA damage measured in the whole dataset for all endpoints. 1a) Tail length; 1b) Tail intensity; 1c) Tail moment; 1d) Visual Scoring. Reference lines corresponds to the overall median values for the specific endpoint. Overall median and interquartile interval is reported with whole and dotted lines, respectively in Figure 1a, 1b, and 1c. All statistics shown in this table refers to unexposed cases only.

Fig. 3.

Estimated variation of Tail intensity by sex. Mean ratios (MR), i.e., the ratio of mean DNA values of males to the mean values of females, with 95 % confidence intervals (95 % CI) are reported for individual studies and for the whole dataset.

Fig. 4.

Estimated variation of Tail intensity for study groups as compared to unexposed controls. Estimates for individual study and for the whole dataset. Mean ratios (MR), i.e., the ratio of mean DNA values of subjects exposed to the mean values of unexposed controls, with 95 % confidence intervals (95 % CI) are reported for individual studies and for the whole dataset.