Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: Historical database and influence of technical aspects

Abstract

There is increased interest in the in vivo comet assay in rodents as a follow-up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin-treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays.

Evaluation of pH effects on genomic integrity in adipose-derived mesenchymal stem cells using the comet assay

“…Studies in which an automatic analysis system is used to determine DNA damage using the comet assay show limited parameters such as only the DNA percentage in the tail (Osipov et al, 2014) or the tail intensity (Priestley et al, 2010;Braz et al, 2011;Costa et al, 2011;Camargo et al, 2013;Valença-Silva et al, 2014), or this parameter is associated with others, such as tail moment and tail length, in the results (Grassi et al, 2007;Kasuba et al, 2012;Aydin et al, 2013;Pant et al, 2014;Timoroglu et al, 2014). However, there is still no consensus regarding the best parameter for MSCs or other biological samples.…”

Oxidative stress, cholinesterase activity, and DNA damage in the liver, whole blood, and plasma of Wistar rats following a 28-day exposure to glyphosate

“…Ethyl methanesulphonate (EMS) served as positive control, being a well-established genotoxicant recommended for in vivo comet assay in rodents (55), which was also dissolved in PBS.…”

Cell-based in vitro models in environmental toxicology: a review

“…Examples for genotoxic positive control test substances are 4-nitroquinoline 1-oxide (4NQO) [123] and ethyl methanesulfonate (EMS) [124], but there is no clear separation from effects observed by carcinogenic compounds, and a parallel loss of basal cell viability has also to be expected in such positive control cells. Carcinogenic substances used as positive controls in in vitro assays are TPA (12-O-tetradecanoylphorbol-13-acetate) [125,126], diethylnitrosamine (DEN) [127], methylnitronitrosoguanidine (MNNG or MNG) [128], benzo[a]pyrene [129] or methylcholanthrene [130].…”