Comparison of Comet assay dose‐response for ethyl methanesulfonate using freshly prepared versus cryopreserved tissues

Abstract

The National Toxicology Program (NTP) is using the Comet assay to evaluate genotoxic potential, and is investigating the integration of this assay into repeat-dose toxicity studies. To reduce sample-to-sample variability, address logistical concerns associated with evaluating multiple tissues from many animals, and accommodate sample collection at geographically distant testing facilities, tissue samples collected for Comet analysis by the NTP are routinely flash-frozen in liquid nitrogen and stored in a -80°C freezer until evaluation. To compare data obtained from frozen tissues to data from freshly isolated tissues, we conducted a dose-response study in male Sprague Dawley rats. Rats (5 per treatment group) were administered ethyl methanesulfonate (EMS; 0, 25, 50, 100, or 200 mg/kg) by gavage twice at an interval of 21 hr; blood, liver, stomach, and colon tissues were harvested 3 hr after the second treatment. Single-cell preparations from each of the four tissues were put into Hank's balanced salt solution with 10% fresh dimethyl sulfoxide. One aliquot of each tissue preparation was used for immediate analysis, while additional aliquots were flash-frozen in liquid nitrogen and stored in a -80°C freezer for 1 or 8 weeks. One set of 8-week frozen samples was shipped roundtrip via air courier from Research Triangle Park, NC to Rochester, NY prior to analysis. For all four tissues, results from frozen, nontransported samples showed a similar dose-response pattern for EMS-induced genotoxicity. We also demonstrated that for three tissues (blood, liver, stomach), air transport did not alter the sensitivity of the Comet assay for detecting DNA damage.

Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure

“…Slides were prepared and analyzed as described previously (Hobbs et al ; Recio et al ) with some modifications. In a laboratory with controlled humidity (≤60%), samples were thawed on ice and a portion of the cell suspension was diluted with 0.5% low melting point agarose (Lonza, Walkersville, MD) dissolved in Dulbecco's phosphate buffer (Ca +2 , Mg +2 , and phenol free) at 37°C and layered onto each well of a 2‐well CometSlide™ (Trevigen, Gaithersburg, MD).…”

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

“…Except for stomach, the fold increase in % tail DNA values was generally lower in cell suspensions that were frozen before slide processing; however, the increase was always statistically significant. Although our results are from a limited number of studies, others have investigated the effect of freezing cell suspensions and subsequent transportation by air courier (Recio et al, 2012). Consistent with our results, they observed that the % tail DNA in liver, blood and stomach from control and EMS-treated rats was increased relative to tissues processed immediately by freezing the cells for 1-8 weeks prior to slide preparation but the level was still within the negative control background 1-8% recommended by JaCVAM.…”

Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay

“…Cells frozen with DMSO (blood cells or cell lines) have previously been used for comet assay validation (2,7–15). Effect of freezing tissues as cell suspensions frozen in 10% DMSO was investigated using rats exposed by repeated oral gavage to ethyl methanesulphonate (16). DNA strand break levels in fresh and frozen blood, liver and stomach samples were similar at all exposure levels collected 3h after exposure, even in tissues that had undergone transportation.…”