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

Abstract

The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor.

MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs

“…After careful evaluation, 3 control samples were excluded due to unusually high levels of DNA strand breaks and apoptotic cells. This is likely due to incorrect thawing procedure, as previously described in Jackson et al (2013). One belonged to the day 3 control group, the last 2 belonged to the 28 days group.…”

Surface modification does not influence the genotoxic and inflammatory effects of TiO2nanoparticles after pulmonary exposure by instillation in mice

“…Sample preparation and comet analysis has previously been described in detail (36). Briefly, BAL cells, lung or liver cell suspensions, were embedded in agarose (0.7% final concentration) on Trevigen 20-Well CometSlides™.…”

“…The day-to-day variation and electrophoresis efficiency was controlled by including phosphate-buffered saline exposed and 60 µM hydrogen peroxide-exposed A549 cells as negative and positive controls, respectively, on all comet slides. Control cells were exposed for 30min at 4°C as described (36). The day-to-day variation for the comet assay analysis on BAL, lung and liver tissue ( n = 19 for each tissue) were 19.3%, 13.1% and 16.7%, for TL, respectively.…”

Limited Link between Oxidative Stress and Ochratoxin A—Induced Renal Injury in an Acute Toxicity Rat Model

“…Frozen liver and kidney tissues were thawed at room temperature during preparation, placed in cylindrical sieves and immersed in 1.5 mL ice-cold Merchant’s medium [60]. Tissues were homogenized by moving a plunger up and down several times to press the tissue through the sieves.…”