Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Toxicology Program, ILS, Inc.

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.

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

Styrene-acrylonitrile Trimer (SAN Trimer), a by-product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2-year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN-RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose-related increases (P < 0.0001) in MN-RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical-related bone marrow toxicity. Results of the Comet assay showed significant, dose-related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical-related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer.

Genotoxicity of styrene-acrylonitrile trimer in brain, liver, and blood cells of weanling F344 rats.

Myricitrin, a flavonoid extracted from the fruit, leaves, and bark of Chinese bayberry (Myrica rubra SIEBOLD), is currently used as a flavor modifier in snack foods, dairy products, and beverages in Japan. Myricitrin is converted to myricetin by intestinal microflora; myricetin also occurs ubiquitously in plants and is consumed in fruits, vegetables, and beverages. The genotoxic potential of myricitrin and myricetin was evaluated in anticipation of worldwide marketing of food products containing myricitrin. In a bacterial reverse mutation assay, myricetin tested positive for frameshift mutations under metabolic activation conditions whereas myricitrin tested negative for mutagenic potential. Both myricitrin and myricetin induced micronuclei formation in human TK6 lymphoblastoid cells under conditions lacking metabolic activation; however, the negative response observed in the presence of metabolic activation suggests that rat liver S9 homogenate may detoxify reactive metabolites of these chemicals in mammalian cells. In 3-day combined micronucleus/Comet assays using male and female B6C3F1 mice, no induction of micronuclei was observed in peripheral blood, or conclusive evidence of damage detected in the liver, glandular stomach, or duodenum following exposure to myricitrin or myricetin. Our studies did not reveal evidence of genotoxic potential of myricitrin in vivo, supporting its safe use in food and beverages.

Genotoxicity evaluation of the flavonoid, myricitrin, and its aglycone, myricetin.

As a part of an international validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals. 

Comet assay evaluation of six chemicals of known genotoxic potential in rats.

Perillaldehyde, a natural monocyclic terpenoid found most abundantly in the herb perilla, has a long history of use as a flavouring ingredient to add spiciness and citrus taste to foods. Previously, it was judged to be safe by several international expert panels. To confirm the safety of flavourings placed on the European Union list of flavourings, perillaldehyde was selected by the European Food Safety Authority as a representative of a subgroup of alicyclic aldehyde flavouring substances to be evaluated for genotoxic potential. Perillaldehyde was tested in a bacterial reverse mutation assay, an in vitro micronucleus assay in human lymphocytes, an HPRT assay in mouse lymphoma cells, and a micronucleus/comet assay in Han Wistar rats. In contrast to previously published results, perillaldehyde induced mutation in Salmonella typhimurium strain TA98 in the absence of metabolic activation. The comet assay was negative for duodenum and weakly positive for liver but only at a hepatotoxic dose of perillaldehyde. All other genotoxicity assays were negative. These data do not provide an indication of any genotoxic potential for perillaldehyde, and they provide the primary basis for recent scientific opinions regarding perillaldehyde genotoxicity announced by several international organizations responsible for safety assessment of food additives and flavourings.

Genotoxicity assessment of the flavouring agent, perillaldehyde.

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products.

Cheryl A. Hobbs

Toxicology Program, ILS, Inc.

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage