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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 in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.

Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol.

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.

The dose-response relationship for the induction of micronuclei (MN) and the impact of glutathione (GSH) detoxication on naphthalene-induced cytotoxicity and genotoxicity were investigated in human TK6 cells. TK6 cells were exposed to 10 concentrations ranging from 0.0625 to 30μM naphthalene in the presence of β-naphthoflavone- and phenobarbital (βNP/PB)-induced rat liver S9 with a nicotinamide adenine dinucleotide phosphate-generating system. Three approaches were used to identify a no-observed-effect level (NOEL) for naphthalene-induced genotoxicity: (1) laboratory criteria of ≥ twofold increase over the concurrent solvent controls (NOEL = 10μM), (2) ANOVA with Bonferroni correction (NOEL = 2.5μM), and (3) the benchmark dose approach (BMCL(10) = 3.35μM). The NOEL and point of departure micronucleus frequency for naphthalene-induced MN are between the tested naphthalene concentrations of 2.5-10.0μM in this experimental system. Supplementation of the exposure system with physiological relevant concentrations of 5mM GSH eliminated naphthalene-induced cytotoxicity and genotoxicity; no increased cytotoxicity or genotoxicity was observed at concentrations of up to 500μM naphthalene in the presence of GSH compared with 2.5-10.0μM in the absence of GSH. Naphthalene bioactivation by βNP/PB-induced rat liver S9 exhibits a nonlinear dose-response for the induction of MN in TK6 cells with a NOEL of 2.5-10μM that in the presence of GSH is shifted upward greater than 50- to 200-fold. These data demonstrate a nonlinear dose-response for naphthalene-induced genotoxicity that is eliminated by GSH, and both observations should be considered when assessing human risk from naphthalene exposures.

Dose-response assessment of naphthalene-induced genotoxicity and glutathione detoxication in human TK6 lymphoblasts.

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.

Furan is a chemical hepatocarcinogen in mice and rats. Its previously postulated cancer mode of action (MOA) is chronic cytotoxicity followed by sustained regenerative proliferation; however, its molecular basis is unknown. To this end, we conducted toxicogenomic analysis of B3C6F1 mouse livers following three week exposures to non-carcinogenic (0, 1, 2mg/kgbw) or carcinogenic (4 and 8mg/kgbw) doses of furan. We saw enrichment for pathways responsible for cytotoxicity: stress-activated protein kinase (SAPK) and death receptor (DR5 and TNF-alpha) signaling, and proliferation: extracellular signal-regulated kinases (ERKs) and TNF-alpha. We also noted the involvement of NF-kappaB and c-Jun in response to furan, which are genes that are known to be required for liver regeneration. Furan metabolism by CYP2E1 produces cis-2-butene-1,4-dial (BDA), which is required for ensuing cytotoxicity and oxidative stress. NRF2 is a master regulator of gene expression during oxidative stress and we suggest that chronic NFR2 activity and chronic inflammation may represent critical transition events between the adaptive (regeneration) and adverse (cancer) outcomes. Another objective of this study was to demonstrate the applicability of toxicogenomics data in quantitative risk assessment. We modeled benchmark doses for our transcriptional data and previously published cancer data, and observed consistency between the two. Margin of exposure values for both transcriptional and cancer endpoints were also similar. In conclusion, using furan as a case study we have demonstrated the value of toxicogenomics data in elucidating dose-dependent MOA transitions and in quantitative risk assessment.

Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan.

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.

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.

Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

Modern testing paradigms seek to apply human-relevant cell culture models and integrate data from multiple test systems to accurately inform potential hazards and modes of action for chemical toxicology. In genetic toxicology, the use of metabolically competent human hepatocyte cell culture models provides clear advantages over other more commonly used cell lines that require the use of external metabolic activation systems, such as rat liver S9. HepaRG™ cells are metabolically competent cells that express Phase I and II metabolic enzymes and differentiate into mature hepatocyte-like cells, making them ideal for toxicity testing. We assessed the performance of the flow cytometry in vitro micronucleus (MN) test and the TGx-DDI transcriptomic biomarker to detect DNA damage-inducing (DDI) chemicals in human HepaRG™ cells after a 3-day repeat exposure. The biomarker, developed for use in human TK6 cells, is a panel of 64 genes that accurately classifies chemicals as DDI or non-DDI. Herein, the TGx-DDI biomarker was analyzed by Ion AmpliSeq whole transcriptome sequencing to assess its classification accuracy using this more modern gene expression technology as a secondary objective.

Leslie Recio

Toxicology Program, ILS, Inc.

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