Cheryl A. Hobbs

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

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 Hanks 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. Environ. Mol. Mutagen., 2012. Published 2011 Wiley Periodicals, Inc.

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

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.

Evaluation of the genotoxicity of the food additive, gum ghatti.

Gum ghatti is a food additive in some parts of the world, serving as an emulsifier, a stabilizer, and a thickening agent. To evaluate its genotoxic potential, we conducted Good Laboratory Practice compliant in vitro and in vivo studies in accordance with the Organisation for Economic Co-operation and Development (OECD) guidelines. No evidence of toxicity or mutagenicity was detected in a bacterial reverse mutation assay using five tester strains evaluating gum ghatti at up to 6 mg/plate, with or without metabolic activation. Gum ghatti also did not induce chromosome structural damage in a chromosome aberration assay using Chinese hamster ovary cells. To assess the ability to induce DNA damage in rodents, a combined micronucleus/Comet assay was conducted in male B6C3F1 mice. Gum ghatti was administered at 1000, 1500, and 2000 mg/kg/day by gavage once daily for 4 days and samples were collected 4h after the final dosing. No effect of gum ghatti was measured on micronucleated reticulocyte (MN-RET) frequency in peripheral blood, or DNA damage in blood leukocytes or liver as assessed by the Comet assay. Our results show no evidence of genotoxic potential of gum ghatti administered up to the maximum concentrations recommended by OECD guidelines.

No increases in biomarkers of genetic damage or pathological changes in heart and brain tissues in male rats administered methylphenidate hydrochloride (Ritalin) for 28 days.

Following a 2005 report of chromosomal damage in children with attention deficit/hyperactivity disorder (ADHD) who were treated with the commonly prescribed medication methylphenidate (MPH), numerous studies have been conducted to clarify the risk for MPH‐induced genetic damage. Although most of these studies reported no changes in genetic damage endpoints associated with exposure to MPH, one recent study (Andreazza et al. [ 2007 ]: Prog Neuropsychopharmacol Biol Psychiatry 31:1282–1288) reported an increase in DNA damage detected by the Comet assay in blood and brain cells of Wistar rats treated by intraperitoneal injection with 1, 2, or 10 mg/kg MPH; no increases in micronucleated lymphocyte frequencies were observed in these rats. To clarify these findings, we treated adult male Wistar Han rats with 0, 2, 10, or 25 mg/kg MPH by gavage once daily for 28 consecutive days and measured micronucleated reticulocyte (MN‐RET) frequencies in blood, and DNA damage in blood, brain, and liver cells 4 hr after final dosing. Flow cytometric evaluation of blood revealed no significant increases in MN‐RET. Comet assay evaluations of blood leukocytes and cells of the liver, as well as of the striatum, hippocampus, and frontal cortex of the brain showed no increases in DNA damage in MPH‐treated rats in any of the three treatment groups. Thus, the previously reported observations of DNA damage in blood and brain tissue of rats exposed to MPH for 28 days were not confirmed in this study. Additionally, no histopathological changes in brain or heart, or elevated serum biomarkers of cardiac injury were observed in these MPH‐exposed rats. Environ. Mol. Mutagen. 2010. Published 2009 Wiley‐Liss, Inc.

Interaction between DNA Polymerase β and BRCA1

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.

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

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.

Transcriptional profiling of male F344 rats suggests the involvement of calcium signaling in the mode of action of acrylamide-induced thyroid cancer

Acrylamide (AA) exposure in 2-year cancer bioassays leads to thyroid, but not liver, adenomas and adenocarcinomas in rats. Hypothesized modes of action (MOAs) include genotoxicity/mutagenicity, or thyroid hormone dysregulation. To examine the plausibility of these two or any alternative MOAs, RNA-sequencing was performed on the thyroids and livers of AA-exposed rats, in parallel with measurement of genotoxicity (blood micronucleus and Pig-a mutant frequency) and serum thyroid hormone levels, following the exposure of male Fischer 344/DuCrl rats to 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. Differentially expressed genes in both tissues provided marginal support for hormonal and genotoxic MOAs, which was consistent with negative/equivocal genotoxicity assay and marginal changes in thyroid hormone levels. Instead, there was a pronounced effect on calcium signaling/cytoskeletal genes in the thyroid. Benchmark dose modeling of RNA-sequencing data for the calcium signaling pathway suggests a point of departure (POD) of 0.68 mg/kg bw-day, which is consistent with a POD of 0.82 mg/kg bw-day derived from the thyroid 2-year cancer bioassay data. Overall, this study suggests a novel MOA for AA-induced thyroid carcinogenicity in male rats centered around perturbation of calcium signaling.

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

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. Environ. Mol. Mutagen. 2012.

Genetic and rat toxicity studies of cyclodextrin glucanotransferase

Highlights • Bacterial cyclodextrin glucanotransferase (CGTase) is used to produce a water soluble form of glycosylated isoquercitrin.• Genotoxicity battery on CGTase and sodium sulfate negative for mutations and DNA damage.• No evidence of systemic toxicity in 90-day rat toxicity study of CGTase.

Transcriptional profiling of male CD-1 mouse lungs and Harderian glands supports the involvement of calcium signaling in acrylamide-induced tumors

ABSTRACT Acrylamide (AA) exposure causes increased incidence of forestomach, lung, and Harderian gland tumors in male mice. One hypothesized mode of action (MOA) for AA‐carcinogenicity includes genotoxicity/mutagenicity as a key event, possibly resulting from AA metabolism to the direct genotoxic metabolite glycidamide. Alternatively, altered calcium signaling (CS) has been proposed as a central key event in the MOA. To examine the plausibility of these proposed MOAs, RNA‐sequencing was performed on tumor target tissues: Harderian glands (the most sensitive tumor target tissue in the rodent 2‐year cancer bioassay) and lungs of AA‐exposed male CD‐1 mice. Animals were exposed to 0.0, 1.5, 3.0, 6.0, 12.0, or 24.0 mg AA/kg bw‐day in drinking water for 5, 15, or 31 days. We observed a pronounced effect on genes involved in CS and cytoskeletal processes in both tissues, but no evidence supporting a genotoxic MOA. Benchmark dose modeling suggests transcriptional points of departure (PODs) of 0.54 and 2.21 mg/kg bw‐day for the Harderian glands and lungs, respectively. These are concordant with PODs of 0.17 and 1.27 mg/kg bw‐day derived from the cancer bioassay data for these tissues in male mice, respectively. Overall, this study supports the involvement of CS in AA‐induced mouse carcinogenicity, which is consistent with a recently proposed CS‐based MOA in rat thyroid, and with other published reports of aberrant CS in malignant tumors in rodents and humans. HighlightsAcrylamide (AA) causes cancer in mouse Harderian glands and lungs.The carcinogenic mode of action (MOA) of AA is poorly understood.Male mice were dosed with 5 doses of AA in drinking water daily for up to 30 days.RNA‐seq on cancer target organs revealed alterations in calcium signaling (CS) genes.The finding is consistent with rat data, suggesting a role for CS in AAs MOA.