Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

Evaluation of the Pig-a, micronucleus, and comet assay endpoints in a 28-day study with ethyl methanesulfonate.

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig-a mutation assay and compared with other well-established in vivo genotoxicity endpoints. Male Sprague-Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre-dose (Day 1) and at various time points up to Day 105. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RET(CD59-) on Day 29 and of RBC(CD59-) on Day 55. The lowest dose producing a statistically significant increase of RET(CD59-) was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC(CD59-) on Day 55. EMS also induced significant increases in % micronucleated RETs (MN-RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBC(CD59-) and/or RET(CD59-) in the Pig-a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints.

Development and validation of an in vitro micronucleus assay platform in TK6 cells.

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.

Differentiating mechanisms of toxicity using global gene expression analysis in Saccharomyces cerevisiae.

Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. Genomic approaches, which monitor gene expressions across large numbers of genes, can serve as a powerful tool for exploring mechanisms of toxicity. Here, using five different agents, we investigated whether the analysis of genome-wide expression profiles in Saccharomyces cerevisiae could provide insights into mechanisms of genotoxicity versus cytotoxicity. To differentiate the genotoxic stress-associated expression signatures from that of a general cytotoxic stress, we compared gene expression profiles following the treatment with DNA-reactive (cisplatin, MMS, bleomycin) and DNA non-reactive (ethanol and sodium chloride) compounds. Although each of the tested chemicals produced a distinct gene expression profile, we were able to identify a gene expression signature consisting of a relatively small number of biologically relevant genes capable of differentiating genotoxic and cytotoxic stress. The gene set includes such upregulated genes as HUG1, ECM4 and previously uncharacterized gene, YLR297W in the genotoxic and GAP1, CGR1 in the cytotoxic group. Our results indicate the potential of gene expression profile analysis for elucidating mechanism of action of genotoxic agents.

Differentiation of DNA reactive and non-reactive genotoxic mechanisms using gene expression profile analysis.

Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. Here, we investigated whether gene expression profiles can differentiate between DNA reactive and DNA non-reactive mechanisms of genotoxicity. We analyzed gene expression profiles and micronucleus levels in L5178Y cells treated with cisplatin and sodium chloride. The assessment of cisplatin genotoxicity (up to six-fold increase in the number of micronuclei) and gene expression profile (increased expression of genotoxic stress-associated genes) was in agreement with cisplatin mode of action as a DNA adduct-forming agent. The gene expression profile analysis of cisplatin-treated cells identified a number of genes with robust up regulation of mRNA expression including genes associated with DNA damage (i.e. members of GADD45 family), early response (i.e. cFOS), and heat shock protein (i.e. HSP40 homologue). The gene expression changes correlated well with DNA damage as measured by DNA-protein crosslinks and platinum-DNA binding. To differentiate the genotoxic stress-associated expression profile of cisplatin from a general toxic stress, we have compared the gene expression profile of cisplatin-treated cells to cells treated with sodium chloride, which causes osmotic shock and cell lysis. Although the sodium chloride treatment caused a two-fold induction of micronuclei, the gene expression profile at equitoxic concentrations was remarkably distinct from the profile observed with cisplatin. The profile of sodium chloride featured a complete lack of expression changes in genes associated with DNA damage and repair. In summary, the gene expression profiles clearly distinguished between DNA reactive and non-reactive genotoxic mechanisms of cisplatin and sodium chloride. Our results suggest the potential utility of gene expression profile analysis for elucidating mechanism of action of genotoxic agents.