RESUMO
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.
Assuntos
Antineoplásicos/toxicidade , Testes para Micronúcleos/métodos , Mutagênicos/toxicidade , Aneugênicos/toxicidade , Biotransformação , Linhagem Celular , Guias como Assunto , HumanosRESUMO
The in vitro micronucleus assay has been extensively used as an in vitro screening tool to identify test articles that might have aneugenic or clastogenic potential. Currently, the Organization for Economic Co-operation and Development (OECD) is working towards a final version of the guideline for the conduct of the in vitro mammalian cell micronucleus Test (MNvit). A few questions regarding appropriate cytotoxicity measurements and cytotoxicity limits to use remain to be answered. In order to resolve the remaining questions, we compared the induction of micronuclei at the top dose (50-60% cytotoxicity) determined by either Relative Cell Counts (RCC), Relative Increase in Cell Counts (RICC), Relative Population Doublings (RPD), or Cytokinesis-Blocked Proliferating Index (CBPI) using weak and strong inducers of micronuclei in both the presence and absence of cytochalasin B (CYB) in Chinese hamster ovary (CHO) and human lymphoblastoid TK6 cells. In order to assess extensive dose-response relationships, we selected expected weak (diazepam, phenolphthalein, quinacrine dihydrochloride dihydrate) and strong (cytosine arabinoside, mitomycin C, vinblastine sulphate) inducers of micronuclei with a variety of different mechanisms of action for testing. The results clearly demonstrated that all six compounds produced positive responses using either cytotoxicity measurement. The outcome from these studies further supports the cytotoxicity measurements and cytotoxicity limits proposed in the draft OECD guideline.
Assuntos
Citotoxinas/toxicidade , Diazepam/toxicidade , Testes para Micronúcleos/métodos , Mutagênicos/toxicidade , Fenolftaleína/toxicidade , Quinacrina/toxicidade , Animais , Células CHO , Cricetinae , Cricetulus , Citocalasina B/farmacologia , Relação Dose-Resposta a Droga , Feminino , Guias como Assunto , HumanosRESUMO
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.
Assuntos
Alquilantes/toxicidade , Perfilação da Expressão Gênica , Mutagênicos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Bleomicina/toxicidade , Cisplatino/toxicidade , Dano ao DNA , Etanol/toxicidade , Genoma Fúngico , Metanossulfonato de Metila/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Cloreto de Sódio/toxicidadeRESUMO
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.
Assuntos
Aneugênicos/farmacologia , Histonas/metabolismo , Linfócitos/efeitos dos fármacos , Testes para Micronúcleos , Mutagênicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Histonas/genética , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Noscapina/farmacologia , Fosforilação , Poliploidia , Tunicamicina/farmacologiaRESUMO
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.
Assuntos
Perfilação da Expressão Gênica , Mutagênicos/toxicidade , Animais , Linhagem Celular Tumoral , Linfoma/genética , CamundongosRESUMO
The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, "misleading" or "irrelevant" positives. We set out to develop a rapid and effective follow-up testing strategy that would predict whether apparent in vitro micronucleus-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp-positive chromatin, cleaved caspase 3-positive chromatin, ethidium monoazide bromide-positive chromatin, polyploid nuclei, phospho-histone H3-positive (metaphase) cells, tetramethylrhodamine ethyl ester-negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ-H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave-one-out approach indicated that a promising base model includes γ-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26-30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results.
Assuntos
Aneugênicos/toxicidade , Biomarcadores/análise , Testes para Micronúcleos/métodos , Mutagênicos/toxicidade , Azidas/química , Caspase 3/metabolismo , Cromatina/química , Citometria de Fluxo , Histonas/química , Humanos , Compostos Orgânicos/química , Ploidias , Poli(ADP-Ribose) Polimerases/metabolismo , Análise de Regressão , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Ensaio Cometa/métodos , Metanossulfonato de Etila/toxicidade , Proteínas de Membrana/genética , Testes para Micronúcleos/métodos , Animais , Colo/citologia , Colo/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Eritrócitos/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Proteínas de Membrana/efeitos dos fármacos , Taxa de Mutação , Ratos , Ratos Sprague-Dawley , Reticulócitos/efeitos dos fármacosRESUMO
The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle control or with the test articles acetyl salicylic acid (ASA), carbendazim (CAR), cyclophosphamide (CP), dimethylhydrazine (DMH), mitomycin C (MMC) or vinblastine sulfate (VIN). The duodenum, colon, and BM were harvested, processed, and analyzed for micronucleus induction. Results from these studies demonstrated differences in the susceptibility for different test compounds in the three tissues tested. When MMC and VIN were dosed by different routes at the same dose levels both compounds produced positive results in all three tissues by intraperitoneal injection but not oral administration. These studies suggest that overall the GI micronucleus assay might be a useful tool for clastogenic and aneugenic compounds that are expected to produce high sustained concentrations in the gastrointestinal tract with little systemic exposure.
Assuntos
Colo/efeitos dos fármacos , Duodeno/efeitos dos fármacos , Testes para Micronúcleos/métodos , Animais , Aspirina/efeitos adversos , Aspirina/farmacologia , Benzimidazóis/efeitos adversos , Benzimidazóis/farmacologia , Carbamatos/efeitos adversos , Carbamatos/farmacologia , Ciclofosfamida/efeitos adversos , Ciclofosfamida/farmacologia , Dano ao DNA/efeitos dos fármacos , Dimetilidrazinas/efeitos adversos , Dimetilidrazinas/farmacologia , Masculino , Mitomicina/efeitos adversos , Mitomicina/farmacologia , Testes de Mutagenicidade/métodos , Mutagênicos/efeitos adversos , Mutagênicos/farmacologia , Ratos , Ratos Sprague-Dawley , Vimblastina/efeitos adversos , Vimblastina/farmacologiaRESUMO
N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.
Assuntos
Dietilnitrosamina/toxicidade , Proteínas de Membrana/genética , Testes de Mutagenicidade , Mutagênicos/toxicidade , Mutação , Animais , Antígenos CD59/genética , Calibragem , Colo/efeitos dos fármacos , Colo/ultraestrutura , Ensaio Cometa/métodos , Ensaio Cometa/normas , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Citometria de Fluxo , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Masculino , Testes para Micronúcleos/métodos , Testes para Micronúcleos/normas , Testes de Mutagenicidade/métodos , Testes de Mutagenicidade/normas , Especificidade de Órgãos , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Padrões de Referência , Reprodutibilidade dos Testes , Reticulócitos/efeitos dos fármacos , Reticulócitos/ultraestrutura , Especificidade da Espécie , Baço/efeitos dos fármacos , Baço/ultraestrutura , Fatores de TempoRESUMO
The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.