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Stephen D Dertinger - One of the best experts on this subject based on the ideXlab platform.
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Aneugen Molecular Mechanism Assay: Proof-of-Concept With 27 Reference Chemicals.
Toxicological sciences : an official journal of the Society of Toxicology, 2019Co-Authors: Derek T. Bernacki, Jeffrey C Bemis, Steven M. Bryce, Stephen D DertingerAbstract:A tiered bioassay and data analysis scheme is described for elucidating the most common molecular targets responsible for chemical-induced in vitro Aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of mitotic kinase(s). To evaluate this strategy, TK6 cells were first exposed to each of 27 presumed Aneugens over a range of concentrations. After 4 and 24 h of treatment, γH2AX, p53, phospho-histone H3 (p-H3), and polyploidization biomarkers were evaluated using the MultiFlow DNA Damage Assay Kit. The assay identified 27 of 27 chemicals as genotoxic, with 25 exhibiting Aneugenic signatures, 1 Aneugenic and clastogenic, and 1 clastogenic. Subsequently, a newly described follow-up assay was employed to investigate the Aneugenic agents' molecular targets. For these experiments, TK6 cells were exposed to each of 26 chemicals in the presence of 488 Taxol. After 4 h, cells were lysed and the liberated nuclei and mitotic chromosomes were stained with a nucleic acid dye and labeled with fluorescent antibodies against p-H3 and Ki-67. Flow cytometric analyses revealed that alterations to 488 Taxol-associated fluorescence were only observed with tubulin binders-increases in the case of tubulin stabilizers, decreases with destabilizers. Mitotic kinase inhibitors with known Aurora kinase B inhibiting activity were the only Aneugens that dramatically decreased the ratio of p-H3-positive to Ki-67-positive nuclei. Unsupervised hierarchical clustering based on 488 Taxol fluorescence and p-H3: Ki-67 ratios clearly distinguished compounds with these disparate molecular mechanisms. Furthermore, a classification algorithm based on an artificial neural network was found to effectively predict molecular target, as leave-one-out cross-validation resulted in 25/26 agreement with a priori expectations. These results are encouraging, as they suggest that an adequate number of training set chemicals, in conjunction with a machine learning algorithm based on 488 Taxol, p-H3, and Ki-67 responses, can reliably elucidate the most commonly encountered Aneugenic molecular targets.
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in vivo pig a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2018Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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Miniaturized flow cytometry-based CHO-K1 micronucleus assay discriminates Aneugenic and clastogenic modes of action.
Environmental and molecular mutagenesis, 2010Co-Authors: Steven M. Bryce, Svetlana L Avlasevich, Jeffrey C Bemis, Stephen D DertingerAbstract:A well recognized advantage of the in vitro micronucleus assay is its ability to detect both Aneugens and clastogens. This laboratory has previously described a flow cytometric approach for scoring in vitro micronuclei (MN)(Avlasevich et al. [2006]: Environ Mol Mutagen 47: 56–66). More recently, based on work with Chinese hamster cells, evidence has accumulated that the multiparametric data acquired by the flow cytometric process is capable of discriminating between Aneugenic and clastogenic modes of action (MOA). That is, in the case of CHO-K1 cells, clastogens are observed to induce MN with minimal effects on the incidence of hypodiploid nuclei or the median size of MN (i.e., fluorescence intensity), whereas Aneugens are observed to affect all three parameters. To systematically test whether these ‘‘signatures’’ are indeed reliable indicators of genotoxic MOA, CHO-K1 cells were treated with eight prototypical clastogens, eight an eugens, and 15 nongenotoxicants. Exposure was continuous (18–24 hrs) with harvest occurring immediately thereafter. Treatment and all subsequent processing and analysis steps occurred in the same 96-well plate, making this an efficient, miniaturized assay. The resulting flow cytometric MN data correlated well with expected in vitro cytogenetics: sensitivity 5 16/16, specificity 5 14/15. In addition, MOA signatures were identified that classified each of the 16 genotoxicants correctly as clastogenic or Aneugenic. Taken together, these data indicate that flow cytometry represents an analytical platform that is capable of rapidly and objectively acquiring MN counts while simultaneously providing information on genotoxic MOA.
Svetlana L Avlasevich - One of the best experts on this subject based on the ideXlab platform.
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in vivo pig a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2018Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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Miniaturized flow cytometry-based CHO-K1 micronucleus assay discriminates Aneugenic and clastogenic modes of action.
Environmental and molecular mutagenesis, 2010Co-Authors: Steven M. Bryce, Svetlana L Avlasevich, Jeffrey C Bemis, Stephen D DertingerAbstract:A well recognized advantage of the in vitro micronucleus assay is its ability to detect both Aneugens and clastogens. This laboratory has previously described a flow cytometric approach for scoring in vitro micronuclei (MN)(Avlasevich et al. [2006]: Environ Mol Mutagen 47: 56–66). More recently, based on work with Chinese hamster cells, evidence has accumulated that the multiparametric data acquired by the flow cytometric process is capable of discriminating between Aneugenic and clastogenic modes of action (MOA). That is, in the case of CHO-K1 cells, clastogens are observed to induce MN with minimal effects on the incidence of hypodiploid nuclei or the median size of MN (i.e., fluorescence intensity), whereas Aneugens are observed to affect all three parameters. To systematically test whether these ‘‘signatures’’ are indeed reliable indicators of genotoxic MOA, CHO-K1 cells were treated with eight prototypical clastogens, eight an eugens, and 15 nongenotoxicants. Exposure was continuous (18–24 hrs) with harvest occurring immediately thereafter. Treatment and all subsequent processing and analysis steps occurred in the same 96-well plate, making this an efficient, miniaturized assay. The resulting flow cytometric MN data correlated well with expected in vitro cytogenetics: sensitivity 5 16/16, specificity 5 14/15. In addition, MOA signatures were identified that classified each of the 16 genotoxicants correctly as clastogenic or Aneugenic. Taken together, these data indicate that flow cytometry represents an analytical platform that is capable of rapidly and objectively acquiring MN counts while simultaneously providing information on genotoxic MOA.
Dorothea K Torous - One of the best experts on this subject based on the ideXlab platform.
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in vivo pig a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2018Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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Practical threshold for micronucleated reticulocyte induction observed for low doses of mitomycin C, Ara-C and colchicine.
Mutagenesis, 2005Co-Authors: Norihide Asano, Stephen D Dertinger, Dorothea K Torous, Carol R Tometsko, Takeshi Morita, Makoto HayashiAbstract:Micronucleus induction was studied for the DNA target clastogens mitomycin C (MMC) and 1-beta-D-arabinofuranosylcytosine (Ara-C), and also the non-DNA target Aneugen colchicine (COL) in order to evaluate the dose-response relationship at very low dose levels. The acridine orange (AO) supravital staining method was used for microscopy and the anti-CD71-FITC based method was used for flow cytometric analysis. In the AO method, 2000 reticulocytes were analysed as commonly advised, but in the flow cytometric method, 2000, 20,000, 200,000 and 1,000,000 reticulocytes were analysed for each sample to increase the detecting power (i.e. sensitivity) of the assay. The present data show that increasing the number of cells scored increases the statistical power of the assay when the cell was considered as a statistical unit. Even so, statistically significant differences from respective vehicle controls were not observed at the lowest dose level for MMC and Ara-C, or the lower four dose levels for COL, even after one million cells were analysed. When the animal was considered as a statistical unit, only the top dose group for each chemical showed significant increase of micronucleated reticulocytes frequency. As non-linear dose-response curves were obtained for each of the three chemicals studied, these observations provide evidence for the existence of a practical threshold for the DNA target clastogens as well as the non-DNA target Aneugen studied.
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An automated method for discriminating Aneugen‐ vs. clastogen‐induced micronuclei
Environmental and Molecular Mutagenesis, 1998Co-Authors: Dorothea K Torous, Stephen D Dertinger, Nikki E Hall, Carol R TometskoAbstract:A flow cytometric (FCM) procedure for quantitating micronucleated reticulocytes in mouse peripheral blood samples was evaluated for its ability to discriminate between Aneugen- and clastogen-induced micronuclei (MN). In this experiment, BALB/c mice were injected with 0.9% saline, the model clastogen methyl methanesulfonate (100 mg/kg bw) or the Aneugen vincristine (0.2 mg/kg bw). Peripheral blood samples were collected 48 hr after injection and were subsequently fixed and stained for flow cytometric analysis. The staining method utilized FITC-conjugated anti-CD71 to differentially label reticulocytes, and the nucleic acid dye propidium iodide to resolve erythrocyte populations with and without micronuclei. The frequency of micronucleated reticulocytes was determined by analyzing 10,000 total reticulocytes per blood sample. A second analysis was performed on each sample whereby the propidium iodide associated fluorescent signals of 250 MN were collected and graphed as a single-parameter histogram. The histogram statistic “median channel” was recorded for each sample and provided a quantitative description of MN distribution according to DNA content. Cumulatively, the results of this study suggest that 1) flow cytometry can be employed to measure the incidence of MN resulting from clastogenic or Aneugenic activity, and 2) MN resulting from Aneugenscan be discriminated from those arising spontaneously or from clastogen treatment based on flow cytometric analysis of DNA content. Environ. Mol. Mutagen. 31:340–344, 1998. © 1998 Wiley-Liss, Inc.
Micheline Kirsch-volders - One of the best experts on this subject based on the ideXlab platform.
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Role of aneuploidy in the carcinogenic process: Part 3 of the report of the 2017 IWGT workgroup on assessing the risk of Aneugens for carcinogenesis and hereditary diseases.
Mutation research, 2019Co-Authors: David Tweats, Micheline Kirsch-volders, Azeddine Elhajouji, Francesca Pacchierotti, Ken-ichi Masumura, David A. Eastmond, Roland Froetschl, Anthony M. Lynch, Francesco Marchetti, Maik SchulerAbstract:Abstract Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical Aneugens; and chemotherapy-related malignant neoplasms. Aneuploidy is seen at various stages in carcinogenesis. However, the relationship between induced aneuploidy occurring after exposure and clonal aneuploidy present in tumours is not clear. Recent evidence indicates that the induction of chromosomal instability (CIN), may be more important than aneuploidy per se, in the carcinogenic process. Down Syndrome, trisomy 21, is associated with altered hematopoiesis in utero which, in combination with subsequent mutations, results in an increased risk for acute megakaryoblastic and lymphoblastic leukemias. In contrast, there is reduced cancer risk for most solid tumours in Down Syndrome. Mouse models with high levels of aneuploidy are also associated with increased cancer risk for particular tumours with long latencies, but paradoxically other types of tumour often show decreased incidence. The Aneugens reviewed that induce cancer in humans and animals all possess other carcinogenic properties, such as mutagenicity, clastogenicity, cytotoxicity, organ toxicities, hormonal and epigenetic changes which likely account for, or interact with aneuploidy, to cause carcinogenesis. Although the role that aneuploidy plays in carcinogenesis has not been fully established, in many cases, it may not play a primary causative role. Tubulin-disrupting Aneugens that do not possess other properties linked to carcinogenesis, were not carcinogenic in rodents. Similarly, in humans, for the tubulin-disrupting Aneugens colchicine and albendazole, there is no reported association with increased cancer risk. There is a need for further mechanistic studies on agents that induce aneuploidy, particularly by mechanisms other than tubulin disruption and to determine the role of aneuploidy in pre-neoplastic events and in early and late stage neoplasia.
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Chemically induced aneuploidy in germ cells. Part II of the report of the 2017 IWGT workgroup on assessing the risk of Aneugens for carcinogenesis and hereditary diseases.
Mutation research, 2019Co-Authors: Francesca Pacchierotti, Micheline Kirsch-volders, Azeddine Elhajouji, Ken-ichi Masumura, David A. Eastmond, Roland Froetschl, Anthony M. Lynch, Maik Schuler, David Tweats, Francesco MarchettiAbstract:Abstract As part of the 7th International Workshops on Genotoxicity Testing held in Tokyo, Japan in November 2017, a workgroup of experts reviewed and assessed the risk of Aneugens for human health. The present manuscript is one of three manuscripts from the workgroup and reports on the unanimous consensus reached on the evidence for Aneugens affecting germ cells, their mechanisms of action and role in hereditary diseases. There are 24 chemicals with strong or sufficient evidence for germ cell Aneugenicity providing robust support for the ability of chemicals to induce germ cell aneuploidy. Interference with microtubule dynamics or inhibition of topoisomerase II function are clear characteristics of germ cell Aneugens. Although there are mechanisms of chromosome segregation that are unique to germ cells, there is currently no evidence for germ cell-specific Aneugens. However, the available data are heavily skewed toward chemicals that are Aneugenic in somatic cells. Development of high-throughput screening assays in suitable animal models for exploring additional targets for aneuploidy induction, such as meiosis-specific proteins, and to prioritize chemicals for the potential to be germ cell Aneugens is encouraged. Evidence in animal models support that: oocytes are more sensitive than spermatocytes and somatic cells to Aneugens; exposure to Aneugens leads to aneuploid conceptuses; and, the frequencies of aneuploidy are similar in germ cells and zygotes. Although aneuploidy in germ cells is a significant cause of infertility and pregnancy loss in humans, there is currently limited evidence that Aneugens induce hereditary diseases in human populations because the great majority of aneuploid conceptuses die in utero. Overall, the present work underscores the importance of protecting the human population from exposure to chemicals that can induce aneuploidy in germ cells that, in contrast to carcinogenicity, is directly linked to an adverse outcome.
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Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of Aneugens for carcinogenesis and hereditary diseases.
Mutation research, 2019Co-Authors: Anthony M. Lynch, Micheline Kirsch-volders, Azeddine Elhajouji, Francesca Pacchierotti, Ken-ichi Masumura, David A. Eastmond, Roland Froetschl, Maik Schuler, Francesco Marchetti, David TweatsAbstract:An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical Aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical Aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of Aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical Aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of Aneugenic agents.
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Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons
Environmental and molecular mutagenesis, 2010Co-Authors: Zoryana Cammerer, Micheline Kirsch-volders, Willi Suter, Martin Schumacher, Azeddine ElhajoujiAbstract:Non-DNA binding genotoxins (e.g., Aneugens), unlike DNA-binding genotoxins, are theoretically expected to show thresholded concentration-effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose-response curves for tubulin interacting agents, a specific class of Aneugens. All experiments with Aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non-linear dose-dependent increase in micronuclei frequencies for all tested Aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg−1, respectively. Environ. Mol. Mutagen., 2010. © 2009 Wiley-Liss, Inc.
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Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to Aneugens after single-dose applications.
Mutagenesis, 2007Co-Authors: Zoryana Cammerer, Micheline Kirsch-volders, Azeddine Elhajouji, Willi SuterAbstract:Detection of clastogenic compounds in the peripheral blood micronucleus test (MNT) in rats is a well-established methodology. However, the results obtained on the induction of micronuclei by Aneugens in rat peripheral blood are controversial. Our aim was a comparative evaluation of the peripheral blood flow cytometry MNT in Wistar Han rat and CD1 mouse exposed to three Aneugens (vinblastine, vincristine and colchicine) after single-dose applications. In addition, the same compounds were tested in the rat bone marrow MNT. The treatment with vinblastine (0.25, 0.5, 1, mg/kg), vincristine (0.025, 0.05, 0.1 mg/kg) or colchicine (0.7, 1, 1.3 mg/kg) induced no statistically significant increase in MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) in rat peripheral blood. In rat bone marrow, a clear statistically significant increase in MN-PCE was found with vincristine and vinblastine. However, colchicine showed a clear increase in MN-PCE frequency without reaching statistically significant level only at 1 mg/kg. The positive effect in the bone marrow MNT shows that the target organ was exposed to the appropriate concentration levels of the respective Aneugens. In mouse, the peripheral blood flow cytometry analysis after the treatment with vinblastine, vincristine and colchicine showed clear statistically significant increase in MN-PCE with all three compounds. The experiments with splenectomized rats treated with vincristine and colchicine were performed and statistically significant increases in MN-PCE were found with 0.05, 0.1, 0.15 mg/kg of vincristine and 0.7 and 1 mg/kg of colchicine. Our results demonstrate that micronucleated cells induced by Aneugens are removed from rat peripheral blood by the spleen due to the large size of micronuclei. Based on our data, it is concluded that the flow cytometry peripheral blood MNT after single-dose applications is an appropriate test system for evaluating the genotoxic effects of Aneugens in mice. However, in rats peripheral blood MNT Aneugen detection might require multiple-dose applications to overwhelm the spleen effect.
Jeffrey C Bemis - One of the best experts on this subject based on the ideXlab platform.
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Aneugen Molecular Mechanism Assay: Proof-of-Concept With 27 Reference Chemicals.
Toxicological sciences : an official journal of the Society of Toxicology, 2019Co-Authors: Derek T. Bernacki, Jeffrey C Bemis, Steven M. Bryce, Stephen D DertingerAbstract:A tiered bioassay and data analysis scheme is described for elucidating the most common molecular targets responsible for chemical-induced in vitro Aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of mitotic kinase(s). To evaluate this strategy, TK6 cells were first exposed to each of 27 presumed Aneugens over a range of concentrations. After 4 and 24 h of treatment, γH2AX, p53, phospho-histone H3 (p-H3), and polyploidization biomarkers were evaluated using the MultiFlow DNA Damage Assay Kit. The assay identified 27 of 27 chemicals as genotoxic, with 25 exhibiting Aneugenic signatures, 1 Aneugenic and clastogenic, and 1 clastogenic. Subsequently, a newly described follow-up assay was employed to investigate the Aneugenic agents' molecular targets. For these experiments, TK6 cells were exposed to each of 26 chemicals in the presence of 488 Taxol. After 4 h, cells were lysed and the liberated nuclei and mitotic chromosomes were stained with a nucleic acid dye and labeled with fluorescent antibodies against p-H3 and Ki-67. Flow cytometric analyses revealed that alterations to 488 Taxol-associated fluorescence were only observed with tubulin binders-increases in the case of tubulin stabilizers, decreases with destabilizers. Mitotic kinase inhibitors with known Aurora kinase B inhibiting activity were the only Aneugens that dramatically decreased the ratio of p-H3-positive to Ki-67-positive nuclei. Unsupervised hierarchical clustering based on 488 Taxol fluorescence and p-H3: Ki-67 ratios clearly distinguished compounds with these disparate molecular mechanisms. Furthermore, a classification algorithm based on an artificial neural network was found to effectively predict molecular target, as leave-one-out cross-validation resulted in 25/26 agreement with a priori expectations. These results are encouraging, as they suggest that an adequate number of training set chemicals, in conjunction with a machine learning algorithm based on 488 Taxol, p-H3, and Ki-67 responses, can reliably elucidate the most commonly encountered Aneugenic molecular targets.
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in vivo pig a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2018Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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In vivo pig‐a and micronucleus study of the prototypical Aneugen vinblastine sulfate
Environmental and Molecular Mutagenesis, 2017Co-Authors: Svetlana L Avlasevich, Carson Labash, Dorothea K Torous, Jeffrey C Bemis, James T. Macgregor, Stephen D DertingerAbstract:The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative Aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days −4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established Aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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Miniaturized flow cytometry-based CHO-K1 micronucleus assay discriminates Aneugenic and clastogenic modes of action.
Environmental and molecular mutagenesis, 2010Co-Authors: Steven M. Bryce, Svetlana L Avlasevich, Jeffrey C Bemis, Stephen D DertingerAbstract:A well recognized advantage of the in vitro micronucleus assay is its ability to detect both Aneugens and clastogens. This laboratory has previously described a flow cytometric approach for scoring in vitro micronuclei (MN)(Avlasevich et al. [2006]: Environ Mol Mutagen 47: 56–66). More recently, based on work with Chinese hamster cells, evidence has accumulated that the multiparametric data acquired by the flow cytometric process is capable of discriminating between Aneugenic and clastogenic modes of action (MOA). That is, in the case of CHO-K1 cells, clastogens are observed to induce MN with minimal effects on the incidence of hypodiploid nuclei or the median size of MN (i.e., fluorescence intensity), whereas Aneugens are observed to affect all three parameters. To systematically test whether these ‘‘signatures’’ are indeed reliable indicators of genotoxic MOA, CHO-K1 cells were treated with eight prototypical clastogens, eight an eugens, and 15 nongenotoxicants. Exposure was continuous (18–24 hrs) with harvest occurring immediately thereafter. Treatment and all subsequent processing and analysis steps occurred in the same 96-well plate, making this an efficient, miniaturized assay. The resulting flow cytometric MN data correlated well with expected in vitro cytogenetics: sensitivity 5 16/16, specificity 5 14/15. In addition, MOA signatures were identified that classified each of the 16 genotoxicants correctly as clastogenic or Aneugenic. Taken together, these data indicate that flow cytometry represents an analytical platform that is capable of rapidly and objectively acquiring MN counts while simultaneously providing information on genotoxic MOA.
