<i>In Vitro</i>
            Mammalian Cell Mutation Assays

Bigger, H.; Moore, Martha M.; Heflich, Robert H.

doi:10.1002/9780470249055.ch5

Cited by 3 publications

(2 citation statements)

References 41 publications

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“…Similar frequencies have been measured in limiting-dilution cloning assays of Pig-a (ProAER-resistant) rat T-cells [Miura et al, 2008b]. These background frequencies are comparable to the background mutant frequency for the X-linked endogenous Hprt gene in lymphocytes and cell lines [e.g., Aidoo et al, 1997;Bigger et al, 2008], and much lower than background mutant frequencies for most transgenic targets [e.g., White et al, 2003]. Based on the proposition that low background mutant frequencies promote the sensitivity of mutation assays, these relatively low background mutant frequencies may represent a major advantage for the Pig-a assay.…”

Section: Observations On the Sensitivity Of The Rat Pig-a Assaysupporting

confidence: 66%

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

Dobrovolsky

Miura

Heflich

et al. 2010

Environ and Mol Mutagen

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116

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The Pig-a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N-acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig-a is the only gene involved in GPI anchor synthesis that is on the X-chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI-anchored cell surface proteins, almost always have mutations in the Pig-a gene. These properties of the Pig-a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig-a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10(-6)) and rats treated with multiple doses of the potent mutagen, N-ethyl-N-nitrosourea, display Pig-a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat-dose toxicology studies.

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Section: Observations On the Sensitivity Of The Rat Pig-a Assaysupporting

confidence: 66%

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

Dobrovolsky

Miura

Heflich

et al. 2010

Environ and Mol Mutagen

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116

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“…Moreover, mouse lymphoma cells have no CYP 2E1 activity, and treatment of mouse lymphoma cells with acrylamide produced no glycidamide‐DNA adducts 30. In addition, it should be noted that the positive mutagenicity responses produced by acrylamide in the mouse lymphoma30 and TK6 cell29 assays were obtained at acrylamide concentrations that exceeded both the maximum concentration of 10 mM established for using in vitro mammalian cell mutation assays for regulatory purposes50 and the plasma levels of acrylamide that can be achieved in vivo 51, 52…”

Section: Discussionmentioning

confidence: 99%

DNA adduct formation and induction of micronuclei and mutations in B6C3F₁/Tk mice treated neonatally with acrylamide or glycidamide

Tungeln

Churchwell

Doerge

et al. 2009

Intl Journal of Cancer

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Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F 1 /Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1-8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F 1 /Tk 1/1 mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F 1 /Tk 1/2 mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine, the major glycidamide-DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1-8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1-8 are due to another mechanism.

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Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays

Elespuru¹,

Agarwal²,

Atrakchi³

et al. 2009

Toxicological Sciences

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With the advent of new technologies (e.g., genomics, automated analyses, and in vivo monitoring), new regulations (e.g., the reduction of animal tests by the European REACH), and new approaches to toxicology (e.g., Toxicity Testing in the 21st Century, National Research Council), the field of regulatory genetic toxicology is undergoing a serious re-examination. Within this context, Toxicological Sciences has published a series of articles in its Forum Section on the theme, "Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation" (beginning with Goodman et al.). As a contribution to the Forum discussions, we present current methods for evaluating mutagenic/genotoxic risk using standard genotoxicity test batteries, and suggest ways to address and incorporate new technologies. We recognize that the occurrence of positive results in relation to cancer prediction has led to criticism of in vitro mammalian cell genetic toxicity assays. We address criticism of test results related to weak positives, associated only with considerable toxicity, only seen at high concentrations, not accompanied by positive results in the other tests of standard test batteries, and/or not correlating well with rodent carcinogenicity tests. We suggest that the problems pointed out by others with these assays already have been resolved, to a large extent, by international groups working to update assay protocols, and by changes in data interpretation at regulatory agencies. New guidances at the U.S. Environmental Protection Agency and the U.S. Food and Drug Administration improve data evaluation and help refocus risk assessment. We discuss the results of international groups working together to integrate new technologies and evaluate new tests, including human monitoring. We suggest that strategies for identifying human health risks should naturally change to integrate new technologies; however, changes should be made only when justified by strong scientific evidence of improvement in the risk assessment paradigm.

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In Vitro Mammalian Cell Mutation Assays

Cited by 3 publications

References 41 publications

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

DNA adduct formation and induction of micronuclei and mutations in B6C3F₁/Tk mice treated neonatally with acrylamide or glycidamide

Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays

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In Vitro Mammalian Cell Mutation Assays

Cited by 3 publications

References 41 publications

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide

Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays

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DNA adduct formation and induction of micronuclei and mutations in B6C3F₁/Tk mice treated neonatally with acrylamide or glycidamide