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The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals.

ABSTRACT: As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their "normal" phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct-acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration-dependent increases in mutant frequency (MF) ranging from 2.6- to 14.4-fold over the control. None of the four nonmutagens, including two misleading, or "false," positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3-nitrobenzanthrone [3-NBA], benzo[a]pyrene [BaP], and aflatoxin B1 [AFB1]) to least (i.e., N-ethyl-N-nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in an in vitro gene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348-360, 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

SUBMITTER: Cox JA

PROVIDER: S-EPMC6593967 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals.

Cox Julie A JA Zwart Edwin P EP Luijten Mirjam M White Paul A PA

Environmental and molecular mutagenesis 20190225 4

As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their "normal" phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gen ...[more]

PMID: 30714215

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Project description:Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

Dataset Information

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals.

Publications

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals.

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