Evaluation of the LacZ reporter assay in cryopreserved primary hepatocytes for In vitro genotoxicity testing.
Environ Mol Mutagen
; 57(9): 643-655, 2016 12.
Article
em En
| MEDLINE
| ID: mdl-27859631
ABSTRACT
Assessment of genotoxic potential is an important step in the safety evaluation of chemical substances. Under most regulatory jurisdictions, the first tier of testing comprises a standard battery of in vitro genotoxicity tests in bacterial and mammalian cells. However, the mammalian cell tests commonly used exhibit a relatively high rate of misleading positive results, which may lead to unnecessary in vivo testing. We previously established a proof-of-concept for the LacZ reporter assay in proliferating primary hepatocytes as a promising alternative genotoxicity test. Here, cryopreserved instead of freshly isolated hepatocytes were used and the assay was evaluated in more detail. We examined the effect of cryopreservation on phenotype and metabolic capacity of the LacZ hepatocytes, and assessed the predictive performance of the assay by testing a set of substances comprising true positive, true negative, and misleading positive substances. Additionally, a historical negative control database was created and the type of mutations induced was analyzed for two of the substances tested. Our findings indicate that proliferating cryopreserved primary hepatocytes derived from LacZ plasmid mice retain their hepatocyte-specific characteristics and metabolic competence. Furthermore, we demonstrate that both gene mutations and genome rearrangements due to large deletions can be detected with the LacZ reporter assay. The assay seems to have a lower rate of misleading positive test results compared to the assays currently used. Together, our findings strongly support the use of the LacZ reporter assay in cryopreserved primary hepatocytes as follow-up to the standard in vitro test battery for genotoxicity testing. Environ. Mol. Mutagen. 57643-655, 2016. © 2016 Wiley Periodicals, Inc.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Criopreservação
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Hepatócitos
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Óperon Lac
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Testes de Mutagenicidade
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
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Risk_factors_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article