Evaluation of the LacZ reporter assay in cryopreserved primary hepatocytes for In vitro genotoxicity testing
© 2016 Wiley Periodicals, Inc..
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. 57:643-655, 2016. © 2016 Wiley Periodicals, Inc.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2016 |
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| Erschienen: |
2016 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:57 |
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| Enthalten in: |
Environmental and molecular mutagenesis - 57(2016), 9 vom: 20. Dez., Seite 643-655 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Luijten, Mirjam [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 09.06.2017 Date Revised 20.12.2017 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/em.22063 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM266311636 |
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| 520 | |a © 2016 Wiley Periodicals, Inc. | ||
| 520 | |a 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. 57:643-655, 2016. © 2016 Wiley Periodicals, Inc | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a DNA damage | |
| 650 | 4 | |a LacZ plasmid | |
| 650 | 4 | |a chromosome aberration | |
| 650 | 4 | |a mutagenicity | |
| 650 | 4 | |a regulatory implementation | |
| 650 | 4 | |a risk assessment | |
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| 700 | 1 | |a Dollé, Martijn E T |e verfasserin |4 aut | |
| 700 | 1 | |a de Pooter, Maaike |e verfasserin |4 aut | |
| 700 | 1 | |a Cox, Julie A |e verfasserin |4 aut | |
| 700 | 1 | |a White, Paul A |e verfasserin |4 aut | |
| 700 | 1 | |a van Benthem, Jan |e verfasserin |4 aut | |
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