Development of a high-throughput human HepG(2) dual luciferase assay for detection of metabolically activated hepatotoxicants and genotoxicants
Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45beta reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG(2) cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45beta promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2009 |
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| Erschienen: |
2009 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:28 |
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| Enthalten in: |
International journal of toxicology - 28(2009), 3 vom: 06. Mai, Seite 162-76 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Liu, Xuemei [VerfasserIn] |
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| Links: |
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| Themen: |
Antigens, Differentiation |
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| Anmerkungen: |
Date Completed 17.08.2009 Date Revised 31.03.2022 published: Print Citation Status MEDLINE |
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| doi: |
10.1177/1091581809337166 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM189472375 |
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| 245 | 1 | 0 | |a Development of a high-throughput human HepG(2) dual luciferase assay for detection of metabolically activated hepatotoxicants and genotoxicants |
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| 500 | |a Date Revised 31.03.2022 | ||
| 500 | |a published: Print | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45beta reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG(2) cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45beta promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants | ||
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| 650 | 7 | |a GADD45B protein, human |2 NLM | |
| 650 | 7 | |a Xenobiotics |2 NLM | |
| 650 | 7 | |a Luciferases |2 NLM | |
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| 700 | 1 | |a Kramer, Jeffrey A |e verfasserin |4 aut | |
| 700 | 1 | |a Hu, Yi |e verfasserin |4 aut | |
| 700 | 1 | |a Schmidt, James M |e verfasserin |4 aut | |
| 700 | 1 | |a Jiang, Jianghong |e verfasserin |4 aut | |
| 700 | 1 | |a Wilson, Alan G E |e verfasserin |4 aut | |
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