A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview

• Published in: Journal of applied toxicology Vol. 44; no. 12; pp. 1868 - 1873
• Main Author: Sahu, Saura C.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01-12-2024
• Subjects: Animal models; assay kit; Assaying; Biocompatibility; Biomarkers; Cell culture; Cell viability; Clone 9 cells; Cytotoxicity; Federal regulation; HepaRG; Hepatocytes; Hepatotoxicity; HepG2; In vitro methods and tests; in vitro screening; In vivo methods and tests; Knowledge bases (artificial intelligence); Liver; liver toxicity; Regulatory approval; Reviews; Screening; Toxicants; Toxicity; toxicity screening; Toxicity testing; Toxicology; Toxins; Xenobiotics; toxicity screening; HepaRG; liver toxicity; liver; hepatotoxicity; HepG2; in vitro screening; Clone 9 cells; assay kit; cytotoxicity
• ISSN: 0260-437X; 1099-1263; 1099-1263
• DOI: 10.1002/jat.4619

The 21st‐century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita‐Orange Cell Viability Assay Kit (Sigma‐Aldrich), CellTiter‐Glo Cell Viability Assay Kit (Promega), CytoTox‐ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma‐Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma‐Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure–activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.
Five commercial cytotoxicity assay kits used for screening hepatotoxins were evaluated using HepG2, HepaRG, and Clone 9 cell cultures as in vitro models. This study supports the hypothesis that a battery of these assays is an excellent tool for predicting in vivo effects of liver toxins.