Mechanistic toxicity profiling of chemicals: A comparative study in 2D and 3D HepaRG models using high-content analysis

Humans and the environment are exposed to a diverse range of chemicals, many of which are introduced through the human activities. This growing chemical burden has given rise to concerns related to their potential adverse effects. Traditionally, chemical toxicity evaluation has relied on animal tests that are time consuming, expensive, pose ethical concerns, and may not be directly extrapolated to humans. This highlights the urgent need to develop New Approach Methodologies (NAMs) to evaluate chemical- associated toxicity. Since the liver is a primary target organ for xenobiotics, our objective is to use both 2D and 3D HepaRG models combined with High-Content Analysis (HCA) for chemical mechanistic toxicity profiling. HepaRG models were exposed to eight chemicals (Chlorothalonil, Tebuconazole, Thioacetamide, Benzoyl peroxide, Diethyl maleate, clofibrate, indeno[1,2,3-cd]pyrene, 3-methylcholanthrene) for 72 h with daily media renewal. Cellular endpoints including cytotoxicity, oxidative stress, mitochondrial dysfunction, lipid accumulation and the pro-inflammatory response were quantified. Additionally, four chemicals were added in this study to test chemicals having different MOA and to demonstrate the sensitivity of the 3D HepaRG spheroid model (FICZ, Diuron, 12-O-Tetradecanoylphorbol-13-acetate, Azoxystrobin). Our findings revealed that 3D HepaRG cells exhibited more pronounced positive responses to chemical treatment compared to the 2D HepaRG cells. In addition to the chemical-specific toxicity profiling generated by HCA, we identified correlations between different cellular alterations such as mitochondrial dysfunction, dysregulated lipid metabolism and oxidative stress in the treated 3D HepaRG spheroids. Overall, this approach proposed in this study provides a rapid, human-relevant in vitro method that enhances the predictive power of NAMs for chemical associated toxicity.