NAM-based analysis of contaminant short-term organ toxicity in HepaRG and RPTEC/TERT1 cells

New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, are promising alternatives to animal testing. To become useable for risk assessment purposes, they have to be applicable for different substance groups. One important group of substances is food contaminants, including synthetic chemicals, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), and natural compounds, such as mycotoxins and pyrrolizidine alkaloids. We tested five known contaminants affecting the liver and/or the kidney - PFOS, PFOA, Aflatoxin B₁ (AB₁), lasiocarpine (Las), and cadmium chloride - using HepaRG and RPTEC/TERT1 cells at non-cytotoxic concentrations for 36 and 72 h. Our NAM-based testing protocol included marker protein analysis for cellular functions and targeted transcriptomics followed by bioinformatics pathway analysis. The effects observed were compared with established in vivo results. Protein analysis indicated various affected pathways in HepaRG cells, with generally fewer effects in RPTEC/TERT1 cells. The strongest transcriptional impact was noted for Las in HepaRG cells. This study demonstrated the test protocol's applicability across different substances, revealing significant differences between HepaRG and RPTEC/TERT1 cell lines. RPTEC/TERT1 cells, while expressing renal-specific CYP enzymes, were less suitable for detecting effects of substances requiring hepatic metabolic activation, like Las and AB₁. Our data supports the concept of specific pathway toxicity, with pathway analysis enabling the prediction of effects based on mechanism rather than target organ. Employing multiple omics techniques provided comprehensive insights into various compound effects, including steatosis, reactive oxygen species production and DNA damage, highlighting the potential of an extended omics approach for advancing toxicological assessments.