Journal
ARCHIVES OF TOXICOLOGY
Volume 96, Issue 1, Pages 211-229Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00204-021-03182-1
Keywords
Steatosis; Mixtures; AOP-wise testing; Relative potency factors; Triglyceride accumulation; Hepatotoxicity
Categories
Funding
- European Unions Horizon 2020 research and innovation program [633172]
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This study utilized an in vitro assay toolbox to investigate the combined effects of mixtures containing differently acting compounds and refine the molecular mechanisms underlying liver steatosis AOP. The results showed dose addition effects in the mixtures, suggesting the validity of the additivity assumption, and proposed further refinement of the AOP to better reflect the diversity of molecular mechanisms causing adverse outcomes.
Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome.
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