Abstract
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.
Highlights
Human exposure to countless chemical substances, occurring as complex mixtures, has become a major concern for regulatory agencies (Escher et al 2017; Rappaport and Smith 2010)
Compounds sharing the same MoA are believed to follow the principle of dose addition when being in mixtures, and, mixture effects may be predicted by the dose addition concept (Backhaus and Faust 2012; Kortenkamp et al 2009)
Based on the transcriptional changes proposed in the liver steatosis adverse outcome pathway (AOP), and based on the nuclear receptors (NRs) activation pattern of propylhexanoic acid (PHX), propylheptanoic acid (PHP), and CTD, we investigated the expression of ACOX1, FASN, MLXIPL, SCD and SREBF1
Summary
Human exposure to countless chemical substances, occurring as complex mixtures, has become a major concern for regulatory agencies (Escher et al 2017; Rappaport and Smith 2010). In response to this issue, different tiered strategies for the risk assessment of combined exposure to multiple chemicals have been proposed and implemented (Rotter et al 2018). In the case of compounds with a different MoA, it is unclear whether the assumption of dose addition is still valid (Borgert et al 2012; EFSA et al 2013; Kortenkamp et al 2009). The dose addition assumption in the case of mixtures involving substances with dissimilar MoA remains to be investigated on a larger scale
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
