Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole

Abstract

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.

Oxidative Stress in NAFLD: Role of Nutrients and Food Contaminants

“…Altogether, these findings indicate that pesticides affect several processes that result in oxidative stress: the CYP450 system, mitochondrial respiration, antioxidant pathways, the stress adaptor system, and scavenger ROS activity. Besides oxidative stress, pesticide exposure may lead to NAFLD through their impact on lipid metabolism by (i) modifying fatty acid uptake and efflux [171] and modulating FFA transport as described for insecticides and herbicides, (ii) increasing lipogenesis ( [173,406] (Table 1), (iii) altering oxidation pathways [175]; insecticides, herbicides, and fungicides affect both β-oxidation and lipogenesis (Tables 1 and 2), (iv) interacting with nuclear receptors [416][417][418][419][420][421] involved in the control of metabolism as demonstrated for pyrethroid and neonicotinoid insecticides [176][177][178], and imidazole or triazole fungicides [158,382] or a pesticide mixture [193], but implication in NAFLD is not proven. Some pesticides may ameliorate lipid metabolism.…”

Cross-species analysis of hepatic cytochrome P450 and transport protein expression

“…Initial 2 weeks of cultivation in William's E medium (Pan-Biotech GmbH, Aidenbach, Germany) containing 10% fetal calf serum (FCS) (PAN-Biotech GmbH, Aidenbach, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05% human insulin (all from PAA Laboratories GmbH, Pasching, Austria) and 50 µM hydrocortisone-hemisuccinate (Sigma-Aldrich, Taufkirchen, Germany) were followed by a 2-week differentiation phase. The differentiation medium contained 1.0% DMSO (days 1-3) and later 1.7% DMSO (days 4-14), in addition to the aforementioned supplements (Gripon et al 2002;Luckert et al 2018). Treatment of HepaRG cells with cyproconazole or prochloraz was then performed for 24 h in phenol red-free William's E medium (Pan-Biotech GmbH, Aidenbach, Germany) which contained all ingredients as the differentiation medium including a final DMSO concentration of 1.7%, but only 2% FCS.…”

More than additive effects on liver triglyceride accumulation by combinations of steatotic and non-steatotic pesticides in HepaRG cells

“…The assays were performed as previously described by Luckert et al (2018). Plasmids, plasmid amount and positive controls were also used as described in the aforementioned paper.…”

“…Therefore, analysis of triglyceride accumulation was performed using combinations of steatotic and non-steatotic substances in the human hepatocarcinoma cell line HepaRG. Previous studies already revealed that HepaRG cells are an appropriate model to study steatosis in vitro (Antherieu et al 2010;Knebel et al 2019a;Lasch et al 2020a;Luckert et al 2018;Tanner et al 2018;Tolosa et al 2016). The following test substances were chosen: fludioxonil (FDO) as a non-steatotic substance, as well as difenoconazole (DIF), propiconazole (PPC) and tebuconazole (TBC) as steatotic substances.…”