Assessment of mixture toxicity of (tri)azoles and their hepatotoxic effects in vitro by means of omics technologies

Abstract

Consumers are constantly exposed to chemical mixtures such as multiple residues of different pesticides via the diet. This raises questions concerning potential combination effects, especially because these substances are tested for regulatory purposes on an individual basis. With approximately 500 active substances approved as pesticides, there are too many possible combinations to be tested in standard animal experiments generally requested for regulatory purposes. Therefore, the development of in vitro tools and alternative testing strategies for the assessment of mixture effects is extremely important. As a first step in the development of such in vitro tools, we used (tri)azoles as model substances in a set of different cell lines derived from the primary target organ of these substances, the liver (human: HepaRG, rat: H4IIE). Concentrations were reconciled with measured tissue concentrations obtained from in vivo experiments to ensure comparable effect levels. The effects of the substances were subsequently analyzed by transcriptomics and metabolomics techniques and compared to data from corresponding in vivo studies. The results show that similar toxicity pathways are affected by substances and combinations, thus indicating a similar mode of action and additive effects. Two biomarkers obtained by the approach, CAR and Cyp1A1, were used for mixture toxicity modeling and confirmed the concentration-additive effects, thus supporting the selected testing strategy and raising hope for the development of in vitro methods suitable to detect combination effects and prioritize mixtures of concern for further testing.

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

“…The doses used in the in vivo experiments were equivalent to the NOAEL and the tenfold of the NOAEL (see also Supplemental Table S1). In vitro concentrations were chosen to reflect the levels of the two compounds reached in the liver after in vivo administration of the above doses as described elsewhere (Seeger et al 2019).…”

The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity

“…Limited evidence is available for activation of the AHR by epoxiconazole (Table 4): weak activation of the receptor in different human cell lines is suggested by findings at the target mRNA and protein levels [26,63,64], whereas no substantial induction of a human AHR-driven reporter assays system has been observed in a human placental cell line [63]. Gene expression and enzyme activity data from in vivo studies and from cultured cells also suggest weak AHR activation by epoxiconazole in the rat [64][65][66].…”

Risk Characterization and Probabilistic Concentration–Response Modeling of Complex Environmental Mixtures Using New Approach Methodologies (NAMs) Data from Organotypic in Vitro Human Stem Cell Assays

“…Due to their lower cost and higher throughput, NAMs and alternative animal models have emerged as a potential approach to substantially advance mixture risk assessment (Blackwell et al 2019;Geier et al 2018;Hayes et al 2020;Hoover et al 2019;Incardona et al 2006;Ruiz et al 2019;Seeger et al 2019). This study adds to the body of the recent advances on using NAMs to characterize the toxicity of mixtures, having applied novel highthroughput in vitro models based on a diverse array of human cells.…”