Fenbendazole, a broad spectrum anthelmintic used especially in veterinary medicine, may impact non-target organisms in the environment. Nevertheless, information about the effects of fenbendazole in plants is limited. We investigated the biotransformation of fenbendazole and the effect of fenbendazole and its metabolites on gene expression in the model plant Arabidopsis thaliana. High-sensitive UHPLC coupled with tandem mass spectrometry, RNA-microarray analysis together with qPCR verification and nanoLC-MS proteome analysis were used in this study. Twelve fenbendazole metabolites were identified in the roots and leaves of A. thaliana plants. Hydroxylation, S-oxidation and glycosylation represent the main fenbendazole biotransformation pathways. Exposure of A. thaliana plants to 5 μM fenbendazole for 24 and 72 h significantly affected gene and protein expression. The changes in transcriptome were more pronounced in the leaves than in roots, protein expression was more greatly affected in the roots at a shorter period of exposure (24 h) and in leaf rosettes over a longer period (72 h). Up-regulated (>2-fold change, p < 0.1) proteins are involved in various biological processes (electron transport, energy generating pathways, signal transduction, transport), and in response to stresses (e.g. catalase, superoxide dismutase, cytochromes P450, UDP-glycosyltransferases). Some of the proteins which were up-regulated after fenbendazole-exposure probably participate in fenbendazole biotransformation (e.g. cytochromes P450, UDP-glucosyltransferases). Finally, fenbendazole in plants significantly affects many physiological and metabolic processes and thus the contamination of ecosystems by manure containing this anthelmintic should be restricted.

Atrazine is an herbicide with several known toxicologically relevant effects, including interactions with other chemicals. Atrazine increases the toxicity of several organophosphates and has been shown to reduce the toxicity of triclosan to D. magna in a concentration dependent manner. Atrazine is a potent activator in vitro of the xenobiotic-sensing nuclear receptor, HR96, related to vertebrate constitutive androstane receptor (CAR) and pregnane X-receptor (PXR). RNA sequencing (RNAseq) was performed to determine if atrazine is inducing phase I-III detoxification enzymes in vivo, and estimate its potential for mixture interactions. RNAseq analysis demonstrates induction of glutathione S-transferases (GSTs), cytochrome P450s (CYPs), glucosyltransferases (UDPGTs), and xenobiotic transporters, of which several are verified by qPCR. Pathway analysis demonstrates changes in drug, glutathione, and sphingolipid metabolism, indicative of HR96 activation. Based on our RNAseq data, we hypothesized as to which environmentally relevant chemicals may show altered toxicity with co-exposure to atrazine. Acute toxicity tests were performed to determine individual LC50 and Hillslope values as were toxicity tests with binary mixtures containing atrazine. The observed mixture toxicity was compared with modeled mixture toxicity using the Computational Approach to the Toxicity Assessment of Mixtures (CATAM) to assess whether atrazine is exerting antagonism, additivity, or synergistic toxicity in accordance with our hypothesis. Atrazine-triclosan mixtures showed decreased toxicity as expected; atrazine-parathion, atrazine-endosulfan, and to a lesser extent atrazine-p-nonylphenol mixtures showed increased toxicity. In summary, exposure to atrazine activates HR96, and induces phase I-III detoxification genes that are likely responsible for mixture interactions.