Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro

Abstract

Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration–response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.

Genotoxic mixtures and dissimilar action: concepts for prediction and assessment

“… AIC Akaike information criterion (small indicates better fit) a Baseline rate is expressed as percentage; , and are estimates of the unknown model parameter θ 1 , θ 2 and d b Published in Ermler et al (2013) c Non-threshold model regression fit (Scholze et al 2001)…”

“…We previously have begun to address this issue in experiments with seven aneugenic benzimidazole pesticides which induce MN in Chinese hamster ovary (CHO)-K1 cells through a common mechanism (Ermler et al 2013). As expected in the light of their mechanistic similarity, CA produced accurate predictions of the joint action of these seven benzimidazoles, on the basis of their individual effects, while IA yielded additivity predictions that fell short of the experimentally observed effects.…”

Impact of Pesticides on Environmental and Human Health

“…They are commonly used as veterinary medicines (anthelmintics) and pesticides. They inhibit microtubule formation when they bind to free β-tubulin monomers at the colchicine-binding site [59].…”

Using transcriptomics to guide lead optimization in drug discovery projects: Lessons learned from the QSTAR project

“…Vinblastine is a known reference compound used in MNT assays as a positive control for the induction of MN formation [30]. Mebendazole and nocodazole are both benzimidazoles that are also considered model compounds for demonstrating thresholded responses of aneugenic compounds [31]. Hence, our identification of MNT-positive compounds using gene expression signatures could also be validated on external data.…”