Pesticides diazinon and diuron increase glutathione levels and affect multixenobiotic resistance activity and biomarker responses in zebrafish (Danio rerio) embryos and larvae

Abstract

Background: Zebrafish have been increasingly used for monitoring and assessing the effects of different contaminants in the aquatic environments. In the present study, zebrafish embryos and larvae were used to study the effects of the insecticide diazinon and the herbicide diuron in regard to occurrence of oxidative stress-related cellular responses, multixenobiotic resistance (MXR)-related efflux transporter activity—which represents the first line of defense against xenobiotics in many aquatic organisms—and responses of different molecular and biochemical biomarkers. Results: The recently established non-invasive quantitative plate assay, which uses the fluorescent probes CM-H 2 DCFDA and CellTracker™ Green CMFDA and measures the fluorescence in whole zebrafish larvae, was applied to assess changes in reactive oxidative species (ROS) and glutathione (GSH) after exposure to the investigated pesticides. The results showed a significant increase of GSH after 1 h exposure of zebrafish larvae to both diazinon and diuron. Regarding the ROS induction, no significant increases in fluorescence could be detected after 2 h exposure to the investigated pesticides. Applying a newly adapted assay for MXR activity, it was determined that diuron caused no change after a 24-h exposure, but caused a significant induction of MXR activity after a 48-h exposure (indicated by a decreased amount of accumulated rhodamine B). On the other hand, diazinon caused an inhibition of MXR activity after both 24 h and 48 h exposure (indicated by an increased amount of accumulated rhodamine B). Regarding the biomarkers, different set-ups and exposure periods were applied and both molecular (gene expression) and biochemical (enzymatic activities) responses were assessed. Diazinon caused an inhibition of carboxylesterase (CES) and acetylcholinesterase (AChE) activity in zebrafish larvae, diuron inhibited AChE activity in in vitro testing, and both pesticides significantly affected gene expression and activities of some of the cytochrome P450 (CYP) family enzymes. Conclusions: The obtained results show various effects of the investigated pesticides and will help to elucidate how aquatic animals cope with pesticides present in their environment. Additionally, the recently developed fluorescence-based assay and the newly adapted MXR activity assay proved to be useful tools for ecotoxicological risk assessment to further investigate pesticide effects.