Clozapine modulation of zebrafish swimming behavior and gene expression as a case study to investigate effects of atypical drugs on aquatic organisms.

Mental illnesses affect more than 150 million people in Europe and lead to an increasing consumption of neuroactive drugs during the last twenty years. The antipsychotic compound, clozapine, is one of the most used psychotropic drugs worldwide, with potentially negative consequences for the aquatic environment. Hence, the objectives of the study presented here were the quantification of clozapine induced changes in swimming behavior of exposed Danio rerio embryos and the elucidation of the molecular effects on the serotonergic and dopaminergic systems. Yolk-sac larvae were exposed to different concentrations (0.2 mg/L, 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L and 6.4 mg/L) of clozapine for 116 h post-fertilization, and changes in the swimming behavior of the larvae were assessed. Further, quantitative real-time PCR was performed to analyze the expression of selected genes. The qualitative evaluation of changes in the swimming behavior of D. rerio larvae revealed a significant decrease of the average swimming distance and velocity in the light-dark transition test, with more than a 36% reduction at the highest exposure concentration of 6.4 mg/L. Furthermore, the total larval body length was reduced at the highest concentration. An in-depth analysis based on expression of selected target genes of the serotonin (slc6a4a) and dopamine (drd2a) system showed an upregulation at a concentration of 1.6 mg/L and above. In addition, a lower increase in expression was detected for biomarkers of general stress (adra1a and cyp1a2). Our data show that exposure to clozapine during development inhibits swimming activity of zebrafish larvae, which could, in part, be due to disruption of the serotonin- and dopamine system.

Effects of the antidepressant mirtazapine on the swimming behaviour and gene expression rate of Danio rerio embryos - Is the sedating effect seen in humans also evident for fish?

In the last decade, mirtazapine has become an important antidepressant in clinical use and has also been found at many different environmental sampling sites. Several homologies between the zebrafish Danio rerio and humans, combined with a number of advantages for behavioural and gene expression research using zebrafish embryos, make their use for the analysis of mirtazapine appropriate. The sedative effect of mirtazapine in humans was also found for a specific concentration range in zebrafish embryos (1333.4 µg/L - 2666.9 µg/L). Specifically, 116 hpf old zebrafish embryos showed a reduced swimming distance when exposed to 1334.4 µg/L mirtazapine. Furthermore, changes at the gene regulatory level could be measured (1333.4 µg/L), in particular in the superordinate regulatory systems. For selected transporters of all regulatory systems, an up regulation of the genes by a factor of more than five times could be measured at the highest mirtazapine exposure concentration that was tested. Finally, studies on the protein levels demonstrated an increase in acetylcholinesterase activity for several exposure concentrations (83.3 µg/L and 666.7 µg/L). The physiological changes in zebrafish embryos caused by mirtazapine demonstrate the relevance of these types of studies in aquatic non-target organisms. Such neuroactive substances could pose a potential risk for aquatic organisms below the previously considered concentration threshold for morphological effects.

Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity.

This study investigated the direct and indirect toxic effects of microplastics and nanoplastics toward zebrafish (Danio rerio) larvae locomotor activity. Results showed that microplastics alone exhibited no significant effects except for the upregulated zfrho visual gene expression; whereas nanoplastics inhibited the larval locomotion by 22% during the last darkness period, and significantly reduced larvae body length by 6%, inhibited the acetylcholinesterase activity by 40%, and upregulated gfap, α1-tubulin, zfrho and zfblue gene expression significantly. When co-exposed with 2µg/L 17 α-ethynylestradiol (EE2), microplastics led to alleviation on EE2's inhibition effect on locomotion, which was probably due to the decreased freely dissolved EE2 concentration. However, though nanoplastics showed stronger adsorption ability for EE2, the hypoactivity phenomenon still existed in the nanoplastics co-exposure group. Moreover, when co-exposed with a higher concentration of EE2 (20µg/L), both plastics showed an enhanced effect on the hypoactivity. Principal component analysis was performed to reduce data dimensions and four principal components were reconstituted in terms of oxidative stress, body length, nervous and visual system related genes explaining 84% of total variance. Furthermore, oxidative damage and body length reduction were evaluated to be main reasons for the hypoactivity. Therefore, nanoplastics alone suppressed zebrafish larvae locomotor activity and both plastic particles can change the larvae swimming behavior when co-exposed with EE2. This study provides new insights into plastic particles' effects on zebrafish larvae, improving the understanding of their environmental risks to the aquatic environment.