Synergistic effects of Pb and repeated heat pulse on developmental neurotoxicity in zebrafish

Pollutant discharges to the aquatic environment often contain multiple environmental stressors, affecting aquatic organisms. To mimic the discharges from nuclear and industry facilities, the combined effects of two independent types of stressors, heavy metal Pb and repeated heat pulse were addressed in this study. We investigated the developmental toxicity of combined treatment, especially its toxic effects on zebrafish neurodevelopment. The normal embryos at 4 hpf were exposed to 0.2 mM of Pb dissolved in the bathing medium with different temperatures (30, 32, and 34 °C) and then maintained in an incubator at 28 °C. After performing above treatment once every 24 h for 6 days, we found that combined treatment significantly affected neural development, including loss of dopaminergic (DA) neurons and brain vasculature, disruption of locomotor activity and neurodevelopmental genes expression in a temperature-dependent manner as compared to the Pb alone exposure group, indicating that repeated heat pulse enhances these negative impacts induced by Pb. In contrast, no apparent toxicity was observed in repeated heat pulse alone groups, suggesting that Pb treatment reduces thermal tolerance in zebrafish, which emphasized the importance to evaluate synergistic effects of Pb and repeated heat pulse. Moreover, repeated heat pulse aggravated Pb-induced apoptosis in the zebrafish brain. Further study of the underlying mechanism suggested that Caspase 3 regulated apoptosis was involved in this process. Taken together, our findings shed light on the full understanding of toxic effects of discharges from industrial applications on living organisms and its environmental impact.