Sustained effects of developmental exposure to inorganic arsenic on hepatic gsto2 expression and mating success in zebrafish.

The impacts of exposure to the pervasive environmental toxicant, inorganic arsenic (iAs), on human and fish health are well characterized and several lines of evidence suggest that some impacts can manifest years after exposure cessation. Using a developmental exposure protocol whereby zebrafish embryos were exposed to 0.5 and 1.5 mM iAs from 4-120 hours post fertilization (hpf) and then removed, we investigated the sustained effects of iAs on gene expression in the liver, survival, reproductive success, and susceptibility to iAs toxicity in the subsequent generation. Persistent exposure to iAs during development had substantial effects on the hepatic transcriptome, with 23% of all expressed genes significantly changed following developmental exposure. The gsto2 gene is involved in iAs metabolism and this gene was significantly downregulated in female livers 9 months after iAs was removed. Developmental exposure to 1.5 mM iAs, but not 0.5 mM, decreased survival by over 50% at 3 months of age. Adults that were developmentally exposed to 0.5 mM iAs had reduced mating success, but their offspring had no differences in observable aspects of development or their susceptibility to iAs toxicity. This demonstrates that developmental exposure of zebrafish to iAs reduces long-term survival, reproductive success and causes sustained changes to gsto2 expression in the liver.

Systematic Evaluation of the Effects of Toxicant Exposure on Survival in Zebrafish Embryos and Larvae.

The use of whole animal models in toxicological studies is essential for understanding the physiological responses caused by chemical exposures. However, such studies can face reproducibility challenges due to unaccounted experimental parameters that can have a marked influence on toxicological outcomes. Zebrafish embryos and larvae are a popular vertebrate animal model for studying cellular, tissue, and organ responses to toxicant exposures. Despite the popularity of this system, standardized protocols that control for the influence of various experimental parameters and culture conditions on the toxicological response in these animals have not been widely adopted, making it difficult to compare findings from different laboratories. Here, we describe a detailed approach for designing and optimizing protocols to assess the impact of chemical exposures on the development and survival of zebrafish embryos and larvae. We first describe our standard procedure to determine two key toxicological thresholds, the maximum tolerable concentration (MTC) and the lethal concentration (LC50 , defined as that in which 50% of larvae die), in response to an exposure that persists from early development through larval maturation. We then describe two protocols to systematically test how key experimental parameters, including genetic background, culture media, animal density, volume, plate material, and developmental stage in which the embryos are exposed, alter the MTC and LC50 . Finally, we provide a step-by-step guide to assess the interaction between two chemicals using this model. These protocols will guide the standardization of toxicological studies using zebrafish and maximize reproducibility. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Zebrafish embryo collection and culture, and establishment of the MTC and LC50 Basic Protocol 2: Evaluation of the impact of culture conditions on toxicant responses of zebrafish embryo and larvae Basic Protocol 3: Identification of the developmental window of sensitivity to toxicant exposure Basic Protocol 4: Testing interaction between multiple toxicants.