Rearing medium dictates variability across replicates in untreated and arsenic challenged zebrafish larvae

Abstract

Reproducibility and consistency are hallmarks of scientific integrity. Biological systems are inherently noisy, posing a challenge to reproducibility. This is particularly relevant to the field of environmental toxicology, where many unaccounted experimental parameters can have a marked influence on the biological response to exposure. Here, we extend the use of zebrafish as a robust toxicological model for studying the effects of inorganic arsenic (iAs) on liver biology. We observed that iAs toxicity in this system is not influenced by important parameters including genetic background, rearing container material or rearing volume but the dose response to iAs is influenced by the rearing medium. We compared mortality as a measure of iAs toxicity to embryos cultured in two standard rearing media: egg water made from dehydrated ocean salts dissolved in water and a defined embryo medium which is a pH adjusted, buffered salt solution. Larvae reared in egg water were more susceptible to iAs compared to those reared in embryo medium. This effect was independent of the pH differences between these solutions. These culture conditions did not cause any difference in the global hepatic transcriptome of control zebrafish. Further, no difference in the expression of genes involved in the unfolded protein response (UPR) in larvae exposed to iAs treatment or in a stress independent system to activate UPR genes by transgenic overexpression of activating transcription factor 6 (nAtf6) in hepatocytes was observed. However, the clutch-to-clutch variation in gene expression was significantly greater in larvae reared in egg water compared to those in embryo medium. These data demonstrate that egg water affects reproducibility across replicates in terms of gene expression and exacerbates iAs mediated toxic response. This highlights the importance of rigorous evaluation of experimental conditions to assure reproducibility.

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

“…During the course of our studies on the toxic effects of iAs (Bambino et al., 2018; Delaney et al., 2020), we identified a difference in susceptibility of embryos to arsenic depending on the embryo culture medium, with those raised in egg water being more sensitive (Delaney et al., 2020). Moreover, we found higher variability in gene expression in livers from larvae reared in egg water (Nair et al., 2020). These results show that rearing medium can have a pronounced effect on the outcome of toxicological studies in zebrafish.…”

Sustained Effects of Developmental Exposure to Inorganic Arsenic on Hepatic Gene Expression and Mating Success in Zebrafish

“…iAs exposure in embryos and adult rearing Embryos were exposed to sodium meta-arsenite (Sigma-Aldrich, USA; henceforth referred to as iAs) by diluting 0.05 M stock solution to final concentrations ranging from 0.5 mM -4 mM in embryo medium from either 4-120 hpf or 96-120 hpf, as indicated in the text. A detailed overview of how treatment parameters were optimized are previously described (Ramdas Nair et al, 2021). For all experiments, mortality was scored daily, dead embryos and larvae were removed, and morphological parameters were recorded at 120 hpffr.…”

“…As in mammals, iAs toxicity is attributed to cell stress responses, including oxidative and proteostatic stress (Delaney et al, 2020;Fuse et al, 2016;Lam et al, 2006;Ramdas Nair et al, 2022;Xu et al, 2013). We optimized the experimental parameters for using zebrafish in toxicology studies (Ramdas Nair et al, 2021) and extended the use of this model for studying iAs toxicity. We discovered that, like humans, iAs accumulates in the zebrafish liver and that the liver is a major source of iAs toxicity (Bambino et al, 2018;Delaney et al, 2023).…”