Arsenic responsive genes recover in the liver by 10 days following washout from acute exposure to 1.5 mM iAs. (A) Treatment scheme of acute treatment (4–5 dpf) to 1.5 mM iAs. At 5 dpf, iAs was washed-out (WO) and zebrafish were reared in the aquaculture system. At the indicated time points, livers were dissected, pooled, and analyzed for gene expression. (B) Heat map of the average log2 fold change (Log2FC) of the expression of genes analyzed in 5–7 livers following iAs WO. All genes have data from two clutches except for cp, fabp10a, and pgm1 which have data from one clutch for both control and treated samples. The average values from one or two clutches are shown (top) with the standard deviation (SD) shown in parentheses.

Treatment scheme to evaluate long-term effects of developmental iAs exposure. (A) Treatment scheme of arsenic exposure where F0 embryos were exposed to 0, 0.5 or 1.5 mM of iAs from 4–120 hpf and then washed out and reared on system. (B) Representative photos of 120 hpf wild-type zebrafish exposed to 0, 0.5 or 1.5 mM iAs from 4–120 hpf. (C) The timeframe of sample collection and parameters assessed.

Developmental iAs exposure induces robust gene expression changes in the liver of zebrafish, increases steatosis and reduces liver size. (A) Volcano plot of RNA-seq data from pools of zebrafish livers following exposure to 1 mM iAs from 4–120 hpf compared to control untreated larvae. The significant (padj<0.05) downregulated genes are labelled in green and upregulated genes are labeled in purple. Selected up and down DEGs for further analysis are labelled. (B) qPCR analysis from seven clutches, with 10 livers pooled per clutch collected after 4–120 hpf exposure to 0, 0.5 and 1.5 mM iAs exposure. Values are expressed as the log2 fold change for each clutch comparing treated to untreated controls. * denotes P-value<0.05, Students t-test. Quantification of the left liver lobe area (C,D) and steatosis (E,F) of 120 hpf (5 dpf) and 168 hpf (7 dpf) larvae that were untreated or exposed to 0.5 mM iAs from 4–120 hpf. Left liver lobe area (arrow) was measured in Tg(fabp10a:CAAX-EGFP) transgenic larvae. Steatosis incidence was measured in Nile Red stained larvae by quantifying the number of livers with more than two lipid spots per clutch divided by the total number of larvae. Steatosis severity was measured by dividing the number of lipid spots by the liver surface area in each larvae. Scale bar: 1000 µm. the number of larvae and clutches are indicated for each condition. * and ** denote P-values<0.05 and <0.01 respectively, by two-way ANOVA.

Developmental exposure to iAs negatively impacts long-term survival but not adult morphology. (A) Survival plot of control (grey), 4–120 hpf 0.5 mM iAs treated larvae (pink) and 4–120 hpf 1.5 mM iAs treated larvae (purple). **** denotes P-value<0.0001, curve comparison (Mantel–Cox). (B) Representative images of 8-month-old male and female zebrafish that were developmentally exposed to 0.5 mM iAs compared to untreated controls. Scale bar: 1 cm. (C) Standard length (cm) of 8-month-old males (left) and females (right). Not significant (ns) by Student's t-test.

Developmental exposure to iAs sustains differential gene expression in gsto2 in adult livers post wash out. qPCR data from adult male and female single livers dissected from age-matched adult zebrafish (9–17 months old). Purple bars are from males and yellow bars are from females from the 0.5 mM 4–120 hpf iAs exposed group. n=7, 2 clutches. Values are expressed as the log2 fold change comparing each individual treated liver to the average of control livers. * denotes P-value<0.05, unpaired t-test between untreated and iAs treated 2^deltaCT values.

Developmental exposure to iAs does not cause DNA hypomethylation. (A) Slot blot hybridization from bulk DNA isolated from whole zebrafish after 0 and 1.5 mM iAs (4–120 hpf) exposure at 5 dpf (n=5 pooled larvae per clutch, three clutches). ns=not significant by Students t-test. (B) Representative brightfield and fluorescent images of 5 dpf tg(fabp10a:Gal4;cmlc2:EGFP; c269^off; 10XUAS:dsRed) zebrafish larvae following 0, 0.5 and 1.5 mM iAs exposure from 4–120 hpf. Heart and liver are annotated. dnmt1s904 zebrafish larvae included as a positive control for hypomethylation. dsRed in the liver indicates the presence of active Gal4 in hepatocytes; GFP in the liver indicates DNA hypomethylation.

Developmental iAs exposure reduces mating success but does not affect the number of fertilized embryos. (A) Percent of mating success between eight single pairs of zebrafish that developed from untreated or 0.5 mM iAs developmentally treated zebrafish. *P-value<0.05 by Chi Square. (B) The number of embryos produced from successful matings of control and 0.5 mM iAs developmentally treated zebrafish. (C) Percent of embryos that were fertilized, unfertilized, and dead from successful mating of control and 0.5 mM iAs developmentally exposed zebrafish. ns: not significant, Students T-test.

Parental development exposure to iAs does not alter the toxicity of iAs in offspring. (A) Representative images of 5 dpf F1 larvae produced from incrosses of untreated parents (left) and parents that were developmentally exposed to 0.5 mM iAs (right). F1 were treated to 0 (top) and 1.5 mM iAs from 4–120 hpf (bottom). (B) Survival curve of F1 produced from incrossing parents exposed to 0 (black) or 0.5 mM iAs from 0–120 hpf (pink) after 4–120 hpf exposure to 0, 0.5, 1.0, 1.5, 2.0 and 4.0 mM iAs (n=20 larvae per clutch per group, five clutches).