Inorganic arsenic causes fatty liver and interacts with ethanol to cause alcoholic liver disease in zebrafish

Abstract

The rapid increase in fatty liver disease (FLD) incidence is attributed largely to genetic and lifestyle factors; however, environmental toxicants are a frequently overlooked factor that can modify the effects of more common causes of FLD. Chronic exposure to inorganic arsenic (iAs) is associated with liver disease in humans and animal models, but neither the mechanism of action nor the combinatorial interaction with other disease-causing factors has been fully investigated. Here, we examined the contribution of iAs to FLD using zebrafish and tested the interaction with ethanol to cause alcoholic liver disease (ALD). We report that zebrafish exposed to iAs throughout development developed specific phenotypes beginning at 4 days post-fertilization (dpf), including the development of FLD in over 50% of larvae by 5 dpf. Comparative transcriptomic analysis of livers from larvae exposed to either iAs or ethanol revealed the oxidative stress response and the unfolded protein response (UPR) caused by endoplasmic reticulum (ER) stress as common pathways in both these models of FLD, suggesting that they target similar cellular processes. This was confirmed by our finding that arsenic is synthetically lethal with both ethanol and a well-characterized ER-stress-inducing agent (tunicamycin), suggesting that these exposures work together through UPR activation to cause iAs toxicity. Most significantly, combined exposure to sub-toxic concentrations of iAs and ethanol potentiated the expression of UPR-associated genes, cooperated to induce FLD, reduced the expression of as3mt, which encodes an arsenic-metabolizing enzyme, and significantly increased the concentration of iAs in the liver. This demonstrates that iAs exposure is sufficient to cause FLD and that low doses of iAs can potentiate the effects of ethanol to cause liver disease.This article has an associated First Person interview with the first author of the paper.

Keywords: Arsenic; Environmental exposure; Ethanol; Fatty liver disease.

Fig. 1.

Exposure to iAs is toxic to zebrafish embryos. (A) Survival analysis of zebrafish treated with iAs from 4 hpf through 6 dpf. Fish were scored daily for mortality (n≥2 clutches, >80 embryos per condition, Table S1). Red arrow indicates addition of iAs. Concentration of iAs used throughout the manuscript is shown in red and indicated by an asterisk. (B) Bright-field images of representative wild-type control (top) and arsenic-exposed (bottom) 5 dpf zebrafish larvae. Arrows indicate arsenic-induced phenotypes, including shortened body length, edema, clustering of pigment cells, under-consumption of yolk, and a small head. (C) Exposure to increasing doses of iAs from 4 hpf to 5 dpf led to an accumulation of phenotypes. The proportion of surviving embryos at 5 dpf that were affected increased with increasing concentrations of iAs (n=2 clutches exposed to 0.1 mM or 0.3 mM, n=3 clutches exposed to 0.6 mM, 1.0 mM or 1.4 mM, >30 fish exposed per treatment condition, Table S1). hpf, hours post-fertilization; dpf, days post-fertilization; iAs, inorganic arsenic.

Fig. 2.

Zebrafish are capable of arsenic metabolism and accumulate iAs in their tissues. (A) Expression of the zebrafish as3mt transcript is dynamic during zebrafish development, as determined by qRT-PCR. as3mt is maternally provided. Expression is enriched in the liver at 120 hpf. Error bars correspond to mean±s.d. L, liver; C, carcass. (B) Representative images of LA-ICP-MS analysis of 10-μm sections of control and iAs-exposed larvae. Following exposure from 4 to 120 hpf, iAs accumulated in the eye (white arrows, white circle in enlarged image), liver (yellow arrows, yellow circle in enlarged image) and in the gut (green arrows, green circle in the enlarged image). Refer to Table S2 for operating parameters. (C) Quantification of total arsenic content in the livers of 5-dpf larvae by ICP-MS. Livers dissected from larvae exposed to 0, 0.1, 0.5 and 1.0 mM iAs from 4 to 120 hpf showed a dose-dependent increase in the total arsenic content per liver (n=3 clutches). Error bars correspond to mean±s.d. hpf, hours post-fertilization; LA-ICP-MS, laser-ablation–inductively-coupled-plasma–mass-spectroscopy; iAs, inorganic arsenic; ICP-MS, inductively-coupled-plasma–mass-spectroscopy.

Fig. 3.

Exposure to iAs causes steatosis in zebrafish larvae. (A) Representative bright-field images of 5-dpf Oil Red O (ORO)-stained control and iAs-exposed (1.0 mM from 4 to120 hpf) larvae. The area around the liver (outlined in black) is enlarged. (B) The percent of larvae with steatosis analyzed by ORO staining of 15 clutches, with an average of 20 larvae per treatment per clutch. The total number of larvae analyzed in each clutch is listed in Table S1. Statistical significance was determined by unpaired, 2-tailed Student's t-test (n=15 clutches, P<0.001, Table S1). Error bars correspond to mean±s.d.

Fig. 4.

Exposure to iAs induces expression of genes involved in metabolic processes and the UPR. MA plot of normalized gene expression in livers from larvae exposed to 1.0 mM iAs from 4 to 120 hpf compared to unexposed siblings (A) and in livers from larvae exposed to 2% ethanol from 96 to 132 hpf compared to unexposed siblings (B). P-values and fold-changes for all significantly differentially expressed genes are included in Table S1. (B) IPA analysis of biological processes based on upregulated genes identified through RNAseq analysis. (C) MA plot of normalized gene expression in larvae exposed to 2% ethanol from 96-132 hpf compared to unexposed larvae. (D) IPA of biological processes based on upregulated genes identified through RNAseq analysis. (E) Plot of genes significantly upregulated in both the iAs and ethanol datasets (upper right quadrant), upregulated in the iAs dataset and downregulated in the ethanol dataset (lower right quadrant), downregulated in both the iAs and ethanol datasets (lower left quadrant), and upregulated in the ethanol dataset and downregulated in iAs dataset (upper left quadrant). (F) IPA of biological processes based on upregulated genes in both iAs and ethanol RNAseq datasets. iAs, inorganic arsenic; IPA, Ingenuity Pathway Analysis.

Fig. 5.

Arsenic and ethanol regulate common cellular pathways. (A) Plot of UPR-associated gene expressions in iAs and ethanol RNAseq datasets. (B) Heat map of the expression of 103 significantly differentially regulated UPR genes. Refer to Table S4 for P-values and fold-changes. iAs, inorganic arsenic.

Fig. 6.

Arsenic potentiates alcohol-induced liver disease in zebrafish larvae. (A) Survival of zebrafish larvae at 120 hpf. Zebrafish larvae were exposed to a range of concentrations of iAs and/or ethanol. Survival was assessed at 120 hpf. Statistical significance was determined by unpaired, 2-tailed Student's t-test, correcting for multiple comparisons (n=3 clutches, P<0.001). Data are presented as mean±s.d. (B) qRT-PCR data from liver cDNA. Data are presented as fold-change vs control. Statistical significance was determined by one-way ANOVA (n=5 clutches, bip: 0.5% ethanol vs 0.5 mM iAs+0.5% ethanol *P=0.0072, 0.5 mM iAs vs 0.5 mM iAs+0.5% ethanol P=0.078; chop: 0.5% ethanol vs 0.5 mM iAs+0.5% ethanol *P=0.0021, 0.5 mM iAs vs 0.5 mM iAs+0.5% ethanol *P=0.0008; atf6: 0.5% ethanol vs 0.5 mM iAs+0.5% ethanol *P=0.0205, 0.5 mM iAs vs 0.5 mM iAs+0.5% ethanol *P=0.0241). (C) qRT-PCR data from liver cDNA. Data are presented as fold-change vs control. Statistical significance was determined by one-way ANOVA (n=5 clutches, 0.5% ethanol vs 0.5 mM iAs+0.5% ethanol *P=0.0013, 0.5 mM iAs vs 0.5 mM iAs+0.5% ethanol *P=0.0158). (D) Quantification of total arsenic content in the livers of 5-dpf larvae by liquid ICP-MS. Statistical significance was determined by one-way ANOVA (n=6 clutches, 0.5 mM iAs vs 0.5 mM iAs+0.5% ethanol *P=0.0003). (E) Steatosis incidence in 120-hpf larvae exposed to 0.5 mM iAs, 0.5% ethanol and 0.5 mM iAs+0.5% ethanol. Statistical significance was determined by one-way ANOVA (n=7 clutches, 0.5% ethanol vs 0.5 mM iAs+0.5% ethanol *P=0.0025, 0.5 mM iAs vs 0.5 mM iAs+0.5% ethanol *P=0.0172). Error bars in all panels correspond to mean±s.d.

Fig. 7.

Model of the mechanism by which iAs and ethanol interact to cause ER stress. iAs and ethanol interact to increase the concentration of iAs in the liver, aggravate ER stress and enhance FLD. This figure was drawn by J. Gregory (Mount Sinai Health System).