Effects of Early Life Oral Arsenic Exposure on Intestinal Tract Development and Lipid Homeostasis in Neonatal Mice: Implications for NAFLD Development

Background: Newborns can be exposed to inorganic arsenic (iAs) through contaminated drinking water, formula, and other infant foods. Epidemiological studies have demonstrated a positive association between urinary iAs levels and the risk of developing nonalcoholic fatty liver disease (NAFLD) among U.S. adolescents and adults. Objectives: The present study examined how oral iAs administration to neonatal mice impacts the intestinal tract, which acts as an early mediator for NAFLD. Methods: Neonatal mice were treated with a single dose of iAs via oral gavage. Effects on the small intestine were determined by histological examination, RNA sequencing, and biochemical analysis. Serum lipid profiling was analyzed by fast protein liquid chromatography (FPLC), and hepatosteatosis was characterized histologically and biochemically. Liver X receptor-alpha (LXRα) knockout (Lxrα−/−) mice and liver-specific activating transcription factor 4 (ATF4)-deficient (Atf4ΔHep) mice were used to define their roles in iAs-induced effects during the neonatal stage. Results: Neonatal mice exposed to iAs via oral gavage exhibited accumulation of dietary fat in enterocytes, with higher levels of enterocyte triglycerides and free fatty acids. These mice also showed accelerated enterocyte maturation and a longer small intestine. This was accompanied by higher levels of liver-derived very low-density lipoprotein and low-density lipoprotein triglycerides, and a lower level of high-density lipoprotein cholesterol in the serum. Mice exposed during the neonatal period to oral iAs also developed hepatosteatosis. Compared with the control group, iAs-induced fat accumulation in enterocytes became more significant in neonatal Lxrα−/− mice, accompanied by accelerated intestinal growth, hypertriglyceridemia, and hepatosteatosis. In contrast, regardless of enterocyte fat accumulation, hepatosteatosis was largely reduced in iAs-treated neonatal Atf4ΔHep mice. Conclusion: Exposure to iAs in neonatal mice resulted in excessive accumulation of fat in enterocytes, disrupting lipid homeostasis in the serum and liver, revealing the importance of the gut–liver axis and endoplasmic reticulum stress in mediating iAs-induced NAFLD at an early age. https://doi.org/10.1289/EHP12381

Table S1.Primers used for RT-qPCR analysis of selected genes.
Table S2.Antibodies used for western blot analysis and immunostaining.S9. iAs, inorganic arsenic; LA, linoleic acid; LGR5, leucine rich repeat containing G protein-coupled receptor 5; PCNA, proliferating cell nuclear antigen; OA, oleic acid.For the adult study, 8 week old male mice were orally treated with vehicle or 10 mg/kg iAs and tissues were collected at 24 h.RT-qPCR of Hmgcs2 (n=3, mean ± SEM, one-way ANOVA).(E)

Figure S2 .
Figure S2.(A) Neonatal hUGT1 mice were orally treated with 10 mg/kg iAs.Small intestine was collected at 4, 24 and 48 h post exposure.Mice treated with vehicle are regarded as control.We conducted RT-qPCR analysis of de novo lipogenesis related genes in the small intestine (n=3, mean ± SEM, one-way ANOVA).(B) Quantitative analysis of intestinal LGR5 and SIS expression at 48 h post oleic acid treatment (n=3, mean ± SEM, two tailed student's t test).Band densities in Figure 2J were quantified (C) Neonatal hUGT1 mice were treated with vehicle or 5 g/kg linoleic acid (LA) for 24 h.RT-qPCR of the small intestinal maturation marker genes (n=3, 5, mean ± SEM, two tailed student's t test).Note: *p<0.05,**p<0.01,***p<0.001,****p<0.0001.Corresponding numeric data and individual p value are listed in Excel TableS9.iAs, inorganic arsenic; LA, linoleic

Figure S3 .
Figure S3.(A-B) Neonatal hUGT1 mice at 13-days-old were orally treated with vehicle (C) or iAs (10 mg/kg).Small intestines were collected 24 h after iAs treatment.RNA samples from 3 mice were combined as one sample, with four samples from each group subjected for RNA sequencing (RNA-seq) analysis.(A) Heat map comparing genes related to lysosome.(B) Heat map comparing genes related to FA β-oxidation.(C) Quantitative analysis of HMGCS2 expression as demonstrated in Figure 3F (n=3, mean ± SEM, one-way ANOVA).(D) Neonatal hUGT1 mice were orally treated with 10 mg/kg iAs.Small intestine was collected at 4, 24 and 48 h post iAs exposure.

Figure
Figure S4.(A) Neonatal hUGT1 mice were orally treated with 10 mg/kg iAs for a single dose or three doses.Serum samples were collected, and ALT and AST levels were measured (n=4, 6, 3, 5, mean ± SEM, one-way ANOVA).(B-D) Neonatal hUGT1 mice were orally treated with 10 mg/kg iAs.Liver tissue was collected at specified time points post iAs exposure.(B) RT-qPCR of de novo lipogenesis related genes (n = 3, mean ± SEM, one-way ANOVA).(C) RT-qPCR of fatty

Table S1 .
Primers used for RT-qPCR analysis of selected genes.

Table S2 .
Antibodies used for western blot analysis and immunostaining.