Chronic subhepatotoxic exposure to arsenic enhances hepatic injury caused by high fat diet in mice

Abstract

Arsenic is a ubiquitous contaminant in drinking water. Whereas arsenic can be directly hepatotoxic, the concentrations/doses required are generally higher than present in the US water supply. However, physiological/biochemical changes that are alone pathologically inert can enhance the hepatotoxic response to a subsequent stimulus. Such a '2-hit' paradigm is best exemplified in chronic fatty liver diseases. Here, the hypothesis that low arsenic exposure sensitizes liver to hepatotoxicity in a mouse model of non-alcoholic fatty liver disease was tested. Accordingly, male C57Bl/6J mice were exposed to low fat diet (LFD; 13% calories as fat) or high fat diet (HFD; 42% calories as fat) and tap water or arsenic (4.9 ppm as sodium arsenite) for ten weeks. Biochemical and histologic indices of liver damage were determined. High fat diet (± arsenic) significantly increased body weight gain in mice compared with low-fat controls. HFD significantly increased liver to body weight ratios; this variable was unaffected by arsenic exposure. HFD caused steatohepatitis, as indicated by histological assessment and by increases in plasma ALT and AST. Although arsenic exposure had no effect on indices of liver damage in LFD-fed animals, it significantly increased the liver damage caused by HFD. This effect of arsenic correlated with enhanced inflammation and fibrin extracellular matrix (ECM) deposition. These data indicate that subhepatotoxic arsenic exposure enhances the toxicity of HFD. These results also suggest that arsenic exposure might be a risk factor for the development of fatty liver disease in human populations.

Figure 1. Effect of high fat diet on growth and organ weight

Body weight over time (A), epididymal fat pad weight (B) and liver weight (C) for tap water or arsenic exposed mice fed with low fat or high fat diet for 10 weeks are shown. Data are means ± SEM (n = 6–10). ^a, p < 0.05 compared to low fat diet.

Figure 2. Effect of high fat diet and arsenic on liver injury

A: Representative photomicrographs (200×) depicting hematoxylin & eosin (H&E) stains are shown. B: Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in tap water or arsenic exposed mice after 10 weeks of diet feeding are shown. Quantitative data are means ±SE> (n = 6–10). ^a, p < 0.05 compared to low fat diet. ^b, p < 0.05 compared to tap.

Figure 3. Effect of high fat diet and arsenic on hepatic lipids

Hepatic levels of free fatty acids (FFA), triglycerides (TG), cholesterol (CHOL) and phospholipids (PPL) in mice exposed to tap water or arsenic water after 10 weeks of low fat or high fat feeding are shown. Data are means ±SEM (n = 6–10). ^a, p < 0.05 compared to low fat diet.

Figure 4. Effect of high fat diet and arsenic on hepatic inflammation

A: Effect of high fat diet and arsenic on hepatic mRNA expressions of tissue necrosis factor-alpha (TNFα), plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and interleukin-10 (IL-10) are shown. Real-time RT-PCR was performed as described in Methods. B: Representative photomicrographs (200×; upper panel) and quantitation (lower panel) of F4/80 immunostains are shown. Quantitative data are means ± SE (n = 6–10) and are expressed as fold of control. ^a, p < 0.05 compared to low fat diet. . ^b, p < 0.05 compared to tap.

Figure 5. Effect of high fat diet and arsenic on fibrin deposition

Representative photomicrographs (200×; upper panel) and quantitation (lower panel) of fibrin immunofluorescence (green color) are shown. Quantitative data are means ± SE (n = 6–10) and are expressed as fold of control. ^a, p < 0.05 compared to low fat diet. . ^b, p < 0.05 compared to tap.

Figure 6. Effect of high fat diet and arsenic on hepatic fibrosis

A: Representative photomicrographs (200×) of Sirius Red stains with 400× insets are shown. B: Effects of high fat diet and arsenic on hepatic mRNA expressions of alpha-smooth muscle actin (αSMA), transforming growth factor beta (TGFβ), Collagen1a1 and prolyl-4-hydroxylase (P4H) are shown. Real-time RT-PCR was performed as described in Methods. Quantitative data are means ± SE (n = 6–10) and are expressed as fold of control. ^a, p < 0.05 compared to low fat diet. ^b, p < 0.05 compared to tap.