Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress

Abstract

Alcoholic liver disease is associated with zinc decrease in the liver. Therefore, we examined whether dietary zinc supplementation could provide protection from alcoholic liver injury. Metallothionein-knockout and wild-type 129/Sv mice were pair-fed an ethanol-containing liquid diet for 12 weeks, and the effects of zinc supplementation on ethanol-induced liver injury were analyzed. Zinc supplementation attenuated ethanol-induced hepatic zinc depletion and liver injury as measured by histopathological and ultrastructural changes, serum alanine transferase activity, and hepatic tumor necrosis factor-alpha in both metallothionein-knockout and wild-type mice, indicating a metallothionein-independent zinc protection. Zinc supplementation inhibited accumulation of reactive oxygen species, as indicated by dihydroethidium fluorescence, and the consequent oxidative damage, as assessed by immunohistochemical detection of 4-hydroxynonenal and nitrotyrosine and quantitative analysis of malondialdehyde and protein carbonyl in the liver. Zinc supplementation suppressed ethanol-elevated cytochrome P450 2E1 activity but increased the activity of alcohol dehydrogenase in the liver, without affecting the rate of blood ethanol elimination. Zinc supplementation also prevented ethanol-induced decreases in glutathione concentration and glutathione peroxidase activity and increased glutathione reductase activity in the liver. In conclusion, zinc supplementation prevents alcoholic liver injury in an metallothionein-independent manner by inhibiting the generation of reactive oxygen species (P450 2E1) and enhancing the activity of antioxidant pathways.

Figure 1

Food and ethanol intakes in MT-KO and WT 129/Sv mice chronically fed ethanol for 12 weeks. Food intake was recorded daily. Results are means ± SD (n = 4 to 6). FI, Food intake; EI, ethanol intake.

Figure 2

Effects of zinc supplementation on hepatic zinc and MT concentrations in MT-KO and WT 129/Sv mice chronically fed ethanol for 12 weeks. Zinc and MT concentrations in the liver were measured by inductively coupled argon plasma emission spectroscopy and by a cadmium-hemoglobin affinity assay, respectively. Results are means ± SD (n = 4 to 6). Significant difference (P < 0.05) is identified by different letters. Cont, Control; EtOH, ethanol.

Figure 3

Effects of zinc supplementation on liver injury in MT-KO and WT 129/Sv mice chronically fed ethanol for 12 weeks. Light microscopy (LM) shows prominent steatosis (arrowhead) and inflammation (arrow) in the liver of ethanol-fed mice, but these histopathological changes were primarily inhibited by zinc. H&E stain. Electron microscopy (EM) reveals ultrastructural alterations in the hepatocytes, including accumulation of lipid droplets (LD), enlargement and degeneration of mitochondria (M), disorganization of rough endoplasmic reticulum (RER), and condensation of chromatin in the nucleus (N). All these ultrastructural changes were suppressed by zinc. Serum ALT activities were measured using a Sigma Diagnostics kit. Hepatic TNF-α levels were measured using an ELISA kit. Results in serum ALT and hepatic TNF-α levels are means ± SD (n = 4 to 6). Significant difference (P < 0.05) is identified by different letters. CV, Central vein; Cont, control; EtOH, ethanol. Original magnifications: [times130 (LM row); ×9800 (EM row).

Figure 4

Effect of zinc supplementation on oxidative stress in the liver of WT 129/Sv mice chronically fed ethanol for 12 weeks. ROS generation was detected by dihydroethidium that yields red fluorescence in the nuclei on oxidation by ROS. Red fluorescence from dihydroethidium was strong in the liver of ethanol-fed mice, but much less in mice treated by ethanol plus zinc. Immunohistochemical stains of 4-HNE and nitrotyrosine in the liver show a strong reactivity in ethanol-fed mice, but a weak reactivity in mice treated by ethanol plus zinc. Lipid peroxidation in the liver was quantitatively measured by biochemical assay of malondialdehyde concentration. Protein oxidation in the liver was quantitatively measured by ELISA assay of protein carbonyl concentrations. Results in lipid peroxidation and protein carbonyl are means ± SD (n = 4 to 6). Significant difference (P < 0.05) is identified by different letters. Cont, Control; EtOH, ethanol; CV, central vein. Original magnifications: ×260 (ROS row); ×130 (4-HNE and nitrotyrosine rows).

Figure 5

Effect of zinc supplementation on alcohol metabolism in WT 129/Sv mice chronically fed ethanol for 12 weeks. CYP2E1 activity in the microsomes was estimated by colorimetrically measuring hydroxylation of p-nitrophenol to 4-nitrocatechol. ADH activity in the cytoplasm was assayed spectrophotometrically using alcohol as substrate by measuring the reduction of NAD+ at 340 nm. ALDH activity in the mitochondria was measured using acetaldehyde as substrate by measuring the reduction of NAD+ at 340 nm. The protein concentrations of CYP2E1 and ADH were quantitatively measured by ELISA. Blood ethanol elimination after an intragastric bolus of ethanol at 3 g/kg was measured to estimate ethanol metabolic rate. Ethanol concentrations were measured using a Sigma Diagnostics Alcohol kit. Results are means ± SD (n = 4 to 6). Significant difference (P < 0.05) is identified by different letters. Cont, Control; EtOH, ethanol.

Figure 6

Effect of zinc supplementation on cytosolic and mitochondrial GSH in the liver of WT 129/Sv mice chronically fed ethanol for 12 weeks. The GSH concentrations in cytosol and mitochondria were measured using a colorimetric Bioxytech GSH-400 assay kit. Activities of GR and GPx were measured using assay kits from Calbiochem. Results are means ± SE (n = 4 to 6). Significant difference (P < 0.05) is identified by different letters. Cont, Control; EtOH, ethanol.