Zinc Supplementation Prevents Alcoholic Liver Injury in Mice through Attenuation of Oxidative Stress
2005; Elsevier BV; Volume: 166; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)62478-9
ISSN1525-2191
AutoresZhanxiang Zhou, Lipeng Wang, Zhenyuan Song, Jack T. Saari, Craig J. McClain, Yin Kang,
Tópico(s)Alcohol Consumption and Health Effects
ResumoAlcoholic 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-α 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. 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-α 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. Zinc depletion in the liver has been well documented in alcoholic patients as well as in animal models of ethanol-induced liver disease.1Kiilerich S Dietrichson O Loud FB Naestoft J Christoffersen P Juhl E Kjems G Christiansen C Zinc depletion in alcoholic liver diseases.Scand J Gastroenterol. 1980; 15: 363-367Crossref PubMed Scopus (51) Google Scholar, 2McClain CJ Antonow DR Cohen DA Shedlofsky S Zinc metabolism in alcoholic liver disease.Alcohol Clin Exp Res. 1986; 10: 582-589Crossref PubMed Scopus (76) Google Scholar, 3Bode JC Hanisch P Henning H Koenig W Richter FW Bode C Hepatic zinc content in patients with various stages of alcoholic liver disease and in patients with chronic active and chronic persistent hepatitis.Hepatology. 1988; 8: 1605-1609Crossref PubMed Scopus (91) Google Scholar Investigation of zinc metabolism in alcoholics has demonstrated that ethanol consumption leads to increased zinc excretion in urine and decreased zinc absorption from intestine.4Dinsmore W Callender ME McMaster D Todd SJ Love AH Zinc absorption in alcoholics using zinc-65.Digestion. 1985; 32: 238-242Crossref PubMed Scopus (29) Google Scholar, 5Valberg LS Flanagan PR Ghent CN Chamberlain MJ Zinc absorption and leukocyte zinc in alcoholic and nonalcoholic cirrhosis.Dig Dis Sci. 1985; 30: 329-333Crossref PubMed Scopus (55) Google Scholar Although zinc depletion has been suggested to play an important role in alcoholic live injury,2McClain CJ Antonow DR Cohen DA Shedlofsky S Zinc metabolism in alcoholic liver disease.Alcohol Clin Exp Res. 1986; 10: 582-589Crossref PubMed Scopus (76) Google Scholar the mechanistic insights into the involvement of zinc depletion in the pathogenesis of alcoholic liver disease have not been achieved. Ethanol is mainly metabolized in the liver through three major pathways with different subcellular locations: alcohol dehydrogenase (ADH) in the cytosol, aldehyde dehydrogenase (ALDH) in the mitochondria, and microsomal ethanol-oxidizing system in the endoplasmic reticulum.6Lieber CS Alcohol metabolism, cirrhosis and alcoholism.Clin Chim Acta. 1997; 257: 59-84Crossref PubMed Scopus (348) Google Scholar All of the three pathways result in reactive oxygen species (ROS) generation. However, the microsomal ethanol-oxidizing system, especially the cytochrome P450 2E1 (CYP2E1), has been shown to play a critical role in ethanol-induced oxidative stress. Long-term ethanol exposure significantly increases the CYP2E1 pathway.7Nanji AA Zhao S Sadrzadeh SM Dannenberg AJ Tahan SR Waxman DJ Markedly enhanced cytochrome P450 2E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats.Alcohol Clin Exp Res. 1994; 18: 1280-1285Crossref PubMed Scopus (234) Google Scholar, 8Morimoto M Zern MA Hagbjork AL Ingelman-Sundberg M French SW Fish oil, alcohol, and liver pathology: role of cytochrome P450 2E1.Proc Soc Exp Biol Med. 1996; 207: 197-205Crossref Scopus (156) Google Scholar, 9Albano E Clot P Morimoto M Tomasi A Ingelman-Sundberg M French SW Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol.Hepatology. 1996; 23: 155-163Crossref PubMed Google Scholar A recent study using CYP2E1 transgenic mice has demonstrated that overexpression of CYP2E1 enhances liver damage by chronic ethanol exposure.10Morgan K French SW Morgan TR Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage.Hepatology. 2002; 36: 122-134Crossref PubMed Scopus (129) Google Scholar Inhibition of CYP2E1 activity by inhibitors reduced ethanol-induced liver injury.11Morimoto M Hagbjork AL Wan YJ Fu PC Clot P Albano E Ingelman-Sundberg M French SW Modulation of experimental alcohol-induced liver disease by cytochrome P450 2E1 inhibitors.Hepatology. 1995; 21: 1610-1617PubMed Google Scholar, 12Gouillon Z Lucas D Li J Hagbjork AL French BA Fu P Fang C Ingelman-Sundberg M Donohue Jr, TM French SW Inhibition of ethanol-induced liver disease in the intragastric feeding rat model by chlormethiazole.Proc Soc Exp Biol Med. 2000; 224: 302-308Crossref PubMed Google Scholar On the other hand, chronic ethanol exposure has little effect on ADH activities,7Nanji AA Zhao S Sadrzadeh SM Dannenberg AJ Tahan SR Waxman DJ Markedly enhanced cytochrome P450 2E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats.Alcohol Clin Exp Res. 1994; 18: 1280-1285Crossref PubMed Scopus (234) Google Scholar although ADH is a major pathway for ethanol metabolism under normal physiological conditions. Thus, the shift from ADH to CYP2E1 in ethanol metabolism under chronic ethanol exposure may account for the generation of ROS under chronic ethanol exposure. Many studies have demonstrated that zinc per se functions as an antioxidant.13Powell SR The antioxidant properties of zinc.J Nutr. 2000; 130: 1447S-1454SPubMed Google Scholar Importantly, zinc has catalytic and structural roles in more than 300 metalloenzymes, as well as regulatory roles in diverse cellular processes such as signaling transduction and gene expression. ADH is a zinc metalloenzyme, and removal of zinc from ADH led to a complete loss of its catalytic activity. Thus, the ethanol-induced zinc depletion is most likely linked to the altered ethanol metabolic pathway, such that a shift from ADH to CYP2E1 results in oxidative stress. Zinc also plays an important role in regulation of cellular glutathione (GSH) that is vital to cellular antioxidant defense.14Parat MO Richard MJ Beani JC Favier A Involvement of zinc in intracellular oxidant/antioxidant balance.Biol Trace Elem Res. 1997; 60: 187-204Crossref PubMed Scopus (64) Google Scholar Therefore, the present study was undertaken to determine whether dietary zinc supplementation can improve hepatic zinc status under chronic ethanol exposure, thereby preventing oxidative liver injury, and the possible mechanisms by which zinc inhibits ethanol-induced oxidative stress, focusing on ethanol metabolic pathway and GSH-related antioxidants. Because zinc is a potent inducer of metallothionein (MT) synthesis and MT plays an important role in zinc homeostasis,15Vallee BL The function of metallothionein.Neurochem Int. 1995; 27: 23-33Crossref PubMed Scopus (259) Google Scholar a MT-knockout (MT-KO) mouse model and the wild-type (WT) 129/Sv controls were used to define whether the action of zinc is MT-dependent or MT-independent. Homozygous MT-KO mice and WT controls were obtained from Jackson Laboratories (Bar Harbor, ME). The MT-KO mice lacking MT-I and MT-II, the major mouse hepatic MT isoforms, were produced on the 129/Sv genetic background by a gene-targeting technique.16Masters BA Kelly EJ Quaife CJ Brinster RL Palmiter RD Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium.Proc Natl Acad Sci. 1994; 91: 584-588Crossref PubMed Scopus (568) Google Scholar All of the mice were treated according to the experimental procedures approved by the Institutional Animal Care and Use Committee. The Lieber-DeCarli ethanol liquid diet has been widely used to establish animal models for alcoholic liver disease. According to our preliminary experiments, a long-term feeding of mice with the Lieber-DeCarli ethanol liquid diet causes decreases in food intake and body weight gain. To eliminate the possible confounding of malnutrition caused by decreased food intake, we established an improved ethanol feeding protocol for mice. In this protocol, a 1-day-stop on the last day of each week was introduced in the ethanol feeding schedule by replacing ethanol diet with control diet. The content of ethanol in the diet (%, w/v) was gradually increased throughout a 12-week feeding period, starting at 3.2, increasing 0.2% every 2 weeks, and reaching 4.2% at the end. Accordingly, the calorie contribution of ethanol started at 23% of total calories, and reached 30% at the end of 12-week feeding. Ten-week-old male MT-KO and WT 129/Sv mice (∼25 g body weight) were used. Animals were pair-fed with the Lieber-DeCarli liquid diet (Bio-Serv, Frenchtown, NJ), containing either ethanol or isocaloric dextrin maltose. Animals were randomly assigned to eight groups using a 2 × 2 × 2 factorial design, MT × ethanol × zinc. Zinc supplementation was conducted by adding zinc sulfate to the liquid diet at 75 mg zinc element/L. Food intake and body weight were recorded daily and weekly, respectively. At the end of the experiment, the mice were anesthetized with sodium pentobarbital (50 mg/kg). Blood was drawn using a heparinized syringe from the dorsal vena cava, and serum was obtained by centrifugation. The liver was perfused with saline and tissue samples were processed for both pathological and biochemical analysis. Blood ethanol levels were examined at 9:00 a.m. in the last week. Blood samples were taken from the tail vein and immediately deproteinized with 6.25% trichloroacetic acid solution. Ethanol concentrations were determined using a Sigma Diagnostics Alcohol kit (procedure no. 332-UV; Sigma, St. Louis, MO). Hepatic zinc concentrations were determined by inductively coupled argon plasma emission spectroscopy (model 1140; Jarrel-Ash, Waltham, MA) after lyophilization and digestion of the tissues with nitric acid and hydrogen peroxide.17Nielsen FH Zimmerman TJ Shuler TR Interactions among nickel, copper, and iron in rats: liver and plasma contents of lipid and trace elements.Biol Trace Elem Res. 1982; 4: 1225-1243Crossref Scopus (74) Google Scholar MT concentrations in the liver were determined by a cadmium-hemoglobin affinity assay.18Eaton DL Cherian MG Determination of metallothionein in tissues by cadium-hemoglobin affinity assay.Methods Enzymol. 1991; 205: 83-88Crossref PubMed Scopus (158) Google Scholar Histopathological and ultrastructural changes in the liver were examined by light and electron microscopy.19Zhou Z Sun X Kang YJ Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress.Exp Biol Med. 2002; 227: 214-222Google Scholar Serum alanine aminotransferase (ALT) activity was colorimetrically measured using a Sigma Diagnostic kit (procedure no. 505, Sigma). Liver tumor necrosis factor (TNF)-α levels were detected by enzyme-linked immunosorbent assay (ELISA) using a murine kit (BioSource Int., Inc., Camarillo, CA).20Zhou Z Wang L Song Z Lambert JC McClain CJ Kang YJ A critical involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha production.Am J Pathol. 2003; 163: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Ethanol-induced oxidative stress in the liver was measured by microscopic detection of ROS, 4-hydroxynonenal (4-HNE), and nitrotyrosine, and by biochemical measurements of lipid peroxidation and protein oxidation. Dihydroethidium was used for in situ detection of ROS in the liver according to a previous report.21Carter WO Narayanan PK Robinson JP Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.J Leukoc Biol. 1994; 55: 253-258Crossref PubMed Google Scholar Nonfluorescent dihydroethidium is oxidized by ROS to yield red fluorescent product that binds to nucleic acids, staining the nucleus a bright fluorescent red. Cryostat sections of liver were cut at 5 μm and incubated with 5 μmol/L dihydroethidium (Molecular Probes, Eugene, OR) at 37°C for 30 minutes. The red fluorescence from dihydroethidium was detected with a Nikon 2000S fluorescence microscope (Nikon, Melville, NY). Lipid peroxidation product, 4-HNE, and protein oxidation product, nitrotyrosine, were detected by immunohistochemical staining. Liver tissues were fixed with 4% paraformaldehyde, and sections were cut at 5 μm. A rabbit anti-4-HNE antibody (Alpha Diagnostic, San Antonio, TX) and a rabbit anti-nitrotyrosine antibody (Upstate, Waltham, MA) in combination with a DAKO EnVision+ horseradish peroxidase-conjugated goat anti-rabbit IgG (DAKO, Carpinteria, CA) were used for recognizing the oxidative products. The negative controls were conducted by omitting the primary antibody. The extent of lipid peroxidation was quantitatively determined by measuring the concentration of thiobarbituric acid-reactive product malondialdehyde.19Zhou Z Sun X Kang YJ Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress.Exp Biol Med. 2002; 227: 214-222Google Scholar Protein peroxidation in the liver was quantified by measuring protein carbonyl content using a sensitive ELISA.22Buss H Chan TP Sluis KB Domigan NM Winterbourn CC Protein carbonyl measurement by a sensitive ELISA method.Free Radic Biol Med. 1997; 23: 361-366Crossref PubMed Scopus (624) Google Scholar Protein derivatization was first performed by incubating 15-μl samples (4 mg protein/ml) and 45 μl of 10 mmol/L dinitro-phenylhydrazide. The detect system was a rabbit anti-dinitrophenylhydrazide antibody (Molecular Probes) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma). The activities of CYP2E1, ADH, and ALDH in the liver and the ethanol metabolic rate were measured. Microsomal CYP2E1 activity was estimated by colorimetrically measuring the hydroxylation of p-nitrophenol to 4-nitrocatechol, a reaction catalyzed specifically by CYP2E1.23Allis JW Robison BL A kinetic assay for p-nitrophenol hydroxlase in rat liver microsomes.Anal Biochem. 1994; 219: 49-52Crossref PubMed Scopus (31) Google Scholar ADH activity was measured by detecting the reduction of NAD+ at 340 nm in a reaction mixture containing 0.5 mol/L Tris-HCl buffer (pH 7.2), 2.8 mmol/L NAD+, and 5 mmol/L ethanol.24Crow KE Cornell NW Veech RL The rate of ethanol metabolism in isolated rat hepatocytes.Alcohol Clin Exp Res. 1977; 1: 43-50Crossref PubMed Scopus (147) Google Scholar Mitochondrial ALDH activity was measured by detecting the reduction of NAD+ at 340 nm in a reaction mixture containing 50 mmol/L sodium pyrophosphate (pH 8.8), 0.5 mmol/L NAD+, 0.1 mmol/L pyrazol, 5 mmol/L acetaldehyde, and 2 μmol/L rotenone.25Tottmar SOC Pettersson H Kiessling KH The subcellular distribution and properties of aldehyde dehydrogenases in rat liver.Biochem J. 1973; 135: 577-586Crossref PubMed Scopus (353) Google Scholar The protein concentrations of CYP2E1 and ADH were quantitatively measured by an ELISA. The detect systems were a rabbit anti-CYP2E1 antibody (Calbiochem, San Diego, CA) or a mouse anti-ADH antibody (Chemicon, Temecula, CA) in combination with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgM (Sigma). Ethanol metabolic rate was estimated by measuring blood ethanol elimination during the last week of feeding. Ethanol diets were replaced with control diets 1 day before the measurement. Mice were given an intragastric bolus of ethanol at 3 g/kg. Blood samples were taken from the tail vein every hour for 4 hours, and the blood ethanol levels were measured using a Sigma Diagnostic Alcohol kit (procedure no. 332-UV, Sigma). For measurement of subcellular GSH levels, cytosolic and mitochondrial fractions were separated from fresh liver tissue. Briefly, the liver was homogenized in 10 mmol/L HEPES buffer, pH 7.4, containing 200 mmol/L mannitol, 50 mmol/L sucrose, 10 mmol/L KCl, and 1 mmol/L ethylenediamine tetraacetic acid using a Dounce glass homogenizer (Kontes, Vineland, NJ). The crude homogenate was centrifuged at 700 × g, and the supernatant was centrifuged further at 15,000 × g to give a cytosolic supernatant and a mitochondrial pellet. The mitochondrial pellet was washed twice and resuspended in the same buffer. Metaphosphoric acid was used for deproteinization at a final concentration of 5%. After centrifuging at 15,000 × g, the supernatants were used as mitochondrial fraction for GSH assay immediately. The GSH concentrations were measured using a Bioxytech GSH-400 assay kit (OXIS, Portland, OR). Glutathione reductase (GR) activity was assayed by measuring the rate of NADPH oxidation at 340 nm in the presence of 2.4 mmol/L GSSG with a glutathione reductase assay kit (Calbiochem). Glutathione peroxidase (GPx) activity was assayed by measuring the rate of NADPH oxidation at 340 nm with a cellular glutathione peroxidase assay kit (Calbiochem) based on the reduction of organic peroxide, tert-butyl hydroperoxide, by GPx in the presence of GSH. All data are expressed as mean ± SE (n = 4 to 6). The data were analyzed by analysis of variance and Newman-Keuls' multiple comparison test. Differences between groups were considered significant at P < 0.05. Daily food intake was monitored in the ethanol-exposure WT and MT-KO mice, and pair-fed mice were adjusted for their food supply accordingly. The dietary ethanol levels were increased by 0.2% (w/v) every 2 weeks and the daily food intake showed an increase during the 12-week feeding period with an average of 445 g/kg/day in WT and 434 g/kg/day in MT-KO mice (Figure 1). Accordingly, the calculated daily ethanol intake (g/kg) was gradually increased. The average daily zinc intake was 33 mg/kg. The blood ethanol levels (mg/dl) measured at 9:00 a.m. in the last week were 221 ± 45 in WT mice and 210 ± 30 in MT-KO mice. The liver:body weight ratios at the end of the experiment significantly increased in the ethanol-fed mice (WT, 4.47 ± 0.36; MT-KO, 4.70 ± 0.48) related to the pair-fed groups (WT, 3.16 ± 0.18; MT-KO, 3.22 ± 0.29). No significant differences in all of the above parameters were found between the MT-KO and WT mice. Zinc supplementation to the ethanol-fed mice had no significant effects on all of the parameters measured above. As shown in Figure 2, chronic ethanol exposure caused a significant decrease in hepatic zinc concentrations in both WT and MT-KO mice with a lower value in the latter. Dietary zinc supplementation did not elevate the basal levels, but prevented ethanol-induced decrease, of the hepatic zinc concentrations in both WT and MT-KO mice. Because MT regulates cellular zinc homeostasis, MT concentrations in the liver were measured. In the WT mice, chronic ethanol exposure significantly decreased MT concentrations in the liver. In contrast, the MT concentrations in the liver of mice chronically fed ethanol were significantly increased by zinc supplementation, although zinc did not increase MT concentrations in the liver of pair-feeding WT mice. Only trace amounts of hepatic MT were detected in all groups of MT-KO mice, which represent the assay background. The results presented in Figure 3 show the effects of zinc supplementation on ethanol-induced liver injury. Examination by light microscopy found that zinc attenuated ethanol-induced histopathological changes, including macrovesicular steatosis and focal inflammation in both WT and MT-KO mice. Severe ultrastructural alterations were also observed in the ethanol-exposed mouse liver, including lipid droplet accumulation, enlargement and degeneration of mitochondria, disorganization of rough endoplasmic reticulum, and chromatin condensation. All these ultrastructural abnormalities of hepatocytes under chronic ethanol exposure were primarily inhibited by zinc supplementation in both WT and MT-KO mice (shown here only the data from WT mice). Corresponding to the pathological changes, the plasma ALT levels were significantly elevated in both WT and MT-KO mice with a greater value in the latter. However, zinc supplementation significantly inhibited ethanol-induced ALT elevation equally in the WT and MT-KO mice (Figure 3). Hepatic TNF-α levels were also increased by ethanol exposure, which was abrogated by zinc supplementation in both WT and MT-KO mice (Figure 3). To understand the possible mechanisms by which zinc prevents ethanol-induced liver injury, the effect of zinc supplementation on ethanol-induced oxidative stress in the liver of WT mice was determined as shown in Figure 4. Chronic ethanol exposure enhanced the red fluorescence from dihydroethidium in the liver, but much less in the liver of ethanol-fed mice with zinc supplementation. Immunoperoxidase stain demonstrated that the immunoreactivities to 4-HNE and nitrotyrosine in the liver were increased by chronic ethanol exposure. The positive stain mainly localized around the central vein. Zinc supplementation markedly diminished ethanol-induced 4-HNE and nitrotyrosine stains in the liver. The negative controls showed no background staining on the sections of all treatments. Quantitative measurements of malondialdehyde and protein carbonyl in the liver revealed that zinc significantly diminished ethanol-induced oxidative lipid and protein damages. Although the data presented here were obtained from WT mice, the same results were also obtained from MT-KO mice (data not shown). To determine whether zinc inhibits oxidative stress through modulating ethanol metabolic pathways, the effect of zinc supplementation on ethanol metabolism was determined in the WT mice. As shown in Figure 5, chronic ethanol exposure caused a significant increase in the hepatic CYP2E1 activity, which was significantly inhibited by zinc supplementation. In contrast, the ADH activity in the liver was not affected by chronic ethanol exposure, but significantly increased by zinc supplementation. The ALDH activities were significantly increased in mice chronically fed ethanol alone or ethanol plus zinc. Next, possible differences in the protein levels of CYP2E1 and ADH between chronic ethanol-exposed and the zinc-supplemented mice were determined. The protein concentrations of CYP2E1 in the liver were significantly increased by chronic ethanol exposure, which was primarily inhibited by zinc supplementation. In contrast, the protein level of hepatic ADH was significantly higher in mice treated with ethanol plus zinc than ethanol alone, although the latter also induced a significant increase in ADH protein level. To determine whether changes in ethanol metabolic pathway affect ethanol metabolic rate, the blood ethanol elimination was determined after an intragastric bolus of ethanol at 3 g/kg. An increased ethanol clearance was found in ethanol-fed mice, and zinc supplementation did not affect the ethanol-elevated elimination rate. To determine the effect of zinc on GSH and GSH-related enzymes, hepatic GSH status, and enzymatic activities of GSH reductase (GR) and GSH peroxidase (GPx) in the WT mice were measured (Figure 6). Chronic ethanol exposure induced a significant decrease in GSH concentrations in both cytosol and mitochondria, which was attenuated by zinc supplementation. The hepatic GR activity was not affected by chronic ethanol feeding. However, zinc supplementation significantly increased the GR activity in the mice chronically fed ethanol. The GPx activity in the liver was decreased in the ethanol-fed mice. Zinc supplementation partially inhibited ethanol-induced decrease in GPx activity. The results obtained demonstrated that dietary zinc supplementation prevented ethanol-induced zinc decrease in the liver and inhibited ethanol-induced oxidative liver injury. The suppression of ethanol-induced oxidative injury by zinc supplementation most likely resulted from inhibition of ROS, in particular, superoxide accumulation. The ROS accumulation apparently resulted from the alcohol-enhanced CYP2E1 pathway, which has been shown to be highly responsible for ROS generation.26Noedmann R Ribiere C Rouach H Implication of free radical mechanisms in ethanol-induced cellular injury.Free Radic Biol Med. 1992; 12: 219-240Crossref PubMed Scopus (556) Google Scholar Zinc supplementation inhibited the activation of the CYP2E1 pathway and at the same time enhanced the ADH pathway, thus leading to the prevention of the ethanol-induced metabolic shift that is in favor of ROS production. Furthermore, zinc also prevented ethanol-induced decreases in GSH and GPx, but increased GR. These results thus indicate that zinc supplementation results in suppression of alcohol-induced metabolic shift favoring ROS generation and enhanced GSH antioxidant capacity, thus inhibits alcohol-induced oxidative stress leading to prevention of alcoholic liver injury. The decrease in zinc concentrations in the liver is one of the most consistent observations in alcoholic patients and animal models of alcoholic liver injury.1Kiilerich S Dietrichson O Loud FB Naestoft J Christoffersen P Juhl E Kjems G Christiansen C Zinc depletion in alcoholic liver diseases.Scand J Gastroenterol. 1980; 15: 363-367Crossref PubMed Scopus (51) Google Scholar, 2McClain CJ Antonow DR Cohen DA Shedlofsky S Zinc metabolism in alcoholic liver disease.Alcohol Clin Exp Res. 1986; 10: 582-589Crossref PubMed Scopus (76) Google Scholar, 3Bode JC Hanisch P Henning H Koenig W Richter FW Bode C Hepatic zinc content in patients with various stages of alcoholic liver disease and in patients with chronic active and chronic persistent hepatitis.Hepatology. 1988; 8: 1605-1609Crossref PubMed Scopus (91) Google Scholar The consequence of zinc depletion has not been well understood, although it has been suggested that zinc deficiency contributes to the pathogenesis of liver disease.2McClain CJ Antonow DR Cohen DA Shedlofsky S Zinc metabolism in alcoholic liver disease.Alcohol Clin Exp Res. 1986; 10: 582-589Crossref PubMed Scopus (76) Google Scholar MT is a metal-binding protein, and the role of MT in cellular zinc homeostasis has long been recognized.15Vallee BL The function of metallothionein.Neurochem Int. 1995; 27: 23-33Crossref PubMed Scopus (259) Google Scholar Under chronic ethanol exposure, MT concentrations in the liver were found to significantly decrease along with zinc depletion in the liver,27Wang J Pierson Jr, RN Distribution of zinc in skeletal muscle and liver tissue in normal and dietary controlled alcoholic rats.J Lab Clin Med. 1975; 85: 50-58PubMed Google Scholar but dietary zinc supplementation significantly increased MT concentrations in the liver exposed to alcohol.28Cabre M Folch J Gimenez A Matas C Pares A Caballeria AJ Paternain JL Rodes J Joven J Camps J Influence of zinc intake on hepatic lipid peroxidation and metallothioneins in alcoholic rats: relationship to collagen synthesis.Int J Vitam Nutr Res. 1995; 65: 45-50PubMed Google Scholar Thus, the hepatoprotective effects by zinc supplementation were ascribed to the MT induction in the liver. However, our recent studies have shown that zinc inhibition of acute alcohol liver injury is independent of MT.29Zhou Z Sun X Lambert JC Saari JT Kang YJ Metallothionein-independent zinc protection from alcoholic liver injury.Am J Pathol. 2002; 160: 2267-2274Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 30Lamb
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