Increase In Metallothionein Content Research Articles

Effect of size on concentrations and cadmium inducibility of metallothionein in the shore crab Carcinus maenas

Metallothionein (MT) plays an important role in protecting organisms from the adverse effects of Cd, Hg, Zn and Cu. Investigations on mammals show variations in metallothionein concentrations and inducibility with age. This has never been investigated in invertebrates, and we determined the concentrations and inducibility of metallothionein in gills and midgut gland of different size classes of shore crabs from uncontaminated areas. Metallothionein concentrations in gills and midgut gland ranged between 30 and 40 μg g−1 dry weight with no differences among the different size classes. Concentrations of cadmium, copper and zinc in the midgut gland increased with increasing size of the crabs when the concentrations were expressed on a dry weight basis; water content in the midgut gland increased with the size and only the cadmium concentration increased with size when concentrations were expressed on wet weight basis. There was an inverse relationship between metallothionein and both copper and cadmium concentrations. Smaller crabs exposed to 1 mg Cd L−1 accumulated higher concentrations of cadmium in midgut gland and gills than larger ones and metallothionein concentrations in the midgut gland were higher in the smaller crabs. However, the increase in metallothionein concentration per accumulated unit of cadmium showed a linear increase with the size of the crabs. The ratio [Cd]midgut/[Cd]gills decreased with the size of the crabs. The overall conclusion is that baseline metallothionein concentrations do not change with age in shore crabs, but that the inducibility of metallothionein upon cadmium challenge does.

Importance of Genetic Polymorphisms in MT1 and MT2 Genes in Metals Homeostasis and Their Relationship with the Risk of Acute Pancreatitis Occurrence in Smokers-Preliminary Findings.

This study was aimed at evaluating the changes in metallothionein (MT) concentration in the blood of patients with acute pancreatitis (AP) and healthy subjects, taking into account the extracellular (plasma) and intracellular (erythrocyte lysate) compartments. The impact of single-nucleotide polymorphisms (SNPs) in the MT1A (rs11640851), MT1B (rs964372) and MT2A (rs10636) genes on MT concentration and their association with the concentration of metals (Cu, Zn, Cd) and ceruloplasmin as Cu-related proteins were analyzed. The concentration of a high-sensitivity C-reactive protein (hs-CRP) and IL-6 as markers of inflammation, and malonyldialdehyde (MDA), superoxide dismutase (SODs) activity and the value of total antioxidant capacity (TAC) as parameters describing the pro/antioxidative balance were also assessed. In the AP patient groups, an increased MT concentration in erythrocyte lysate compared to healthy subjects was shown, especially in individuals with the GG genotype for rs964372 in the MT1B gene. A Zn concentration was especially decreased in the blood of smoking AP patients with the AA genotype for SNP rs11640851 in the MT1A gene and the GC genotype for SNP rs10636 in MT2A, compared to non-smokers with AP, which was accompanied by an increase in the value of the Cu/Zn ratio. The exposure to tobacco smoke xenobiotics increased the risk of AP occurrence in subjects with the CC genotype for SNP rs11640851 in the MT1A gene by more than fourfold. The investigated polymorphisms, rs11640851 in the MT1A gene, rs964372 in the MT1B gene and rs10636 in the MT2A gene, seem to be an important factor in maintaining homeostasis in an organism under oxidative stress conditions.

Biomarker responses in African sharptooth catfish, Clarias gariepinus (Burchell, 1822), as indicators of potential metal and organic pollution along the Vaal River system, South Africa

The Vaal River is of considerable economic and environmental importance. It flows through five provinces within South Africa and it is subject to numerous recreational and industrial activities, which often prove detrimental to the health of the system. To determine the extent of the impact that these anthropogenic stressors have on the river system, physiological effects in Clarias gariepinus were assessed during a 2016/2017 sample survey using a suite of biomarkers (exposure and effect). Inhibition of acetylcholinesterase and increased metallothionein concentrations were recorded in response to the increase in organic pollutants and metals. Oxidative stress biomarkers, such as superoxide dismutase, showed significant increases in response to possible recent pesticide exposures, whereas lipid peroxidation and protein damage were evident with increased malondialdehyde and protein carbonyl concentrations respectively. The data provide a novel account of the use of biomarkers to assess negative anthropogenic effects observed along the entire length of the Vaal River system.

Effect of Zinc on the Oxidative Stress Biomarkers in the Brain of Nickel-Treated Mice.

The overexposure to nickel due to the extensive use of it in modern technology remains a major public health concern. The mechanisms of pathological effects of this metal remain elusive. The present study was devoted to evaluate the effect of nickel on the oxidative state of the brain cells of mice and to assess whether zinc as redox state modulator could efficiently protect cells against nickel's neurotoxicity. As oxidative stress biomarkers in the present study, we have measured the concentrations of reduced glutathione, metallothioneins, and malondialdehyde and the activity of the enzyme δ-aminolevulinate dehydratase. For the single metal exposure, mice were i.p. injected once with solutions of NiCl2 and/or ZnSO4; repeated exposure was performed i.p. injecting metal salt solutions for 14 days (once a day). The control mice received i.p. injections of saline. Results of our study demonstrate that single and 14 days of Ni2+ exposure decreased reduced glutathione and increased malondialdehyde contents in the brain of mice. Repeated Ni2+ administration significantly inhibited δ-aminolevulinate dehydratase while increasing brain metallothionein concentration at both exposure periods. Zinc exhibited a protective effect against nickel-induced glutathione and lipid peroxidation in brain cells of mice at both intervals of time, while repeated exposure to this metal significantly raised the brain metallothionein content. Repeated Zn2+ pretreatment protected δ-aminolevulinate dehydratase from Ni2+-induced inhibition and significantly increased metallothionein concentration at both investigated time intervals.

Protective effects of dietary selenium on abaloneHaliotis discus hannaiagainst the toxicity of waterborne cadmium

This study was conducted to investigate protective effects of dietary selenium (Se) on abalone Haliotis discus hannai Ino against the toxicity of cadmium (Cd). A 60‐day feeding trial was conducted with abalone (initial weight: 3.17 ± 0.01 g), which were exposed to 0.34 mg/L of waterborne Cd. During a feeding trial, abalone were fed graded levels of Se at 0.10 (controls), 1.31, and 4.20 mg/kg diet respectively. Results showed that there was no significant difference in specific growth rate and survival rate of abalone among the three treatments. Compared with the controls, dietary Se significantly decreased Cd concentrations in serum, muscle, mantle, gill, mantle, and hepatopancreas of abalone. Besides, compared with the controls, dietary Se significantly increased metallothionein concentration in the hepatopancreas of abalone. Additionally, compared with the controls, dietary Se significantly decreased concentrations of malondiadehyde and protein carbonyl in hepatopancreas of abalone. Meanwhile, compared with the controls, dietary Se significantly increased activities of glutathione peroxidase, thioredoxin reductase, and thioredoxin peroxidase, and concentration of glutathione in the hepatopancreas of abalone. Based on the data above, in abalone, dietary Se showed protective effects against waterborne Cd.

Tolerance and stress response of sclerotiogenic Aspergillus oryzae G15 to copper and lead.

Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50-100mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.

Seasonal effects to metallothionein responses to metal exposure in a naturalised population of Ruditapes philippinarum in a semi-enclosed estuarine environment

The Manila clam (Ruditapes philippinarum), an invasive species in Northern Europe, can be used as a bioindicator of metal pollution. Seasonal effects on metallothionein (MT) production have not been considered in this species at the northernmost extent of its European distribution. This study assesses the annual seasonal effects on MT and metal concentrations in R. philippinarum from Poole Harbour, UK. R. philippinarum were collected in winter, spring, summer, and autumn throughout 2015, and MT and metal concentrations, as well as biotic and abiotic variables, were quantified. During winter, linear regression analysis showed significant positive relationships between tissue metal and MT concentrations. However, during spring and summer, these relationships were mostly insignificant. MT concentrations during spring had significant positive relationships with tissue and whole weight. Significant positive relationships were also observed between MT and condition index, during summer. During spring and summer, biotic factors seem to override the role of MT as a detoxification mechanism for metal exposure in this species. This is probably due to an increase in MT concentration in spring caused by gametogenesis, associated with increased tissue weight as the gonads expand. A depletion of energy resources, or physical stressors such as heat, may be attributed to the reduced MT production in clams of poor body condition in summer. The evidence from this study suggests that MT may only be a useful biomarker of metal pollution during winter in R. philippinarum in the UK. This verifies the natural variability of MT in this species at high latitudes, and highlights the potential and limits to a widely available bioindicator of metal pollution.

Multivariate analysis of potential biomarkers of oxidative stress in Notopterus notopterus tissues from Mahanadi River as a function of concentration of heavy metals

In this study, investigation were done on the Mahanadi River water and health of dwelling Indian Knife fish Notopterus notopterus from three sites along the course of the river in an around Cuttack city (Odisha). Oxidative stress biomarker assays such as thiobarbituric acid reactive substances, protein carbonyls, protein and non-protein thionyls, reduced glutathione, metallothionein, superoxide dismutase, catalase, glutathione peroxidase/reductase couple, glutathione-S-transferase, and tissue metal (Fe, Cu, Ni, Cd, Pb and Zn) levels along with water quality assessments were assayed to measure the impacts on fish health. Results indicate that except Fe all other metals studied were within approved limits for fish liver and gill as approved by FAO/WHO. However, the muscle tissue do not have any metal beyond the permissible limit. A site and tissue specific response of the above mentioned oxidative biomarkers as well as metal accumulation in the fish tissues were noticed. Lipid peroxidation and protein carbonylation were increased gradually in the fish tissues collected from experimental sites along the course of the River in comparison to upstream reference site. Glutathione-S-transferase, glutathione peroxidase/reductase couple, reduced glutathione and non-protein thiol content were significantly decreased in fish tissues from experimental sites. An increase in metallothionein content was observed while superoxide dismutase and catalase showed tissue specific responses. Multivariate (Discriminant Function) analysis revealed that lipid peroxidation, protein carbonylation and superoxide dismutase have highest association as predictors of impact in the muscle and liver while that for gill is protein carbonylation, superoxide dismutase and glutathione reductase.

In situ impact assessment of wastewater effluents by integrating multi-level biomarker responses in the pale chub (Zacco platypus)

The integration of biomarker responses ranging from the molecular to the individual level is of great interest for measuring the toxic effects of hazardous chemicals or effluent mixtures on aquatic organisms. This study evaluated the effects of wastewater treatment plant (WWTP) effluents on the freshwater pale chub Zacco platypus by using multi-level biomarker responses at molecular [mRNA expression of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and metallothionein (MT)], biochemical (enzyme activities of CAT, SOD, GST, and concentration of MT), and physiological [condition factor (CF) and liver somatic index (LSI)] levels. The mRNA expression levels of GST and MT in Z. platypus from a site downstream of a WWTP significantly increased by 2.2- and 4.5-fold (p<0.05) when compared with those from an upstream site. However, the enzyme activities of CAT, SOD, and GST in fish from the downstream site significantly decreased by 43%, 98%, and 13%, respectively (p<0.05), except for an increase in MT concentration (41%). In addition, a significant increase in LSI (46%) was observed in Z. platypus from the downstream site (p<0.05). Concentrations of Cu, Zn, Cd, and Pb in the liver of Z. platypus were higher (530%, 353%, 800%, and 2,200%, respectively) in fish from a downstream site than in fish from an upstream location, and several multi-level biomarker responses were significantly correlated with the accumulated metals in Z. platypus (p<0.05). Integrated biomarker responses at molecular, biochemical, and physiological levels (multi-level IBR) were much higher (about 4-fold) at the downstream site than at the upstream site. This study suggests that the multi-level IBR approach is very useful for quantifying in situ adverse effects of WWTP effluents.

The influence of biological and environmental factors on metallothionein concentration in the blood

The concentration of metallothionein (MT), a low-molecular-weight protein, is regulated by many factors, primarily metals (zinc, cadmium, copper), cytokines, glucocorticoides and free radicals. These factors are determined by such aspects of human biology as gender, pregnancy and age, as well as by environmental factors including the use of oral contraceptives and cigarette smoking, all which may affect MT levels in the body.The aim of this study was to investigate the influence of these biological and environmental factors on MT concentrations in erythrocyte lysate and in plasma.MT concentrations were determined by a two-step direct enzyme-linked immunosorbent assay. Evaluation of exposure to cigarette smoking was performed by checking cotinine levels in the plasma of subjects.The studies showed higher MT concentrations in both the erythrocyte lysate and plasma of women when compared to men. Furthermore, pregnancy causes an increase of MT concentration in plasma, while oral contraceptives cause an elevated concentration of MT in erythrocyte lysate. Age impacts plasma MT concentrations in men, whereas it does not affect concentrations of MT in erythrocyte lysate.

OSMOLALITY, METALLOTHIONEIN AND NA/KATPASE ACTIVITY IN A MANGROVE CRAB, UCIDES CORDATUS, SUBMITTED TO ACUTE AND CHRONIC COPPER EXPOSURE IN THE WATER

Ucides cordatus were submitted to acute (24h and 96h) and chronic copper (Cu) exposure (15 days), containing 5mg/L CuSO4 in the water. Anterior and posterior gills, hepatopancreas (HP), and aliquots of haemolymph and urine were collected to measure osmolality, metallothionein (MT) concentration and Na/K‐ATPase activity (NKA). Haemolymph showed an increase in osmolality for crabs submitted to 24h Cu exposure, while animals submitted for 96h and 15 days to Cu demonstrated a reduction in osmolality compared to control. There was no change in urine osmolality for animals exposed to Cu for 24h and 15 days, while in the 96h group, there was a small reduction in the osmolality. MT synthesis increased in the anterior gills in crabs exposed to 24h, 96h and 15 days of Cu exposure, while posterior gills showed increased MT concentration in the 24h and 15 days groups, but not in the 96h group, suggesting that there is a regulation between MT concentration and time of exposure to water Cu. There was an increase of NKA in the anterior gills, while in the posterior gills there was reduction in the 24h group and an increase in the 96h and 15 days group compared to controls. For HP, there was an increase of activity for 96h group, while for the 15 days group the values were similar to control. Animals exposed to Cu show a relationship between time of exposure and higher MT concentration and increased NKA in both organs as well as alterations in osmolality.

Metallothionein is up-regulated in molluscan responses to cadmium, but not aluminum, exposure

Metallothionein (MTs) is a family of low molecular weight peptides with high cysteine content. In aquatic invertebrates, MTs play an important role in the detoxification of metals and they are often cited as useful biomarkers for toxic metal stress. The change in concentrations of metallothionein in response to aluminum and cadmium exposure was investigated in the soft tissues of two freshwater molluscs: Lymnaea stagnalis and Dreissena polymorpha that have contrasting feeding behavior and therefore metal accumulation profiles. The majority of added Al had precipitated in the bottom of tanks within 24h, with an average of only 15.4% and 26.2% remaining in the water column of tanks containing snails and mussels, respectively. In contrast, most of the Cd did not precipitate but remained in solution (81.1% and 82.7%, respectively) between water changes. Both metals accumulated in the soft tissues of both animals, but only cadmium exposure led to an increase in metallothionein concentrations in the soft tissues. However, no significant difference was observed in the level of total protein in both tested molluscs upon exposure to Al and Cd compared to the corresponding control. Therefore, care should be taken in the use of metallothioneins as biomarkers for metal stress, since not all metallic stressors induce their expression.

Metallothionein in the freshwater gastropod Melanopsis dufouri chronically exposed to cadmium: A methodological approach

Previous studies have demonstrated that the use of differential pulse polarography (DPP) for metallothionein (MT) determination in marine gastropod tissues, particularly the digestive gland, requires taking into account the presence of heat-stable high molecular weight compounds that exhibit polarographic signal. In the present paper, similar compounds were identified in tissues from the freshwater snail Melanopsis dufouri which also interfere with MT determination by DPP and, due to their silver binding capacity, also interfere in the silver assay for MT quantification. Ultrafiltration seems to be effective in removing these high molecular weight compounds from heat-denatured homogenate supernatant allowing direct MT quantification by DPP. A fully validated procedure for metallothionein determination in M. dufouri is described. In spite of a considerable accumulation of cadmium in the visceral complex of M. dufouri following exposure to 100 μg Cd L −1 for 8 weeks (up to 37 μg g −1) only a small increase in MT concentration was found.

Cadmium detoxification effect of Chinese parsley Coriandrum sativum in liver and kidney of rainbow trout Oncorhynchus mykiss

Although fish feed supplemented with Chinese parsley (CP), Coriandrum sativum, does not affect the health of rainbow trout Oncorhynchus mykiss, it has the remarkable effect of reducing the uptake of Cd (12–17% compared with controls without CP). Here, we measured the change in the amount of metallothionein (MT) in the liver and kidney using gel permeation chromatography-high-performance liquid chromatography (TSK GEL SW 3000 column). The increase in MT content over time in the Cd-fed groups corresponded to the change in Cd accumulation. Gel permeation was performed with soluble fractions from the fish liver and kidney, and changes in the Cd, Cu, and Zn concentrations over time for each fraction, and the relationship between the form of Cd that accumulated in tissues and its toxicity were measured. A nontoxic MT fraction of 7,000 Da and a high-molecular-weight fraction of approximately 60,000 Da were isolated from the soluble fractions. The induction of nontoxic MT–Cd was higher in the CP-supplemented groups than in the control groups. The synergetic actions of several compounds in CP may contribute to bind MT, thereby preventing accumulation of harmful heavy metals and essential metals in fish.

DIFFERENTIAL METALLOTHIONEIN INDUCTION PATTERNS IN FED AND STARVED CARP (CYPRINUS CARPIO) DURING WATERBORNE COPPER EXPOSURE

Starved and fed carp (Cyprinus carpio) were exposed to sublethal waterborne copper exposure (1 microM) for 28 d in softened Antwerp, Belgium, city tap water. Copper accumulation in liver and gill tissues was determined, and changes in branchial Na+/K+-adenosine 5'-triphosphatase (ATPase) activity and metallothionein (MT) induction in gill and liver tissues were investigated following 28-d copper exposure. Gill Na+/K+-ATPase activity in exposed fish, both starved and fed, was at its lowest values after 3 d of exposure, after which it slowly recovered to preexposure values. No significant differences in branchial Na+/K+-ATPase activity were found between starved and fed fish. Copper accumulation in the liver and gills of the exposed starved carp was significantly higher than that in the exposed fed carp. The highest MT induction was found in liver tissues. Different patterns of MT induction were observed in the starved and fed carp during copper exposure. Before exposure, MT concentrations in the livers of the starved fish were significantly higher than those in the fed ones. Copper exposure significantly increased MT concentration in the liver of the fed fish, but no changes occurred in the starved fish. In contrast, copper exposure increased MT concentrations in the gills of the starved fish during the first week of exposure, whereas only a slight increase in MT concentration in the gills of the fed fish was observed. When taking into account the role of feeding status in MT induction, hepatic MT was a more relevant indicator for long-term monitoring of copper pollution in carp, but gill MT provided useful information regarding short-term copper toxicity.

Accumulation and effects of metals in caged carp and resident roach along a metal pollution gradient

Metal accumulation and effects on plasma Ca 2+, alanine transaminase (ALT) and fish condition factor were examined in caged carp ( Cyprinus carpio) and resident roach ( Rutilus rutilus) at four locations along the Grote Nete River system (Belgium). Cadmium and zinc accumulation were found in carp and roach, with highest concentrations at the most contaminated site (dissolved Cd: 1.82 µg/l, Zn: 967 µg/l). On the tissue level, highest cadmium concentrations were measured in kidneys of carp and roach, followed by gills, intestine and liver, while low concentrations were observed in carcass and muscle. For zinc, a similar pattern was observed (intestine > kidney > gills > liver > carcass > muscle). Comparison between species showed higher cadmium concentrations in feral roach, while zinc levels were lower, owing to the high zinc concentrations in control carp. Furthermore, comparison of metal concentrations between two sampling periods (2005 and 2000–2001) revealed a drastic decrease in cadmium concentration in gills, liver and muscle of roach, similar to the reduction in waterborne cadmium concentrations, while differences for zinc were much less pronounced. In addition to metal accumulation, increased metallothionein concentrations (~ 2×) were found in carp and roach, while no metal-related effects were found on ALT, Ca 2+or condition factor. However, negative effects on fish community structure, as assessed by the index of biotic integrity (IBI), were found along the pollution gradient and indicated long-term adverse effects of metal pollution.

Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea

Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 microM Cd and/or 15.3 microM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd + Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.

Effects of malathion and cadmium on acetylcholinesterase activity and metallothionein levels in the fish Seriola dumerilli

The potential use of acetylcholinesterase (AChE) and metallothionein (MT) responses as biomarker of organophosphorous (OPs) and trace metal were assessed in fish Seriola dumerilli exposed to 0, 4, 6 mg/kg of malathion for 2, 7 and 13 days, and to 0, 50, 100, 250 mug/kg of Cd for 2 days. Brain AChE was significantly inhibited after 2 and 7 days of malathion exposure, in a dose-response manner, but no inhibition was observed after 13 days of exposure. When exposed to Cd for 2 days, S. dumerelli presented an increase in AChE activity at a concentration of 50 mug/kg, but a strong and dose-dependent AChE inhibition at 100 and 250 mug/kg. Cd treatment also caused a rapid increase in MTs concentration in liver, even at the lower concentration. Our experiments indicate that the measurement of hepatic MT concentration and brain AChE activity in S. dumerilli would be useful biomarkers of OP and Cd exposure and/or effects.

USING OF ELECTROCHEMICAL METHODS FOR STUDYING OF METALLOTHIONEIN CONTENT IN THE HUMAN BLOOD SERUM OF A PATIENT POISONED BY LEAD AND TREATED BY PLATINUM

Metallothioneins belong to the group of intracellular, high molecular and cysteine-rich proteins whose content increase with increasing concentration of a heavy metal. Here we applied the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothionein in human blood serum of patient poisoned by lead and/or treated by platinum. The increased metallothionein concentrations in both cases were observed.

Zinc Supplementation Prevents Alcoholic Liver Injury in Mice through Attenuation of Oxidative Stress

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.