Augmented metalloproteinase activity and acute lung injury in copper-deficient rats.

Dietary copper is required for normal function of >30 mammalian enzyme systems. Copper deficiency causes a number of cardiovascular defects as well as impaired immune cell function. Little is known regarding the effects of copper deficiency on acute inflammatory responses, but this topic is relevant because many members of the Western population receive less than the recommended dietary allowance of copper. In the current studies, we investigated the effects of dietary copper deficiency on acute lung injury induced by intrapulmonary deposition of IgG immune complexes. Weanling male Long-Evans rats were fed diets either adequate (5.6 microg/g) or deficient (0.3 microg/g) in copper. IgG immune complex lung injury was greatly increased in copper-deficient rats as determined by lung vascular leakage of albumin and histopathology. However, no change was observed in either the lung content of tumor necrosis factor-alpha or lung neutrophil accumulation. Lungs from copper-deficient rats had much higher levels of matrix metalloproteinase (MMP)-2 and MMP-9 than did copper-adequate control animals. This increased activity was not attributable to alveolar macrophages or neutrophils. These data suggest that the augmented lung injury caused by copper deficiency is due to increased pulmonary MMP-2 and MMP-9 activity and not a generalized amplification of the inflammatory response.

Isolated ventricular myocytes from copper-deficient rat hearts exhibit enhanced contractile function.

Dietary copper deficiency leads to cardiac hypertrophy, cardiac fibrosis, derangement of myofibrils, and impaired cardiac contractile and electrophysiological function. The purpose of this study was to determine whether impaired cardiac function from copper deficiency is due to depressed contractile function at the single myocyte level. Male Sprague-Dawley rats were fed diets that were either copper adequate (5.59-6.05 microg copper/g body wt; n = 11) or copper deficient (0.29-0.34 microg copper/g body wt; n = 11) for 5 wk. Ventricular myocytes were dispersed and mechanical properties were evaluated using the SoftEdge video-based edge-detection system. Intracellular Ca2+ transients were examined using fura 2-acetoxymethyl ester. Myocytes were electrically stimulated to contract at 0.5 Hz. Properties evaluated included peak shortening (PS), time to peak shortening (TPS), time to 90% relengthening (TR90), and maximal velocities of shortening and relengthening (+/-dL/dt). Myocytes from the copper-deficient rat hearts exhibited significantly enhanced PS values associated with shortened TR90 measurements compared with those from copper-adequate rat hearts. The +/-dL/dt values were enhanced and the intracellular Ca2+ transient decay rate was depressed in myocytes from copper-deficient rats. These data indicate that impaired cardiac contractile function that is seen in copper-deficient whole hearts might not be due to depressed cardiac contractile function at the single cell level but rather to other mechanisms such as cardiac fibrosis.

Leukocyte-endothelial adhesion is impaired in the cremaster muscle microcirculation of the copper-deficient rat.

Dietary copper deficiency impairs the function of both the vascular endothelium and circulating leukocytes. In the current study, leukocyte-endothelium adhesion was observed in the in vivo cremaster muscle microcirculation of copper-adequate and copper-deficient rats. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (5.6 microg/g) or deficient (0.3 microg/g) in copper. Adhesion was stimulated with the inflammatory mediators tumor necrosis factor-alpha and bradykinin, and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. Intravascular adhesion of leukocytes to the vascular endothelium was significantly attenuated in the copper-deficient group in response to all three agonists. These results occurred without any difference in intravascular wall shear rate between the dietary groups. Based on previous work, we propose that the attenuated response is caused by either decreased expression of adhesion molecules on leukocytes and endothelial cells or by inhibition of the endothelial cell calcium signaling associated with copper deficiency.

Copper deficiency and cardiovascular disease: role of peroxidation, glycation, and nitration.

Dietary copper deficiency causes a variety of cardiovascular deficits. Systemic effects include high blood pressure, enhancement of inflammation, anemia, reduced blood clotting, and possibly arteriosclerosis. Effects on specific organs or tissues include weakened structural integrity of the heart and blood vessels, impairment of energy use by the heart, reduced ability of the heart to contract, altered ability of blood vessels to control their diameter and grow, and altered structure and function of circulating blood cells. In some instances, the cause of a defect can be directly attributed to reduced activity of a specific copper-dependent enzyme. However, three nonspecific mechanisms of damage have been implicated in cardiovascular defects of copper deficiency. They are peroxidation, the interaction of oxygen-derived free radicals with lipids and proteins (possibly DNA); glycation, the nonenzymatic glycosylation of proteins; and nitration, the interaction of nitric oxide and its metabolites with peptides and proteins. Though independently these mechanisms present great potential for damage, the possibility that they may interact presents an added reason for concern. Furthermore, the fact that at least two of these mechanisms are associated with diabetes and aging suggests that copper deficiency may exacerbate deficits associated with these two conditions.

Metallothionein inhibits myocardial apoptosis in copper-deficient mice: role of atrial natriuretic peptide.

Dietary copper restriction causes heart hypertrophy in animal models. Several studies have indicated that this cardiomyopathy is mediated by oxidative stress. Metallothionein (MT), a low molecular weight and cysteine-rich protein, functions in protecting the heart from oxidative injury. We therefore used a cardiac-specific MT-overexpressing transgenic mouse model to test the hypothesis that MT inhibits copper deficiency-induced heart hypertrophy. Dams of both transgenic pups and non-transgenic littermates were fed a copper-adequate or copper-deficient diet, starting on the fourth day post-delivery, and the weanling mice were continued on the dams' diets until they were killed. Heart hypertrophy developed in copper-deficient pups by the fourth week of the combined pre- and post-weaning feeding and aggressively progressed until the end of the experiment (6 weeks). MT overexpression did not prevent the occurrence of heart hypertrophy, but inhibited the progression of this cardiomyopathy, which correlated with its suppression of cardiac lipid peroxidation. Corresponding to the progression of heart hypertrophy, myocardial apoptosis and atrial natriuretic peptide (ANP) production in the left ventricle were detected in non-transgenic copper-deficient mice; these effects were significantly suppressed in transgenic copper-deficient mice. Measurement of apoptosis by TUNEL assay and Annexin V-FITC confocal microscopy in primary cultures of cardiomyocytes revealed that ANP was largely responsible for the myocyte apoptosis and that MT inhibited ANP-induced apoptosis. The data clearly demonstrate that elevation of MT in the heart inhibits oxidative injury and suppresses the progression of heart hypertrophy in copper deficiency, although it does not block its initiation. The results suggest that MT inhibits the transition from heart hypertrophy to failure by suppressing apoptosis through inhibition of both cardiac ANP production and its apoptotic effect.

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

Alterations in hypertrophic gene expression by dietary copper restriction in mouse heart.

Dietary copper (Cu) restriction causes a hypertrophic cardiomyopathy similar to that induced by work overload in rodent models. However, a possible change in the program of hypertrophic gene expression has not been studied in the Cu-deficient heart. This study was undertaken to fill that gap. Dams of mouse pups were fed a Cu-deficient diet (0.35 mg/kg diet) or a Cu-adequate control diet (6.10 mg/kg) on the fourth day after birth, and weanling mice continued on the dams' diet until they were sacrificed. After 5 weeks of feeding, Cu concentrations were dramatically decreased in the heart and the liver of the mice fed the Cu-deficient diet. Corresponding to these changes, serum ceruloplasmin concentrations and hepatic Cu,Zn-superoxide dismutase activities were significantly (P<0.05) depressed. The size of the Cu-deficient hearts was greatly enlarged as estimated from the absolute heart weight and the ratio of heart weight to body weight. The abundances of mRNAs for atrial natriuretic factor, beta-myosin heavy chain, and alpha-skeletal actin in left ventricles were all significantly increased in the Cu- deficient hearts. Furthermore, Cu deficiency activated the expression of the c-myc oncogene in the left ventricle. This study thus demonstrated that a molecular program of alterations in embryonic genes, similar to that shown in the work-overloaded heart, was activated in the hypertrophied heart induced by Cu deficiency.

Cardiovascular measurements relevant to heart size in copper-deficient rats.

Dietary copper deficiency in animals is often associated with cardiac enlargement and anemia. In this study we examined the hypothesis that anemia leads to a high cardiac output state that results in work-induced (physiological) cardiac hypertrophy. Blood pressure was measured by carotid cannulation and cardiac output was measured by aortic flow probe in anesthetized, open-chested rats that had been subjected to various degrees of dietary copper deficiency for five weeks. Cardiac output was unaffected by dietary copper deficiency. However, the components of cardiac output were found to vary reciprocally, heart rate decreasing and stroke volume increasing with copper deficiency. Further, total peripheral resistance, calculated as the ratio of mean arterial blood pressure and cardiac output, was depressed by dietary copper deficiency. These findings suggest that bradycardia and depression of vascular resistance induced by copper deficiency contribute to increased venous filling and a resultant increase in stroke volume; these factors may lead to cardiac hypertrophy. A significant correlation between stroke volume and heart weight in rats of varying copper status supports this conclusion.

Metallothionein inhibits ischemia-reperfusion injury in mouse heart.

Oxidative stress is believed to play a major role in ischemia-reperfusion injury to the heart. Metallothionein (MT), a potential free radical scavenger, may function in cardiac protection against ischemia-reperfusion damage. To test this hypothesis, a specific cardiac MT-overexpressing transgenic mouse model was used. The hearts isolated from these animals were subjected to 50 min of warm (37 degrees C) zero-flow ischemia followed by 60- or 90-min reflow. Compared with the nontransgenic controls, the transgenic mouse hearts with MT concentrations approximately 10-fold higher than normal showed significantly improved recovery of contractile force postischemia (69.2 +/- 4.2 vs. 26.0 +/- 6.0% at the end of 60-min reperfusion, P < 0.01). Efflux of creatine kinase from these transgenic hearts was reduced by more than 50% (P < 0.01). In addition, the zone of infarction induced by ischemia-reperfusion at the end of 90-min reperfusion was suppressed by approximately 40% (P < 0.01) in the transgenic hearts. The results strongly indicate that MT provides protection against ischemia-reperfusion-induced heart injury.

Relationship between dietary copper concentration and acetylcholine-induced vasodilation in the microcirculation of rats.

Dietary copper deficiency has been shown to significantly reduce acetylcholine (Ach)-induced vascular smooth muscle relaxation. The current study was designed to examine the relative relationship between dietary copper and the vasodilator response to Ach in the microcirculation of the rat. Male weanling rats were fed a purified basal diet supplemented with 6.0, 3.0, 1.5 or 0.0 microg Cu/g diet for 4 weeks to provide an adequate, two marginal, and deficient intakes of dietary copper. Arteriole dilation in response to increasing concentrations of acetylcholine (10(-7) to 10(-4) M) was measured in the in vivo cremaster muscle microcirculation for each dietary group. Liver copper and both aortic and erythrocyte Cu,Zn-SOD activity were used as indices of systemic copper status. Dilation to the increasing concentrations of Ach was only different in the 0 microg Cu supplemented group compared to the copper-adequate control values. However, the combined results showed an exponential increase in 10(-5) M Ach-induced vasodilation as liver copper concentration increases from 0 microg Cu/g dry wt. This relationship suggests that dilation is attenuated at liver Cu concentrations below 5 microg/g dry wt. The results indicate that Ach-induced vasodilation is copper-dependent but that the pathway is not very sensitive to short-term marginal restriction of copper intake.

Cardiovascular effects of dietary copper deficiency.

Dietary copper deficiency may impair cardiovascular health by contributing to high blood pressure, enhancement of inflammation, anemia, reduced blood clotting and arteriosclerosis. The purpose of this review is to compile information on the numerous changes of the heart, blood and blood vessels that may contribute to these cardiovascular defects. These alterations include weakened structural integrity of the heart and blood vessels, impairment of the use of energy by the heart, reduced ability of the heart to contract, altered ability of blood vessels to control their diameter and to grow, and altered structure and function of circulating blood cells. The fundamental causes of these changes rest largely on reduced effectiveness of enzymes that depend on copper for their activity.

Early and advanced glycation end-products are increased in dietary copper deficiency.

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.

Repression of hypoxia-reoxygenation injury in the catalase-overexpressing heart of transgenic mice.

Hypoxia-reoxygenation injury results at least in part from reactive oxygen free radicals. Catalase is a major enzyme involved in detoxification of hydrogen peroxide. The activity of catalase per gram of tissue in the heart is very low, being only about 2% that of liver in rodents and humans, which may be responsible for the high sensitivity of the heart to hypoxia-reoxygenation injury. The present study was undertaken to determine whether elevation of catalase specifically in the heart of transgenic mice could provide protection against hypoxia-reoxygenation injury. Transgenic mice with elevated cardiac catalase 60-fold higher than normal were selected, and the effects of catalase elevation on hypoxia-reoxygenation induced functional and morphological changes in isolated atria were determined. Catalase overexpression ameliorated reductions in contractile force and heart rate caused by hypoxia-reoxygenation, and eliminated reoxygenation-induced arrhythmia. The catalase-overexpressing transgenic atria were also highly resistant to hypoxia-reoxygenation-induced morphological alterations, as examined by electron microscopy. Use of cardiac catalase-overexpressing transgenic mice thus demonstrates that hydrogen peroxide is involved in hypoxia-reoxygenation cardiotoxicity, and that this mouse model provides a useful tool for study of free radical mechanism in the heart damage.

Catalase-overexpressing transgenic mouse heart is resistant to ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury is at least partially mediated by oxygen-derived free radicals. Catalase is a major enzyme involved in the detoxification of hydrogen peroxide. The activity of catalase in the heart is very low, which may be a factor responsible for the high sensitivity of the heart to ischemia-reperfusion injury. The present study was undertaken to determine whether elevation of catalase specifically in the heart of transgenic mice can provide protection against ischemia-reperfusion injury. Hearts isolated from transgenic mice in which catalase in the heart was elevated approximately 60-fold higher than that in nontransgenic heart and from the non-transgenic littermates were subjected to 50 min of warm (37 degrees C) zero-flow ischemia followed by 90 min reflow. Compared with nontransgenic controls, transgenic hearts showed significantly improved recovery of contractile force (75 vs. 25% at the end of 90 min reperfusion, P < 0.01). Efflux of creatine kinase was reduced by approximately 50%, and the zone of myocardial infarction as demarcated by triphenyltetrazolium at the end of reperfusion was reduced by approximately 40% in transgenic hearts compared with nontransgenic controls. These findings support the view that hydrogen peroxide is an important cause of ischemia-reperfusion damage and suggest that protection may be provided by elevation of catalase activity.

Von Willebrand factor restores impaired platelet thrombogenesis in copper-deficient rats.

Dietary copper restriction reduces microvascular thrombogenesis. We have now examined the roles of shear forces and von Willebrand factor (vWF) in in vivo thrombus formation in the cremaster microcirculation of copper-deficient rats. Male weanling Sprague-Dawley rats were fed purified diets that were either copper-adequate (6.3 mg Cu/kg) or copper-deficient (0.3 mg Cu/kg) for 4 wk. Intravascular fluorescein isothiocyanate tagged to bovine serum albumin was activated with 450-490 nm light to induce thrombus formation in microvessels. Thrombus initiation time was significantly prolonged in copper-deficient rats; after thrombus appearance, however, vessel occlusion was significantly accelerated. The greater shear rates of arterioles compared with venules significantly increased the thrombus initiation time in both groups. However, vessel occlusion time and thrombus growth time were independent of shear rate. Intravascular vWF (0.2 u/100 g body wt) decreased thrombus initiation time in the CuD group without affecting thrombus growth time. The data suggest that decreased thrombogenesis in copper-deficient rats is not a result of altered rheological factors or arteriolar-venular differences, but appears to result from decreased platelet-to-endothelial cell adhesion.

Arteriolar dilation to endotoxin is increased in copper-deficient rats.

We have previously reported that there is an altered response to mast cell-mediated inflammation in copper-deficient rats. In the current study we determined the microvascular reactivity to inflammatory stimuli with lipopolysacccharide (LPS) during dietary copper restriction. Male Sprague-Dawley rats were fed purified diets which were either copper-adequate (CuA, 6 micrograms Cu/g) or copper-deficient (CuD, 0.4 micrograms Cu/g) for 4 weeks. Rats were anesthetized and the cremaster muscle was prepared for in vivo television microscopy. Arteriolar diameters were measured and then 2.5 mg/kg LPS was injected i.p. In separate groups, animals were pretreated with the NO-synthase inhibitor L-NAME (2 x 10(-4) M), the cyclooxygenase inhibitor ibuprofen (9.6 x 10(-5) M) or the histamine receptor antagonist diphenhydramine (DPH, 10(-6) M). LPS caused arteriolar dilation in both dietary groups with the response being significantly greater in the CuD group. Ibuprofen and DPH but not L-NAME, each significantly reduced but did not block the dilation in the CuD group. Ibuprofen and DPH together blocked the dilation. These results suggest that dietary copper deficiency increases arteriolar dilation to LPS. The mechanism appears to involve a greater response to arachidonic acid metabolites and histamine but not NO.

Platelet aggregation and adhesion during dietary copper deficiency in rats.

The role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 micrograms copper/g of diet) or copper-deficient (CuD, 0.3 microgram/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rate endothelial cells. After a one hour incubation at 37 degrees C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 x 10(-4)M) and measured in a turbidometric aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from ++CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.

Defects of copper deficiency in rats are modified by dietary treatments that affect glycation.

We examined the hypothesis that nonenzymatic glycosylatin of proteins (glycation) contributes to the defects of copper deficiency. We studied copper-adequate and -deficient rats while altering two factors known to affect glycation: type of dietary carbohydrate and amount of food intake. Copper deficiency caused cardiac enlargement and anemia, decreased erythrocyte osmotic fragility, enhanced heart lipid peroxidation, increased the percentage of glycated hemoglobin (Hb A1) and reduced staining of lens crystallins on SDS-PAGE gels (suggestive of glycation). Increasing dietary sucrose reduced organ copper concentration, exacerbated the rise in Hb A1 and worsened the anemia caused by copper deficiency. Food restriction ameliorated heart and erythrocyte defects, reduced the percentage of glycated hemoglobin and heart peroxidation and also improved heart and liver copper status in copper-deficient rats. These findings indicate that copper deficiency enhances glycation and that sucrose may exacerbate some defects of copper deficiency by enhancing glycation. Inhibition of defects of copper deficiency by food restriction suggests that glycation and/or peroxidation may contribute to those defects.

A role for dietary copper in nitric oxide-mediated vasodilation.

OBJECTIVE: This study was designed to investigate the role of dietary copper in nitric oxide-mediated arteriolar dilation. METHODS: Male weanling Sprague-Dawley rats were fed a purified diet that was either copper-adequate (6.0 micrograms Cu per g diet) or copper-deficient (0.3 microgram Cu per g diet) for a period of 4 weeks. Each rat was anesthetized with pentobarbital and its cremaster muscle was positioned in a Krebs'-filled bath to which graded concentrations of vasoactive agents were added. In the first series, responses to norepinephrine (NE 10(-9)-10(-6) M) and acetylcholine (ACH 10(-7)-10(-4) M) were compared in third-order arterioles. Second, the dilator response to 10(-5) M ACH in the absence and presence of 240 U/ml Cu, Zn-superoxide dismutase (SOD) was determined. Third, arteriolar dilation was determined in response to NO-independent stimulation of soluble guanylate cyclase with hydrogen peroxide (10(-7)-10(-5) M) and to dibutyryl cGMP (10(-6)-10(-4) M), dibutyryl cAMP (10(-6)-10(-4) M), and papaverine (10(-4) M). RESULTS: The arteriole constrictor response to NE and the dilator response to hydrogen peroxide, dibutyryl cGMP and cAMP, and papaverine were not different between the dietary groups. Copper deficiency attenuated the ACH-induced dilation, but the response was restored in the presence of SOD. CONCLUSIONS: The inactivation of cytosolic Cu, Zn-SOD by restriction of dietary copper results in the depression of NO-mediated vascular smooth-muscle relaxation probably by interaction of NO with superoxide.

Expression of gamma-glutamylcysteine synthetase in the liver of copper-deficient rats.

Copper deficiency in rats increases hepatic glutathione concentration. The present study was undertaken to determine the biochemical and molecular basis for the glutathione elevation. Weanling Sprague-Dawley rats were fed a purified diet deficient in copper (0.4 micrograms/g diet) or one containing adequate copper (5.7 micrograms/g diet) for 4 weeks. Hepatic glutathione concentration, the activity of the rate-limiting enzyme in glutathione biosynthesis, gamma-glutamylcysteine synthetase (gamma-GCS) and the relative amount of mRNA for the enzyme were determined. Hepatic glutathione concentration in copper-deficient rats was significantly elevated (6.6 vs 5.6 mumol/g). The activity of hepatic gamma-GCS was 1.6 times higher in the copper-deficient than in the copper-adequate rats (58.0 vs 35.9 nmol NADH/min.mg protein). The steady-state amount of mRNA for gamma-GCS was increased 5-fold in the copper-deficient rat liver. The findings demonstrate that the elevated hepatic glutathione concentration in copper-deficient rats results from upregulation of gamma-GCS activity. This study provides further understanding of changes in hepatic glutathione metabolism induced by copper deficiency.