Copper-induced Oxidative Stress Research Articles

Characterization of the HMA7 gene and transcriptomic analysis of candidate genes for copper tolerance in two Silene vulgaris ecotypes

Silene vulgaris possesses ecotype-specific tolerance to high levels of copper in the soil. Although this was reported a few decades ago, little is known about this trait on a molecular level. The aim of this study was to analyze the transcription response to elevated copper concentrations in two S. vulgaris ecotypes originating from copper-contrasting soil types - copper-tolerant Lubietova and copper-sensitive Stranska skala. To reveal if plants are transcriptionally affected, we first analyzed the HMA7 gene, a known key player in copper metabolism. Based on BAC library screening, we identified a BAC clone containing a SvHMA7 sequence with all the structural properties specific for plant copper-transporting ATPases. The functionality of the gene was tested using heterologous complementation in yeast mutants. Analyses of SvHMA7 transcription patterns showed that both ecotypes studied up-regulated SvHMA7 transcription after the copper treatment. Our data are supported by analysis of appropriate reference genes based on RNA-Seq databases. To identify genes specifically involved in copper response in the studied ecotypes, we analyzed transcription profiles of genes coding Cu-transporting proteins and genes involved in the prevention of copper-induced oxidative stress in both ecotypes. Our data show that three genes (APx, POD and COPT5) differ in their transcription pattern between the ecotypes with constitutively increased transcription in Lubietova. Taken together, we have identified transcription differences between metallifferous and non-metalliferous ecotypes of S. vulgaris, and we have suggested candidate genes participating in metal tolerance in this species.

Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells

Transition metal copper (Cu) can exist in oxidized or reduced states in cells, leading to cytotoxicity in cancer cells through oxidative stress. Recently, copper complexes are emerging as a new class of anticancer compounds. Here, we report that a novel anticancer copper complex (HYF127c/Cu) induces oxidative stress-dependent cell death in cancer cells. Further, transcriptional analysis revealed that oxidative stress elicits broad transcriptional changes of genes, in which autophagy-related genes are significantly changed in HYF127c/Cu-treated cells. Consistently, autophagy was induced in HYF127c/Cu-treated cells and inhibitors of autophagy promoted cell death induced by HYF127c/Cu. Further analysis identified that the MAPK11/12/13/14 (formerly known as p38 MAPK) pathway was also activated in HYF127c/Cu-treated cells. Meanwhile, the MAPK11/12/13/14 inhibitor SB203580 downregulated autophagy by inhibiting the transcription of the autophagy genes MAP1LC3B, BAG3, and HSPA1A, and promoted HYF127c/Cu-induced cell death. These data suggest that copper-induced oxidative stress will induce protective autophagy through transcriptional regulation of autophagy genes by activation of the MAPK11/12/13/14 pathway in HeLa cells.

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidant properties of Penicillium thomii PT95 strain

Penicillium thomii PT95 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation and antioxidant properties of PT95 strain. The results showed that the time of exudates initiation, sclerotial initiation and sclerotial maturation of PT95 strain were advanced in 1-2 days under the copper-induced oxidative stress growth conditions. The analytical results of sclerotial biomass, carotenoids content in sclerotia showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids. Under the copper-induced oxidative stress growth conditions, the total phenolics content and DPPH free radical scavenging activity of sclerotia of this fungus were decreased as compared with the control. However, the oxidative stress induced by a lower amount of CuSO₄ in media could enhance significantly the reducing power of sclerotia.

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidants contents ofPenicillium thomiiQ1

Penicillium thomii Q1 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation, biosynthesis of some endogenous antioxidants, and the activities of some antioxidative enzymes of Q1 strain. The results showed that the oxidative stress induced by copper was clearly dependent on the CuSO4 concentrations in media, and characterized by the initiation of lipid peroxidation. Under the copper-induced oxidative stress conditions, the time of exudates initiation, sclerotial initiation and sclerotial maturation of Q1 strain were advanced in 1-2 days. The analytical results of sclerotial biomass, carotenoids, and ascorbate contents showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids and ascorbate. The oxidative stress induced by a lower amount of CuSO4 in media could enhance significantly the superoxide dismutase and catalase activities of Q1 strain.

Zinc ameliorates copper-induced oxidative stress in developing rice (Oryza sativa L.) seedlings

Rice (Oryza sativa L.) seedlings were treated with different concentrations of copper (Cu) either in presence or absence of zinc (Zn), and different events were investigated to evaluate the ameliorative effect of Zn on Cu stress. In presence of high Cu concentration, growth of both root and shoots were considerably reduced. Decline in elongation and fresh mass was observed in root and shoot. Zn alone did not show any considerable difference as compared to control, but when supplemented along with high concentration Cu, it prompted the growth of both root and shoot. After 7 days, root growth was 9.36 and 9.59 cm, respectively, at 200 and 500 μM of Cu alone as compared to 10.59 and 12.26 cm at similar Cu concentrations, respectively, in presence of Zn. Cu accumulation was considerably high after 7 days of treatment. In absence of Zn, significant accumulation of Cu was observed. Zn supplementation ameliorated the toxic impact of Cu and minimized its accumulation. Cu treatment for 1 and 7 days resulted in a dose-dependent increase in hydrogen peroxide (H2O2). When Cu was added in presence of Zn, the H2O2 production in root and shoot was reduced significantly. The increase in H2O2 production under Cu stress was accompanied by augmentation of lipid peroxidation. In absence of Zn, Cu alone enhanced the malondialdehyde (MDA) production in both root and shoot after 1 and 7 days of treatment. The MDA content drastically reduced in root and shoot as when Zn was added during Cu treatment. The activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) were elevated under Cu stress both in root and shoot. Addition of Zn further stimulated the activities of these enzymes. Both ascorbate (AsA) and glutathione (GSH) contents were high under Cu stress either in presence or absence of Zn. The results suggests that Zn supplementation improves plant survival capacity under high Cu stress by modulating oxidative stress through stimulation of antioxidant mechanisms and restricts the accumulation of toxic concentrations of Cu.

Copper stress and filamentous fungusHumicola lutea103 — ultrastructural changes and activities of key metabolic enzymes

Humicola lutea 103 is a copper-tolerant fungal strain able to grow in the presence of 300 μg·mL(-1) Cu(2+) under submerged cultivation. To prevent the consequences of copper overload, microorganisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. In spite of this avoidance strategy, high heavy-metal concentrations caused distinct and widespread ultrastructural alterations in H. lutea. The mitochondria were the first and main target of the toxic action. The effect of copper on activities of the key enzymes (hexokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase) included in the 3 main metabolic pathways, glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, was investigated. High metal concentrations exhibited a dramatic negative effect on hexokinase, while the other 3 enzymes showed a significant and dose-dependent stimulation. On the basis of the present and previous results we concluded that the copper-induced oxidative stress plays an important role in the fungal tolerance to high Cu (2+) concentrations.

Copper-Induced Oxidative Stress and Responses of the Antioxidant System in Roots of Medicago sativa

The effects of various copper (Cu) concentrations on the antioxidative system in the roots of Medicago sativa were explored. The results indicated that the Cu content of the roots reached a value of 854 μg g−1 DW at 10 μm Cu and a value of 4415 μg g−1 DW at 100 μm Cu, suggesting that M. sativa has better ability to tolerate and accumulate Cu than other Cu-bioaccumulators, and is a potential plant for phytoremediation. Treatment with Cu resulted in a significant increment in the levels of H2O2, O2˙− and OH˙. The reduced form of ascorbate and glutathione reached a peak at 30 μm Cu, and was followed by a sharp depletion to a lower level than that of the control. In contrast, the levels of the oxidised forms of ascorbate and glutathione showed a progressive increment with increasing Cu concentrations, suggesting that the antioxidant system was unable to cope with Cu stress at higher Cu levels. Under the Cu concentrations tested, the activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) increased at lower Cu concentrations, and then decreased, reaching a maximum at 30 μm of Cu for APX and GR, at 10 μm for CAT, whereas the activities of guaiacol peroxidase (POD, EC 1.11.1.7) were gradually increased with increasing Cu concentrations. PAGE analysis of superoxide dismutase (SOD, EC 1.1.5.1.1) revealed that one band is a Mn-SOD and five bands are identified as Cu, Zn-SOD, whereas Fe-SOD isoforms were not found in the roots of alfalfa. Cu at 10–100 μm increased the intensity of constitutive isozymes of CAT, APX and POD, whereas it decreased the intensity of isozymes of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) significantly. The activities of lipoxygenases (LOX, EC 1.13.11.12) were gradually augmented with increasing Cu concentrations, demonstrating that LOXs are probably involved in production of lipid hydroperoxides and superoxide anion. There was a continuous and pronounced enhancement in the activity of esterase (EST, EC 3.1.1.1) in roots treated with 10–30 Cu μm, whereas EST activity in roots exposed to above 30 μm Cu declined, suggesting that EST plays a protective role under lower Cu concentrations stress.

Copper-induced oxidative stress in Alternanthera philoxeroides callus

The callus of Alternanthera philoxeroides, an emerging plant, was subjected to different concentrations of Cu2+ (0, 0.05, 0.1, 0.2, 0.6, 0.8, and 1 mM) for a period of 5 days and the contents of soluble protein and malondialdehyde (MDA), activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase (G-POD, EC 1.11.1.7), levels of reactive oxygen species (ROS, such as O 2 −∙ and H2O2) were determined. Treatment with 0.05 mM Cu2+ enhanced the content of soluble protein and reduced levels of O 2 −∙ and H2O2. As the Cu2+ concentration exceeded 0.1 mM, the content of soluble protein decreased greatly and the amounts of two polypeptides (apparent molecular weights 45 and 39 kDa, respectively) became visible in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); SOD activity was enhanced markedly owing to the accumulation of O 2 −∙ , while POD and CAT activities increased alternately in response to increased H2O2 content. Because of the congenerous function of POD and CAT, the H2O2 content dropped to a low level finally. An interesting result was that the MDA content decreased with the increasing concentration of Cu2+. The results of this study indicated that antioxidant enzymes (SOD, POD, and CAT) protected A. philoxeroides callus against copper stress efficiently, especially for SOD. Whether there was another protective mechanism in A. philoxeroides callus or not requires further research. The critical value of copper on A. philoxeroides callus was 0.1 mM.

Epibrassinolide induces changes in indole-3-acetic acid, abscisic acid and polyamine concentrations and enhances antioxidant potential of radish seedlings under copper stress

In the present study, the effects of epibrassinolide (EBL) on indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamine (PA) tissue concentrations and antioxidant potential of 7-day-old Raphanus sativus L. cv. 'Pusa chetki' seedlings grown under Cu stress were investigated. EBL treatment alone or in combination with Cu enhanced free and bound IAA titers when compared with the metal alone. Modest increases in free and bound ABA contents were observed for EBL treatment alone. However, the combination of EBL with Cu caused major increases in both forms of ABA, over Cu alone. Among the PAs analyzed, only putrescine and cadaverine concentrations were enhanced by EBL treatment alone. By contrast, a significant decline in putrescine and spermine contents was found in seedlings treated with EBL plus Cu. EBL treatments alone or in combination with Cu enhanced activities of guaiacol peroxidase (EC1.11.1.7), catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) and protein contents in comparison with metal and control treatments. A major decrease in malondialdehyde content was also recorded for EBL treatments with or without Cu. An increase in phytochelatin content was also observed in seedlings treated with EBL alone or in combination with Cu. Major improvement in radical scavenging activities, as attested by the antioxidant activity assay using DPPH (1,1-diphenylpicrylhydrazyl), and elevated deoxyribose and reducing powers, along with increased contents of ascorbic acid, total phenols and proline, also suggest a major influence of EBL application in mitigating copper-induced oxidative stress in radish seedlings.

Differential responses to copper-induced oxidative stress in the marine macroalgae Lessonia nigrescens and Scytosiphon lomentaria (Phaeophyceae)

In order to help explain the absence of the brown kelp Lessonia nigrescens from a coastal environment chronically enriched with copper, we characterized the biochemical responses induced by copper stress in this kelp and compared them with those displayed by the copper tolerant brown alga Scytosiphon lomentaria. These algae were cultivated with increasing concentrations of copper (20, 40 and 100 μg L −1) for 96 h and the temporal production of hydrogen peroxide, superoxide anions and lipoperoxides as well as the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GP), ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and the activity of the defense enzyme lipoxygenase (LOX) were determined. In L. nigrescens and S. lomentaria, a single peak of hydrogen peroxide was detected, with similar maxima after 3 h of copper exposure, although in L. nigrescens buffering took longer. Superoxide anions, on the other hand, were only detected in L. nigrescens. The production of lipoperoxides in L. nigrescens increased steadily at higher copper levels, in a pattern clearly different to their rapid stabilization in S. lomentaria. We suggest that the accumulation of lipoperoxides might be related to LOX, whose activity also increases with exposure time. Furthermore, activities of the antioxidant enzymes CAT, GP, AP and DHAR were lower in L. nigrescens than in S. lomentaria, and GP and DHAR were completely inhibited at higher copper concentrations. Since these enzymes also detoxify fatty acid hydroperoxides, their inhibition, together with the activation of LOX, may explain the persistent and copper-dependent levels of lipoperoxides in L. nigrescens. Based on terrestrial plant models demonstrating toxic effects of lipoperoxides, and on our results on organellar ultrastructural changes, we suggest that copper toxicity induced an uncontrolled lipoperoxide accumulation which may lead to cell damage and dysfunction in L. nigrescens, explaining at least partially, the absence of this kelp in a copper-enriched coastal environment.

Copper-induced growth inhibition, oxidative stress and ultrastructural alterations in freshly grown water lettuce ( Pistia stratiotes L.)

The effects of increasing concentrations of copper on the growth, ultra-structure and on certain biochemical parameters of water lettuce ( Pistia stratiotes L.) were investigated under controlled conditions in the nutrient solutions containing increased copper sulfate concentrations ranging from 0 to 100 μM. Copper treatment for 12, 18 or 24 h resulted in inhibition of roots and leaves dry biomass. Atomic absorption spectrometry analysis of roots and leaves showed that copper accumulation increased with increase in concentration and duration of metal treatment. It is seen that copper resulted in increased production of hydrogen peroxide and superoxide radical in both roots and leave cells, showed a significant change after 24 h of treatment. Also, the significant decrease in the contents of total protein and photosynthetic pigments was observed. The antioxidant enzymes, viz., peroxidase (POX, E.C.1.11.1.7), catalase (CAT, E.C.1.11.1.6) and superoxide dismutase (SOD, E.C.1.15.1.1) showed significant variation with the increase in lipid peroxidation. Increasing trends was observed in levels of ascorbate and glutathione. The rapid inducibility of some of these enzymes are useful early and sensitive indicators of heavy metal toxicity. The results demonstrated that exposure to elevated concentration of Cu had a remarkable effect on the biochemistry and physiology, induced oxidative stress in water lettuce characterized by the initiation of lipid peroxidation that inhibited growth and disintegration of major antioxidant systems. To cite this article: R.K. Upadhyay, S.K. Panda, C. R. Biologies 332 (2009).

ATM-mediated Transcriptional Elevation of Prion in Response to Copper-induced Oxidative Stress

Increasing evidence suggests that the cellular prion protein (PrP(C)) plays a protective role in response to oxidative stress, but the molecular mechanism is unclear. Here, we demonstrate that murine neuro-2a and human HeLa cells rapidly respond to an increase of intracellular copper concentration by up-regulating ataxia-telangiectasia mutated (ATM)-mediated transcription of PrP(C). Copper stimulation activates ATM by phosphorylation at Ser-1981, which leads to phosphorylation of p53 at Ser-15 and the initiation of the mitogen-activated protein kinase kinase/extracellular-related kinases/extracellular-related kinases (MEK/ERK)/Sp1 pathway. As results, Sp1 and p53 bind to the PrP promoter, leading to increase PrP(C) expression. Elevated PrP(C) correlates with reduction of intracellular copper concentration and suppression of Cu(II)-induced accumulation of reactive oxygen species and cell death. Depletion of PrP(C), ATM, p53, and/or Sp1 further demonstrates that ATM is a key regulatory protein to promote activation of p53 and Sp1 leading to PrP(C) elevation, which is required to reduce Cu(II) toxic effects and may play an important role in modulation of intracellular copper concentration.

Mechanism of Copper-Activated Transcription: Activation of AP-1, and the JNK/SAPK and p38 Signal Transduction Pathways

Copper is an essential metal that is able to produce reactive oxygen species and to induce intracellular oxidative stress. Several studies have examined the effects of excessive copper and oxidative stress on various organisms and tissues, but few have addressed the molecular mechanisms by which copper affects transcription. Our results demonstrated that, in COS-7 cells, copper treatment caused an increase in the binding of nuclear proteins to activating protein-1 and antioxidant response elements. The level of copper-inducible nuclear protein binding was modulated by increasing or decreasing the level of intracellular oxidative stress. Copper exposure also led to an increase in the steady-state levels of c-fos, c-jun, and c-myc mRNAs. Exposure to copper resulted in an increase in the levels of phosphorylation and activation of the c-Jun N-terminal kinase/stress-activated protein kinase and p38 pathways. The activation of these pathways resulted in a concomitant increase in c-Jun phosphorylation. We investigated the hypothesis that copper-induced oxidative stress leads to the formation of stable lipid peroxidation by-products that activate mitogen-activated protein kinase (MAPK) pathways, ultimately affecting transcription. While exposure did result in the production of 4-hydroxynonenal, the timing of the increased levels of proto-oncogene mRNA, phosphorylation of c-jun, and phosphorylation and activation of MAPKs, as well as the inability of the lipophilic antioxidant vitamin E to abrogate MAPK phosphorylation, suggest that the formation of stable lipid peroxidation by-products may not be the primary mechanism by which copper activates MAPKs. These results further elucidate the effects of copper on signal transduction pathways to alter gene expression.

Effect of copper intoxication on rat liver proteasome activity: Relationship with oxidative stress

Copper toxicity is associated with formation of reactive oxygen species, which are capable to oxidize proteins. The selective removal of the latter by the 20S proteasome is considered an essential part of the cell antioxidant defense system. The aim of the present study was to investigate whether peptidase activities of rat liver proteasomes were affected by chronic (40 mg CuSO(4)/rat/daily with the drinking water for 2 weeks) and acute (20 mg/kg CuSO(4), s.c.) copper treatment. To evaluate the role of proteasome, its inhibitor MG132 was also used. The degree of copper-induced oxidative stress (OS), established by measuring lipid peroxidation, protein oxidation, and cellular glutathione level, as well as activities of antioxidant enzymes--catalase, superoxide dismutase, and gultathionine peroxidase, depended on the mode of copper administration. Chronic copper administration (mild oxidative stress) did not affect proteasome activities, whereas acute copper treatment (severe oxidative stress) caused a decline in chymotryptic- and tryptic-like activities. The treatment of copper-loaded animals with MG132 did not change copper-induced alterations in the tested indices, except an additional increase in protein oxidation and inhibition of glutathionine peroxidase activity. The results suggested that the in vivo copper-induced oxidative stress was associated with changes in the catalytic activity of proteasome.

Copper-induced oxidative stress in rainbow trout gill cells

Copper is known to pose a serious threat to aquatic organisms. However, the mechanisms of its toxicity still remain unclear. Cu is known to exert its toxicity partly due to the formation of reactive oxygen species (ROS). The purpose of this work was therefore to link the exposure to copper at pH 6 and 7 to cellular formation of ROS and effects like cell viability and genotoxicity using the rainbow trout gill cell line RTgill-W1. To relate effects to bioavailable copper, free Cu 2+ concentrations in the medium were calculated using the programm ChemEQL 3.0. 2′,7′-Dichlorodihydrofluorescein-diacetate (H 2DCF-DA) was used as cell-permeant indicator of ROS formation. Cell viability was assessed using the fluorogenic probe 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). DNA strand breaks were assessed using the comet assay, and lipid peroxidation was investigated using the thiobarbituric acid-reactive substances assay (TBARS). Copper treatment resulted in a dose-dependent elevation in cytotoxicity and formation of cellular ROS. Cell viability was significantly reduced at total copper (Cu T) concentrations of 5 μM (corresponding to a free Cu 2+ of 0.11 μM at pH 7) and higher, resulting in an EC 50 of Cu T = 29.2 μM (Cu 2+ = 0.63 μM, pH 7). Neither an impairment concerning the viability of control cells due to growth at pH 6 was observed nor significant differences for cytotoxicity in cells exposed to the same nominal Cu T concentrations at pH 6 compared to pH 7. Cellular ROS concentrations increased significantly and decreased with loss of cell viability. After normalizing ROS formation to cell viability, ROS induction up to 25–35-fold compared to the control was detected, but mainly for rather high concentrations (Cu T ≥ 100 μM; Cu 2+ ≥ 2.2 μM, pH 7). ROS formation rates were slightly higher when cells were exposed to Cu at pH 6 compared to pH 7, correlating with the higher free Cu 2+ concentrations. A significant induction of DNA strand breaks was noted at Cu T of 1 and 2.5 μM with greater effects at pH 6 due to higher free Cu 2+ concentrations than at pH 7. No effects on lipid peroxidation were observed. These results lead to the hypothesis that copper-induced loss in viability and genotoxicity in trout gill cells are partially triggered by the generation of ROS and related to the free Cu 2+.

Copper-Induce Change in Antioxidative System in Maize (Zea mays L.)

Maize seedlings treated with various concentrations (25-100 microM) of copper for 15 days. A progressive decrease of root length and biomass with increasing Cu in nutrient solution was observed. The roots accumulated significantly higher amounts of Cu than the above ground parts. Accumulation of copper resulted in more active lipid peroxidation in both roots and shoots, which was attributed to copper-induced additional oxidative stress. Activities of APX (ascorbate peroxidase), GPX (guaiacol peroxidase), GR (glutathione reductase) and CAT(catalase) were higher in both roots and shoots in response to copper accumulation. Changes in lipid peroxidation and antioxidant enzyme activities suggest that oxidative damage may be involved in copper toxicity.

Copper decreases gene expression of TNF-α, IL-10, and of matrix metalloproteinases MMP-2 and MMP-9 in isolated perfused rat livers

Copper is known to induce oxidative stress in a number of models. It was shown that many pathophysiological events were associated with oxidative stress. Further, oxidative stress can increase gene expression of cytokines and of metalloproteinases. We previously found that copper toxic effects in isolated perfused rat livers were associated with significant oxidative stress (as assessed by lipid peroxidation, protein oxidation and oxidative DNA damage, particularly at concentration of 0.03 mM of Cu2+ in the perfusate). Here we investigated gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10); matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in frozen liver tissue samples by the real-time PCR assay. Compared to controls, copper at concentration of 0.01 mM did not affect gene expression of TNF-α, IL-10, MMP-2 and MMP-9, whereas copper at concentration of 0.03 mM significantly decreased gene expression of all the four TNF-α, IL-10, MMP-2 and MMP-9 by 69%, 81%, 43%, and 62%, respectively. These results suggest that copper-induced oxidative stress in the isolated rat liver can lead to the suppression of gene expression. Because TNF-α and metalloproteinases are involved also in liver regeneration, the suppression of these genes by copper may be one of the mechanisms by which acute intoxication of animals and humans with copper may impair regenerative capability of the liver.

Gene expression profiling of liver cells after copper overload in vivo and in vitro reveals new copper-regulated genes

Copper toxicity in the liver is mediated by free-radical generation, resulting in oxidative stress. To prevent toxic accumulation of copper, liver cells adapt to high copper levels. Here, we used microarray analysis to compare the adaptive responses on global gene expression in liver cells exposed to high copper levels in vitro and in vivo. In HepG2 cells we identified two clusters of upregulated genes over time, an "early" cluster that comprised metallothionein genes and a "late" cluster, highly enriched in genes involved in proteasomal degradation and in oxidative stress response. Concomitant with the "late" cluster, we detected a significant downregulation of several copper metabolism MURR1 domain (COMMD) genes that were recently implicated in copper metabolism and inhibition of nuclear transcription factor kappaB (NF-kappaB) signaling. As metal-induced oxidative stress increases NF-kappaB activity, our data suggest a role for reduced COMMD protein levels in prolonged activation of NF-kappaB, thus inducing cell survival. Mice exposed to a copper diet that highly exceeded normal daily intake accumulated only twofold more hepatic copper than control mice. Although a moderate, but significant upregulation of a set of 22 genes involved in immunity, iron and cholesterol metabolism was detected, these cannot account for direct mechanisms involved in copper excretion. In conclusion, we identified a novel set of genes that represent a delayed response to copper overload, thus providing insight into the adaptive transcriptional response to copper-induced oxidative stress.

Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle

Copper, though essential, is potentially toxic heavy metal at supraoptimal level and has widespread contamination. The present investigation was carried out to study the responses induced by lower as well as higher doses of copper (0.1–25 μM) in an aquatic macrophyte, Hydrilla verticillata (L.f.) Royle for a period of 1–7 days. The plants accumulated copper in high amount with a maximum of 770 μg g −1 dw on day 7 at 25 μM. Biomass and photosynthetic pigments showed less alteration up to 1 μM while at higher concentrations, significant decline occurred. Malondialdehyde (MDA) content and electrical conductivity (EC) also showed sharp increase at higher concentrations indicating oxidative stress. In response to copper exposure, plants showed significant induction of proteins and enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR), however, only up to moderate exposures. Total non-protein thiols (NP-SH) and cysteine levels increased significantly up to 5 μM copper exposure while at 25 μM, their level declined drastically. Reduced glutathione (GSH) showed decrease at all concentrations while oxidized glutathione (GSSG) simultaneously increased. Phytochelatins (PCs) were also induced significantly at studied concentrations of 1 and 5 μM on day 4 in comparison to control. However, copper chelation depicted by PC-SH to copper ratio was found to be low (6.5% at 1 μM and 2.4% at 5 μM) suggesting that PCs play only a part in integrated mechanisms of copper homeostasis and detoxification. Tolerant response of plants to moderate copper exposures and high accumulation potential warrants their suitability for remediation of moderately copper polluted water bodies.

The Greater Susceptibility of North Ronaldsay Sheep Compared with Cambridge Sheep to Copper-induced Oxidative Stress, Mitochondrial Damage and Hepatic Stellate Cell Activation

Sheep of the semi-feral North Ronaldsay (copper-sensitive) and domesticated Cambridge (copper-tolerant) breeds were compared in respect of pathological changes and protein expression in the liver as a result of excessive dietary copper. Acute mitochondrial damage and hepatic stellate cell (HSC) activation with collagen synthesis occurred in response to moderate copper overload in North Ronaldsay but not in Cambridge sheep. Mitochondrial degradative changes occurred either as ballooning degeneration and rupture with subsequent autophagic degradation or as mitochondrial matrical condensation (pyknosis). In North Ronaldsay sheep prolonged exposure to copper produced mitochondrial hyperplasia and hypertrophy, and nuclear damage with necrosis. Cytosolic isocitrate dehydrogenase (IDH), an enzyme responsive to oxidative stress, was induced in the liver of Cambridge sheep receiving a Cu-supplemented diet but was undetectable in the non-supplemented control sheep. Conversely, IDH was detected at similar levels in both control and copper-supplemented North Ronaldsay sheep, indicating a lower threshold response, and an enhanced susceptibility, to oxidative stress. "Upregulation" of mitochondrial thioredoxin-dependent peroxidase reductase (antioxidant protein-1) in the hepatic cytosol of the North Ronaldsay (but not Cambridge) sheep affirmed the increased susceptibility of the mitochondria to Cu-induced oxidative stress in this breed. Likewise the upregulation of cathepsin-D indicated increased lysosomal activity and HSC activation. The findings may be relevant to copper toxicosis in human infants.