Modulation of copper accumulation and copper-induced toxicity by antioxidants and copper chelators in cultured primary brain astrocytes

Copper is essential for several important cellular processes, but an excess of copper can also lead to oxidative damage. In brain, astrocytes are considered to play a pivotal role in the copper homeostasis and antioxidative defence. To investigate whether antioxidants and copper chelators can modulate the uptake and the toxicity of copper ions in brain astrocytes, we used primary astrocytes as cell culture model. These cells accumulated substantial amounts of copper during exposure to copper chloride. Copper accumulation was accompanied by a time- and concentration-dependent loss in cell viability, as demonstrated by a lowering in cellular MTT reduction capacity and by an increase in membrane permeability for propidium iodide. During incubations in the presence of the antioxidants ascorbate, trolox or ebselen, the specific cellular copper content and the toxicity in copper chloride-treated astrocyte cultures were strongly increased. In contrast, the presence of the copper chelators bathocuproine disulfonate or tetrathiomolybdate lowered the cellular copper accumulation and the copper-induced as well as the ascorbate-accelerated copper toxicity was fully prevented. These data suggest that predominantly the cellular content of copper determines copper-induced toxicity in brain astrocytes.     

Copper/zinc and copper/selenium ratios,and oxidative stress as biochemical markers in recurrent aphthous stomatitis

ProjectRecurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent, painful oral aphthae, and oxidative stress presumably contributes to its pathogenesis. The aim of this study is to scrutinize the relationship between oxidative stress and serum trace elements (copper, Cu; zinc, Zn; selenium, Se), and to evaluate the ratios of Cu/Zn and Cu/Se in this disorder.ProcedurePatients with RAS (n = 33) and age- and sex-matched healthy control subjects (n = 30) were enrolled in this study. Malondialdehyde (MDA) concentrations in plasma and the activities of superoxide dismutase (SOD1; CuZnSOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocyte were determined as spectrophotometric. Also, the levels of Se, Zn and Cu in serum were determined on flame and furnace atomic absorption spectrophotometer using Zeeman background correction.Results and conclusionsOxidative stress was confirmed by the significant elevation in plasma MDA, and by the significant decrease in CAT, SOD1, and GPx (p < 0.05). When compared to controls, Zn and Se levels were significantly lower in patients, whereas Cu levels was higher in RAS patients than those in controls (p < 0.05). In addition, the correlation results of this study were firstly shown that there were significant and positive correlations between Se–CAT, Se–GPx, and Cu–MDA parameters, but negative correlations between Se–Cu, Se–MDA, Cu–CAT, Cu–SOD1 and Cu–GPx parameters in RAS patients. Furthermore, the ratios of Cu/Zn and Cu/Se were significantly higher in the patients than the control subjects (p < 0.05). Our results indicated that lipid peroxidation associated with the imbalance of the trace elements seems to play a crucial role in the pathogenesis of RAS. Furthermore, the serum Cu/Zn and Cu/Se ratios may be used as biochemical markers in these patients.     

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H₂O₂, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.     

Increased oxidative stress and altered serum macro-minerals and trace elements levels are associated with coronary artery disease

BackgroundThis study was designed to evaluate the serum malondialdehyde (MDA), non-enzymatic antioxidants (vitamin A and C), macro-minerals (magnesium and calcium), and trace elements (zinc, copper, and iron) levels in patients with coronary artery disease (CAD) and to explore their role in disease progression.MethodsThis prospective case-control study was comprised of 40 CAD patients and 40 healthy volunteers as cases and control subjects, respectively. The level of lipid peroxidation was assessed by measuring the serum MDA level using a UV spectrophotometer. The levels of vitamins A and C were determined by high-performance liquid chromatography (HPLC) and UV spectrophotometric method, respectively. Atomic absorption spectroscopy (AAS) was used to measure serum macro-minerals (Mg and Ca) and trace elements (Zn, Cu, and Fe) concentrations.ResultsThe mean age of CAD patients and control subjects was 53.90 ± 2.22 and 37.03 ± 1.50 years, respectively. This study revealed significantly higher concentrations of MDA (p < 0.01) and lower concentrations of vitamin A (p < 0.01), and vitamin C (p < 0.05) in the CAD patients than in control subjects. The mean values of Mg, Cu, Zn, Ca, and Fe were 11.67 ± 0.64, 1.17 ± 0.03, 0.43 ± 0.02, 107.38 ± 1.81, and 1.66 ± 0.04 μg/mL, respectively for the CAD patients and 19.38 ± 0.65, 1.07 ± 0.02, 0.87 ± 0.02, 94.29 ± 1.89, and 1.52 ± 0.05 μg/mL, respectively for the controls and the differences were significant (p < 0.05) between the patients and controls.ConclusionFrom these findings, we can suggest that there is a strong association of CAD with an elevated level of MDA, depleted levels of antioxidants, and altered macro-minerals and trace elements concentrations.     

Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.     

Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma

Copper (Cu) deficiency decreases the activity of Cu-dependent antioxidant enzymes such as Cu,zinc-superoxide dismutase (Cu,Zn-SOD) and may be associated with increased susceptibility to oxidative stress. Iron (Fe) overload represents a dietary oxidative stress relevant to overuse of Fe-containing supplements and to hereditary hemochromatosis. In a study to investigate oxidative stress interactions of dietary Cu deficiency with Fe overload, weanling male Long–Evans rats were fed one of four sucrose-based modified AIN-93G diets formulated to differ in Cu (adequate 6 mg/kg diet vs. deficient 0.5 mg/kg) and Fe (adequate 35 mg/kg vs. overloaded 1500 mg/kg) in a 2×2 factorial design for 4 weeks prior to necropsy. Care was taken to minimize oxidation of the diets prior to feeding to the rats. Liver and plasma Cu content and liver Cu,Zn-SOD activity declined with Cu deficiency and liver Fe increased with Fe overload, confirming the experimental dietary model. Liver thiobarbituric acid reactive substances were significantly elevated with Fe overload (pooled across Cu treatments, 0.80±0.14 vs. 0.54±0.08 nmol/mg protein; P<.0001) and not affected by Cu deficiency. Liver cytosolic protein carbonyl content and the concentrations of several oxidized cholesterol species in liver tissue did not change with these dietary treatments. Plasma protein carbonyl content decreased in Cu-deficient rats and was not influenced by dietary Fe overload. The various substrates (lipid, protein and cholesterol) appeared to differ in their susceptibility to the in vivo oxidative stress induced by dietary Fe overload, but these differences were not exacerbated by Cu deficiency.     

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.     

Mitochondrial localization of Smad5 in a human chondrogenic cell line

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily and regulate the formation of cartilage and bone tissues as well as other key events during development. TGF-beta superfamily signaling is mediated intracellularly by Smad proteins, some of which can translocate into the cell nucleus and influence gene expression. Although much progress has been made in understanding how TGF-beta superfamily signaling regulates expression of target genes, little formal proof has been presented regarding the intracellular distribution of the Smad proteins before their entry into the nucleus. In the literature, non-nuclear Smad proteins are generally referred to as cytoplasmic. Using confocal microscopy, we here show for the first time that immunofluorescent labeling of Smad5, one of the Smad proteins associated with BMP signaling, colocalizes with the mitochondrion-specific probe MitoTracker, demonstrating a mitochondrial distribution of Smad5 in non-stimulated chondroprogenitor cells.     

On the potential increase of the oxidative stress status in patients with abdominal aortic aneurysm

Abstract

Background

Abdominal aortic aneurysm (AAA) is a major cause of preventable deaths in older patients. Oxidative stress has been suggested to play a key role in the pathogenesis of AAA. However, only few studies have been conducted to evaluate the blood oxidative stress status of AAA patients.

Methods and results

Twenty seven AAA patients (mean age of 70 years) divided into two groups according to AAA size (≤50 or >50 mm) were compared with an age-matched group of 18 healthy subjects. Antioxidants (vitamins C and E, β-carotene, glutathione, thiols, and ubiquinone), trace elements (selenium, copper, zinc, and copper/zinc ratio) and markers of oxidative damage to lipids (lipid peroxides, antibodies against oxidized patients, and isoprostanes) were measured in each subject. The comparison of the three groups by ordinal logistic regression showed a significant decrease of the plasma levels of vitamin C (P = 0.011), α-tocopherol (P = 0.016) but not when corrected for cholesterol values, β-carotene (P = 0.0096), ubiquinone (P = 0.014), zinc (P = 0.0035), and of selenium (P = 0.0038), as AAA size increased. By contrast, specific markers of lipid peroxidation such as the Cu/Zn ratio (P = 0.046) and to a lesser extent isoprostanes (P = 0.052) increased.

Conclusion

The present study emphasizes the potential role of the oxidative stress in AAA disease and suggests that an antioxidant therapy could be of interest to delay AAA progression.     

Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence

Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations ( 180 µM) of hydrogen peroxide (H₂O₂). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 µM H₂O₂. Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide orm-chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less ° cells was also significantly increased by exposure to low concentrations (e.g. 180 µM) of H₂O₂. These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria.     

Exploring the role of copper and selenium in the maintenance of normal thyroid function among healthy Koreans

BackgroundSelenium and iodine are trace elements well known to have important roles in the synthesis and metabolism of thyroid hormones. However, the effects of other trace elements on thyroid hormones are still inconclusive. We investigated the association between several trace elements and thyroid hormones.MethodsThe data of 448 subjects who were measured for both, trace elements and TSH/free T4, at the Heath Checkup Center were retrospectively reviewed. The presence of thyroiditis (from thyroid echogenicity) and thyroid nodules were reviewed in the subjects who underwent thyroid ultrasonography.ResultsBlood concentrations of manganese, copper, selenium, and molybdenum were associated with TSH or free T4. After adjusting for age, sex, BMI, smoking, and alcohol consumption, blood copper levels were positively associated with free T4 in both sexes and selenium levels were positively associated with free T4 in women. There was no association between trace elements and thyroiditis. Blood copper concentration had a weak non-linear association with the presence of thyroid nodules.ConclusionsThis study demonstrated that blood concentrations of copper and selenium were significantly associated with free T4 in healthy Korean subjects with sufficient iodine intake suggesting their role in maintaining normal thyroid function.     

The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line

PURPOSE: Topotecan, a semisynthetic water-soluble derivative of camptothecin exerts its cytotoxic effect by inhibiting topoisomerase I and causes double-strand DNA breaks which inhibit DNA function and ultimately lead to cell death. In previous studies it was shown that camptothecin causes ROS formation. The aim of this study was to investigate if Topotecan like camptotecin causes oxidative stress in MCF-7 human breast cancer cell line. Determining the oxidant effect of Topotecan may elucidate a possible alternative mechanism for its cytotoxicity. EXPERIMENTAL DESIGN: MCF-7 cells were cultured and exposed to Topotecan for 24 h at 37 degrees C. The viability of the cells (% of control) was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation (TBARS), protein oxidation (carbonyl content), sulfhydryl, glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were determined in MCF-7 cells with and without Topotecan incubation. RESULTS: We found the IC(50) concentration of Topotecan as 0.218 microM in MCF-7 cells. This concentration of Topotecan was used in the incubations of the cells. Our data indicated increased oxidative status, as revealed by increased lipid peroxidation and protein oxidation, and decreased GSH and sulfhydryl levels in MCF-7 cells exposed to Topotecan compared to control cells. In contrast, there was a slight increase in SOD and a significant increase in GPx and catalase activity in MCF-7 cells incubated with Topotecan compared to the control. CONCLUSIONS: These results support our hypothesis that Topotecan increases oxidative stress in MCF-7 cells.     

Modulation of copper toxicity-induced oxidative damage by excess supply of iron in maize plants

In this study, we examined the modulation of Cu toxicity-induced oxidative stress by excess supply of iron in Zea mays L. plants. Plants receiving excess of Cu (100 μM) showed decreased water potential and simultaneously showed wilting in the leaves. Later, the young leaves exhibited chlorosis and necrotic scorching of lamina. Excess of Cu suppressed growth, decreased concentration of chloroplastic pigments and fresh and dry weight of plants. The activities of peroxidase (EC 1.11.1.7; POD), ascorbate peroxidase (EC 1.11.1.11; APX) and superoxide dismutase (EC 1.15.1.1; SOD) were increased in plants supplied excess of Cu. However, activity of catalase (EC 1.11.1.6; CAT), was depressed in these plants. In gel activities of isoforms of POD, APX and SOD also revealed upregulation of these enzymes. Excess (500 μM)-Fe-supplemented Cu-stressed plants, however, looked better in their phenotypic appearance, had increased concentration of chloroplastic pigments, dry weight, and improved leaf tissue water status in comparison to the plants supplied excess of Cu. Moreover, activities of antioxidant enzymes including CAT were further enhanced and thiobarbituric acid reactive substance (TBARS) and H₂O₂ concentrations decreased in excess-Fe-supplemented Cu-stressed plants. In situ accumulation of H₂O₂, contrary to that of O₂ ^·− radical, increased in both leaf and roots of excess-Cu-stressed plants, but Cu-excess plants supplied with excess-Fe showed reduced accumulation H₂O₂ and little higher of O₂ ^·− in comparison to excess-Cu plants. It is, therefore, concluded that excess-Cu (100 μM) induces oxidative stress by increasing production of H₂O₂ despite of increased antioxidant protection and that the excess-Cu-induced oxidative damage is minimized by excess supply of Fe.     

The antioxidant effect of Aronia melanocarpa extract in rats oxidative stress induced by cisplatin administration

BackgroundThe reactive oxygen species generated by numerous xenobiotic substances has as consequences the impairment of different organs normal function. Many plants pose antioxidant activity to counteract oxidative stress, among them being the chokeberry (Aronia melanocarpa). The purpose of present study was to determine if the use of A. melanocarpa extract can counteract the oxidative stress induced by cisplatin administration in rats.Material and methodsThe study was made on forty Wistar rats divided in four groups as follows: C (control): receiving i.p. 1 mL of saline solution; E1: receiving cisplatin 20 mg/kg bw, i.p.; E2: receiving cisplatin 20 mg/kg bw, i.p and A. melanocarpa berry 6 % aqueous extract as drinking water, and CB (control blank): i.p 1 mL saline solution and A. melanocarpa 6 % aqueous extract for four weeks. Results. Administration of Cisplatin was followed by the increase of serum superoxide dismutase (+21.18 %, P < 0.05), catalase (+25.44 %, P < 0.001), glutathione peroxidase (+17.88 %, P < 0.05) and thiobarbituric reactive substances (+28.17 %, P < 0.01) but significantly decreased glutathione reductase (−22.35 %, P < 0.001) level comparative to control, pointing out that administration of cisplatin induced oxidative stress in rats. In groups that received A. melanocarpa extract as drinking water, we noted that the levels of the oxidative stress biomarkers tended to be restored almost to normal levels, which could be a possible good antioxidant used in condition to cisplatin use. Also, we noted a significant (P < 0.001) decrease of total antioxidant capacity in liver and kidney of rats exposed to cisplatin, recovered in those that received chokeberry. Studied trace elements important for the stress oxidative enzymes (Cu, Zn, Fe and Mn) were decreased in cisplatin exposed groups compared to control and mainly all were almost to normal level in groups receiving A. melanocarpa. Conclusion. A. melanocarpa extract due to its antioxidants content could offer protection against free radicals produced as a consequence of cisplatin use.     

Measurement of oxidative stress in human liver by EPR spin-probe technique

A method for the measurement of reactive oxygen species (ROS) in human hepatic tissue has been developed. The method is based on the EPR detection of the nitroxide radical produced by reaction of the hydroxylamine spin-probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate with ROS generated under pseudo-physiologic conditions in fine needle biopsies of healthy (10 controls) and diseased (22 patients) human liver. Measures of malonaldehyde in 9 liver biopsies (3 controls and 6 patients) have also been obtained by high pressure liquid chromatography and values parallel those obtained by the spin-probe technique. The amount of ROS found in healthy human liver (median = 1.8 × 10^-11 mol/mg) was significantly lower than values found in liver affected by hepatitis B (median = 5.8 × 10^-10 mol/mg; p < 0.02) or by hepatitis C (median = 2.7 × 10^-9 mol/mg; p < 0.003) as well as compared to some other non-viral liver diseases (NVLD): autoimmune hepatitis, primary biliary cirrhosis, primary schlerosing cholangitis (median = 9.8 × 10^-9 mol/mg; p < 0.005). NVLD also showed significantly higher ROS levels compared to hepatitis B (p < 0.04) and hepatitis C (p < 0.04).

The mechanism, potentiality and limitations of our method are discussed.     

Partial characterization of human choriocarcinoma cell line JAR cells in regard to oxidative stress

Characterization of free radical-induced cell injury processes of placenta cells is of vital importance for clinical medicine for the maintenance of intrauterine fetal life. The present study has analyzed cell injury processes in cells of the choriocarcinoma cell line JAR treated with menadione, an anticancer drug, and H(2)O(2) in comparison to osteosarcoma 143B cells using electron microscopic and flow cytometric techniques. Flow cytometry on JAR cells exposed to 100 muM menadione and double-stained with Annexin V and propidium iodide (PI) detected apoptotic cells reaching the maximum after 4 h of incubation with a rapid decrease thereafter. Viable cells became decreased to 46% of the control after 2 h of incubation, reaching 5% after 4 h. Cells stainable with both Annexin V and PI began to increase distinctly after 2 h of incubation, reaching 55% after 4 h. Electron microscopy showed that cells stainable with both dyes specified above had condensed nuclei and swollen cytoplasm, suggesting that they were undergoing a switch of the cell death mode from apoptosis to necrosis. On the other hand, 90% of 143B cells remained intact after 4 h of menadione treatment although the intracellular levels of superoxide were always higher than those of JAR cells treated with the drug. In contrast, JAR cells were more resistant than 143B cells to H(2)O(2)-induced cytotoxicity. These results may suggest that cytotoxicity of menadione cannot be explained simply by oxygen free radicals generated from the drug. The resistance of JAR cells to oxygen free radical-induced cytotoxicity may be advantageous for intrauterine fetal life.     

A study of dose response and organ susceptibility of copper toxicity in a rat model

Copper (Cu) in higher concentration is toxic and results in various organ dysfunction. We report Cu concentration in liver, brain and kidney in the rat model following chronic exposure of oral copper sulphate at different subtoxic doses and correlate the tissue Cu concentrations with respective organ dysfunction. Fifty-four male wistar rats divided in 3 groups, the control group received saline water and the experimental group (Group-IIA and IIB) received oral copper sulphate in dose of 100 and 200 mg/kg Body Weight. At the end of 30 days, 60 days and 90 days of exposure, six rats were sacrificed from each group. The maximum peak force in grip strength, latency to fall in rotarod and percentage attention score in Y-maze were significantly reduced in the copper sulphate exposed rats compared to the controls at all time points and these were more marked in Group-IIB compared to Group-IIA. Cu concentration was significantly higher in liver, kidney and brain in the Group-II compared to the Group-I. The Cu concentration was highest in the liver (29 folds) followed by kidney (3 folds) and brain (1.5 folds). Serum ALT, AST and bilirubin correlated with liver Cu, BUN with kidney Cu, and grip strength, rotarod and Y-maze findings correlated with brain Cu level. In rats, chronic oral copper sulphate exposure at subtoxic level results in neurobehavioral abnormality and liver and kidney dysfunctions due to increased Cu concentration in the respective organs. Liver is the most vulnerable organ and copper toxicity increases with increasing dose and duration of exposure.     

Preventive effects of a water-soluble derivative of chroman moiety of vitamin E on lipid hydroperoxide-induced cell injuries and DNA cleavages through repressions of oxidative stress in the cytoplasm of human keratinocytes

ChrCrx (6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid) is a water-soluble analog in which 4', 8', 12'-trimethyltridecyl chain is deleted from an alpha-tocopherol molecule known as a hydrophobic antioxidant. Cell viability of human skin epidermal keratinocytes HaCaT was lowered by treatment with tert-butylhydroperoxide (t-BuOOH) of 50 microM for 48 h, designated as a subacute cytotoxicity, which was prevented by previous administration with ChrCrx in a dose-dependent manner as estimated by mitochondrial function-based WST-1 assay and cell morphological microscopy. In contrast an acute cytotoxicity due to treatment with t-BuOOH as dense as 200 microM for a period as short as 2 h could be also prevented with ChrCrx that was administered before and after, but was eliminated during, treatment with t-BuOOH. In contrast alpha-tocopherol was not cytoprotective against t-BuOOH. DNA strand cleavages were induced with t-BuOOH in the keratinocytes, and could be prevented by ChrCrx more effectively than alpha-tocopherol as assayed by TUNEL stain. The intracellular reactive oxygen species (ROS) was accumulated in a manner dependent on periods of t-BuOOH treatment in the cytoplasm more abundantly rather than the nucleus of keratinocytes, and was markedly diminished by ChrCrx as shown by fluorography using the redox indicator dye. Thus t-BuOOH-induced cell injuries and DNA cleavages of the keratinocytes can be prevented at least in part through efficient diminishment of ROS generated in the cytoplasm, to which the preferred distribution of ChrCrx may be advantageous over to the nucleus or membrane owing to its molecular hydrophilicity relative to alpha-tocopherol.