THE ROLE OF REACTIVE OXYGEN SPECIES IN COPPER TOXICITY TO TWO FRESHWATER GREEN ALGAE1

The role of reactive oxygen species (ROS) in copper (Cu) toxicity to two freshwater green algal species, Pseudokirchneriella subcapitata (Korshikov) Hindák and Chlorella vulgaris Beij., was assessed to gain a better mechanistic understanding of this toxicity. Cu‐induced formation of ROS was investigated in the two algal species and linked to short‐term effects on photosynthetic activity and to long‐term effects on cell growth. A light‐ and time‐dependent increase in ROS concentrations was observed upon exposure to environmentally relevant Cu concentrations of 50 and 250 nM and was comparable in both algal species. However, effects of 250 nM Cu on photosynthesis were different, leading to a 12% reduction in photosynthetic activity in P. subcapitata, but not in C. vulgaris. These results indicate that differences in species‐specific sensitivities measured as photosynthetic activity were not caused by differences in the cellular ROS content of the algae, but probably by different species‐specific ROS defense systems. To investigate the role of ROS in Cu‐mediated inhibition of photosynthesis, the ROS scavenger N‐tert‐butyl‐α‐phenylnitrone (BPN) was used, resulting in a reduction of Cu‐induced ROS production up to control level and a complete restoration of photosynthetic activity of Cu‐exposed P. subcapitata. This finding implied that ROS play a primary role in Cu toxicity to algae. Furthermore, we observed a time‐dependent ROS release process across the plasma membrane. More than 90% of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient method of cellular protection against oxidative stress.     

Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.     

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Cadmium-dependent generation of reactive oxygen species and mitochondrial DNA breaks in photosynthetic and non-photosynthetic strains of Euglena gracilis

The photosynthetic strain Z of Euglena gracilis is more susceptible to cadmium chloride (Cd) than the non-photosynthetic strain SMZ. We investigated the correlation of intracellular reactive oxygen species (ROS) levels with Cd-induced cellular damage. Flow cytometry with dihydrorhodamine 123 showed that strain Z generated higher levels of ROS, probably H(2)O(2) and/or ONOO(-), than strain SMZ, and that this difference between the two strains became more pronounced with increasing Cd dose. The levels of ROS increased at cytotoxic concentrations of Cd, at over 10 microM Cd for Z and 50 microM Cd for SMZ. These results show an association of Cd cytotoxicity with ROS generation. Considering that strain SMZ is non-photosynthetic, the higher levels of ROS in strain Z might be due to blockage of photosynthetic electron flow by Cd. Using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling analysis in combination with 4',6-diamidino-2-phenylindole, dihydrochloride staining, we observed DNA breaks in the mitochondria of both strains after Cd exposure. The results suggest that the mitochondrion is the primary target organelle of Cd in E. gracilis cells.     

Dopaminergic modulation of oxidative stress and apoptosis in human peripheral blood lymphocytes: evidence for a D1-like receptor-dependent protective effect

Dopamine (DA) is a neurotransmitter in the central and peripheral nervous system, which can be either cytotoxic or cytoprotective under selected conditions. Such effects involve oxidative mechanisms and are likely to play a role in neurodegenerative disorders. Because increasing evidence points to peripheral blood lymphocytes (PBL) as a feasible model for studying DA-related mechanisms of cell death and survival, we have explored in these cells the effects of DA on oxidative metabolism and apoptosis. Our results show that, whereas DA 100-500 microM resulted in increased intracellular reactive oxygen species (ROS) levels and apoptotic cell death through oxidative stress, DA 0.1-5 microM decreased ROS levels and apoptosis. DA (both 1 and 500 microM) partially counteracted the decrease in Cu/Zn superoxide dismutase levels observed in untreated PBL. However, whereas the effect of the low dose lasted for the whole incubation period (24 h), the effect of DA 500 microM was transient. DA-dependent reduction of ROS levels and apoptosis was prevented by D1-like (but not D2-like) receptor antagonism. The present findings add knowledge about the sensitivity of PBL to DA and strengthen the rationale for exploiting these cells as an easily accessible peripheral model for the ex vivo investigation of oxidative stress-related dopaminergic mechanisms underlying human neurodegenerative diseases.     

Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2

The aim of this study was to evaluate melatonin cytotoxicity by measuring its effects on various cellular targets. Cell viability, intracellular reduced glutathione (GSH) level, and reactive oxygen species (ROS) production were assessed in the human liver cell line (HepG2), after incubation with increasing melatonin concentrations (0.1-10,000 microM). The incubation times tested were 24, 72, and 96 h for cell viability and intracellular GSH level, and 15 and 45 minutes for ROS production. Cellular target evaluations were possible in living cells by means of a new microplate cytofluorimeter. This technology was suitable for the assessment of cell viability, GSH level, and ROS overproduction with, respectively, neutral red, monochlorobimane (mBCl), and 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probes. At the lowest melatonin concentrations (0.1-10 microM) and for a relatively short incubation time (24 h), the antioxidant effect of melatonin was revealed by an increased intracellular GSH level, associated to cell viability improvement. In contrast, after longer incubation (96 h), cell viability significantly decreased with these lowest melatonin concentrations (0.1-10 microM). Moreover, high melatonin concentrations (1,000-10,000 microM) induced GSH depletion. This oxidative stress is associated with ROS overproduction from 10 microM after only 15 minutes of incubation. This dual effect is strong evidence that, in vitro, melatonin can be both antioxidant and prooxidant on the human liver cell line, depending on the concentration and incubation time.     

Copper-deficient rat embryos are characterized by low superoxide dismutase activity and elevated superoxide anions

The teratogenicity of copper (Cu) deficiency may result from increased oxidative stress and oxidative damage. Dams were fed either control (8.0 microg Cu/g) or Cu-deficient (0.5 microg Cu/g) diets. Embryos were collected on Gestational Day 12 for in vivo studies or on Gestational Day 10 and cultured for 48 h in Cu-deficient or Cu-adequate media for in vitro studies. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GR) activities were measured in control and Cu-deficient embryos as markers of the oxidant defense system. Superoxide anions were measured as an index of exposure to reactive oxygen species (ROS). No differences were found in GPX or GR activities among treatment groups. However, SOD activity was lower and superoxide anion concentrations higher in Cu-deficient embryos cultured in Cu-deficient serum compared to control embryos cultured in control serum. Even so, Cu-deficient embryos had similar CuZnSOD protein levels as controls. In the in vitro system, Cu-deficient embryos had a higher frequency of malformations and increased staining for superoxide anions in the forebrain, heart, forelimb, and somites compared to controls. When assessed for lipid and DNA oxidative damage, conjugated diene concentrations were similar among the groups, but a tendency was observed for Cu-deficient embryos to have higher 8-hydroxy-2'-deoxyguanosine concentrations than controls. Thus, Cu deficiency resulted in embryos with malformations and reduced SOD enzyme activity. Increased ROS concentrations in the Cu-deficient embryo may cause oxidative damage and contribute to the occurrence of developmental defects.     

The Involvement of Copper Transporter in Lead-induced Oxidative Stress in Astroglia

Lead (Pb), depositing primarily in astroglia in the brain, is a well-known neurotoxicant and a risk factor for neurologic disorders. Pb has been reported to induce oxidative stress by probably the disturbance of copper (Cu) homeostasis in astroglia. Thus, we hypothesized that Pb-induced oxidative stress is initiated by interfering with Cu transporter in astroglia. In this study, we observed Pb-induced oxidative stress as indicated by reactive oxygen species (ROS) augmentation and GRP78 and GRP94 protein induction, and it was parallel to Cu accumulation intracellularly by Pb. To further address Cu transporter as a potential Pb target, a heavy metal-binding (HMB) domain of Cu-transporting ATPase (Atp7a) was overexpressed and purified. Evidence showed that one molecule of HMB chelated 11 Pb ions or seven Cu ions and that Pb competed with Cu for binding to HMB. These findings suggest that Pb-induced oxidative stress results from the impairment of Cu metabolism by Pb targeting of Atp7a.     

Metal catalyzed oxidation of tyrosine residues by different oxidation systems of copper/hydrogen peroxide

Metal-catalysed oxidation (MCO) reactions result in the formation of reactive oxygen species (ROS) in biological systems. These ROS cause oxidative stress that contributes to a number of pathological processes leading to a variety of diseases. Tyrosine is one residue that is very susceptible to oxidative modification and the formation of dityrosine (DT) and 3,4-dihydroxyphenylalanine (DOPA) have been widely reported in a number of diseases. However, the mechanisms of MCO of tyrosine in biological systems are poorly understood and require further investigation. In this study we investigated the mechanism of DT and DOPA formation by MCO using N-acetyl tyrosine ethyl ester as a model for tyrosine in proteins and peptides. The results showed that DT formation could be observed upon Cu2+/H2O2 oxidation at pH 7.4. Our results indicate that it is unlikely to be via Fenton chemistry since Cu+/H2O2 oxidative conditions did not lead to the formation of DT.     

Susceptibility to oxidative stress and modulated expression of antioxidant genes in the copper-exposed polychaete Perinereis nuntia

To identify and evaluate potentially useful biomarkers for oxidative stress as early warning indices in the polychaete, Perinereis nuntia, we exposed P. nuntia to copper (Cu) and measured several biomarker enzymes (glutathione S-transferase; GST, glutathione peroxidase; GPx, Metallothionein-like protein; MTLPs, and catalase; CAT) and genes (Pn-GSTs, Pn-CAT, and Pn-MT) with a cellular oxidative index, reactive oxygen species (ROS) level. Accumulated Cu concentrations in P. nuntia increased in a time-dependent manner. Intracellular ROS reached high levels 6h after exposure in P. nuntia with an increase of GST activity and glutathione (GSH) content. Particularly, GSH in polychaetes showed a positive correlation with Cu contents accumulated in P. nuntia. Messenger RNA expressions of GST sigma and GST omega showed relatively high expressions at 50 μg/L of Cu exposure, even though the moderate increase of rest of GST isoforms was also observed. Also regarding long-term exposure, we reared P. nuntia in sediments for 15 days, and found that there was an obvious increase of Pn-GSTs, Pn-CAT, and Pn-MT genes with elevated concentrations of Cu and Cd in polychaete body, compared to initial levels, suggesting that P. nuntia in sediment was affected by metals as well as by other organic pollutants to induce oxidative stress genes and enzymes. These findings suggest that oxidative stress is a potential modulator of defense system of P. nuntia. Several potential biomarker genes are available as early warning signals for environmental biomonitoring.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 microM, GE prevents oxidative stress and MPT induced by supraphysiological Ca2+ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca2+. GE also inhibits Ca2+ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca2+ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Amyloid beta-Cu2+ complexes in both monomeric and fibrillar forms do not generate H2O2 catalytically but quench hydroxyl radicals

Oxidative stress plays a key role in Alzheimer's disease (AD). In addition, the abnormally high Cu(2+) ion concentrations present in senile plaques has provoked a substantial interest in the relationship between the amyloid beta peptide (Abeta) found within plaques and redox-active copper ions. There have been a number of studies monitoring reactive oxygen species (ROS) generation by copper and ascorbate that suggest that Abeta acts as a prooxidant producing H2O2. However, others have indicated Abeta acts as an antioxidant, but to date most cell-free studies directly monitoring ROS have not supported this hypothesis. We therefore chose to look again at ROS generation by both monomeric and fibrillar forms of Abeta under aerobic conditions in the presence of Cu(2+) with/without the biological reductant ascorbate in a cell-free system. We used a variety of fluorescence and absorption based assays to monitor the production of ROS, as well as Cu(2+) reduction. In contrast to previous studies, we show here that Abeta does not generate any more ROS than controls of Cu(2+) and ascorbate. Abeta does not silence the redox activity of Cu(2+/+) via chelation, but rather hydroxyl radicals produced as a result of Fenton-Haber Weiss reactions of ascorbate and Cu(2+) rapidly react with Abeta; thus the potentially harmful radicals are quenched. In support of this, chemical modification of the Abeta peptide was examined using (1)H NMR, and specific oxidation sites within the peptide were identified at the histidine and methionine residues. Our studies add significant weight to a modified amyloid cascade hypothesis in which sporadic AD is the result of Abeta being upregulated as a response to oxidative stress. However, our results do not preclude the possibility that Abeta in an oligomeric form may concentrate the redox-active copper at neuronal membranes and so cause lipid peroxidation.     

15d-Deoxy-Delta12,14-prostaglandin J2 modulates collagen type I synthesis in human hepatic stellate cells by inducing oxidative stress

15 deoxy-Delta(12,14)-prostaglandin(2) (15d-PGJ(2)) is known to inhibit the proliferation of hepatic stellate cells (HSCs), major cellular components that cause hepatic fibrosis, in vitro. It also induces oxidative stress, which results in hepatic myofibroblast death. On the other hand, oxidative stress generally induces HSC proliferation and collagen synthesis in vitro, and liver fibrogenesis in vivo. In this study, we evaluated the effects of 15d-PGJ(2) at various concentrations on the viability and collagen synthesis of HSCs. 15d-PGJ(2) increased intracellular reactive oxygen species (ROS), and reduced the viability of human HSCs at concentrations 5 microM by inducing apoptotic cell death. In addition, the antioxidants alpha-tocopherol and N-acetylcysteine (NAC) blocked 15d-PGJ(2)-induced HSC death. Collagen I synthesis was increased 1.5-fold by 0.5 microM 15d-PGJ(2) treatment, but was reduced to 30% of the control level by 10 microM 15d-PGJ(2), and NAC pretreatment prevented these changes in collagen production by 15d-PGJ(2). We conclude that 15d-PGJ(2) may either induce or prevent hepatic fibrogenesis depending on its concentration.     

NaCl胁迫对西瓜嫁接苗叶片抗氧化酶活性及光合特性的影响

以耐盐性较强的葫芦品种‘超丰抗生王’为砧木,耐盐性较弱的西瓜品种‘秀丽’为接穗,采用营养液水培法,研究了NaCl胁迫对西瓜自根苗和嫁接苗保护酶活性、膜质过氧化及光合特性的影响。结果表明,NaCl胁迫下,嫁接苗和自根苗生物量显著下降,但嫁接苗下降幅度小于自根苗;NaCl胁迫抑制了西瓜自根苗和嫁接苗的气体交换参数,但是嫁接苗的净光合速率(Pn)、气孔导度(Gs)、胞间CO2浓度(Ci)及叶绿素含量显著高于自根苗;NaCl胁迫下西瓜嫁接苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著高于自根苗,丙二醛含量较自根苗低。上述结果表明,NaCl胁迫下嫁接苗通过维持较高的抗氧化酶活性来提高清除活性氧(ROS)的能力,从而降低氧化损伤,并保持较高光合速率,从而增强西瓜幼苗对盐胁迫的耐性。     

VIRAL INFECTION OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) LEADS TO ELEVATED PRODUCTION OF REACTIVE OXYGEN SPECIES1

The effect of viral infection of Emiliania huxleyi (Lohman) Hay and Mohler on the concentration of intracellular reactive oxygen species (ROS), hydrogen peroxide (H₂O₂) excretion and cell photosynthetic capacity (CPC) was examined. During the crash of an E. huxleyi culture induced by viruses intracellular ROS concentrations were generally elevated and reached levels of approximately double those observed in non‐infected control cultures. H₂O₂ concentrations also increased in the media of the infected cultures from background levels of around 130 nM to approximately 580 nM while levels in the controls decreased. These data suggest that oxidative stress is elevated in infected cells. Although the precise mechanism for ROS production was not identified, a traditional defense related oxidative burst was ruled out, as no evidence of a rapid intracellular accumulation of ROS following addition of the virus was found. CPC declined substantially in the infected culture from a healthy 0.6–0 arbitrary units. Clearly infection disrupted normal photosynthetic processes, which could lead to the production of ROS via interruption of the electron transport chain at the PSII level. Alternatively, ROS may also be a necessary requirement for viral replication in E. huxleyi, possibly due to a link with viral‐induced cell death or associated with general death processes.