Cu-induced Oxidative Stress Research Articles

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.

Changes in antioxidant and lignifying enzyme activities in sunflower roots ( Helianthus annuus L.) stressed with copper excess

Treatment with 50 μM CuSO 4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed. To cite this article: H. Jouili, E. El Ferjani, C. R. Biologies 326 (2003).

Nitric oxide-dependent pro-oxidant and pro-apoptotic effect of metallothioneins in HL-60 cells challenged with cupric nitrilotriacetate.

Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl(2)-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl(2)-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.

Nitric oxide-dependent pro-oxidant and pro-apoptotic effect of metallothioneins in HL-60 cells challenged with cupric nitrilotriacetate

Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl2-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl2-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.

Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte: Response of antioxidant enzymes and antioxidants

Copper-induced oxidative stress, response of antioxidative enzymes and substances were investigated in Ceratophyllum demersum L. (Coontail), a free floating macrophyte to understand the mechanisms of Cu resistance. Bioconcentration of Cu in plants treated with 2 and 4 μM Cu for 24 h was dependent on external Cu concentration. Cu uptake decreased the chlorophyll content and increased both lipid peroxidation and leakage of ions which were attributed to Cu-induced oxidative stress. Plants responded to Cu-induced oxidative stress by modulating antioxidative enzymes and antioxidants. Cu (2 and 4 μM) considerably increased the activities of ascorbate peroxidase, catalase and superoxide dismutase. The extent of increase with 4 μM Cu was lower compared to 2 μM Cu for catalase and superoxide dismutase, while for ascorbate peroxidase the activity remained virtually identical. Cu also altered the antioxidants level such as GSH and other non-protein thiols. At 2 μM Cu, both GSH and other non-protein thiols were increased and the extent of increase varied among them. However, 4 μM Cu decreased their levels, possibly due to the greater oxidation in the presence of Cu compared to the rate of synthesis. This decrease could also partly due to limited activity of antioxidant enzymes fostering oxidation. The results suggest that Cu accumulation in C. demersum induces oxidative stress and the level of tolerance depends on the antioxidative capacity of the plants.

Thiol pools and glutathione redox ratios as possible indicators of copper toxicity in the green macroalgae Enteromorpha spp. from the Scheldt Estuary (SW Netherlands, Belgium) and Thermaikos Gulf (Greece, N Aegean Sea)

Defence mechanisms against Cu toxicity were examined in two dominant Enteromorpha species from two coastal water types. The macroalgae were collected at three locations in the eulittoral of the Scheldt Estuary (Netherlands, Belgium) and the Thermaikos Gulf (Greece). For 10 days E. prolifera (Scheldt) and E. linza (Thermaikos) were incubated in seawater media of different salinities: 6, 9, 23 psu and 25, 30, 35 psu, respectively. In one series, media were enriched with 100 μg Cu l-1; responses were compared with those in controls with no extra Cu added. Enteromorpha, which is frequently used as a monitor species for heavy metal contamination, had relatively high Cu tissue levels (0.5–3.8 μmol Cu gdwt-1). Cu levels in E. prolifera controls (Scheldt) decreased with salinity; this was not the case with Cu levels in E. linza controls (Thermaikos). During the 10-d incubation algal protein contents and tissue Cu were rather stable. In E. linza (Thermaikos) algal protein contents were significantly lower than those of E. prolifera (Scheldt), although there was no indication for nitrogen limitation in E. linza. E. linza also had much lower glutathione pools than E. prolifera. Only under acute Cu stress (metal addition) did E. prolifera synthesise metal-binding thiols (phytochelatins). Phytochelatin pools are not suitable as an indicator of the Cu levels in these algae. The glutathione redox ratio GSH:(GSH + 0.5GSSG) was used as an indicator of (Cu-induced) oxidative stress. In E. prolifera (Scheldt) this ratio decreased with algal Cu content (P <0.05), from ~0.5 to ~0.2. The average glutathione ratios in Enteromorpha from the Scheldt and Thermaikos showed some oxidative stress induction with increasing algal Cu contents, however more clearly if Cu was added. As this redox ratio can also be influenced by environmental factors such as irradiance and desiccation, it may not be useful as an indicator for Cu-induced oxidative stress in situ.

HPLC analysis of nonprotein thiols in planktonic diatoms: pool size, redox state and response to copper and cadmium exposure

A sensitive method was developed to analyze low molecular weight thiols involved in metal homeostasis and detoxification in phytoplankton. The aims of this study were to (1) separate and measure all relevant thiols in a single HPLC run, (2) measure redox states of the thiols and (3) identify specific responses of thiols (pools, redox) to heavy metals by testing diatoms with different metal tolerances (Ditylum brightwellii, Phaeodactylum tricornutum, Skeletonema costatum andThalassiosira pseudonana). Copper or cadmium were dosed at a maximum tolerable, species-dependent level, to exponential phase cells growing in artificial medium (14‰ salinity). Loss of cell viability was monitored by the decrease of fluorescein fluorescence after a 24-h metal exposure. Thiols in extracts of exposed cells and controls were labeled with monobromobimane. Picomoles of cysteine, glutathione, γ-glutamylcysteine and phytochelatins (PCs) were detected and separated by reversed-phase high performance liquid chromatography. Cysteine increased in all species after metal exposure. The species with the largest glutathione pools in the control (P. tricornutum) synthesized the largest PC pools upon metal exposure, however, at a ∼ 40% loss of glutathione. A considerable increase of glutathione was observed inD. brightwellii upon metal exposure. However, it produced little PC (and only with Cu). In controls (∼ 3 pM Cu2+), PC2 was detectable inS. costatum, P. tricornutum andT. pseudonana. Oxidized thiol fractions were recovered by the reductants DTT and TCEP, both performing identically. Compared to the other thiols, cysteine had low redox ratios. InD. brightwellii glutathione and PC redox ratios were lower than inP. tricornutum, S. costatum andT. pseudonana. It was expected that Cu-induced oxidative stress would decrease the thiol redox ratios, however, this was not observed.

Oxidative stress induced by copper: defense and damage in the marine planktonic diatom Ditylum brightwellii, grown in continuous cultures with high and low zinc levels

Light-limited cultures of the planktonic diatom Ditylum brightwellii (West) Grunow were grown at 14‰ salinity. Cells were subjected to oxidative stress induced by copper, in the presence of zinc. In two continuous cultures with total Zn levels of 40 and 140 nM, respectively, dissolved Cu levels were increased from 3 to 126 nM. This resulted in an increased Cu adsorption capacity of the cell walls, probably due to an increase of surface area and roughness. Sexual reproduction (auxospore formation) was accelerated but was considered as a non-specific stress response. Cu-induced oxidative stress was indicated by a decrease of reduced glutathione (GSH), and a removal of superoxide anions monitored as an increasing activity of superoxide dismutase (SOD). Although Zn has no oxidative potential per se, cell division rates and chlorophyll c contents were lower in the culture with high Zn levels. In both cultures, the pro-oxidant Cu caused a decrease of chlorophyll a, decreasing photosynthetic O2 evolution and cell devision rates, and a growing number of deformed and broken cells.