Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.     

Stress Responses of Zea mays to Cadmium and Mercury

A hydroponic experiment was carried out to characterize the oxidative stress responses of maize seedlings (Zea mays L. cv. Dekalb DK604) to cadmium (Cd) and mercury (Hg). Plants were grown hydroponically for 7 days in a nutrient solution supplemented with several concentrations of Cd and Hg: 0.0 (control), 6 or 30 μM. Growth was inhibited by both metals. The effect was more severe in plants exposed to Hg. Oxidative stress was caused by the exposure to the metals, as quantified by malondialdehyde and carbonyl accumulation, by-products of lipid peroxidation and protein oxidation, respectively. The activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD), enzymes involved in the scavenging of reactive oxygen species, were measured upon metal treatment. We found an activation of a cytosolic APX isoform, as identified by using a specific polyclonal antiserum. However, there were negligible changes in SOD activity. Analysis of thiol-peptides revealed that at 6 μM Cd a remarkable increase in root reduced glutathione (GSH) content occurred, and little effect on the relative content of oxidised glutathione (GSSG) was observed. However, at 30 μM Cd and in plants exposed to 6 and 30 μM of Hg, GSH root content either remained stable or decreased significantly, while the proportion of GSSG increased. Moreover, only Cd was able to induce accumulation of phytochelatins at both assayed concentrations. Apparently, Hg was more toxic than Cd, as inferred from the magnitude of the changes found in the physiological parameters tested.     

Metal uptake,antioxidant status and membrane potential in maize roots exposed to cadmium and nickel

Root growth of the seedlings of maize cultivars Premia and Blitz exposed to 2 μM cadmium (Cd), nickel (Ni) or both metals acting simultaneously (Cd + Ni) for 72 h was significantly reduced but not ceased. The effect was more pronounced in the seedlings of the cv. Blitz. The heavy metals (HMs) contents increased significantly in the roots. Simultaneous application of metals had an antagonistic effect on either Cd or Ni uptake in Premia but not in Blitz. In control roots the contents of ascorbic acid (AsA) and dehydroascorbic acid (DHA) were lower and gluthatione (GSH) content was higher in Premia than in Blitz. A decrease of AsA content was induced by all metal treatments in Premia but only by Cd + Ni in Blitz while an increase was induced by single metals in this cultivar. All metal treatments increased DHA contents in both cultivars. GSH content decreased significantly in Premia treated with Cd or Cd + Ni, and in Blitz treated with Ni. Unlike the contents of AsA, DHA and GSH, the increased metal concentrations in root cells did not affect the membrane potential (E _M). The changes in antioxidant contents depended on both, maize genotypes and HMs treatments. Nevertheless, the results indicated a role of antioxidative system in minimizing the effects of oxidative stress and protecting cell membranes in both maize cultivars.     

Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells

In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.     

Root uptake, transport, and metabolism of externally applied glutathione in Phaseolus vulgaris seedlings

The most abundant thiol in beans (Phaseolus vulgaris L. cv. Saxa) is the tripeptide homoglutathione (hGSH) rather than glutathione (GSH). At the whole-plant level the GSH content is less than 0.5% of the hGSH content. In the present study GSH was supplied to the roots of bean seedlings to test whether GSH can be taken up by roots and transported to the shoot. Therefore, 12-day-old plants were exposed to 1 mmol/L GSH for 4, 8 and 24 h prior to harvest. In response to this GSH exposure, elevated GSH contents were found in all tissues. After 4 h the GSH content increased in the roots from 1 +/- 1 to 22 +/- 2 nmol GSH g(-1) fresh weight (FW), in the leaves from 2 +/- 1 to 9 +/- 4 nmol GSH g(-1) FW, and in the apex from 30 +/- 5 to 75 +/- 4 nmol GSH g(-1) FW. These data indicate that GSH is taken up by bean roots and is transported to above above-ground parts of the plants. Roots exposed to GSH for 24 h contained 2-fold higher cysteine (Cys) and hGSH contents than the controls. Apparently, GSH taken up by the roots is not only loaded into the xylem but also partially degraded and used for hGSH synthesis.     

Glutathione is the antioxidant responsible for resistance to oxidative stress in V79 Chinese hamster fibroblasts rendered resistant to cadmium.

By manipulation of Cd and Zn concentrations in the medium, several phenotypes, differing in the contents of glutathione (GSH) and metallothionein (Mt), were derived from a parental clone of V79 Chinese hamster fibroblast. In some of these phenotypes, resistance to Cd and cross-resistance to oxidative stress was developed. The highest levels of GSH and Mt were found in cells which were rendered resistant to Cd by stepwise increases of Cd and Zn in the cell medium for over 50 passages. Upon removal of Cd/Zn from the medium of these cells or addition of Cd/Zn to the parental cell medium, changes of cellular GSH and Mt levels occurred to different extents. At the same time, changes in the resistance to Cd and H2O2 were observed. Good linear correlations were observed for Mt levels x resistance to Cd and for GSH levels x resistance to H2O2. Poor linear correlations were found for Mt levels x resistance to H2O2 or for GSH levels x resistance to Cd. Moreover, addition of Zn to the medium produced an increase in Mt content without affecting the GSH content. In this case no cross-resistance to oxidative stress was developed. Therefore, Mt which has been shown to be an excellent antioxidant in in vitro experiments, does not seem to play any major role against oxidative stress in Zn and Cd challenged cells. Most of the cross-resistance to oxidative stress in Cd challenged cells seems to be accounted for by the parallel increase in GSH.     

Novel mode of resistance to Fusarium infection by a mild dose pre-exposure of cadmium in wheat.

Exposure of healthy wheat seeds (Triticum aestivum var Sonalika) to mild dose of cadmium (Cd(2+)) given as 50 microM CdCl(2) for 48 h and then washed off Cd(2+) offered resistance to the subsequent infection by Fusarium oxysporum inoculum. Seven days old seedlings having two primary leaves were aseptically inoculated with fungus, F. oxysporum (1 x 10(6)) spores. The seedlings pre-exposed to low level of Cd(2+) survived the Fusarium infection, while plantlets without Cd(2+) stress wilted and then perished due to Fusarium infection. The stress associated proteins induced by Cd(2+) (50 microM), F. oxysporum and by the co-stress (50 microM Cd(2+) and then with F. oxysporum) treatments were observed to be of same molecular weight (51 kDa). Antibody was raised against the purified Cd(2+)-stress associated protein (CSAP). Immuno-gold labeling of wheat seedling root tissue showed the presence of this CSAP in Cd(2+) pre-exposed and in co-stressed tissues and to be located predominantly on the inner linings of the cell membranes. We also observed that the anti-CSAP-antibody also labeled the root tissue of only Fusarium inoculated seedlings and the gold labeling was intensely located on the membrane. This cross-reaction of anti-CSAP suggests that Fusarium-induced stress protein (FISP) possibly has close homology to CSAP. We thus show for the first time the over expression of a high molecular mass protein by mild dose of Cd(2+) pre-exposure to wheat seeds which subsequently provided protection against Fusarium infection. This mode of resistance developed by an abiotic stress-causing agent against pathogen infection is novel.     

镉、汞暴露在中华绒螯蟹卵巢中的富集及其氧化应激反应

研究以中华绒螯蟹(Eriocheir sinensis)为实验对象, 用不同浓度的Cd2+ 溶液(0、0.63、1.26、2.52、5.04和10.07 mg/L)或Hg2+ 溶液(0、0.06、0.11、0.23、0.46和0.91 mg/L)对其分别进行染毒, 6d后解剖取其卵巢, 检测卵巢中Cd和Hg的富集量以及氧化应激相关指标的变化情况。结果显示, 随着Cd2+或Hg2+染毒浓度的升高, 卵巢中Cd和Hg的富集量显著增加; H₂O₂、MDA和NO含量、MT mRNA表达量及NOS活性均随着Cd2+和Hg2+浓度的增加出现上升的趋势, 但SOD、CAT和GPx活性呈先升后降的趋势。这说明镉、汞均可引起卵巢组织产生过量的自由基和脂质过氧化, 并诱导氧化应激酶及MT mRNA的表达以抵抗不良的环境。总之, MT对中华绒螯蟹卵巢中重金属的解毒发挥着重要作用, 抗氧化酶能有效地消除活性氧自由基(ROS), 维持机体的氧化还原处于平衡状态, 共同保护膜脂免受氧化损伤。     

A reassessment of substrate specificity and activation of phytochelatin synthases from model plants by physiologically relevant metals

Phytochelatin synthases (PCS) catalyze phytochelatin (PC) synthesis from glutathione (GSH) in the presence of certain metals. The resulting PC-metal complexes are transported into the vacuole, avoiding toxic effects on metabolism. Legumes have the unique capacity to partially or completely replace GSH by homoglutathione (hGSH) and PCs by homophytochelatins (hPCs). However, the synthesis of hPCs has received little attention. A search for PCS genes in the model legume Lotus (Lotus japonicus) resulted in the isolation of a cDNA clone encoding a protein (LjPCS1) highly homologous to a previously reported homophytochelatin synthase (hPCS) of Glycine max (GmhPCS1). Recombinant LjPCS1 and Arabidopsis (Arabidopsis thaliana) PCS1 (AtPCS1) were affinity purified and their polyhistidine-tags removed. AtPCS1 catalyzed hPC synthesis from hGSH alone at even higher rates than did LjPCS1, indicating that GmhPCS1 is not a genuine hPCS and that a low ratio of hPC to PC synthesis is an inherent feature of PCS1 enzymes. For both enzymes, hGSH is a good acceptor, but a poor donor, of gamma-glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd2+, Zn2+, Cu2+, and Fe3+, but not by Co2+ or Ni2+, in the presence of 5 mm GSH and 50 microm metal ions. Activation of both enzymes by Fe3+ was proven by the complete inhibition of PC synthesis by the iron-specific chelator desferrioxamine. Plants of Arabidopsis and Lotus accumulated (h)PCs only in response to a large excess of Cu2+ and Zn2+, but to a much lower extent than did with Cd2+, indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

Antioxidant effect of vitamin E treatment on some heavy metals-induced renal and testicular injuries in male mice

Toxic heavy metals in water, air and soil are global problems that are a growing threat to humanity. Heavy metals are widely distributed in the environment and some of them occur in food, water, air and tissues even in the absence of occupational exposure. The antioxidant and protective influences of vitamin E on a mixture of some heavy metals (Pb, Hg, Cd and Cu)-induced oxidative stress and renal and testicular injuries were evaluated in male mice. Exposure of mice to these heavy metals in drinking water for seven weeks resulted in statistical increases of plasma creatinine, urea and uric acid concentrations. The levels of glutathione (GSH) and superoxide dismutases (SOD) in kidney and testis tissues were significantly declined. Moreover, the histopathological evaluation of kidney and testis showed severe changes in mice treated with these heavy metals. Administration of vitamin E protected the kidney and testis of mice exposed to heavy metals as evidenced by appearance of normal histological structures, insignificant changes in the values of plasma creatinine, urea and uric acid, and the levels of kidney GSH and SOD, while the levels of testis GSH and SOD were notably decreased. These data suggest that the administration of vitamin E protects against heavy metals-induced renal and testicular oxidative stress and injuries.     

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

Involvement of glutathione in heat shock– and hydrogen peroxide–induced cadmium tolerance of rice (Oryza sativa L.) seedlings

The role of reduced glutathione (GSH) in heat shock (HS)- and H₂O₂-induced protection of rice (Oryza sativa L., cv. Taichung 1) seedlings from Cd stress was investigated. HS- and H₂O₂-pretreatment resulted in an increase in GSH content in leaves of rice seedlings. Addition of exogenous GSH under non-HS conditions, which resulted in an increase in GSH in leaves, enhanced subsequent Cd tolerance of rice seedlings. Pretreatment with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, which effectively inhibited GSH content induced by HS and H₂O₂, reduced subsequent Cd tolerance. Furthermore, the effect of BSO on HS- and H₂O₂-induced GSH accumulation and toxicity by subsequent Cd stress can be reversed by the addition of GSH. The time-course analyses of HS in rice seedlings demonstrated that the accumulation of H₂O₂ preceded the increase in GSH. Based on the data obtained in this study, it could be concluded that the early accumulation of H₂O₂ during HS signals the increase in GSH content, which in turn protects rice seedlings from oxidative damage caused by Cd.     

Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.     

镉致黑斑蛙肝脏氧化损伤与金属硫蛋白含量的变化

为观察镉对黑斑蛙(Rana nigromaculata)肝脏脂质过氧化产物和金属硫蛋白含量的影响,将黑斑蛙暴露于10.0mg·mL-1浓度的镉溶液中30d,分别测定了黑斑蛙在暴露4、10d和30d时肝脏组织中镉(Cd)、还原型谷胱甘肽(GSH)、金属硫蛋白(MT)和过氧化产物丙二醛(MDA)的含量。实验结果表明,黑斑蛙肝脏中镉的积累量、GSH和MT含量均随着镉暴露时间的延长而显著升高,具有明显的时间-效应关系;在镉暴露的第10天,肝MDA含量明显高于对照组。提示镉可对黑斑蛙肝脏造成氧化损伤,而GSH、MT含量的升高则可能是机体抗氧化损伤的机理之一。     

Insulin neuroprotection against oxidative stress in cortical neurons--involvement of uric acid and glutathione antioxidant defenses

In this study we investigated the effect of insulin on neuronal viability and antioxidant defense mechanisms upon ascorbate/Fe2+-induced oxidative stress, using cultured cortical neurons. Insulin (0.1 and 10 microM) prevented the decrease in neuronal viability mediated by oxidative stress, decreasing both necrotic and apoptotic cell death. Moreover, insulin inhibited ascorbate/Fe2+-mediated lipid and protein oxidation, thus decreasing neuronal oxidative stress. Increased 4-hydroxynonenal (4-HNE) adducts on GLUT3 glucose transporters upon exposure to ascorbate/Fe2+ were also prevented by insulin, suggesting that this peptide can interfere with glucose metabolism. We further analyzed the influence of insulin on antioxidant defense mechanisms in the cortical neurons. Oxidative stress-induced decreases in intracellular uric acid and GSH/GSSG levels were largely prevented upon treatment with insulin. Inhibition of phosphatidylinositol-3-kinase (PI-3K) or mitogen-induced extracellular kinase (MEK) reversed the effect of insulin on uric acid and GSH/GSSG, suggesting the activation of insulin-mediated signaling pathways. Moreover, insulin stimulated glutathione reductase (GRed) and inhibited glutathione peroxidase (GPx) activities under oxidative stress conditions, further supporting that insulin neuroprotection was related to the modulation of the glutathione redox cycle. Thus, insulin may be useful in preventing oxidative stress-mediated injury that occurs in several neurodegenerative disorders.