Oxidative Stress Induced by Cadmium in the C6 Cell Line: Role of Copper and Zinc

In this report, we have investigated the role of copper (Cu) and zinc (Zn) in oxidative stress induced by cadmium (Cd) in C6 cells. Cells were exposed to 20 μM Cd, 500 μM Cu, and 450 μM Zn for 24 h. Then, toxic effects, cellular metals levels, oxidative stress parameters, cell death, as well as DNA damage were evaluated. Cd induced an increase in cellular Cd, Cu, and Zn levels. This results not only in the inhibition of GSH-Px, GRase, CAT, and SOD activities but also in ROS overproduction, oxidative damage, and apoptotic cell death not related to Cu and Zn mechanisms. The thiol groups and GSH levels decreased, whereas the lipid peroxidation and DNA damage increased. The toxicity of Zn results from the imbalance between the inhibition of antioxidant activities and the induction of MT synthesis. The increase in Cu and Zn levels could be explained by the disruption of specific transporter activities, Cd interference with signaling pathways, and metal displacement. Our results suggest that the alteration of Cu and Zn homeostasis is involved in the oxidative stress induced by Cd.     

The role of the kinase OXI1 in cadmium‐ and copper‐induced molecular responses in Arabidopsis thaliana

The hypothesis that mitogen‐activated protein kinase (MAPK) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of oxidative signal‐inducible kinase (OXI1) in metal‐induced oxidative signalling, the responses of oxi1 knockout lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared with those of wild‐type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase‐1 may be a downstream component of OXI1 signalling. Metal‐specific differences in OXI1 action were observed. For example, OXI1 was required for the up‐regulation of antioxidative defences such as catalase in leaves and Fe‐superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1‐MKK2‐WRKY25 cascade. Moreover, the induction of Cu/Zn superoxide dismutases in Cu‐exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1‐independent pathways but rather involves changes in components mediating intracellular communication.     

Relation between lipoic acid and cell redox status in wheat grown in excess copper

Cell redox status and lipoic acid contents were analysed in wheat (Triticum durum Desf. cv. Creso) plants treated with 150 μM Cu to elucidate the role of the antioxidant lipoic acid against oxidative stress. In comparison with shoots, roots suffered a higher oxidative stress showing a decrease in NADPH contents and an oxidation of glutathione and ascorbate. Shoots did not evidence a clear oxidative damage since Cu was translocated in small amounts. Lipoic acid as reduced (DHLA) or oxidised (LA) form was present in both leaves and roots of wheat. Analysis of the cell sap showed that this antioxidant was present also as free form. The analyses showed that stroma contained significant amounts of free LA and that, after acidic hydrolysis, higher amounts of LA and DHLA were released. However, lipoic acid was undetectable in both thylakoids and microsomal membranes. Cu treatment did not determine changes in the contents of total LA and DHLA in roots, they being likely involved in Cu chelation. In contrast, in leaves after 48 h of treatment the metal induced an increase in DHLA, which could in part explain the reduction in the oxidised glutathione levels. In leaves free lipoic acid was more prone to be oxidised compared to the bound form, and the reduced form disappeared in both leaves and roots after Cu treatment.     

Part of the series: from dietary antioxidants to regulators in cellular signalling and gene expression. Role of reactive oxygen species and (phyto)oestrogens in the modulation of adaptive response to stress

There is increasing evidence that reactive oxygen species (ROS) are not only toxic but play an important role in cellular signalling and in the regulation of gene expression. We, here, discuss two examples of improved adaptive response to an altered cellular redox state. First, differences in longevity between males and females may be explained by a higher expression of antioxidant enzymes in females resulting in a lower yield of mitochondrial ROS. Oestrogens are made responsible for these phenomena. Oestradiol induces glutathione peroxidase-1 and MnSOD by processes requiring the cell surface oestrogen receptor (ER) and the activation of pathways usually involved in oxidative stress response. Second, oxygen radicals produced during moderate exercise as performed during training up-regulate the expression of antioxidant enzymes in muscle cells. An increased level of these enzymes might prevent oxidative damage during exhaustive exercise and should, therefore, not be prevented by antioxidants. The relevance of these findings is discussed in the context with observations made in transgenic animals overexpressing MnSOD or catalase.     

The Effects of Cadmium-Zinc Interactions on Biochemical Responses in Tobacco Seedlings and Adult Plants

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.     

Selenium alleviates cadmium toxicity by preventing oxidative stress in sunflower (Helianthus annuus) seedlings

The present study investigated the possible mediatory role of selenium (Se) in protecting plants from cadmium (Cd) toxicity. The exposure of sunflower seedlings to 20 μM Cd inhibited biomass production, decreased chlorophyll and carotenoid concentrations and strongly increased accumulation of Cd in both roots and shoots. Similarly, Cd enhanced hydrogen peroxides content and lipid peroxidation as indicated by malondialdehyde accumulation. Pre-soaking seeds with Se (5, 10 and 20 μM) alleviated the negative effect of Cd on growth and led to a decrease in oxidative injuries caused by Cd. Furthermore, Se enhanced the activities of catalase, ascorbate peroxidase and glutathione reductase, but lowered that of superoxide dismutase and guaiacol peroxidase. As important antioxidants, ascorbate and glutathione contents in sunflower leaves exposed to Cd were significantly decreased by Se treatment. The data suggest that the beneficial effect of Se during an earlier growth period could be related to avoidance of cumulative damage upon exposure to Cd, thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity.     

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. This revised version was published online in September 2006 with corrections to the Cover Date.     

干旱与氧化胁迫对小麦根氧化还原状态和叶片ABA积累的影响

通过分析一氧化氮(nitric oxide,NO)、活性氧(reactive oxygen species,ROS)和干旱胁迫对小麦根氧化还原状态和叶片脱落酸(abscisic acid,ABA)积累的影响,探讨了干旱胁迫下NO和H2O2调节ABA合成的可能机制。结果表明:干旱胁迫处理初期小麦根还原型谷胱甘肽含量降低、抗氧化酶活性发生振荡变化,细胞氧化还原状态向氧化型转变。NO和H2O2能模拟干旱胁迫的作用使细胞状态向氧化型转变,还可以使小麦叶片ABA积累量上升。干旱胁迫下NO和H2O2对ABA合成的调节作用可能是通过调节细胞氧化还原状态进行。     

Zinc-metallothionein genoprotective effect is independent of the glutathione depletion in HaCaT keratinocytes after solar light irradiation

UV radiations are the major environmental factors that induce DNA damage of skin cells either by direct absorption (UVB), or after inducing an oxidative stress (UVA and UVB). Cells maintain a reducing intracellular environment to avoid genomic damage. MTs have been expected not only to control metal homeostasis but also counteract the glutathione (GSH) depletion induced by oxidative stress because of their high thiol content. Induction and redistribution of MTs in cultured human keratinocytes (HaCaT) in response to SSL, is an important cellular defense mechanism against DNA damage. Reduced glutathione (GSH) is another way of cellular protection against UV-induced oxidative stress. This study which extend our previous finding focused on the relation between intracellular GSH and Zn genoprotective effects after solar irradiation. HaCaT cells, depleted or not in GSH by a chemical treatment were used to compare MTs induction by Northern blot, expression by Western blot and localization using immunocytochemistry. Zn genoprotection experiments after SSL irradiation was carried out by the comet assay. We demonstrated that in absence of GSH, Zn-MTs could protect DNA after SSL irradiation and that GSH depletion has no effect on MTs induction and localization. Nuclear Zn-MTs could be responsible for this observed genoprotection in GSH depleted cells. So the GSH/Zn and the MT/Zn systems could be two independent but interacting mechanisms of cellular protection against SSL injury.     

铜、镉胁迫下外源NO介导的番茄解毒途径

一氧化氮(NO)作为信号分子,在抵御重金属胁迫中起重要作用,但对不同离子胁迫下的解毒机制尚缺乏研究.本研究采用营养液培养法,研究了铜(Cu)、镉(Cd)单一或复合胁迫下,番茄幼苗对Cu、Cd的吸收转运特性及对外源NO的响应机制.结果表明: 50 μmol·L^-1的Cu²⁺、Cd²⁺均显著抑制番茄植株的生长,其中Cd胁迫对生长的抑制效应远高于Cu胁迫.Cu、Cd单一或复合胁迫均使番茄根系Cu、Cd含量显著升高,但根系对Cu、Cd吸收存在严格选择性.根细胞对必需元素Cu表现出“奢侈吸收”的现象,而对毒性较强的Cd则吸收相对较少,胞内Cd浓度仅为Cu的1/10左右.外源NO处理可不同程度地缓解Cu、Cd胁迫,其中缓解Cd胁迫的效能更强.番茄对被动进入细胞的Cu、Cd具有相似的解毒机制:一方面,Cu、Cd胁迫诱导细胞质中产生谷胱甘肽(GSH)、植物螯合肽(PCs)和金属硫蛋白(MTs),络合过多的Cu、Cd离子,降低其生物毒性;另一方面,过多的Cu、Cd离子或螯合物被转运至液泡区隔化.外源NO通过调控GSH-GSSG(氧化型谷胱甘肽)氧化还原状态及GSH-PCs代谢方向的改变,促进Cu、Cd离子转运至液泡区隔化来缓解胁迫抑制;NO还可诱导植株叶片或根系表达更多的金属硫蛋白、GSH和PCs,而且上述响应普遍存在叠加效应.这可能是NO介导番茄对Cu、Cd胁迫的另一主要解毒途径.     

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks

Background Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H₂O₂ generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules.Scope This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid β-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H₂O₂ can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes.Conclusions Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.     

Unravelling cadmium toxicity and tolerance in plants: Insight into regulatory mechanisms

The occurrence of heavy metals in soils may be beneficial or toxic to the environment. The biota may require some of these elements considered essentials (like Fe, Zn, Cu or Mo) in trace quantities, but at higher concentrations they may be poisonous. Due to the difficulty in controlling environmental metal accumulation, organisms have to cope with exposure to unwanted chemical elements, specially those considered biologically nonessential. Cadmium (Cd) belongs to this latter group. The effect of Cd toxicity on plants has been largely explored regarding inhibition of growth processes and decrease of photosynthetic apparatus activity. This article reviews current knowledge of uptake, transport and accumulation of Cd in plants and gives an overview of Cd-detoxification mechanisms, Cd-induced oxidative damage and antioxidant defenses in plants. It also presents a picture of the role of reactive oxygen and nitrogen species in Cd toxicity; signalling and gene regulation are topics critically discussed. This review aspires to pinpoint new avenues of research that may contribute to a more differentiated view of the complex mechanisms underlying Cd toxicity in target tissues.     

Evidence of Drought Stress Memory in the Facultative CAM,Aptenia cordifolia: Possible Role of Phytohormones

Although plant responses to drought stress have been studied in detail in several plant species, including CAM plants, the occurrence of stress memory and possible mechanisms for its regulation are still very poorly understood. In an attempt to better understand the occurrence and possible mechanisms of regulation of stress memory in plants, we measured the concentrations of phytohormones in Aptenia cordifolia exposed to reiterated drought, together with various stress indicators, including leaf water contents, photosynthesis and mechanisms of photo- and antioxidant protection. Results showed that plants exposed to drought stress responded differently if previously challenged with a first drought. Gibberellin levels decreased upon exposure to the first drought and remained lower in double-stressed plants compared with those exposed to stress for the first time. In contrast, abscisic acid levels were higher in double- than single-stressed plants. This occurred in parallel with alterations in hydroperoxide levels, but not with malondialdehyde levels, thus suggesting an increased oxidation state that did not result in oxidative damage in double-stressed plants. It is concluded that (i) drought stress memory occurs in double-stressed A. cordifolia plants, (ii) both gibberellins and abscisic acid may play a role in plant response to repeated periods of drought, and (iii) changes in abscisic acid levels in double-stressed plants may have a positive effect by modulating changes in the cellular redox state with a role in signalling, rather than cause oxidative damage to the cell.