Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Effects of chelating agents on the cadmium burden of cells in culture

The effects of some new chelating agents on the cadmium burden of CHO cells in culture were investigated. The chelators were sodium-N-(4-methoxybenzyl)-D-glucamine-dithiocarbamate (MeOBG-DTC), sodium-N-benzyl-D-glucaminedithiocarbamate (BG-DTC) and di-isopropylmeso-2,3-dimercapto succinate (DiP-DMSA). The results were compared with the effect of the well known dimercaptopropanol (BAL).The derivates of dithiocarbamate are much less toxic than DiP-DMSA and BAL. All chelators effectively prevent Cd uptake into the cells. Mobilization of intracellular Cd, however, is more effective by the DTC-derivatives than by DiP-DMSA or BAL. Within the cell the major fraction of Cd after 48 hours incubation is found in the nuclei and cytosol and very little in the peroxisomes. The chelating agents remove the metal mostly from nuclei and cytosol. Incubation of the cells with cadmium leads to the induction of a Cd binding protein of an apparent molecular weight of 12500 Da, presumably metallothionein. MeOBG-DTC is more effective in removing the metal from this protein than BG-DTC.Abbreviations MeOBG-DTC Na-N(4-methoxybenzyl)-D-glucaminedithiocarbamate - BG-DTC Na-N-benzyl-D-glucaminedithiocarbamate - DiP-DMSA di-isopropyl-2,3-dimercaptosuccinate - BAL 2,3-dimercaptopropane-1-o1 - Da dalton - MEM minimum essential medium - IU international units - FBS fetal bovine serum - CD unbroken cells and debris - N nuclei - ML mitochondria, and lysosomes - P peroxisomes - HMW high molecular weight - MT metallothionein     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.     

Studies on the role of iron in the reversal of cadmium toxicity in chicks

Studies were conducted to determine the effect of dietary iron (Fe) levels ranging from a deficiency to an excess on the toxicity of cadmium (Cd) in chicks. In Fe-deficient animals, cadmium was found to be more toxic than in Fe supplemented animals as measured by growth. The liver Cd burdens were increased significantly in the presence of dietary Fe supplementation, and there was a significant Cd−Fe interaction in the Cd concentration of the kidney, indicating that iron deficiency increased the concentration of Cd in the kidneys of those chicks receiving this element. Cd tended to reduce the Fe concentration in both the liver and kidney. The absorption of Cd as measured by the amount of¹⁰⁹Cd that disappeared from an isolated duodenal segment in one h was not affected by the Fe content of the diet, but the amount of isotope appearing in the liver compared to the amount present in the blood was increased in the Fe supplemented chicks. Separation of the Cd binding ligands by column chromatography revealed that more of the Cd in the liver, but not the kidney, was associated with ligands which eluted in a column volume that contained metallothionein in those chicks receiving Fe than in the livers from Fe deficient animals. The inverse relationship between the amount of Cd bound to the metallothionein containing fraction and toxicity may be related causally. Paper No. 10538 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names in this publication does not imply endorsement by the NC Agricultural Research Service of the products named nor criticism of similar ones not mentioned.     

Lack of evidence for activation of a serum factor in protease-induced differentiation of mouse erythroleukemia cells

summary The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induced up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B⁺). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B⁺ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B⁺ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B⁺ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B⁺ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells. This study was supported by U.S. Public Health Service Grants CA 24403 and CA 37874; American Cancer Society Grant CH-303; the Spingold Foundation; the Chemotherapy Foundation, Inc; the Gar Reichman Foundation; an Irving Alpert Cancer Research Award; and institutional general research funds. Part of this work was presented at the 1984 meeting of the American Society of Biological Chemists and American Association of Immunologists, St. Louis, MO (21).     

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 μmol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 μmol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by K _m of 1.65±0.54 μmol/L and V _max of 3.9±0.3 pmol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 μmol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 μmol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content. This revised version was published online in July 2006 with corrections to the Cover Date.     

Subcellular distribution of metallothionein and cadmium in the liver and kidneys of bank voles (Clethrionomys glareolus) exposed to dietary cadmium

Metallothionein (MT) and cadmium (Cd) contents were determined in the subcellular fractions of the liver and kidneys of bank voles exposed for 6 weeks to elevated levels of dietary Cd-40 and 80 g g^-1 dry weight. Hepatic and renal MT was detected exclusively in the cytosol, while Cd was found in the cytosol (73–79% of the total content), nuclei (14–18%) and particulates (4–9%). The concentration of MT in the cytosol as well as Cd content in the particular subcellular fractions appeared to be a dose-dependent. The absence of MT in the nuclear and particulate fractions implied that Cd present in these compartments was not bound to the protein that is considered to provide protection against the toxic metal. Therefore, it is assumed that this component of intracellular Cd could be responsible for the histopathological changes that occurred in the liver (granuloma and focal hepatocyte swelling) and kidneys (focal degeneration of proximal tubules) of bank voles exposed to the higher level of dietary Cd.     

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: γ‐glutamylcysteine synthetase (γ‐GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over‐express either γ‐GCS or GS and found that γ‐GCS over‐expression resulted in inclusion body formation and impaired cell physiology, whereas GS over‐expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over‐expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild‐type cells in size (2–5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ≈2.5 times that attained in the wild‐type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli‐based biosynthesis offers a more energy‐efficient and eco‐friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 M cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 M cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.     

超积累植物伴矿景天镉耐受基因SpMT2的分离及功能鉴定

超积累植物由于其对重金属具有地上部超积累以及超耐受等特性,不仅是研究植物离子转运及毒性耐受的理想模式,而且在植物修复的发展和应用中具有不可替代的作用。伴矿景天是近年在我国境内发现的一种景天科镉(Cd)/锌(Zn)超积累植物。为鉴定其富集和耐受Cd的关键基因,笔者构建了其酵母表达cDNA文库,利用酵母的遗传互补系统筛选到一个极大提高了酵母抗Cd能力的基因SpMT2。SpMT2属于富含半胱氨酸(Cys)的金属硫蛋白(Metallothionein)家族。亚细胞定位表明SpMT2表达于酵母细胞质中,并特异地提高酵母对Cd的抗性。进一步研究发现SpMT2的表达显著降低了酵母液泡中Cd含量,但酵母吸收的总Cd含量无显著变化。推测抗性增加是由于SpMT2在酵母细胞质中通过螯合Cd从而降低Cd对酵母的毒害。qRT-PCR分析表明SpMT2在伴矿景天的根和地上部都高丰度表达,且不受Cd诱导变化。鉴于SpMT2也定位于植物细胞质中,结合上述结果,推测SpMT2可能在伴矿景天细胞质中螯合Cd,在降低Cd毒害的同时可能还保持Cd在细胞质中的流动性,从而在Cd长途转运过程中也发挥重要作用。     

Tissue partitioning of cadmium in transgenic tobacco seedlings and field grown plants expressing the mouse metallothionein I gene

Since agricultural crops contribute >70% of human cadmium (Cd) intake, modification of crops to reduce accumulation of this pollutant metal during plant growth is desirable. Here we describe Cd accumulation characteristics of seedlings and field grown tobacco plants expressing the Cd-chelating protein, mouse metallothionein I. The objective of the transformation is to entrap Cd in roots as Cd-metallothionein and thereby reduce its accumulation in the shoot. Transformed and control seedlings were exposed for 15 days in liquid culture at a field soil-solution-like Cd concentration of 0.02 μm. Transformed seedlings ofNicotiana tabacum cultivar KY 14 contained about 24% lower Cd concentration in shoots and about 5% higher Cd concentration in roots than control seedlings. Dry weights of transformed and control tissues did not differ significantly. In the field in 1990, mature transformedN. tabacum cv. KY 14 plants exposed only to endogenous soil Cd contained about 14% lower leaf lamina Cd concentration than did controls. Differences were significant at thep≤0.1 level in 13 of 16 leaf positions. Leaf dry weight did not differ significantly but transformed field plants had 12% fewer leaves and were 9% shorter than the controls. Copper (Cu) concentration was significantly higher (ca10%) in the bottom nine leaf positions of transformed plants suggesting that reduced leaf number and plant height may be due to Cu deficiency or toxicity. Alternatively, somaclonal variation or gene position effects may be involved. No differences were found in zinc levels. WithN. tabacum cv. Petit Havana, transformed seedlings contained no less Cd in shoots but 48% higher Cd concentration in roots. However, dry weights of shoots and roots of transformed seedlings were 25% and 26%, respectively, greater than in controls. In the field, transformed and control plants of this cultivar showed little significant differences in leaf Cd content, plant height or leaf number. Although comparison of additional metallothionein-expressing tobaccos and other plants is needed, results obtained with cultivar KY 14 support the hypothesis that sequestration of Cd in roots as Cd-metallothionein may have potential for reducing Cd content of above root tissues of certain plants.     

柱状田头菇菌丝对镉胁迫的抗氧化响应

研究了不同浓度Cd处理对柱状田头菇菌丝抗氧化酶及谷胱甘肽含量的影响.结果表明,在低浓度范围内随着Cd处理浓度的增加,菌丝抗氧化酶的活力上升,过氧化氢酶(CAT)与超氧化物歧化酶(SOD)的活性分别在Cd浓度为0.1和0.4mmol·L-1时达最大值;过氧化物酶(POD)、谷胱甘肽还原酶(GR)和脂氧合酶(LOX)的活性在Cd浓度为0.2mmol·L-1时达到峰值.而在高Cd浓度处理时,柱状田头菇菌丝抗氧化酶系(POD、CAT、SOD等)显著受到抑制.0.4~1.6mmol·L-1Cd处理可显著提高菌丝体内还原型谷胱甘肽(GSH)水平,却不影响氧化型谷胱甘肽(GSSG)含量.在整个试验过程中,均未检测到抗坏血酸及抗坏血酸过氧化物酶(APX)的活性.用聚丙烯酰胺凝胶电泳分析Cd胁迫下柱状田头菇菌丝抗氧化酶的同工酶谱发现,0.1~0.8mmol·L-1Cd处理可诱导过氧化物酶(POD)、酯酶(EST)和脂氧合酶(LOX)新同工酶的表达,提高组成型过氧化氢酶(CAT)、超氧化物歧化酶(SOD)同工酶的表达强度;1.6mmol·L-1Cd处理显著抑制POD、CAT、SOD等的表达.     

Rat primary hepatocyte cultures are a good model for examining metallothionein-induced tolerance to cadmium toxicity

Summary The effect of Zn-induced metallothionein (MT) on the toxicity, uptake, and subcellular distribution of cadmium (Cd) was examined in rat primary hepatocyte cultures and compared to results obtained earlier in this laboratory from intact animals. Hepatocytes were isolated and grown in monolayer culture for 22 h and subsequently treated with ZnCl₂ (100 μM) for 24 h, which increased MT concentration about 15-fold. After Zn pretreatment, hepatocytes were exposed to Cd for 24 h. Cytotoxicity was assessed by enzyme leakage, intracellular potassium loss, and cellular glutathione content. The toxicity of Cd was much less in Zn-pretreated cells than in control cells, similar to that previously demonstrated in the intact animal. Zn pretreatment had no appreciable effect on the hepatocellular uptake of¹⁰⁹Cd, but markedly altered its subcellular distribution, with more Cd accumulating in the cytosol and less in the nuclear, mitochondrial, and microsomal fractions. In the cytosol of Zn-pretreated cells, Cd was associated mainly with MT; in contrast, cytosolic Cd in control cells was mainly associated with non-MT macromolecules. Zn-induced changes in the subcellular distribution of Cd in vitro are identical to those observed in vivo in Zn-pretreated rats challenged with Cd. In summary, Zn pretreatment of rat primary hepatocyte cultures protects cells against Cd toxicity. Protection seems to be due to MT-promotes sequestration of Cd and reduction of the amount of Cd associated with critical organelles and proteins. These observations are similar to those noted in the whole animal. These results indicate that cultured hepatocytes are an ideal model for examining MT-induced tolerance to Cd hepatotoxicity. This work was supported by grant ES-01142, and WCK was supported by training grant ES-07079, both from the Public Health Service, Department of Health and Human Services.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

Oxidation of dimethyl sulfide by various aromatic compound oxygenases from bacteria

As a result of the determination of dimethyl sulfide (DMS) oxidizing activity of bacterial aromatic compound oxygenases, multicomponent monooxygenases (DmpKLMNOP from Pseudomonas sp. CF600, AphKLMNOP from Comamonas testosteroni TA441, and TodABCDEF from Pseudomonas sp. JS150), single component monooxygenases (TfdB from Pseudomonas putida EST4011 and XylMA from Pseudomonas putida mt-2), and dioxygenases (CumA1A2A3A4 from Pseudomonas fluorescens IP01 and PahAaAbAcAd from Pseudomonas putida OUS82) showed DMS-oxidizing activity, while CarAaAcAd from Pseudomonas sp. CA10 and SoxC from Rhodococcus sp. IGTS8 did not. These results indicate the possibilities that these oxygenases might oxidize DMS to DMSO under the natural condition in the environment.Present address: Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan     

Melatonin increases tissue accumulation and toxicity of cadmium in the bank vole (<Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>)

Recent study has shown that a short photoperiod increases the accumulation and toxicity of cadmium (Cd) in the bank vole as compared to a long photoperiod. Since many of the effects of photoperiod on physiological processes in small mammals are transduced by the pineal gland and its hormone melatonin, in this study the effect of subchronic melatonin injection (7 mol/kg/day for 6 weeks) on the hepatic, renal and intestinal Cd accumulation in the bank voles raised under a long photoperiod and exposed to dietary Cd (0.9 mol/g) was examined. Simultaneously, histological examinations of the liver and kidneys, and analyses of metallothionein (MT) and lipid peroxidation were carried out. Melatonin co-treatment brought about a significant increase in the hepatic (61%), renal (79%) and intestinal (77%) Cd concentrations as compared to those in the Cd alone group. However, the concentrations of MT in the liver and kidneys of the Cd + melatonin co-treated bank voles did not differ from those in the Cd alone group. Also, histopathological changes in the liver (infiltration of leukocytes) and kidneys (glomerular swelling and a focal tubular cell degeneration) as well as an increase (2-fold) in the renal lipid peroxidation occurred only in animals from the Cd + melatonin group. These data indicate that (1) subchronic melatonin injection has similar effect on the tissue accumulation and toxicity of Cd to that produced by a short photoperiod and (2) the Cd-induced toxicity in the liver and kidneys of melatonin co-treated bank voles is probably due to increased Cd accumulation and decreased synthesis of MT.     

Synthesis of phytochelatins and modulation of antioxidants in response to cadmium stress in Cuscuta reflexa--an angiospermic parasite

Effect of cadmium on growth, antioxidative enzymes namely catalase, peroxidase, glutathione reductase, level of glutathione and phytochelatin synthesis was investigated in callus and seedlings of Cuscuta reflexa. A time, concentration and tissue dependent response of Cd was observed. Cd inhibited the growth of callus and seedlings by 50% at 300 and 500 micromol/L concentrations, respectively. Shorter exposure of low concentration of Cd led to augmentation of antioxidant activity, both in callus and seedlings, while longer exposure and high concentration of Cd led to a concentration dependent decrease in callus. Analysis of phytochelatin (PC) synthesis in callus and seedlings of C. reflexa revealed both quantitative and qualitative changes. Cd at low concentrations led to synthesis of predominantly PC4, while at higher concentrations, PC3 was the major form being synthesized. Amelioration of antioxidative systems of C. reflexa in response to Cd stress might be playing a protective role, alleviating the damaging effects of ROS, generated during Cd stress. Concomitantly, chelation and sequestering of toxic Cd ions in this parasite was mediated by synthesis of PC. The response to Cd stress shown by this holoparasitic plant was found to be similar to those of non-parasitic plants (hosts).     

Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium

The effects of excess Cd on the contents of free cysteine, total glutathione and phytochelatin (PC) were measured in roots of intact maize seedlings. Exposure to 3 /tmM Cd for 15 min caused PCs to appe substrates for formation of longer PCs. Total glutathione levels declined with PC synthesis, free cysteine contents changed little. The reactions to excess Cd differed along the length of roots. In the 1 cm apical region a high production of PCs occurred with a moderate loss of total glutathione. In the mature region, PC content was 2.5-fold less than in apices, several unidentified thiols accumulated, and total glutathione levels declined drastically. Exposure to 0.05 μM Cd for 24 h induced PCs, the contents rose as Cd concentrations were increased. The roots produced PCs in excess of that required to chelate the Cd present, as if some PCs were compartmentalized or had not yet formed Cd-PC complexes. Phytochelatin formation was stimulated most effectively by Cd, less by Zn and Cu and negligibly by Ni. Total glutathione declined with Cd and Zn exposure, however, with excess Cu the roots contained 45% more total glutathione than did the controls.