J BIOCHEM MOLECULAR TOXICOLOGY Volume 18, Number 1, 2004
The Effect of Superoxide Dismutase Deficiency on Cadmium Stress Paula D. B. Adamis,1 D´ebora Silva Gomes,1 Marcos Dias Pereira,1 Joelma Freire de Mesquita,2 Maria Lucia Couto C. Pinto,3 Anita D. Panek,1 and Elis C. A. Eleutherio1 1
Deptartamento de Bioqu´ımica - I.Q. - UFRJ – Rio de Janeiro, RJ, Brazil; E-mail:
[email protected] Departamento de Bioqu´ımica - I.Q. - USP – S˜ao Paulo, SP, Brazil 3 Deptartamento de Qu´ımica Anal´ıtica - I.Q. - UFRJ – Rio de Janeiro, RJ, Brazil 2
Received 10 July 2003; revised 12 September 2003; accepted 30 September 2003
ABSTRACT: Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transC 2004 Wiley Periodicals, Inc. J Biochem Mol Toxiport. col 18:12–17, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20000 KEYWORDS: Superoxide
Dismutase; Cadmium; Glutathione; Metallothionein; Saccharomyces cerevisiae
Correspondence to: Elis C. A. Eleutherio. Contract Grant Sponsor: FAPERJ. Contract Grant Sponsor: CNPq. c 2004 Wiley Periodicals, Inc.
INTRODUCTION Widespread pollution by heavy metals has important consequences for human health and the quality of the environment. With the increased use of cadmium, e.g. in alloy preparations, metal plating, and manufacture of batteries, particular attention has been paid to this metal, which is very toxic even at low concentrations. The concern arises because it accumulates in particular food species, with potential consequences for human health [1]. Cadmium has been correlated with hypertension, reduced life span, prostate cancer, suppression of testicular function, and disruption of a number of enzyme systems [2]. Cadmium constitutes a typical example of a nonredox metal ion whose toxicity depends on its ability to form complexes. However, the basis for its toxicity is not clearly understood. Yeast is an excellent model for studying detoxification pathways due to the ease of its genetic manipulation and the availability of the complete Saccharomyces cerevisiae genomic sequence. Living organisms use several mechanisms to counter cadmium toxicity. In bacteria, efflux pumps are able to export toxic ions out of the cell [3]. In eukaryotes, cadmium is sequestrated by a ligand and, in some cases, the metal is subsequently compartmentalized as a ligand complex [4]. The molecules involved with cytoplasmic sequestration of cadmium are glutathione, phytochelatins, and metallothioneins [5–7]. In the form of glutathione or phytochelatin complexes, cadmium is transported into vacuole, limiting the cytoplasmic concentrations of this heavy metal [4]. In Saccharomyces cerevisiae Ycf1 mediates the vacuolar accumulation of cadmium-glutathione complexes in this organelle [5]. Recently, we have shown strong evidence that, in yeast cells, the level of the cadmium-glutathione complex controls cadmium uptake through a zinc transporter [8]. 12
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The major effect of cadmium on a living cell is oxidative stress, particularly lipid peroxidation [9]. This stress arises when cellular defenses and/or repair mechanisms against oxidative damage are either compromised or overwhelmed by excessive generation of reactive oxygen species (ROS). In response to the destructive nature of ROS, aerobically growing organisms have evolved multiple defense mechanisms, which include enzymes that remove and repair the products of oxidatively damaged components [10]. The antioxidant enzyme superoxide dismutase is involved in the conversion of superoxide anion to dioxygen and hydrogen peroxide, which is further degraded by catalases or peroxidases [11]. Cells of Saccharomyces cerevisiae harbor two isoforms, the cytoplasmic Cu/Zn Sod1 and the mitochondrial Mn Sod2. In this study, Saccharomyces cerevisiae sod mutant strains were used to study cadmium absorption and the oxidative stress produced by exposure of cells to this metal. We have previously observed that the redox state of the cell seems to affect its capacity of absorbing cadmium [12]. Therefore, the use of mutants defective in the antioxidant defense systems, like sod cells, could help to elucidate the mechanisms which regulate cadmium transport and how cells protect themselves against this metal.
MATERIALS AND METHODS Yeast Strains and Growth Conditions Studies were performed with the control strain Eg103 (Mat, leu2, his3-11, trp1-289, ura3-52) [11]. The disruptants used in this study were isogenic derivatives of Eg103. The mutant Eg118 (sod1::URA3-52) and Eg110 (sod2::TRP1-289) were a kind gift from Dr. E. Gralla, University of California, USA. Stocks of the SOD strains were maintained on solid 2% YPD (1% yeast extract, 2% glucose, 2% peptone, and 2% agar) under appropriate conditions to avoid selection of petites or suppressors. For all experiments cells were grown up to stationary phase in liquid YPD medium using an orbital shaker at 28◦ C and 160 rpm, with the ratio of flask volume/medium of 5/1.
Cadmium Stress Cells were harvested by centrifugation and washed twice with 50 mM sodium phosphate buffer, pH 6.0. Thereafter, 200 mg of cells (dry weight) were resuspended in 50 mL of the same buffer either containing or not 48 M of CdSO4 and maintained at 28◦ C/160 rpm. Aliquots were withdrawn at time intervals to analyze
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cadmium absorption and to perform some investigations with the pellet [8].
Atomic Absorption Cadmium absorption was estimated by determining the difference in metal content between control medium without cells and test medium containing cells. The metal contents were determined with an atomic absorption spectrophotometer (Perkin Elmer 3100) as previously reported [8].
Fluorescence Assays The oxidant-sensitive probe 2 ,7 -dichlorofluorescein was used to measure the levels of oxidation developed during exposure to cadmium [8,12]. Fluorescence was measured using a PTI (Photo Technology International) spectrofluorimeter set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm. Dichlorofluorescein was added to a cell suspension prepared as described above from a fresh 5 mM stock in ethanol to a final concentration of 10 M. Incubation continued for 15 min at room temperature for the uptake of the probe. Cadmium was added at the appropriate concentration and incubation continued. After 24 h, cells (50 mg) were harvested by centrifugation and washed twice with water. The pellet was resuspended in 0.5 mL of water and 1.5 g of glass beads were added. The samples were lyzed by three cycles of 1-min agitation on a vortex mixer followed by 1-min on ice. The supernatant solution was obtained after centrifugation at 25,000 × g for 5 min, diluted 6-fold with water and, then, fluorescence was measured. As control, fluorescence was measured in cells not exposed to cadmium.
RT-PCR Analysis Total mRNA from cells was extracted with a QuickPrep micro mRNA purification kit (Amersham Pharmacia Biotech). One microgram mRNA was used to perform RT-PCR. For RT-PCR analysis, cDNA preparation and amplification were performed using a ready-to-go RT-PCR beads kit from Amersham Pharmacia Biotech. The experiments were carried out using specific primers for either CUP1 (sense primer: 5 -TGAAGGTCATGAGTGCCAATGC-3 ; antisense primer: 5 -CCAGAGCAGCATGACTTCTTGGTT-3 ), YCF1 (sense primer: 5 -AGCTCTGTAGATCTCCTGAAGGGT-3 ; antisense primer: 5 -CAAATGCCTTCGTAGGTGTGTC-3 ), or PDA1 (sense primer: 5 -CTTGCTGCTTCATTCAAACGCC-3 ; antisense primer: 5 -TGGCCTCCTGACCAACAGAT-3 ). PDA1
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RT-PCR product was used as internal standard. Imageanalysis of the amplified fragments was carried out by using Quantity One software (Bio-Rad). The relative expression of CUP1 or YCF1 mRNA was calculated by the quotient of the area of CUP1 or YCF1 and the area of the internal standard PDA1.
Determination of Glutathione Reduced glutathione (GSH) concentrations were determined spectrophotometrically, in neutralized trichloroacetic acid extracts [13], before and after 24 h metal treatment, by following the glyoxylase catalyzed production of S-lactoyl-GSH at 240 nm [14]. Oxidized glutathione (GSSG) was determined in the same cuvette by addition of NADPH and glutathione reductase, and then following the change in absorbance at 340 nm [14]. The redox ratio was expressed as the relation between GSSG and GSH contents.
RESULTS AND DISCUSSION Cadmium Absorption and Oxidative Stress in sod Mutants In this paper we bring a deeper insight into the mechanisms that control absorption of cadmium, whose toxicity seems to involve an oxidative stress. In Saccharomyces cerevisiae, Sod enzymes have been shown to play important roles in protection against oxidation [11]. Cells deficient in SOD1 exhibit a series of defects, including reduced rates of aerobic growth and altered metal homeostasis [15]. This intriguing behavior makes the sod1 mutant an interesting model for the investigation of uptake and oxidative stress caused by cadmium. The yeast physiological state is one of the most important factors, which influence metal absorption activity. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of stress tolerance [16]. Cells of a wild type strain harvested in stationary growth phase have demonstrated a higher capacity of cadmium absorption than exponential cells [12]. Then, we chose to work with resting cells. As can be seen in Figure 1, deletion of the SOD1 gene increased the absorption of cadmium from the medium in 24 h. These cells were able to remove about 40% of the initial metal, while control cells removed only 20%. On the other hand, the SOD2 deficiency impaired cadmium uptake. Since dead cells of Saccharomyces cerevisiae are not capable of absorbing cadmium
FIGURE 1. Atomic absorption analyses of cadmium were conducted in buffer solution containing stationary phase cells. The concentration of cadmium in the medium was determined at the beginning of the experiment and after 1, 3, 5, 7, and 24 h. The experiments were done as described in Methods. The results represent the mean ± standard deviation of at least three independent experiments. Bars for some points may be smaller than plot symbols.
[12], the low metal uptake demonstrated by sod2 could be related to reduced tolerance. However, by plating cells on solid medium containing cadmium, we verified that sod2 strain showed no growth inhibition at 48 M CdSO4 (results not shown). Intracellular oxidative stress produced by cadmium was monitored by measuring changes in fluorescence resulting from an oxidation-sensitive probe. The molecule 2 ,7 -dichlorofluorescein (DCF) can permeate cell membrane by passive diffusion and, once inside the cell, it becomes susceptible to attack by ROS, producing a more fluorescent compound [17]. The intensity of fluorescence can be measured and is the basis of a common assay for quantifying oxidative stress. Oxidation of DCF may be mediated by a number of oxidants. The particular species responsible for oxidation following CdSO4 treatment have not yet been ascertained [18]. Table 1 presents the relation between the fluorescence of cadmium stressed cells and nonstressed cells. According to the results, while crude extracts from control strain, Eg103, showed a 1.6-fold increase in fluorescence over a 24 h period, fluorescence of extracts obtained from the deleted strains, Eg118 (sod1) and Eg110 (sod2) strains, did not change significantly. Cadmium TABLE 1. Enhancement of Intracellular Oxidation Measured as Fold Increase in Fluorescence Strain Eg103 (control strain) Eg118 (sod1)b Eg110 (sod2)b
Relative Fluorescence a
1.6 ± 0.3 1.0 ± 0.1 0.8 ± 0.2
Note: Different letters are statistically different by Student’s-t test ( p ≤ 0.05). Relation between fluorescence of cadmium stressed cells for 24 h and nonstressed cells. The experiments were done as described in Methods. The results represent the mean ± standard deviation of at least three independent experiments.
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seemed to induce an oxidative stress in the control strain, which might be responsible for the toxic effects of this metal. The low oxidation exhibited by sod2 cells is in agreement with their low metal absorption. On the other hand, we would have expected that such a high cadmium intake, as occurred in the sod1 strain, would produce severe damage. However, no increase in endogenous oxidative species could be detected in the Eg118 strain as compared to the control strain. It seems that the deletion in SOD1 triggers some other defense mechanisms for cellular protection. In Saccharomyces cerevisiae, deficiency in Sod1 leads to amplification of the number of vacuole [19], a compartment that plays an important role in cadmium detoxification [4]. Furthermore, sod1 cells exhibited altered expression of metallothionein under oxidative stress conditions [20].
Sequestration of Cadmium by Metallothionein and Glutathione We have proposed a model for cadmium uptake based on the existing literature, as well as, on our own results [2,8]. Cadmium is a nonessential heavy metal, which seems to be absorbed by yeast cells through essential metal transporters. Saccharomyces cerevisiae
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zrt1 mutant cells did not remove cadmium from the medium, suggesting that cadmium absorption by yeast cells is dependent on the Zrt1 transporter [8]. Once inside the cell, protection against cadmium involves its sequestration by peptides or proteins. Metallothionein (MT) is critical for metal detoxification [1]. Like the mammalian metallothioneins, the Cup1 protein from yeast mediates resistance through its ability to chelate metals [7]. When expressed at physiological levels, Cup1 functions to detoxify copper, and when overexpressed it can also protect against cadmium [21]. The detoxification potential of Cup1 is not markedly different from that of human metallothionein, which is best documented to offer protection against cadmium, zinc, and copper [1]. Moreover, it has been shown that the expression of yeast MT, in the presence of copper, suppresses a number of growth defects of mutant strains lacking the Cu-Zn dismutase [22]. We, therefore, asked the question of whether a deletion in the SOD1 gene would alter transcription of metallothionein, thus preventing the oxidative stress produced by cadmium. The level of CUP1 mRNA was analyzed by RT-PCR in control and sod1 cells growing exponentially on glucose as well as after depletion of this substrate. According to Figures 2A and 2B, the expression of CUP1 gene, in cells lacking Sod1, was higher
FIGURE 2. RT-PCR analysis of CUP1 and YCF1 mRNA levels: (A) The mRNA templates used in the RT-PCR reactions were obtained from cells growing exponentially on glucose (lanes 1, 2, 5, and 6) or after depletion of this substrate (lanes 3, 4, 7, and 8). CUP1 primers were used in reactions 1–4. YCF1 primers were used in reactions 5–8. In each case, PDA1 served as an internal control. Relative expression of CUP1 (B) or YCF1 (C) mRNA. The CUP1 or YCF1 mRNA levels are normalized to the expression of the internal standard PDA1 by calculating the quotient of the area of the CUP1 (or YCF1) and the area of PDA1 band.
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than in the control strain. Repressed cells growing on glucose showed a lower level of CUP1 expression than derepressed cells (Figures 2A and 2B), suggesting that CUP1 is subjected to catabolic repression. These results could explain the low oxidation exhibited by the sod1 strain, although it had shown the highest capacity of removing cadmium from the medium. The exposure to cadmium did not change the level of CUP1 mRNA as a consequence of the metal stress in nonproliferating cells (results not shown). In addition to MT-mediated sequestration of the metal, several studies show that heavy metals in humans and yeasts can be eliminated via glutathione conjugation [5,23]. Strains Eg103 (control) and Eg118 (sod1) exhibited similar levels of GSSG/GSH when harvested in stationary phase (Figure 3). As expected, after cadmium treatment, there was a significant enhancement of the intracellular GSSG content in the control strain (Figure 3), since in yeast cells, the main defense system against cadmium consists of binding the metal with GSH and transporting this complex into the vacuole [5]. When yeast is exposed to cadmium, most of the sulfur assimilated is converted into GSH [24]. In the Sod2 deficient strain, the level of GSSG was higher than in the other strains, demonstrating that these cells were more oxidized even during cellular growth (Figure 3). When this strain was exposed to cadmium, no changes in this ratio were observed. Cells of the sod2 mutant removed little metal from the medium (Figure 1), corroborating previous results, which showed that increased levels of oxidized glutathione reduce cadmium uptake [8]. However, in the Sod1 deficient mutant, the presence of the metal did not produce any change in GSSG/GSH ratio, which is in accordance to the intracellular oxidation levels observed in Table 1. These results suggest that the higher levels of MT expression found in sod1 strain might affect the redox status of cells, favoring
FIGURE 3. Glutathione determinations: GSSG/GSH ratio of cells collected in stationary growth phase ( ) and cells exposed to 48 M CdSO4 during 24 h ( ). The experiments were done as described in Methods. The results represent the mean ± standard deviation of at least three independent experiments.
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cadmium absorption by relieving GSH under cadmium stress. Another point that must be considered is that the absence of the antioxidant enzyme Cu, Zn-Sod1 was shown to cause vacuolar fragmentation in Saccharomyces cerevisiae [19]. In wild-type yeast cells, 1–3 large vacuoles are to be seen, whereas, cells of the sod1 strain showed as many as 50 smaller vacuoles. Electron micrographs demonstrated that the electron density of a smaller vacuole from sod1 cells was comparable to a full-size vacuole from wild-type strain, indicating that the vacuolar concentration of proteins was similar [19]. Yeast deficient in the mitochondrial manganese Sod2 showed vacuole morphology undistinguishable from that of wild-type strain [19]. Therefore, a large number of vacuole inside the cell of a sod1 strain might increase the number of vacuolar membrane proteins, such as Ycf1. Then, in sod1 cells, the traffic of glutathione Sconjugates—Cd(GS)2 —into the vacuole through Ycf1 might be done more effectively than in the control strain. Corroborating this hypothesis, the sod1 strain showed a higher level of YCF1 expression, which did not change significantly with glucose depletion (Figures 2A and 2C). Recently, it has been demonstrated that Cd(GS)2 controls cadmium absorption [8]. Thus, the Cd(GS)2 level in the cytosol of sod1 cells would always be lower than in the control strain, signaling for cadmium uptake. Since we determined total GSSG/GSH ratio (including that present in vacuole), an increase in this ratio after cadmium exposure would be expected even in sod1 cells. However, this mutant showed similar GSSG/GSH ratios before and after metal stress (Figure 3). In the literature no information is available about cadmium storage in vacuole. In plants, the degradation of glutathione S-conjugates is catalyzed by a vacuolar carboxypeptidase [25]. Glutathione conjugates seem to be a transport form but not a storage form of xenobiotic molecules. In yeast, glutathione catabolism appears to be mediated by gamma-glutamyl transpeptidase and cysteinylglycine dipeptidase (both located in vacuolar membrane) generating L-glutamate, L-cysteine, and glycine in the cytoplasm [26,27]. Therefore, a higher number of vacuole could increase glutathione turnover as well as cadmium uptake, since the level of Cd(GS)2 in cytoplasm regulates cadmium entrance [8]. In sum, our results show that Sod1 deficiency is beneficial for cadmium absorption by increasing metallothionein and Ycf1 expressions, both conditions would lead to high levels of reduced glutathione, facilitating metal uptake. The study of how cadmium-glutathione complex is stored and recycled might help to elucidate the mechanisms of metal detoxification, bringing broad applications for bioremediation and human health.
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ACKNOWLEDGMENTS We thank the technicians Adailton Or¸cai Fialho and Matias Ramos de Azevedo (Dep. Qu´ımica Anal´ıtica– I.Q./UFRJ, Brazil) for measuring cadmium concentration as well as Prof. Ricardo Chaloub and Prof. Marcoaurelio Almenara (Depto. de Bioqu´ımica– I.Q./UFRJ, Brazil) for the use of the spectrofluorimeter.
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