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Department of Chemistry, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
Rolf A. Andersen
Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
Eiliv Steinnes
Department of Chemistry, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
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ABSTRACT |
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The induction of defense systems against metal exposure was investigated in 48 wild-growing fruiting bodies of the king bolete (Boletus edulis) from two areas polluted with several transition metals from smelters, as well as five reference areas. To determine the degree of metal exposure, cadmium (Cd), zinc (Zn), and copper (Cu) were determined in caps of fruiting bodies by atomic absorption spectrophotometry (AAS), whereas mercury (Hg) was determined by cold vapor atomic fluorescence spectrometry (CVAFS). Caps were analyzed further with respect to relative activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as concentrations of total glutathione (GSHTOT = GSH + GSSG) and relative concentrations of heat shock protein 70 kDa (HSP70). The results showed that concentrations of the four metals, as well as SOD, CAT and HSP70, were significantly elevated in the exposed group (Mann-Whitney, P 
0.001). In contrast, GSHTOT was significantly lowered in the exposed group (P 0.05). Significant positive correlations were established between concentrations of Cd, Zn, Hg, or Cu and activities of SOD (Spearman’s P 0.01 for the association between SOD and Cd, P 0.001 for all other metal exposure parameters), CAT (P 0.001 for all exposure parameters), or expression of HSP70 (P 0.001 for all exposure parameters). Significant negative correlations were found between total GSH and Cd (P 0.001), Zn (P 0.001), or Hg (P 0.05). We conclude that antioxidant enzymes are induced in wild-growing B. edulis exposed to environmentally relevant concentrations of potentially toxic transition metals; whereas the net consumption of GSH that occurs with increasing metal exposure may reflect GSH consumption by mechanisms of metal detoxification. Finally, the induction of HSP70 suggests that the antioxidant response and the mechanisms in which GSH is consumed are insufficient for protection against the harmful effects of severe metal stress.
Key words: catalase, glutathione, heat shock protein 70 kDa, heavy metal, mushroom, superoxide dismutase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Leyval et al 1997).
Studies on effects and responses on the molecular level to transition metals in macromycetes, as well as speciation studies have focused on edible, economically important saprophytic species, such as the Agaricales (Meisch et al 1983, Esser and Brunnert 1986, Meisch and Schmitt 1986) and the oyster mushroom (Pleurotus ostreatus) (Baldrian and Gabriel 2002). Among the mycorrhizal species that have received some attention are the edible Suillus luteus (Colpaert et al 2000) and the poisonous species Paxillus involutus (Jacob et al 2001, Ott et al 2002) and Hebeloma crustuliniforme (Frey et al 2000). Previous studies in our laboratory have shown high concentrations of Cd, Zn, Hg and Cu in the edible, ectomycorrhizal king bolete (penny bun, Boletus edulis) growing in uncontaminated and polluted areas (Allen and Steinnes 1978, Collin-Hansen et al 2002).
Metal-binding compounds and mechanisms for metal sequestration constitute a first line of defense against metal toxicity. The second line includes a range of indirect mechanisms such as cellular antioxidants, heat shock proteins (HSPs, also known as "stress proteins"), and damage repair systems.
Mechanisms for sequestration and immobilization of certain potentially toxic metals within living cells may allow organisms to accumulate these elements to high concentrations in the cells. In a previous study from our laboratory Cd-binding capacities in cytosols were determined in B. edulis collected near the Outukumpu Norzinc Zn smelter at Odda, S.-W. Norway and the former Cu smelter at Sulitjelma, N. Norway. The Cd-Chelex affinity assay (Bartsch et al 1990) revealed Cd-binding capacities differing more than 250-fold among different samples collected near the Zn smelter and 24-fold among samples collected near the former Cu smelter (Collin-Hansen et al 2002). In a follow-up study of B. edulis exposed to Cd from the same Zn smelter, a novel Cd-containing protein was isolated and its N-terminal sequence determined (Collin-Hansen et al 2003).
Transition metals that may undergo redox cycling, such as Cu, Fe, Co and Mn, may act as potent catalysts in some of the reactions generating reactive oxygen species (ROS), notably the Haber-Weiss reaction (Halliwell 1992). Increasing evidence points to the involvement of ROS in the toxicities of Cd and Hg as well, yet the molecular mechanisms by which Cd and Hg induce ROS formation are not understood (Schützendübel et al 2001, Hartwig et al 2002).
Cd toxicity is associated with increased cellular oxidative stress, the formation and disruption of sulfhydryl and metal thiolate bonds and alterations in secondary protein structure, and interference with essential metal uptake, transport and metabolism (Brennan and Schiestl 1996, Meplan et al 1999, Sandalio et al 2001). By competing with essential metals in protein binding sites, Cd can induce the release of Fe and Cu, causing increased generation of ROS and increased oxidative stress (Pruski and Dixon 2002). Eventually Cd can induce cell death either by necrotic or apoptotic mechanisms.
Hg exposure is known to be associated with increased oxidative damage to biomolecules and linked to increased activity of ROS (Stacey and Kappus 1982).
The most important enzymes for removal of ROS in the cell are superoxide dismutase (SOD) and catalase (CAT). SOD catalyzes the dismutation of superoxide anion to hydrogen peroxide and molecular oxygen (Fridovich 1986), whereas CAT mediates the cleavage of hydrogen peroxide evolving molecular oxygen (Scandalios 1993). Increased SOD and/or CAT synthesis is correlated with increased tolerance to oxidative stress in bacteria (Ma and Eaton 1992), yeast (Pereira et al 2001) and plants (Arisi et al 1998, Mittler 2002). By contrast, null mutants of SOD in Saccharomyces cerevisiae are associated with several biochemical defects (Tamai et al 1993). These studies clearly indicate that SOD and/or CAT protect eucaryotic metabolic enzymes against damage by ROS. Few studies have investigated the relationship between metal exposure and antioxidant enzymatic activity.
During normal metabolism the tripeptide glutathione (GSH) protects cell constituents against the damaging effects of endogenous ROS, among its many important roles in metabolism (Rauser 1999). During metal stress GSH functions in cellular protection, partly because of its antioxidant properties. Yeast strains lacking GSH or altered in their GSH redox state are sensitive to oxidative stress induced by peroxides and the superoxide anion, demonstrating the important role played by GSH as an antioxidant in these organisms (Grant et al 1996, Stephan and Jamieson 1996, Turton et al 1997). GSH also functions as a strong complexing agent for "sulfurphilic" metal ions such as Cd2+ and Hg2+ (Penninckx and Elskens 1993). Furthermore, several plant and yeast species are able to use GSH as building stone in the synthesis of phytochelatins (PCs). PCs are enzymatically synthesized, Cd-binding oligopeptides consisting of repeating units of 
-glutamylcysteine followed by a C-terminal glycine ([-Glu-Cys]n-Gly) (Rauser 1999). Whereas depletion of tissue GSH levels enhances the acute toxicity of Cd in animals, elevation of GSH levels protects against this element (Singhal et al 1987). Under normal conditions, GSH exists primarily in its reduced form, but during oxidative stress GSH may be consumed to yield glutathione disulfide (GSSG). To prevent loss of GSH, glutathione reductase may regenerate GSH from GSSG at the expense of NADPH (Penninckx and Elskens 1993).
One of the responses of pro- and eukaryotes to a variety of toxicants is represented by the increased transcription of genes encoding the highly conserved heat shock proteins (HSPs), also known as stress proteins. In unstressed cells, HSPs function as molecular chaperones, contributing to the folding and assembly of nascent polypeptides and to protein transport and degradation, as well as preventing stress-mediated accumulation of misfolded or damaged proteins. The cellular abundance and protective effects of the 70 kDa HSP (HSP70) make these proteins of particular interest in studies on the effects of stressful conditions on several levels of biological organization (Kiang and Tsokos 1998). A number of environmental stressors, including exposure to certain transition metals, have been shown to induce the expression of HSP70 in a wide range of organisms and cell lines (Yenari et al 1999).
The present study was aimed at testing differences between concentrations of four metals (Cd, Zn, Hg, and Cu) and four response parameters (activities of SOD and CAT as well as concentrations of GSH and HSP70) between two groups of B. edulis fruiting bodies (one group exposed to emissions from smelters and one reference group). Furthermore, correlations between exposure and response parameters in the whole study selection were tested. The results obtained were expected to reveal whether the defense systems in question were induced in fruiting bodies of B. edulis during different degree of metal exposure.
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MATERIALS AND METHODS |
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cm–1 deionized water provided by a Milli-Q system [Millipore, Watford, UK]). All buffers were prepared in Milli-Q water. Field sampling and sampling sites.— – Forty-eight young (4–14 cm tall, cap diam 3–17 cm) and seemingly healthy fruiting bodies of B. edulis were sampled in Sep 2000 and Sep 2002 from six areas, two of which are polluted by metals from smelter emissions and the remaining four are relatively unpolluted.
Six samples were collected from a birch forest area at altitudes between 150 and 500 meters above sea level (masl) near a former Cu smelter at Sulitjelma, northern Norway (67°07'N 16°05'E). Until the smelter was shut down in 1987, the smelting of the sulphide ore resulted in emissions of metals such as Cu, Zn, Pb, Cd and Hg. The annual emissions of SO2 were about 20 000 tons, making it the largest single SO2 source in Norway at the time. A previous study from our group revealed substantially increased concentrations of Cu, Zn and Cd in topsoil and five macromycete species, among them B. edulis, close to the smelter, steadily leveling off with increasing distance from the smelter (Collin-Hansen et al 2002).
Twenty-four samples were collected from spruce plantations at altitudes between 20 and 500 masl and at different distances from the Outukumpu Norzink smelter at Odda (60°04'N, 6°33'E), southwestern Norway. The still-operating smelter has dispersed metals such as Zn, Cd, Hg and Cu. In our previous study, we reported elevated concentrations of Zn, Cd and Cu in B. edulis and four other species of macromycetes sampled near this smelter (Collin-Hansen et al 2002). These 30 samples comprised the "exposed" group.
In addition, 18 samples ("reference" group) were collected from four relatively unpolluted sites situated within a 20 km radius from the city of Trondheim (central Norway). The four reference sites were Børsa (63°18'N, 10°00'E), Vassfjellet (63°19'N, 10°25'E), Bymarka (63°25'N, 10°18'E) and Ha°en (63°07'N, 10°30'E). Short-range deposition of transition metals emitted from Trondheim (population approx. 154 000) might have affected the reference sites closest to the city (sites Vassfjellet and Bymarka), whereas reference sites Børsa and Ha°en probably are influenced insignificantly by such deposition.
Sample preparation.— – Immediately upon arrival at the laboratory, fruiting bodies were weighed, cap diameter was measured, and fruiting bodies were thoroughly checked with respect to parasites and contamination by soil and plant material. If any part of the fruiting body was infected by insect larvae the whole fruiting body was rejected. A clean plastic brush was used to remove foreign material (e.g. soil particles and spruce needles) from the sample surface. Each fruiting body was divided into cap and stipe, and then the cap of each fruiting body was further divided using a stainless steel knife. The present study reports data obtained from whole caps, frozen (–80 C) in individual clip-lock polyethene (PE) bags for metal determinations and molecular biological studies.
Samples for metal determinations.— –
Cap tissue to be analyzed with regard to metal concentrations was weighed and freeze-dried (GT 2A, Leybold-Heraeus, Hanau, Germany) to constant mass. Freeze-dried samples were weighed again for calculations of water content, then transferred to clean PE bags with clip-lock and pulverized in the closed bags with a wooden hammer and manual grinding. Powdered mushroom tissue (0.2 g) was transferred to Teflon flasks and wet-digested by applying microwaves (Multiwave digestion system, Perkin Elmer/Anton Paar) with nitric acid (65%, 4 mL). After cooling samples, water (>18 M cm–1, Milli-Q) was added to a total volume of 100 mL, resulting in a HNO3 concentration of approximately 0.5 M. 10 mL was transferred to a PE flask for determinations of Cd, Zn and Cu by atomic absorption spectrophotometry (AAS).
Conservation of samples for determination of mercury.— – To the remaining 40 mL of sample, HCl (33% v/v, 7.5 mL), KBrO3/KBr mixture (0.1 N, 2.0 mL) and water (Milli-Q, 0.5 mL) was added to oxidize all forms of Hg to the Hg(II) oxidation state. PE flasks were sealed. The following steps in the determination of Hg were performed within the next 48 h.
Neutralization of samples for determination of mercury.— – The halogen mixture was neutralized by addition of hydroxylamine hydrochloride (NH2OH x HCl, 12%, 60 µL) no more than 3 h before analysis.
Element determination.— –
Sample blanks, spiked samples and standard reference material (Bovine liver [1577 a and b] and Tomato leaves 1573 from the US National Institute of Standards and Technology) were included, and randomly chosen samples were reanalyzed. Recoveries of metals in randomly selected samples were calculated from the ratio of the amount of the elements recovered after spiking to the amount added. Recovery of added standard in the analyses fell within the range of 90–105% for Cd (91% of the samples 
95% recovery), 84–101% for Zn (44% of the samples 95% recovery), 89–107% for Cu (96% of the samples 95% recovery), and 84–104% for Hg (80% of the samples 95% recovery). Maximum allowable relative standard deviation between three replicates was set to 5%. Metal concentrations in standard reference materials deviated from the average by at most –2% for Cd, –10% for Zn, +5% for Cu and –4% for Hg and normally fell within the limits of the certified values.
Atomic absorption spectrophotometry (AAS).— – Concentrations of Cd, Zn and Cu were determined by flame AAS (Model 1100B, Perkin-Elmer), with deuterium background correction for Cd and Zn.
Cold vapor atomic fluorescence spectrometry (CVAFS).— – Concentrations of Hg were determined by cold vapor atomic fluorescence spectrometry (CVAFS) with a Merlin atomic fluorescence detector (Model 10.04 Flow Module, Model 10.023 Fluorescence Detector and Merlin Absorption Accessory). Following neutralization the sample was mixed with SnCl2 to reduce the Hg from the Hg(II) to the Hg(0) oxidation state. The Hg(0) vapor then passed through a gas/liquid separator by a stream of argon, through a membrane dryer and to the AFS detector.
Total glutathione concentration.— –
Total glutathione (GSHTOT = GSH + GSSG) was assessed by the 5,5' dithiobis-(2-nitrobenzoic acid)-glutathione reductase-coupled assay (Anderson 1985). Frozen (–80 C) tissue (approx. 1 g) was thawed on ice, cut into pea-sized pieces with a scalpel and homogenized in 10 times its volume of metaphosphoric acid (5% [w/v], 10 mL, Merck) with three sequential pulses of 5 s each using a Heidolph DIAX 900 tissue homogenizer equipped with a 6G tool (Heidolph, Kelheim, Germany). Cell debris and precipitated proteins were removed by centrifugation (3000 g, 4 C, 10 min), and the supernatant was assayed with the Total Glutathione Determination Colorimetric Microplate Assay Kit (Oxford Biomedical Research, Oxford, Michigan) according to the manufacturer’s instructions to determine the concentration of total glutathione, expressed as GSH equivalents, from a standard curve.
Preparation of tissue extracts.— – Frozen (–80 C) tissue (approx. 3 g) was thawed on ice and homogenized as described above, with two modifications: Samples were homogenized with 3x its volume of Tris buffer (30 mM, 250 mM NaCl, pH 7.6) using an ice bath. Extreme care was taken to avoid inactivation of enzymes by overheating the homogenates. Following centrifugation (12 000 g, 15 min, 4 C), aliquots (1.5 mL) of supernatant were frozen at –80 C. Aliquots were thawed on ice and used in the methods described in the following.
Total protein concentration.— –
Protein concentrations in supernatants were determined by the Bradford method (Bradford 1976) by using Coomassie blue reagents (Bio-Rad, Munich, Germany). Bovine serum albumin (BSA) (fraction V) was used as an external standard.
Relative SOD activity.— – Relative activities of superoxide dismutase in cytosols were determined by a specific colorimetric inhibition activity method, using the Superoxide Dismutase (SOD) Assay Kit (Kamiya Biomedical, Seattle, Washington) according to the manufacturer’s instructions.
Relative CAT activity.— –
CAT activity was measured spectrophotometrically by a modification of the method originally developed by Johansson and Borg (1988). This method utilizes the peroxidatic function of catalase with methanol as the hydrogen donor. The resulting formaldehyde is determined with the chromogen 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, also known as Purpald (Cayman, Ann Arbor, Michigan). Phosphate buffer (250 mM KH2PO4, 10 mM EDTA, 1% BSA, pH 7.5, 100 µL) was added to each well with a multi-channel pipette, then a sample (20 µL) of tissue extract, blank solution or formaldehyde standard was added. For the remaining steps, a multi-channel pipette was used to add each reagent. Following addition of methanol (100% (w/v), 30 µL) and hydrogen peroxide (0.12% (v/v), 20 µL), samples were incubated with continuous shaking for 20 min at room temperature (20°C). After termination of the enzymatic reaction with KOH (10 M, 30 µL), Purpald (34.2 mM in 0.5 M HCl, 30 µL) was added to the wells, and samples were incubated (10 min, 20 C). The product of the reaction between formaldehyde and purpald was oxidized by KIO4 (65.2 mM in 0.5 M KOH, 10 µL). The absorbance was measured at 540 nm and the CAT activity calculated from the standard curve.
Relative HSP70 concentration.— – The NuPAGE system (Invitrogen, Carlsbad, California) for Western blotting was used to determine relative concentrations of HSP70. Tissue extracts were thawed on ice. A volume of extract containing 14 µg total protein as determined by the Bradford assay was mixed with NuPAGE LDS Sample Buffer 4X (8.5 µL), NuPAGE Sample Reducing Agent 10X (5 µL) and homogenization buffer (see Ch. 2.3) to a total volume of 50 µL and heated (70 C, 10 min).
Samples (25 µL) or NuPAGE SeeBlue Plus2 Pre-Stained Standards (5 µL) were loaded onto gels and separated by SDS-PAGE using an XCell SureLock Mini-Cell (Invitrogen) in combination with precast NuPAGE 10% Bis-Tris gels (Invitrogen) following the manufacturer’s instructions. On each gel, a randomly chosen sample was run as an internal control. Following electrophoresis, proteins were transferred onto nitrocellulose membranes (0.2 uM pore size, Invitrogen) using an Xcell II Blot Module (Invitrogen). Membranes were subsequently blocked (20 C, 1 h) using a Detector Block kit (KPL, Gaithersburg, Maryland) and incubated (1 h) with rabbit anti-human HSP70 polyclonal antibody (Calbiochem, California), diluted 1 : 10 000 in Detector Block solution. After washing (7 x 7 min) in Tris-buffered saline/Tween-20 (TBST) (10 mM Tris, 150 mM NaCl, 0.05% (w/v) Tween-20, pH 8.0), membranes were probed with a 1 : 5000 dilution of alkaline phosphatase-conjugated Anti-Rabbit IgG secondary antibody (Calbio-chem) in Detector Block (20 C, 1 h), washed again (7 x 7 min) in TBST and then developed for approx. 15 min in 10 mL of alkaline phosphatase substrate solution (0.404 mM nitroblue tetrazolium, 0.384 mM 5-bromo-4-chloro-3-indolyl phosphate, 5 mM MgCl2, 100 mM Tris, 100 mM NaCl, pH 9.5). The color reaction was terminated by transferring the membranes to an EDTA solution (5 mM EDTA, 20 mM Tris, pH 8.0). Blots were digitized on a ScanJet 3300C page scanner (Hewlett Packard, Palo Alto, California), and relative optical densities of immunoreactive bands were quantified by means of image-analysis software (Digital Science 1D 2.0, Eastman Kodak, New Haven, Connecticut).
Normalization of data.— – All measurements were carried out in duplicate. Before statistical analyses, data of fungal metal concentrations were normalized using the fresh-weight data, whereas relative activities of SOD and CAT as well as concentrations of GSHTOT and relative concentrations of HSP70 were normalized using total protein concentrations. Soil metal concentrations were not normalized.
Statistical treatment of data.— – Distribution analysis revealed, on the one hand, that the data for HSP70 and metal concentrations were nearly log-normally distributed, except from the case of Zn concentrations. The data for SOD, CAT and GSHTOT, on the other hand, were nearly normally distributed. The nonparametric Mann-Whitney U-test for two independent samples was used to determine statistical significance of differences in parameters between exposed and reference samples, since logarithmic transformation failed to normalize the distributions of Zn concentration. Furthermore, because the bivariate scatter-plots suggested nonlinear relationships between several of the variables, Spearman’s correlation analysis was chosen to indicate the degree of monotonic, but not necessarily linear, correlation. P = 0.05 was chosen as the level of statistical significance throughout.
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RESULTS |
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). Not surprisingly, concentrations of Zn and the chemically related Cd were high in B. edulis growing near the Zn smelter at Odda, whereas Cu concentrations were highest in samples collected near the former Cu smelter at Sulitjelma. Concentrations of Hg are similar in these two areas. The essential metals Zn and Cu are present in substantial concentrations in samples from all areas, whereas the difference between exposed versus reference sites are larger for the unessential metals Cd and Hg. Concentrations of all four metals in cap tissues were significantly higher in the exposed group compared with the reference group (Mann-Whitney, P 0.001).
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0.001 for all bivariate correlations). The results from the molecular biological analyses show that all of the four response parameters (SOD, CAT, GSH, HSP70) were readily detectable in caps of B. edulis from heavily polluted as well as relatively unpolluted areas.
Highly significant differences were established between the exposed group and the reference group with regard to relative activities of SOD and CAT, as well as in relative concentrations of HSP70. These three defense parameters were elevated in the exposed group (Mann-Whitney, P 0.001). Concentrations of GSHTOT were lower in the exposed relative to the reference group (Mann-Whitney, P 0.05).
Highly significant positive correlations were established between ranks of metal concentrations on one side, and ranks of expression of SOD, CAT, or HSP70, on the other (TABLE II, FIG. 1A, B, D), indicating the concerted involvement of these proteins in the defense toward potentially toxic metals in B. edulis. Highly significant non-parametric correlations were found between the concentrations of GSHTOT and Cd, Zn, or Hg as well, however, these associations were negative (TABLE II, FIG. 1C).
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), whereas FIG. 2 provides an example of the bands representing HSP70 on the nitrocellulose membrane after western blotting and immunostaining.
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DISCUSSION |
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and Blaudez et al (2000). The results presented here strongly indicate that the proteins SOD, CAT and HSP70 are induced in B. edulis exposed to potentially toxic transition metals. The tripeptide GSH, by contrast, seems to be consumed at a rate exceeding synthesis, possibly as a direct consequence of detoxification mechanisms discussed in some detail below.
The highly significant bivariate correlations established between concentrations of individual metals in fruiting bodies were expected findings, considering that the two exposed sampling areas chosen for the present study have been polluted with a range of metals, whereas concentrations of metals are low in soil from the four reference areas. This multi-element exposure regime contrasts the many controlled single- or dual-element exposure experiments using plants or fungi that can be grown under controlled conditions. Mycorrhizal fungi are notoriously difficult to grow in the lab and apparently, all attempts to grow B. edulis under controlled conditions have been unsuccessful (Hall et al 1998). The high degree of covariation between concentrations of the metals determined in the present study limits the value of considerations of dose-response relationships between the individual metals and the response of interest. However, this covariation allows for a simple and intuitive treatment of the metal exposure data by seeing them as a whole.
Differences in size and age, both of the mycelium and of the individual fruiting body, might be expected to significantly affect metal concentrations, gene expression and protein concentrations in fruiting bodies. Furthermore, from experiments with fungi and plants it is known that enzyme activities may change greatly in actively dividing tissues (Wilson 1975, Dubey and Pessarakli 1995). To minimize effects arising from differences in size and age upon the measured parameters, it was decided to sample specimens within a relatively low size range.
Only two previous studies have dealt with the responsiveness of SOD or CAT to metal exposure in macromycetes. Kojo and Lodenius (1989) reported a significant positive correlation between Hg concentrations and CAT activity for 21 fruiting bodies of at least 15 different species collected near Helsinki, whereas the correlation between Cd and CAT activity was not significant. More recently, Ott et al (2002) demonstrated the induction of SOD, but not CAT, in isolates of the ectomycorrhizal macromycete Paxillus involutus exposed to Cd.
Responsiveness of antioxidants to pollutants is generally difficult to predict. Several animal studies report a high degree of response variability depending on class of chemicals, kind of exposure and phase of the biological cycle (Ribera et al 1989, Viarengo et al 1991a, b). Increased activities of SOD and CAT are often attributed to stressful conditions that increase the intracellular levels of ROS such as superoxide anion and/or hydrogen peroxide (Koricheva et al 1997, Cho and Park 2000). High intracellular concentrations of Cu, Cd and Hg are known to enhance the intracellular formation of such ROS species. Thus, the highly significant positive relationships established in the present study between metal exposure and activities of SOD or CAT probably reflects increased production of ROS species such as superoxide anion and hydrogen peroxide due to metal exposure.
Mitochondria generally are regarded as the primary source of ROS production in unstressed cells. Mitochondria are able to accumulate Cd and Hg, and mitochondrial generation of ROS may be enhanced by the presence of excessive levels of metals such as Cd (Wang et al 2004). Accumulation of Cd by mitochondria may result also in the inhibition of oxidative phosphorylation (Tang and Shaikh 2001), suggesting a negative impact of Cd exposure on the energy available for growth, reproduction and detoxification of exo- and endogenous compounds. Wang et al (2004) showed that complex III (ubiquinol:cytochrome c oxidoreductase) in the mitochondrial electron transport chain may be the only one of the mitochondrial enzyme complexes involved in the Cd-inducible production of ROS.
Although previous studies suggest that Cd may induce SOD activity (Ott et al 2002) and Hg may induce CAT activity (Kojo and Lodenius 1989) in macromycetes, in vitro experiments have shown that Cd inhibits SOD (Hussain et al 1987) and CAT (Casalino et al 2002), probably via direct interaction with these enzymes. Effects arising from enzyme inhibition therefore would not be unexpected in specimens of B. edulis with high concentrations of these metals.
It is possible that such inhibition of enzyme activity at high Cd concentrations occurs for SOD (FIG. 1A). On the one hand, these data suggest a curved dose-response relationship between Cd and SOD activity, although the possibility of interference from other metals cannot be excluded. The data for CAT, on the other hand, do not support such an exponential dose-response relationship with Cd (FIG. 1B). It should be noted that although the scatter-plots shown here are semi-logarithmic, the non-parametric (rank-based) Spearman correlation test is unaffected by log-transformation of the data.
Significant effort has been put into revealing the mechanism underlying the inhibitory effect of Cd on Cu,Zn-SOD activity. The possibility has been advanced that Cd can replace the chemically related Zn (Bauer et al 1980, Hussain et al 1987), however, recent data contradicts this mechanism (Casalino et al 2002). Alternative mechanisms are inactivation caused by free radical-mediated fragmentation of the enzyme (Kwon et al 2000) or topographic perturbation of the channel where the active site is localized, possibly due to Cd interacting with the enzyme (Casalino et al 2002). For mammalian Mn-SOD the inhibitory effect of Cd was attributed to the substitution of Cd for Mn, whereas in the same study binding of Cd to the unprotonated N delta of the His-74 imidazole residue was suggested as a probable mechanism for CAT inhibition (Casalino et al 2002).
A recent study from our laboratory showed elevated oxidative damage to lipids and DNA’s bases in 16 fruiting bodies of B. edulis growing near the Outukumpu Norzinc Zn smelter at Odda relative to 15 reference samples from central Norway (Collin-Hansen et al 2005). First, these results point to a weakness of the direct and indirect defense systems of this species at effectively detoxifying metals. Second, the established associations between metal exposure and structural alterations in DNA may provide a mechanism that, at least partly, explains the reduced expressions of SOD and GSH that are associated with high metal exposure in the present study. Genetic mutations would be expected to impair proteins involved in defense mechanisms (e.g. anti-oxidant enzymes and GSH synthetase, the enzyme that generates GSH), thus contributing to cumulative oxidative injury to cellular components.
Previous studies of organisms grown under controlled conditions have shown a rapid accumulation of GSH following Cd exposure in P. involutus (Ott et al 2002) and S. cerevisiae (Dormer et al 2000, Vido et al 2001), as well as in plants (Arisi et al 2000, Schützendübel et al 2001). However, in other studies of plants and yeasts grown under controlled conditions, Cd exposure was linked to a decline in intracellular GSH (Grill et al 1987, Tukendorf and Rauser 1990, Meuwly and Rauser 1992, Klapheck et al 1994), which predisposes the cell to oxidative damage.
This decline in GSH often is attributed to the ability of several plant and certain yeast species to generate phytochelatins (PCs). In a recent study, the zygomycete Mucor racemosus was shown to synthesize PCs in response to exposure to Cd, but not to Zn or Cu. As might be expected, this Cd-induced PC production induced a significant concurrent decrease in GSH levels (Miersch et al 2001).
There are large gaps in the current knowledge of the distribution in the fungal kingdom of the ability to synthesize PCs. However, PCs are more widespread in fungi than was once thought, and it is now recognized that the fission yeast Schizosaccharomyces pombe, Candida glabrata, S. cerevisiae and Neurospora crassa all express PCs upon Cd exposure. Preliminary experiments using liquid chromatography-tandem mass spectrometry (LC-MS/MS) have shown that B. edulis synthesize PCs, and that more complex PCs are induced in specimens growing in Cd-polluted areas than in areas polluted mainly with Cu. PCs were not found in specimens from reference aeras (C. C.-H., R. A. A. and E. S., unpublished). Thus, it seems plausible that PC synthesis is at least partly responsible for the observed negative correlations between exposure to Cd, Zn or Hg and concentrations of GSHTOT in the present study.
Part of the irreversible loss of GSH also may be due to the inhibition of GSH reductase by Hg (Zalups and Lash 1996), which is used to "recycle" oxidized GSH and return GSH to the pool of available antioxidants. An alternative, or additional, mechanism underlying the observed decline in GSH with increasing metal exposure, is immobilization of GSH through complexation by metals such as Cd and Hg, rendering GSH undetected. It is well known that GSH has a high affinity for these metals. Hg can bind irreversibly to GSH to form Hg-(GS)2, causing the loss of up to two GSH molecules per molecule of Hg. Hg also inhibits GSH synthetase. Because Hg promotes formation of ROS and lipid peroxides, it is evident that Hg may induce an imbalance in the oxidative/antioxidant ratio. Cd-(GS)2 is transported readily into the vacuoles of plants and yeasts (Li et al 1997, Cobbett and Goldsborough 2000), where it may condense to dense granules with a high CdS content. The finding by Ott et al (2002) of vacuolar electron-dense bodies displaying a high degree of correlation between Cd and S concentrations in Paxillus involutus is in accordance with this mechanism, and suggests that complexation of Cd to GSH and/or PCs and subsequent sequestering to vacuoles is an important detoxification mechanism also in macromycetes.
Although binding of Cd to PCs is believed to be a spontaneous process, recent studies of S. cerevisiae have shown that the formation of Cd-GSH complexes depends on the action of GSH-S-transferases (GST), a group of enzymes essential in detoxification of detrimental compounds, such as endogenous molecules that have experienced damage by ROS (Adamis et al 2004, Vuilleumier and Pagni 2002).
Further speculations regarding the observed relationship between metal exposure and GSH concentration can be attached to the use of GSH in repair of ROS-induced damage to biomolecules. In agreement with this, hydrogen peroxide exposure causes a reduction in GSH levels in S. cerevisiae, reflecting increased levels of oxidized (GSSG or GS-protein) and extracellular GSH (Grant et al 1998). Furthermore, administering of compounds that are good substrates for GST has been demonstrated to lower liver GSH levels in rats (Boyland and Chasseaud 1970). The decline in GSH with increasing metal exposure observed in the present study thus seems consistent with the role of GSH both as a complexing agent for metals such as Cd and Hg, a free radical scavenger, and a cofactor for several antioxidant enzymes.
In order to evaluate the combined detoxifying effect of mechanisms constituting the first line of defense (i.e. metal-binding compounds and sequestration mechanisms) as well as the antioxidant response, it was decided to investigate the responsiveness of a general stress marker to metal exposure. HSP70 was chosen as stress marker due to its claimed ubiquity (Kiang and Tsokos 1998) and eventually, because the anti-HSP70 polyclonal antibody from Calbiochem proved suitable for B. edulis. It has been long recognized that in many biological systems, including yeast, the heat shock response is inducible by a large variety of chemicals. However, only recently have HSPs been used as biomarkers of environmental pollution exposure (Kiang and Tsokos 1998). To our knowledge, the present study is the first to suggest the induction of HSPs in macromycetes by metals.
The finding of highly significant positive bivariate correlations between Cd, Zn, Hg or Cu and HSP70 expression indicates that the induction of cellular antioxidants such as SOD and CAT is neither in itself nor in combination with possible metal complexing and/or sequestering systems capable of providing a satisfactory response toward the stress imposed by the metal exposure.
To investigate whether a similar pattern of induction of HSP70 expression could be detected in a selection of B. edulis from background areas, a second round of bivariate Spearman correlation analyses was performed with the 18 reference samples from central Norway. Of all metal parameters tested, HSP70 was significantly associated only with Zn, showing a positive correlation (rSp = 0.544, P = 0.020), suggesting that other defense mechanisms are sufficient in handling toxic effects caused by low concentrations of Cd, Hg and Cu. Collectively, these observations indicate that HSP70 may be important in protection against metal stress when the primary and secondary defense systems give insufficient protection, e.g. under drastic environmental conditions such as severe metal stress.
In conclusion, the present study suggests that activities of SOD, CAT, GSH and HSP70 reflect the degree of exposure to harmful metals in B. edulis collected in uncontaminated or metal-polluted areas, indicating a close connection between metal exposure and increased generation of ROS. Although activities of SOD or CAT increase in a highly significant manner with increasing concentrations of the indivdual metals (Cd, Zn, Hg or Cu), the scatter-plot for the association between Cd concentration and SOD activity (FIG. 1A) indicates that enzyme inhibition occurs at high concentrations of Cd.
Significant negative correlations observed between GSH and Cd, Zn, or Hg suggest that GSH is being consumed in response to metal exposure at a rate exceeding GSH synthesis. Furthermore, the highly significant positive correlations of the four elements with concentration of HSP70 indicate that sufficient protection is not achieved through these antioxidant systems alone. Further studies are necessary to test whether the combined action of the defense systems expressed in B. edulis toward metals is effective at protecting biomolecules of this species, or its host plants, against damage.
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FOOTNOTES |
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1 Corresponding author. E-mail: Christian.Collin-Hansen{at}yale.edu
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LITERATURE CITED |
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