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Yale University, School of Medicine, CMHC, 34 Park Street, New Haven, Connecticut 06519
Sindre A. Pedersen
Rolf A. Andersen
Department of Biology, Norwegian University of Technology and Science, Høgskoleringen 5, 7491 Trondheim, Norway
Eiliv Steinnes
Department of Chemistry, Norwegian University of Technology and Science, Høgskoleringen 5, 7491 Trondheim, Norway
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ABSTRACT |
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Some species of macromycetes (mushrooms) consistently are found to contain high concentrations of toxic metals such as cadmium (Cd) and mercury (Hg), and consumption of wild-growing mushrooms is acknowledged as a significant source for Cd and Hg in humans. Yet little is known about the speciation of Cd and Hg in mushroom tissues. Here we present the first evidence of peptides of the phytochelatin family being responsible for binding a large fraction of Cd in caps of the macromycete Boletus edulis exposed to excess metals. Concentrations of Cd, Zn, Cu and Hg, as well as cytosolic Cd-binding capacity (CCBC), glutathione (GSH) and free proline (Pro) were quantified in fruiting bodies of B. edulis differentially exposed to a wide range of metals. Metal distribution among cytosolic compounds were investigated by size exclusion chromatography (SEC), followed by metal determinations with atomic absorption chromatography (AAS) and HR-ICP-MS. Cd-binding compounds in SEC elutates were investigated further by high performance liquid chromatography-mass spectrometry (HPLC-MS). CCBC was > 90 times higher in the exposed group relative to the reference group (Mann-Whitney’s P < 0.001), whereas concentrations of free Pro were almost identical for the two groups. For the whole study selection, CCBC correlated positively with metal exposure (Spearman’s P < 0.001 for all four metals), suggesting dose-dependent induction of Cd-binding compounds by exposure to these metals, possibly as a defense mechanism. The presence of phytochelatins (PCs), a family of cystein-rich oligopeptides, was confirmed in Cd-containing SEC fractions by HPLC-MS. The appearance of more complex PCs was coupled to declining concentrations of GSH. To our knowledge this is the first report demonstrating the presence of PCs in a macromycete.
Key words: Cadmium, glutathione, heavy metal, HPLC-MS, mercury, metallothionein, mushroom, Superdex
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFINAL REMARKSLITERATURE CITED |
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, Hall et al 1998). The extensive use of B. edulis fruiting bodies both commercially and domestically is in part a result of their appealing taste, their sometimes frequent occurrence and the fact that they are both voluminous (mass < 3 kg) and easily recognizable. B. edulis apparently has a high tolerance for generally fungitoxic metals such as Cd and Cu in the substrate because it thrives on soils high in these metals (e.g. near metal smelters) (Collin-Hansen et al 2002, 2003). Studies have shown that B. edulis possesses a strong potential for concentrating metals such as Cd, Zn, Hg, Fe, Zn, Cu and Mn, as well as Cs, Se and Po from the substrate to above-ground edible tissues (Allen and Steinnes 1978; Bauer Petrovska 1999; Skwarzec and Jakusik 2003; Collin-Hansen et al 2005a, b). Accumulation of potentially toxic metals in fruiting bodies of macromycetes that are consumed by animals or humans might constitute a significant route for transfer of these elements from soil to terrestrial food chains (Kala
and Svoboda 2000).
Although several investigations have reported concentrations of potentially toxic metals in fruiting bodies of edible fungi, detailed studies of metal distribution among cytosolic compounds are scarce in edible, mycorrhizal species, especially in specimens exposed to high metal concentrations. Wuilloud et al (2004) investigated fractionation patterns of Cd and Hg, as well as Pb, Ag, As and Sn in specimens of B. edulis and two other edible macromycete species with low to moderate concentrations of potentially toxic elements such as Cd, Ag and As. Such studies are important from the view of consumption by humans. However to investigate fungal defense mechanisms against excess metals, investigations must be performed with specimens that are exposed to high levels of the metal, or metals, of interest. Furthermore, as concluded by Wuilloud et al (2004), recent advances in multidimensional fractionation techniques and biological mass spectrometry call for the application of such techniques in the field of macromycetal responses to environmental stressors.
As noted by several authors, there is a severe lack of knowledge regarding the mechanisms responsible for the high metal tolerance of many macromycete species (Ross 1994, Frey et al 2000). Peculiarly it is often stated that metallothioneins (MTs) constitute a prime mechanism in direct fungal defense against exposure to certain metals, however this appears generally incorrect in the case of macromycetes. To the knowledge of the authors MTs have only been confirmed in one macromycete species, namely the "pizza mushroom" Agaricus bisporus, a saprotrophic species (Münger and Lerch 1985).
Recent investigations have shown that phytochelatins (PCs), a family of oligopeptides known to serve in metal detoxification in plants, are induced after Cd exposure in some species of lower fungi as well (Mehra and Winge 1991, Miersch et al 2001). PC synthesis is believed to constitute the dominant pathway for Cd detoxification in the yeast Schizosac-charomyces pombe (Clemens and Simm 2003). One important similarity between MTs and PCs is their high content of cystein (Cys), an amino acid containing a sulfur (S) atom, to which Cd may bind tightly. However, whereas MTs are ribosomally transcribed proteins with molecular mass of approximately 7 kDa, PCs are enzymatically synthesized peptides with molecular masses which may vary, but which, according to the current literature, hardly seem to exceed 2 kDa in lower fungi. The ubiquitous cellular tripeptide glutathione (GSH) serves as a building stone in PC synthesis (Rauser 1999, Cobbett 2000).
A recent report from our group revealed an inverse relationship between concentrations of GSHTOT (= GSH + GSSG) and metal exposure in B. edulis fruiting bodies (Collin-Hansen et al 2006). These findings suggest a reduction in the rate of synthesis of GSH and/or an increase in the rate of its consumption by increasing metal load.
It is well documented that the effect of metal exposure on intracellular GSH concentration is highly species dependent and also affected by the experimental conditions. Controlled exposure studies have reported increased (Arisi et al 2000, Schützendübel et al 2001) or decreased (Grill et al 1987, Tukendorf and Rauser 1990) concentrations of GSH in plants and yeasts after Cd exposure. In the cases where a decline in intracellular GSH is reported, this decline often is attributed to GSH consumption by PC synthesis. Thus our aforementioned observation of negative correlations between GSHTOT and metal exposure in B. edulis called for a closer investigation of the importance of low-molecular, sulfur-containing chelating agents such as PCs in this species. If PC synthesis is induced in this species during metal exposure, this could also account for the observation that commonly as much as 70% of the cytosolic Cd in fruiting bodies is not precipitated (our unpublished results) together with the cytosolic proteinaceous compounds by the ethanol precipitation procedure followed in our previous study (Collin-Hansen et al 2003).
In several plant and microorganism species, increased intracellular concentrations of free proline (Pro) have been implicated in ameliorating environmental stress, including exposure to Cd and certain other toxic metals (Delauney and Verma 1993, Xiong and Zhu 2002). In plants subjected to metal stress, intracellular accumulation of Pro is associated with reduced damage to membranes and proteins (Alia et al 1997, Shah and Dubey 1998, Verma 1999), possibly by scavenging of hydroxyl radicals (Smirnoff and Cumbes 1989) and singlet oxygen (Alia et al 2001) by free Pro. Hare and Cress (1997) suggested that increased Pro levels might alleviate cytoplasmic acidosis, stabilize the NADP+/NADPH ratio at values compatible with metabolism and provide reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP for recovery from stress-induced damage. The Pro-dependent increase of cytoplasmic GSH in Cd-treated Chlamydomonas algae was shown to aid sequestration and detoxification of Cd as phytochelatin conjugates (Siripornadulsil et al 2002).
The goal of the present study was to determine whether MTs, PCs or Pro are induced in B. edulis as a response to elevated metal concentrations in the substrate. Unlike many other techniques, high performance liquid chromatography-mass spectrometry (HPLC-MS) offers the promise of identifying compounds with high accuracy, often without demanding numerous other experiments. However, due to the complexity of the fungal matrix, size exclusion chromatography (SEC) was used as an initial separation step before HPLC-MS. This two-dimensional approach also aided a more detailed study of the metal-binding compounds present in the cytosolic fraction of B. edulis.
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MATERIALS AND METHODS |
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Site description.— – The exposed study area is located at Odda, southwestern Norway (60°04'N, 6°33'E). Odda is a small town (population approximately 7500) located at the head of the Sørfjord, a north-south trending extention of the Hardangerfjord. The town of Odda is shielded from strong winds by mountains ranging up to 1000 m on the W and E sides of the Sørfjord, and local wind patterns are dominated by circulating winds, causing entrapment of air pollutants within the valley. Annual precipitation at Odda averages 1500 mm.
The Outokumpu Norzink Zn smelter, the only Zn smelter in Scandinavia, has been the major contributor to the dispersion of metals to the marine and terrestrial environment, the release of metals (predominantly Zn and Pb released to the fjord) from this factory alone exceeding 10 tons per day during 1975–1986 (Melhuus et al 1978, Storaas and Skei 1996). Substantial amounts of Zn, Pb, Cd, Hg, Cu and a range of other elements also have been emitted into the air since production started in 1929 (Storaas and Skei 1996). Since 1986 extensive efforts have been devoted to reducing the emissions to the fjord and the atmosphere. Four sample sites (designated from north to south Odda 1, 2, 3 and 4) were chosen in spruce plantations at altitudes of 20–500 masl within a 2.5 km radius from the smelter, along the western coast of the Sørfjord (see FIG. 1).
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Sampling and sample preparation.— –
Sampling at Odda ("exposed group", n = 24) was performed in Sep 2000 and Sep 2002. Reference samples ("reference group", n = 18) were collected in Sep 2002. Young, seemingly healthy fruiting bodies of Boletus edulis were sampled according to our previous study (Collin-Hansen et al 2002), put individually in clean paper bags and taken to the laboratory in Trondheim within 20 h.
After inspection of fruiting bodies for parasites and contamination by foreign material, a stainless-steel knife was used to prepare subsamples ("cake slices") of the cap by cutting radially. These subsamples were treated as aliquots of the sample and will be referred to as "whole cap" below. Samples were stored individually in clean PE clip-lock bags (–80 C), and samples from the 2000 and 2002 sampling rounds were randomized for further analyses.
Element determinations.— –
Tissue aliquots ("whole cap") for metal determinations were digested in nitric acid, and metal concentrations were determined by AAS (in the case of Cd, Zn and Cu) or CVAFS (in the case of Hg) according to our previous report (Collin-Hansen et al 2005b). The same methods were used to analyze chromatographic fractions of the samples from the "exposed group" with respect to Cd, Zn, Cu and Hg, whereas chromatographic fractions from reference samples were analyzed by high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) using a Thermo (Finnigan) model element instrument with settings given in Erikson et al (2004).
Control of analytical procedures.— – To check for possible drift in the instruments used in metal determinations, standard solutions with known elemental concentrations were analyzed every 10 samples. In addition blank samples and standard reference material (bovine liver [1577a, b] and tomato leaves [1573], US National Institute of Standards and Technology) were analyzed every 10 samples. Randomly chosen samples were re-analyzed.
Recoveries in metal determinations.— –
Recoveries of the four metals in randomly selected samples were determined by the method of standard addition and calculated from the ratio of the amount of the elements recovered after spiking to the amount added. Recoveries were 93–99% for Cd (94% of the samples 
95% recovery), 87–101% for Zn (50% of the samples 95% recovery), 93–103% for Cu (98% of the samples 95% recovery), 84–101% for Hg (85% of the samples 95% recovery). Metal concentrations in standard reference materials deviated from the average by at most –2% for Cd, –12% for Zn, + 3% for Cu and –3% for Hg and were normally well within the limits of the certified values.
Data presentation.— – The ArcView GIS version 3.3 software (ESRI, Redlands, California) was used to create maps showing metal concentrations in fruiting bodies (whole cap) at Odda. Grouping of the samples was done with the NATURAL BREAKS function in the software to minimize the variation within each group, whereas the variation between groups is maximized.
Preparation of cytosolic extracts.— – Frozen (–80 C) samples (whole cap) were thawed on ice and homogenized with 3x its mass of Tris buffer (30 mM, 250 mM NaCl, pH 7.6) with three sequential pulses of 5 s each using a Heidolph DIAX 900 tissue homogenizer equipped with a 6G tool (Heidolph, Kelheim, Germany). After removal of cell debris by centrifugation (30 000g, 4 C, 15 min), aliquots (1.5 mL) of supernatant were frozen at –80 C. Aliquots were thawed on ice and used in the methods described below, except for determinations of free Pro.
Total cytosolic protein concentration.— –
To normalize CCBC values to total protein concentration in cytosol, cytosolic extracts prepared from whole cap or segments were analyzed with respect to total cytosolic protein according to Bradford (1976), by using Coomassie blue reagents (Bio-Rad, Munich, Germany). Bovine serum albumin (BSA) was used as standard. The standard curve was linear 50–300 µg protein mL–1 sample, and recoveries were 90–98%.
Cytosolic Cd-binding capacity (CCBC).— –
Cytosolic Cd-binding capacities (CCBCs) of heat-stable Cd-binding molecules, such as MT, PC and GSH in cytosolic extracts of B. edulis (whole cap or segments), were estimated by the cadmium-Chelex assay (Bartsch et al 1990). In this assay Cd is allowed to distribute between the cation exchanger Chelex-100 and heat-stable cytosolic compounds. The fraction of Cd remaining in the supernatant after removal of Chelex-100 by centrifugation gives a measure of CCBC. Due to the lack of any kind of MT reference material of macromycete origin, rat liver MT II isolated in our laboratory was used as standard. The calibration curve was linear throughout the studied interval (~4 to ~80 µg MT mL–1 sample). Recoveries were 90–94%. Results were expressed as mg MT equivalents g–1 total cytosolic protein, assuming a molar ratio of seven Cd atoms per MT molecule and a molecular weight of the protein of 7.0 kDa (Hamer 1986, Gadd 1993).
Free proline in cytosol.— –
Concentrations of free Pro in cytosolic extracts were determined according to Bates (1973) with minor modifications concerning only the preparation of cytosolic extracts. L-Pro was used to make the standard curve and for recovery measurements. The standard curve was linear 0.025–4.0 µmol Pro mL–1 sample. Recoveries fell within 86–93%. Results were expressed as µmoles Pro g–1 fresh weight material.
Size exclusion chromatography (SEC).— – One single step of SEC was used to separate Cd-binding compounds in all samples (whole cap) from Odda and a selection of three reference samples (randomly chosen among the samples from site Håen) before analysis of a subset of samples by HPLC-MS.
Cytosolic extracts of all 42 samples were prepared in Tris buffer (30 mM, 250 mM NaCl, pH 7.6) as described above and filtered through a 0.45 µm mesh. Sample (500 µL) was loaded onto a Superdex 30 gel filtration column (Ø: 16 mm, h: 60 cm) connected to an Äkta Prime chromatography system (Amersham Biosciences, Piscataway, New Jersey) and eluted at 0.5 mL min–1 with Tris buffer (30 mM, 250 mM NaCl, pH 7.6). The elutate was monitored at 254 nm wavelength before passing through to the fraction collector. Fractions (2 mL) were collected. Two parallel runs were performed consecutively for each sample, resulting in a final volume of 4 mL in each fraction tube. Due to the low endogenous Cd concentrations in the reference samples, parallel runs of samples spiked with the radiotracer 109Cd were performed for the three reference samples as well as for a selection of the exposed samples. The column was calibrated under the same conditions by running mixtures of the pure standards aprotinin (6512 Da), glutathione (307 Da) and L-phenylalanine (165.2 Da). The elution volumes (Ve) of these molecular-mass markers were used to estimate the molecular masses of fungal compounds by interpolation from the standard curve, according to Trathnigg (2000). A linear relationship (Pearson’s r = 1.00) was established between log MW and Ve for the standards.
To achieve complete digestion of organic compounds before metal determinations, nitric acid (65%, p.a., 1 mL per 1.5 mL elutate) was added to each fraction tube. Tubes were sealed and heated (70 C, 18 h). Concentrations of Cd and Zn were determined by flame AAS as described above. Cu determinations were omitted due to interference of the Tris buffer with the analyte at the wavelength of interest, possibly due to the high salt concentration in the buffer. Parallel runs for Hg determinations in elutate fractions resulting from five consecutive parallel runs were performed for a selection of samples. Cd-containing fractions from parallel SEC runs were filtered through a 0.20 µm mesh, subsamples were pooled, and individual fractions or pooled samples were separated further and analyzed by HPLC-MS without acidification, preconcentration or desalting.
High-performance liquid chromatography-mass spectrometry (HPLC-MS).— – Presence of individual PCs in the Cd-containing fractions from SEC were investigated for a selection of 10 samples (five randomly chosen exposed samples from Odda and five reference samples randomly selected from sites Vassfjellet and Håen) by reversed phase HPLC-MS on an Agilent 1100 series HPLC-MS (Agilent, Palo Alto, California) equipped with an MSD IonTrap SL and the ChemStation software (Agilent). Sample (15 µL) was transferred onto a Brownlee New Guard Aquapore (15 x 0.32 mm) capping column (Applied Biosystems, Foster City, California) connected directly to a Brownlee Spheri-5, RP-18 analytical column (100 x 4.6 mm, Perkin Elmer). The HPLC column elutate was monitored at 200 nm and positively ionized by electron spray ionization, as analyses of both samples and PC standards revealed considerably higher sensitivity in the positive mode compared to negative ionization. The mobile phase was delivered at 1.0 mL min–1. Peptides were eluted with a gradient from buffer A (0.1% trifluoracetic acid [TFA], 99.9% Milli-Q water) to buffer B (0.1% TFA, 20% acetonitrile, 79.9% Milli-Q water). After an initial 2 min wash with buffer A, peptides were eluted with a linear gradient of 0–100% buffer B for 10 min. Identification of GSH and individual PCs was based on comparisons of elution times and m/z ratios with those obtained for standards in parallel runs.
Normalization of data.— – Samples were analyzed in random order. All measurements were carried out in duplicate. Before statistical analyses, data of elemental concentrations in fungal samples were normalized with the fresh-weight data, whereas CCBC values were normalized using total cytosolic protein concentrations.
Statistical treatment of data.— – Distribution analysis of the data revealed that they were generally not normally distributed. Furthermore the bivariate correlations could not necessarily be assumed to be linear. Consequently nonparametric Spearman’s correlation analysis was chosen to indicate the degree of monotonic, but not necessarily linear, correlation. Nonparametric Mann-Whitney U-test was used for comparisons between means of variables in the two groups ("exposed" vs. "reference"). Before statistical analyses mean values were calculated from the determined metal concentrations and CCBC in segments of three different fruiting bodies from each of the sites Odda 1 (exposed site) and Vassfjellet (reference site). P < 0.05 was chosen as the level of statistical significance throughout.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFINAL REMARKSLITERATURE CITED |
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). CCBC for the 24 specimens of the exposed group (22 ± 5.6 mg g–1) was greatly increased compared to the 18 reference samples (0.24 ± 0.12 mg g–1). The difference was statistically significant at P < 0.001.
Using Spearman correlation analysis, significant bivariate correlations were found among CCBC and all of the four metals (P < 0.001 for all correlations). The highest rSp for association with CCBC was found for Zn (rSp = 0.82) followed by Cd (rSp = 0.80), Cu (rSp = 0.77) and finally Hg (rSp = 0.65). The results are shown graphically for Cd and Zn (FIG. 2).
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SEC analysis of cytosolic extracts from all 24 samples (whole cap) from Odda and three reference samples (from site Håen) showed similar elution profiles regarding absorbances at 254 nm of the elutates of all 27 samples. Metal determinations in the elutate however revealed large intersample variations in metal profiles. The elution of almost all cytosolic Hg in the void volume (V0, eluted around 50 mL, see FIG. 3), was common for all 27 samples. Varying fractions of Cd and Zn were eluted here as well. Estimated molecular masses of the compounds in V0 are > 12 000 Da.
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). Noticing the elution volumes (Ve) for the molecular mass standards, it becomes evident that the second Zn peak (Ve = 103 mL) coincides with the elution of the glutathione (GSH) marker in parallel runs. This indicates that Zn is associated with GSH, or a compound (or compounds) with estimated molecular mass similar to that of GSH, in these fractions. The identity of the compound(s) eluted in the third Zn peak (at approximately 113 mL) is still uncertain.
In all samples from Odda, the elution of a second, broad Cd peak (starting at Ve = 92 mL in FIG. 3) begins before the second Zn peak, indicating the association of Cd with molecules of slightly larger molecular mass than that of GSH. However parts of this Cd peak coincides with the second Zn peak, as well as with the elution of GSH in parallel runs. From this it is reasonable to speculate that the Zn-binding compound, or compounds, eluted around Ve = 103 mL, exhibit(s) a significant affinity for Cd as well. This is in accordance with our suggestion that GSH is involved in complexation of Zn in B. edulis. Molecular masses of the Cd-binding compounds eluted in the Cd peak starting at Ve = 92 mL were estimated by interpolation and were 300–1000 Da. A significant negative correlation was observed for the 24 samples from Odda between Ve of the first fraction of this Cd peak and Cd concentration in whole cap (rSp = –0.59, P < 0.01).
Of note, the second Cd peak (eluted around Ve = 92, FIG. 3) appears broader for the exposed samples compared to the reference samples. More specifically the substantially narrower Cd peak observed for the reference samples is eluted in the same fractions as the second Zn peak (i.e. not skewed to the left). Due to the lower endogenous Cd concentrations in the reference samples, parallel runs of cytosolic extracts that were spiked with the radiotracer 109Cd and incubated 30 min before application onto the column, were performed for a selection of samples. This confirmed the skewing of the second Cd peak to the left in exposed samples relative to reference samples (data not shown).
For the exposed samples a final fraction of Cd is eluted as a very wide peak, at a Ve of approximately 135–180 mL. The elution of this peak at a significantly higher Ve compared to L-phenylalanine in parallel runs suggests the Cd eluted here exists mainly as "free" (i.e. hydrated) Cd2+ ions. Cd was not detected in these fractions for unspiked reference samples, and only very low activities of the radiotracer were recorded in these fractions for spiked samples (data not shown).
HPLC-MS analysis of the fractions constituting the second Cd peak from SEC (pooled samples of the fractions eluted at 92–107 mL for the sample in FIG. 3) revealed the presence of phytochelatins (PCs) as well as GSH and GSSG in these fractions for the five exposed samples (from Odda) that were analyzed. FIGURE 4 shows selected MS spectra from HPLC-MS analysis of the sample from site Odda 1 for which the metal distribution in SEC eluate is displayed (in FIG. 3). Compounds with m/z ratios identical to positively ionized PCs elute synchronously with PCs from a standard mixture are shown (FIG 4A). A closer investigation of the mass spectra obtained for the exposed sample revealed the presence of PCs ranging in complexity up to PC 4, well above the background (FIG. 4B–D). These results demonstrate that GSH and PCs were ionized by absorbing one H+. Molecular masses of the detected compounds are 307 Da for GSH (protonated ion (m + H+) detected at m/z 308), 539 Da for PC 2 (m + H+ at m/z 540), 771 Da for PC 3 (m + H+ at m/z 772), and 1003 Da for PC 4 (m + H+ at m/z 1004).
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). PC 2 and PC 3 were detected in all samples from Odda, except one in which PC 2 was the only PC detected. GSH and GSSG were detected in all samples analyzed from Odda. In Odda samples HPLC-MS analyses of individual fractions from SEC showed a trend toward decreasing complexity (and hence decreasing molecular mass) with increasing Ve from SEC. GSH and GSSG, but no PCs, were detected in SEC elutates from the five reference samples. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFINAL REMARKSLITERATURE CITED |
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) only six fruiting bodies of B. edulis were collected from three sites at Odda. To obtain more material for statistical analyses, more material was collected from Odda in 2000 and 2002.
The results of the metal determinations demonstrate increasing concentrations of Cd, Zn, Hg and Cu in B. edulis fruiting bodies approaching the Out-okumpu Norzink Zn smelter at Odda (FIG. 1). Metal concentrations in fruiting bodies seem high compared to corresponding data reported for B. edulis and a range of other macromycete species collected near smelters (Svoboda et al 2000, Kala et al 2004). Important in this respect is the fact that the present study investigated metal concentrations in whole caps, while previous studies regarding metal uptake in different macromycete species have dealt mainly with whole fruiting bodies.
The decision to analyze whole caps and stipes separately was made primarily because in B. edulis the size of the cap relative to that of the stipe is highly variable. In addition from a previous study we were aware of the substantially higher concentrations of Cd, Zn and Cu, as well as CCBC, in whole cap compared to the stipe of this species (Collin-Hansen et al 2002). Similar observations have been made for the distribution of Hg in B. edulis (Kala and 
lapetová 1997). These differences in elemental composition between cap and stipe tissues are reflected by the SEC elution profiles for the metals included in this study. When SEC was performed with whole fruiting bodies rather than caps, the UV chromatogram as well as the metal elution profiles were strongly dependent on the mass ratios between cap and stipe (data not shown). Recent advances in the field of proteomics have made it clear that analysis of individual tissues or cell types can be critical to reduce the complexity of biological samples before identification of biomolecules by HPLC-MS. Given the problems associated with growing ectomycorrhizal fungi (or cells from those organisms) in the lab we think that studies focusing on their molecular biology should focus more on individual tissues.
The mechanisms underlying uptake of Cd in mycorrhizal fungi deserve further exploration, partly due to the possibility of transfer of Cd from soil to humans or grazing animals by mycorrhizal fungi. The considerable number of studies suggesting increased tolerance of Cd toxicities and certain other metals in plants conferred by ectomycorrhizal associations (Brown and Wilkins 1985, Denny and Wilkins 1987, Colpaert and van Assche 1992) should spur further investigation of the mechanisms involved in Cd uptake in mycorrhizal fungi. Of importance, it is now recognized that the amelioration of metal stress in plants by ectomycorrhizal symbiosis is not a general phenomenon and that whether such protective effects do occur is highly dependant on the plant and fungal species, as well as the metal of interest (Jones and Hutchinson 1986, Galli et al 1994).
CCBC.— –
Our previous finding of much higher CCBC in B. edulis fruiting bodies growing near the Outokumpu Norzinc smelter at Odda compared to reference samples lead us to investigate further the relationship between CCBC and metal exposure in this species, using a larger sample number than in our previous study (Collin-Hansen et al 2002). The finding in the present study of a > 90-fold increase in CCBC in the exposed samples compared to the reference samples, as well as an apparent dose-response relationship (FIG. 2), confirm our previous data. Taken together these observations strongly indicate that Cd-binding compounds are synthesized in this species when exposed to high concentrations of metals such as these present at Odda.
Free Pro.— –
To our knowledge the possible association between metal exposure and concentration of free Pro has not been investigated previously in macromycetes. Siripornadulsil et al (2002) demonstrated a role for free Pro in Cd detoxification in algae by maintenance of a more reducing redox state of the cell, thus securing that the GSH:GSSG ratio remains compatible with PC synthesis. The lack of significant differences in concentrations of free Pro between the exposed group vs. the reference group in the present study indicates that induction of free Pro does not serve as an important mechanism for stabilizing GSH in B. edulis.
SEC.— –
Knowledge of speciation and binding forms of toxic metals is important to understand the mechanisms involved in fungal tolerance of excessive metal concentrations. SEC has been used in some studies to describe the speciation of metals in certain mycorrhizal macromycetes (Esser and Brunnert 1986, Galli et al 1993, Howe et al 1997). In our opinion Superdex 30 is a column material that under optimized conditions gives a superior separation of compounds with a low and medium molecular mass compared with the column materials used in these previous studies. Therefore we decided to conduct a rather detailed study of the distributions of Cd, Zn and Hg in B. edulis by SEC with this column material before HPLC-MS. Preliminary runs of cytosolic extracts or standards showed that a relatively high salt concentration of 250 mM NaCl in the mobile phase was necessary to obtain sufficient separation of fungal compounds, as well as a linear standard curve. Deviations among parallel SEC runs of aliquots of the same sample were negligible due to the automated procedure for sample application and elution allowed by the Äkta Prime apparatus. Consequently several parallel runs could be collected in the same test tubes to obtain enough elutate for metal determinations without compromising the chromatographic separation.
Superdex 30 provides a molecular mass cut-off of 12 kDa. Thus cytosolic molecules with an apparent molecular mass above this value will be eluted in V0, including a major fraction of the cytosolic proteins. The observation that almost all Hg and a significant fraction of the Cd were eluted in V0 (FIG. 3) might reflect the interference of these nonessential metals with biomolecules, such as enzymes and structural proteins.
Notably, redistribution of metals between cellular compounds, likely to occur during sample preparation and SEC, introduces major uncertainties to the discussion of the relevance of the SEC results to conditions in vivo. Nevertheless, depending on the biological roles of the presumably wide range of compounds eluted in V0, one may speculate that binding of Cd and/or Hg by some of these compounds occurs in the living cell and might be harmful. Studies of several enzymes and structural proteins containing Zn have demonstrated that substitution of Cd or Hg for the chemically related Zn may take place, often with devastating effects on the structure and/or biological function of the protein (Hussain et al 1987, Hanas and Gunn 1996).
Relevant in this respect is our recent observation of elevated oxidative damage to DNA and lipids in B. edulis collected at Odda, compared to reference samples (Collin-Hansen et al 2005b). It is well recognized that one mechanism by which Cd and Hg exert their toxicity is by increasing cellular levels of reactive oxygen species (ROS). However the mechanisms by which this occurs are still unclear. It seems probable that interference of Cd and/or Hg with antioxidant enzymes may contribute to such oxidative damage (Hussain et al 1987, Casalino et al 2002).
It is likely that the Cd-binding protein isolated from B. edulis in a previous report from our group (Collin-Hansen et al 2003) is eluted in V0. However further research is needed to identify the compounds that bind Cd, Zn and Hg in V0.
At this point in the experimental work, several observations from SEC suggested the involvement of phytochelatins in detoxification of Cd in B. edulis collected at Odda. These observations were the breadth of the second Cd peak in these samples and the fact that the start of elution of this peak was both before the Zn peak in the same run, as well as before the GSH peak in parallel runs. In addition the recent observation of negative correlations between concentrations of metals and GSHTOT in whole cap (Collin-Hansen et al 2005a) could be explained by the induction of PCs. The negative correlations established between metal concentration and GSHTOT were significant at P < 0.001 for Cd and Zn and at P < 0.05 for Hg but not significant for Cu (Collin-Hansen et al 2005a).
HPLC-MS.— –
To our knowledge the present paper is the first to report the presence of PCs in a macromycete. Previous research has shown the expression of PCs in response to metal exposure in some lower fungi, including yeasts (Winge et al 1998, Kneer et al 1992) and the zygomycete Mucor racemosus (Miersch et al 2001). In these species the major role exerted by GSH in metal detoxification appears to be indirect (i.e. that it is converted to PCs before playing a significant role in metal binding). In other species however GSH plays a direct role in the defense against elevated concentrations of transition metals (Li et al 1997, Winge et al 1998). Our results indicate that a truncated PC species, Des-Gly PC 2, also is expressed in B. edulis as a response to metal toxicity, but more data is needed to confirm a role for this species in metal handling in macromycetes.
With histochemical staining Morselt et al (1986) found SH-containing compounds being induced by Cd in hyphae of B. edulis. These researchers suggested the results could be due to induction of MTs, however the results from the present study indicate the observations could be explained by an induction of PCs.
After binding of Cd to GSH or PC these complexes may be readily transported into the vacuoles of plants and yeasts (Li et al 1997, Cobbett and Goldsborough 2002). Inside the vacuole the complexes may condense to form CdS-rich granules. A recent study by Ott et al (2002) demonstrated the formation of vacuolar electron-dense bodies displaying a high degree of correlation between Cd and S concentrations in mycelium of Paxillus involutus. This finding suggests that sequestering of complexes of GSH or PCs with Cd to vacuoles might be an important detoxification mechanism in macromycetes as well. Thus the vacuole might function as a cellular sink for Cd, allowing for an extensive uptake of this element, provided that sulfur is not a limiting factor and that synthesis of GSH and PCs is sufficient.
The finding of PCs in B. edulis exposed to a range of metals from the Outokumpu Norzinc smelter at Odda provides a reasonable explanation to our recent observation that GSHTOT concentrations are correlated negatively with metal exposure in this species (Collin-Hansen et al 2005a), given the essential role played by GSH as a building stone in PC synthesis. When viewed together these results offer a consistent picture showing that GSH synthesis may be a rate-limiting step in the synthesis of PCs in this species under metal stress. These findings are in accordance with results obtained by Miersch et al (2001) from controlled Cd exposure experiments with the zygomycete Mucor racemosus, which was found to respond to Cd exposure by synthesizing PCs on the expense of cellular GSH.
It is interesting to note that one single step of SEC before HPLC-MS offered sufficient removal of compounds interfering with the HPLC-MS analysis. The observed trend toward more complex PCs being eluted from SEC before smaller PCs and GSH for the samples from Odda also demonstrates the high resolution of the Superdex-30 column used in SEC. Several other methods were tested as preceding steps before HPLC-MS, such as anion exchange chromatography on a DEAE Sephadex A-25 column, as well as a combination of SEC and anion exchange chromatography (with SEC as the first or the last step). These methods provided results similar to those showed here for SEC alone.
Preliminary studies by SEC (followed by metal determinations) and HPLC-MS of B. edulis growing on soil polluted by a former Cu smelter indicate low expression of PCs in these samples and that GSH seems to be the main complexing agent for Cu, Cd and Zn. Hg however elutes from the Superdex 30 SEC column in V0 also for these samples, indicating its association with high molecular-mass compounds, which is similar to the results for B. edulis from Odda.
These observations are in good agreement with the results obtained by recalculation of the data from the GSHTOT determinations in our recent study (Collin-Hansen et al 2005a). The nonparametric Mann-Whitney test revealed significantly lower GSHTOT concentrations in the samples from Odda when compared to the samples collected near the former Cu smelter (P < 0.01) or to reference samples (P < 0.001). No significant difference was found however between the samples exposed to emissions from a Cu smelter and the reference samples. Thus, even though these data suggest an important role for GSH as a complexing agent for Cu, Zn, and Cd in B. edulis exposed to high concentrations of Cu as well as considerable concentrations of Zn and Cd, they do not suggest the induction of GSH synthesis by Cu.
Metal determinations in SEC elutate failed to indicate the presence of MTs in any of the samples. If MTs (molecular mass approximately 7 kDa) were present they were expected to elute from SEC at a Ve somewhat lower than for the aprotinin marker in parallel runs. No significant peaks in UV absorbance, endogenous metals or, in spiked samples, the radiotracer 109Cd were observed in these fractions for any of the samples (FIG. 3). Thus it seems evident that in B. edulis the MT-like proteins determined in a previous paper from our group (Collin-Hansen et al 2002) are not true MTs. The results from HPLC-MS in the present study, in conjunction with our recent observation of a negative correlation between metal exposure and GSHTOT, strongly indicate that PCs, rather than MTs (or GSH) are responsible for the dose-dependent increase in CCBC noted for this species.
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ACKNOWLEDGMENTS |
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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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———, ———, Matysik J. 2001. Effect of proline on the production of singlet oxygen. Amino Acids 21:195–200.[CrossRef][Medline]
Allen RO, Steinnes E. 1978. Concentrations of some potentially toxic metals and other trace elements in wild mushrooms from Norway. Chemosphere 4:371–378.
Arisi ACM, Mocquot B, Lagriffoul A, Mench M, Foyer CH, Jouanin L. 2000. Responses to cadmium in leaves of transformed poplar overexpressing gamma-glutamylcysteine synthetase. Physiol Plant 109:143–149.[CrossRef]
Bartsch R, Klein D, Summer KH. 1990. The Cd-chelex assay: a new sensitive method to determine metallothionein containing zinc and cadmium. Arch Toxicol 64:177–180.[CrossRef][Medline]
Bates LS. 1973. Rapid determination of free proline from water-stress studies. Plant Soil 39:205–207.[CrossRef]
Bauer Petrovska B. 1999. Mineral composition of some Macedonian edible mushrooms. Acta Pharmaceut 49: 59–64.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.[CrossRef][Medline]
Brown MT, Wilkins DA. 1985. Zinc tolerance of mycorrhizal Betula. New Phytol 99:101–106.[CrossRef]
Casalino E, Calzaretti G, Sblano C, Landriscina C. 2002. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 179:37–50.[CrossRef][Medline]
Clemens S, Simm C. 2003. Schizosaccharomyces pombe as a model for metal homeostasis in plant cells: the phytochelatin-dependent pathway is the main cadmium detoxification mechanism. New Phytol 159:323–330.[CrossRef]
Cobbett CS. 2000. Phytochelatins and their roles in heavy metal detoxification. Plant Physiol 123:825–832.
———, Goldsborough PB. 2002. Phytochelatins and metal-lothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53:159–182.[CrossRef][Medline]
Collin-Hansen C, Yttri KE, Andersen RA, Berthelsen BO, Steinnes E. 2002. Mushrooms from two metal-contaminated areas in Norway: occurrence of heavy metals and metallothionein-like proteins. Geochem: explor, enviro, analy 2:121–130.[CrossRef]
———, Andersen RA, Steinnes E. 2003. Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis. J Phys IV 107:311–314.
———, ———, ———. 2005a. Molecular defense systems are expressed in the king bolete (Boletus edulis) growing near metal smelters. Mycologia 97:973–983.
———, ———, ———. 2005b. Damage to DNA and lipids in Boletus edulis exposed to metals. Mycol Res 109: 1386–1396.[CrossRef][Medline]
Colpaert JV, van Assche JA. 1992. Zinc toxicity in ectomycorrhizal Pinus sylvestris. Plant Soil 143:201–211.[CrossRef]
Delauney A, Verma DPS. 1993. Proline biosynthesis and osmoregulation in plants. Plant J 4:215–223.[CrossRef]
Denny HJ, Wilkins DA. 1987. Zinc tolerance in Betula spp. IV. The mechanism of ectomycorrizal amelioration of zinc toxicity. New Phytol 106:545–553.
Erikson KM, Syversen T, Steinnes E, Aschner M. 2004. Globus pallidus: a target brain region for divalent metal accumulation associated with dietary iron deficiency. J Nutr Biochem 15:335–341.[CrossRef][Medline]
Esser J, Brunnert H. 1986. Isolation and partial purification of cadmium-binding components from fruiting bodies of Agaricus bisporus. Environ Pollut 41:263–275.
Frey B, Zierhold K, Brunner I. 2000. Extracellular complexation in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abies–Hebeloma crustuliniforme ectomycorrhizas. Plant Cell Environ 23:1257–1265.[CrossRef]
Gadd GM. 1993. Interactions of fungi with toxic metals. New Phytol 124:25–60.[CrossRef]
Galli U, Meier M, Brunold C. 1993. Effects of cadmium on nonmycorrhizal and mycorrhizal Norway spruce seedlings (Picea abies [L.] Karst.) and its ectomycorrhizal fungus Laccaria laccata (Scop. Ex Fr.) Bk. & Br.: sulphate reduction, thiols and distribution of the heavy metal. New Phytol 125:837–843.[CrossRef]
———, Schüepp H, Brunhold C. 1994. Heavy metal binding by mycorrhizal fungi. Physiol Plant 92:364–368.[CrossRef]
Grill E, Winnacker EL, Zenk MH. 1987. Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proc Nat Acad Sci USA 84:439–443.
Hall IR, Lyon AJE, Wang Y, Sinclair L. 1998. Ectomycorrhizal fungi with edible fruiting bodies—2. Boletus edulis. Econ Bot 52:44–56.
Hamer D. 1986. Metallothionein. Ann Rev Biochem 55:913–951.[Medline]
Hanas JS, Gunn CG. 1996. Inhibition of transcription factor IIIA-DNA interactions by xenobiotic metal ions. Nucleic Acid Res 24:924–930.
Hare PD, Cress WA. 1997. Metabolism implications of stress-induced proline accumulation in plants. Plant Growth Regul 21:79–102.[CrossRef]
Howe R, Evans RL, Ketteridge SW. 1997. Copper-binding proteins in ectomyccorrhizal fungi. New Phytol 135: 123–131.[CrossRef]
Hussain T, Shukla GS, Chandra SV. 1987. Effects of cadmium on superoxide dismutase and lipid peroxidation in liver and kidney of growing rats: in vivo and in vitro studies. Pharmacol Toxicol 60:355–358.[Medline]
Jones MD, Hutchinson TC. 1986. The effect of mycorrhizal infection on the response of Betula papyrifera to nickel and copper. New Phytol 102:429–442.[CrossRef]
Kala P, lapetová M. 1997. Mercury contents in fruiting bodies of wild growing edible mushrooms. Potrav Ve dy 15:405–410.
———, Svoboda L. 2000. A review of trace element concentrations in edible mushrooms. Food Chem 69: 273–281.[CrossRef]
———, ———, Havlíc ková B. 2004. Contents of cadmium and mercury in edible mushrooms. J Appl Biomed 2: 15–20.
Kneer R, Kutchan TM, Hochberger A, Zenk MH. 1992. Saccharomyces cerevisiae and Neurospora crassa contain heavy metal sequestering phytochelatin. Arch Microbiol 157:305–310.[CrossRef][Medline]
Li ZS, Lu YP, Zhen RG, Szczypka M, Thiele DJ, Rea PA. 1997. A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium. Proc Nat Acad Sci USA 94:42–47.
Mehra RK, Winge DR. 1991. Metal ion resistance in fungi: molecular mechanisms and their regulated expression. J Cell Biochem 45:30–40.[CrossRef][Medline]
Melhuus A, Seip KL, Seip HM, Myklestad S. 1978. A preliminary study of the use of benthic algae as biological indicators of heavy metal pollution in Sorfjorden, Norway. Environ Pollut 15:101–107.[CrossRef]
Miersch J, Tschimedbalshir M, Bärlocher F, Grams Y, Pierau B, Schierhorn A, Krauss GJ. 2001. Heavy metals and thiol compounds in Mucor racemosus and Articulospora tetracladia. Mycol Res 105:883–889.[CrossRef]
Morselt AFW, Smits WTM, Limonard T. 1986. Histochemical demonstration of heavy metal tolerance in ectomycorrhizal fungi. Plant Soil 96:417–420.[CrossRef]
Münger K, Lerch K. 1985. Copper metallothionein from the fungus Agaricus bisporus: chemical and spectroscopic proerties. Biochemistry 24:6751–6756.[CrossRef]
Ott T, Fritz E, Polle A, Schützendübel A. 2002. Characterisation of antioxidative systems in the ectomycorrhiza-building basidiomycete Paxillus involutus (Bartsch) Fr. and its reaction to cadmium. FEMS Microbiol Ecol 42: 359–366.[Medline]
Rauser WE. 1999. Structure and function of metal chelators produced by plants. Cell Biochem Biophys 31:19–48.[CrossRef][Medline]
Ross IS. 1994. Uptake of zinc by fungi. In: Winkelmann G, Winge DR, eds. Metal Ions in Fungi. Mycology series, vol 11. New York: Dekker. p 237–257.
Schützendübel A, Schwanz P, Teichmann T, Gross K, Langenfeld-Heyser R, Godbold DL, Polle A. 2001. Cadmium induced changes in antioxidative systems, H2O2 content, and differentiation in Scots pine roots (Pinus sylvestris). Plant Physiol 127:887–898.
Shah K, Dubey RS. 1998. Effect of cadmium on proline accumulation and ribonuclease activity in rice seedlings: role of proline as a possible enzyme protectant. Biol Plant 40:121–130.
Siripornadulsil S, Traina S, Verma DPS, Sayre R. 2002. Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. Plant Cell 14:2837–2847.
Skwarzec B, Jakusik A. 2003. 210Po bioaccumulation by mushrooms from Poland. J Environ Monit 5:791–794.[CrossRef][Medline]
Smirnoff N, Cumbes Q J. 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28:1057–1060.[CrossRef]
Storaas R, Skei J. 1996. Ei miljøhistorie frå Sørfjorden. Odda: Vestnorsk Industristadmuseum. 57 p. (in Norwegian).
Svoboda L, Zimmermannová K, Kala P. 2000. Concentrations of mercury, cadmium, lead and copper in fruiting bodies of edible mushrooms in an emission of a copper smelter and a mercury smelter. Sci Tot Environ 246:61–67.[CrossRef]
Trathnigg B. 2000. Size-exclusion chromatography of polymers. In: Meyers R, ed. Encyclopedia of Analytical Chemistry. Chichester, UK: Wiley & Sons. p 8008–8034.
Tukendorf A, Rauser WE. 1990. Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium. Plant Sci 70:155–166.
Verma DPS. 1999. Osmotic stress tolerance in plants: role of proline and sulfur metabolisms. In: Shinozaki K, Yamaguchi-Shinozaki K, eds. Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants. Austin: R.G. Landers. p 153–168.
Wang Y, Sinclair L, Hall IR. 1995. Boletus edulis sensu lato: a new record for New Zealand. N Z J Crop Hort 23:227–231.
Winge DR, Sewell AK, Yu W, Thorvaldsen JL, Farrell H. 1998. Metal ion stress in yeast. In: Silver S, Walden W., eds. Metal Ions in Gene Regulation. New York: Chapmann & Hall. p 279–315.
Wuilloud RG, Kannamkumarath SS, Caruso JA. 2004. Speciation of essential and toxic elements in edible mushrooms: size-exclusion chromatography separation with on-line UV-inductively coupled plasma mass spectrometry detection. Appl Organometal Chem 18: 156–165.[CrossRef]
Xiong L, Zhu JK. 2002. Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ 25: 131–139.[CrossRef][Medline]
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