Damage to DNA and lipids in Boletus edulis exposed to heavy metals

Mycol. Res. 109 (12): 1386–1396 (December 2005). f The British Mycological Society

1386

doi:10.1017/S0953756205004016 Printed in the United Kingdom.

Damage to DNA and lipids in Boletus edulis exposed to heavy metals

Christian COLLIN-HANSEN1*, Rolf A. ANDERSEN2 and Eiliv STEINNES1 1

Department of Chemistry, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway. Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway. E-mail : [email protected]

2

Received 22 September 2004; accepted 6 August 2005.

This study investigates the potential of emissions from a zinc smelter to induce oxidative damage to DNA and lipids in Boletus edulis, the king bolete. Concentrations of cadmium, zinc, copper, and mercury were determined in 16 fruit bodies collected near the smelter (exposed group), as well as in 15 reference samples. Frequency of apurinic/apyrimidinic (AP) sites in DNA (a pre-mutagenic DNA base modification) and concentration of lipid hydroperoxides were chosen as damage parameters. Concentrations of the four metals, as well as oxidative damage to DNA and lipids were significantly elevated in the exposed group (Mann–Whitney, P<0.001). Both damage parameters correlated positively with concentrations of cadmium, zinc or copper in fruiting bodies (Spearman’s P<0.01). Frequency of AP sites correlated significantly with mercury in the fruit bodies (P<0.05), whereas the association between lipid hydroperoxides and mercury was insignificant. Frequency of AP sites correlated positively with concentration of lipid hydroperoxides (P<0.001). Negative trends for the associations between concentrations of metals and AP sites or lipid hydroperoxides in the reference group (significant only for mercury and lipid hydroperoxides ; P<0.05) suggest that in B. edulis low concentrations of mercury, possibly also of other of the metals determined in the present study, may induce dose-response relationships of a hormetic (‘J-shaped ’) nature.

INTRODUCTION Production of reactive oxygen species (ROS) is inevitable during aerobic metabolism. ROS generation may, however, increase during exposure to a range of chemicals such as certain transition metals, or adverse environmental conditions. The damaging effects of ROS are manifold, ranging from damage of nucleic acids, via lipid peroxidation, to inactivation of certain metabolic enzymes. Damage of DNA includes breaking of its sugar-phosphate backbone and alterations of its bases (Demple & Harrison 1994). Abasic sites, also called apurinic/apyrimidinic (AP) sites, are among the most prevalent DNA lesions. An AP site is formed when a DNA base such as A, C, G or T is removed, either by spontaneous depurination or deamination followed by removal of the deaminated base (Lhomme, Constant & Demeunynck 1999). The generation of AP sites in unstressed cells can be significant ; in mammalian cells under normal physiological conditions it was estimated that approx. 9000 AP * Corresponding author.

sites are generated per cell per day (Nakamura et al. 1998). Increased levels of ROS and alkylating agents may, however, greatly enhance this number. Detection of AP sites and examination of genetic integrity is therefore of prime importance in DNA diagnosis, since these nonfunctional sites are mutagenic and cytotoxic, and thus constitute a potential cause of DNA alterations, cell death and carcinogenesis (Sun, Qian & Yokota 2001). To our knowledge, damage to DNA by metals in macromycetes has not been investigated previously. On one hand, the protective effects of various antioxidants and radical scavengers strongly suggest the involvement of ROS in the generation of DNA damage induced by cadmium (Cd) (Filipicˇ & Hei 2004). On the other hand, growing evidence indicates that the carcinogenic potential of Cd is partly due to its ability to inactivate proteins involved in specific DNA repair events (Hartwig et al. 1998, Asmuss et al. 2000, Jin et al. 2003). Notably, Cd2+ was shown to inhibit the activity of AP endonuclease 1 (Ape1), the major mammalian abasic endonuclease (McNeill et al. 2004). Ape1 accounts for >95 % of the total AP site incision activity

C. Collin-Hansen, R. A. Andersen and E. Steinnes in mammalian cells, in addition to serving other important tasks in the repair of strand-breaks and certain 3k-mismatched nucleotides (Suh, Wilson & Povirk 1997, Chou & Cheng 2002). Lipid hydroperoxides are non-radical lipid intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters, and cholesterol itself. They are formed enzymatically or in non-enzymatic reactions involving ROS (Luft 1997). Binding of trace metal ions to fatty acids in biomembranes may result in peroxidation of membrane lipids, which may again induce alterations in membrane structure and viscosity, increased leakage, inactivation of membrane-associated enzymes, and generation of cytotoxic secondary products (Ochiai 1987, Luft 1997). Several studies have shown that Cd enhances lipid peroxidation in animals (Ge´ret et al. 2002, Me´ndezArmenta et al. 2003) and plants (Wu, Zhang & Dominy 2003), but to our knowledge no studies have addressed this issue in macromycetes. Repair of peroxidized lipids includes the hydrolytic action of phospholipase before the damage can be reversed by glutathione peroxidase. Phospholipase may, however, be inhibited by ROS (McHowat, Swift & Sarvazyan 2001) or directly by Cd2+ replacing Ca2+ in the active site of the enzyme (Yu, Berg & Jain 1993). There are also several studies demonstrating an inhibiting effect of Cd on glutathione peroxidase activities in various test systems (Splittgerber & Tappel 1979, Shukla et al. 1989). Cd is a multitarget toxicant for most organisms studied, and it is a well established human carcinogen. Unlike transition metals such as iron (Fe) and copper (Cu) that may undergo redox cycling and act as potent catalysts in some of the reactions generating ROS (Halliwell 1992), Cd does not undergo redox cycling. However, Cd may induce increased production of ROS by interfering with metalloproteins involved in cellular redox or electron transfer processes (Stohs & Bagchi 1995, Schu¨tzendu¨bel et al. 2001), and in this manner Cd may induce oxidative damage to cellular macromolecules. In vivo and in vitro studies suggest that mercury (Hg) toxicity involves increased formation of superoxide anion (O2x) and hydrogen peroxide (H2O2) by mitochondria (Castoldi et al. 2001, Møller 2001). Lipid peroxidation seems to be a major mechanism involved in the toxicity of Hg in both animals (Berntssen, Aatland & Handy 2003) and plants (Cho & Park 2000). Excessive zinc (Zn) may be cytotoxic, although Zn plays critical roles in cellular defense systems against ROS. At high intracellular concentrations, Zn has been shown to increase oxidative stress in plants (Clijsters, Cuypers & Vangronsveld 1999) and to lower nutrient assimilation in plants and microorganisms (Nies 1999, Blaudez, Botton & Chalot 2000). When administered alone, Zn2+ may inhibit enzymes crucial for DNA repair, although to a lesser extent than Cd2+ (Zharkov & Rosenquist 2002). However, when Cd and Zn are

1387 administered in combination, the inhibitory effect of Cd2+ upon nucleotide excision repair is reversible to some extent by Zn2+ (Hartwig et al. 1998), suggesting a protective effect of Zn2+ in Cd genotoxicity. Furthermore, Zn serves a function in protecting against programmed cell death (apoptosis) induced by a range of agents, including Cd (Szuster-Ciesielska et al. 2000, Wa¨tjen et al. 2002). Thus, both deficiency and excess of Zn may result in apoptosis (McCabe, Jiang & Orrenius 1993, Haase & Beyersmann 1999, Haase, Wa¨tjen & Beyersmann 2001). Boletus edulis (king bolete, penny bun, cep, ce`pe, steinpilz, porcino) is one of the most popular edible mushrooms throughout Europe. This is due to its appealing taste, its voluminous, recognizable and often relatively parasite-free fruit bodies (basidiocarps), and the fact that it may be quite abundant. Attempts to grow B. edulis under controlled conditions have so far been unsuccessful (Hall et al. 1998). Previous studies have shown high concentrations of certain metals, especially Zn and Cd, in soil near the Outokumpu Norzink Zn smelter at Odda, S.-W. Norway. In an earlier study, Cd concentrations as high as 126 mg gx1 (D.W.) were recorded in caps of B. edulis from the same area (Collin-Hansen et al. 2002). In another study, a Cd-binding protein was isolated from fruiting bodies of B. edulis collected near the same smelter, and its N-terminal sequence determined (Collin-Hansen, Andersen & Steinnes 2003). Studies of Cd levels in soils at Odda have revealed high concentrations, consistently exceeding 5 mg kgx1 in topsoil and humic layer samples collected several km away from the smelter in the N and S direction (La˚g 1974, Collin-Hansen et al. 2002). The present study was aimed at testing the hypothesis that metals from smelter emissions induce damage to macromolecules of B. edulis fruit bodies near the Outokumpu Norzink smelter at Odda.

MATERIALS AND METHODS All chemicals were from Sigma–Aldrich (St Louis, MO) unless otherwise indicated. All equipment used in the steps proceeding metal determinations was acid washed automatically (Miele G 7735 CD) or manually (6 M HNO3, >18 h, followed by rinsing >6 times with >18 MV cmx1 deionized water provided by a Milli-Q system ; Millipore). All buffers were prepared in Milli-Q water purified with a water-purification system (Millipore, Watford). Sampling sites and field sampling 16 fruit bodies of Boletus edulis (4–10 cm tall, cap 6–17 cm diam) were sampled as described earlier (Collin-Hansen et al. 2002) from spruce plantations in the vicinity of the Outokumpu Norzink Zn smelter at Odda (60x 04k N, 6x 33k E), south-west Norway

Damage to DNA and lipids in Boletus edulis (exposed group). In addition, 15 reference samples (7–12 cm tall, cap 7–17 cm diam) were collected from four relatively unpolluted spruce forests near Trondheim, central Norway (reference group). Only young and seemingly healthy fruiting bodies were included in the study. Fruit bodies were put individually in clean paper bags and brought to the laboratory in Trondheim within 20 h. Voucher specimens are preserved in the reference collections in TRH under collection numbers Collin-Hansen 2004 : Boletus edulis 1000–1024. The four reference sites were: Børsa (63x 18k, 10x 00k E), Vassfjellet (63x 19k N, 10x 25k E), Bymarka (63x 25k N, 10x 18k E), and Ha˚en (63x 07k N, 10x 30k E). Short-range deposition of transition metals emitted from the city of Trondheim (population approx. 154 000) was considered likely to significantly affect the reference sites closest to the city (sites Vassfjellet and Bymarka), whereas reference sites Børsa and Ha˚en probably are insignificantly influenced by such deposition.

1388 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 microwave-assisted digestion (Multiwave digestion system, Perkin Elmer/ Anton Paar) with nitric acid (65 %, 4 ml). After cooling of samples, water (>18 MV cmx1, Milli-Q) was added to a total volume of 100 ml, resulting in a HNO3 concentration of approx. 0.5 M. 60 ml of this solution was transferred to a PE flask for determinations of Cd, Zn, and Cu by 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) were 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. Element determination

Sample preparation Immediately upon arrival at the laboratory, fruit bodies were weighed, cap diameter was measured, and thoroughly checked with respect to parasites and contamination by soil and plant material. To minimize interference from growth-dilution or other effects arising from differences in size of fruit bodies, specimens within a limited range of fruit body size were sampled. If any parts of the fruit bodies were infected by insect larvae, the whole fruit 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 fruit body was divided into cap and stipe, then the cap of each fruiting body was cut into two symmetric halves using a stainless steel knife. One of these halves was further divided in four. One of the latter parts was kept at 4 xC for use in molecular biological analyses in the present study, whereas the remaining parts of the cap, along with the undivided stipe, were frozen (x80 x) in individual clip-lock polyethene (PE) bags for determination of metal concentrations, and for subsequent molecular biological studies. In the present study, all molecular biological analyses (determination of oxidative damage to DNA or lipids) were performed on fresh (i.e. unfrozen) cap tissue within 36 h after sampling, because of the possibility that freezing and thawing of samples could significantly affect the integrity of DNA and lipids. Samples for metal determinations Samples of cap tissue to be analysed with regard to metal concentrations were weighed and freeze dried (GT 2A, Leybold-Heraeus, Hanau) to constant mass. Freeze-dried samples were weighed again for calculations of water content, then transferred to clean PE

Sample blanks, spiked samples, and standard reference material (Bovine liver 1577 a and b and Tomato leaves 1573 US National Institute of Standards and Technology) were included, and randomly chosen samples were re-analysed. 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 92–103 % for Cd (93 % of the samples o95 % recovery), 84–99 % for Zn (37 % of the samples o95 % recovery), 89–107% for Cu (94 % of the samples o95 % recovery), and 86–104 % for Hg (82 % of the samples o95 % recovery). Maximum allowable relative standard deviation between three replicates was set to 5%. Metal concentrations in standard reference material deviated from the average at most by x3 % for Cd, x13 % for Zn, +6% for Cu, and x4 % for Hg, and were normally well 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 vapour atomic fluorescence spectrometry (CVAFS) Concentrations of Hg were determined by cold vapour atomic fluorescence spectrometry (CVAFS) with a Merlin atomic fluorescence detector (Model 10.04 Flow Module, Model 10.023 Fluorescence Detector and Merlin Absorption Accessory). Hydroxylamine hydrochloride (NH2OHrHCl, 12%, 60 ml) was added to the

C. Collin-Hansen, R. A. Andersen and E. Steinnes samples no more than 3 h before analysis. The sample was mixed with SnCl2 to reduce Hg from the Hg(II) to the Hg(0) oxidation state, and the Hg(0) vapour then passed through a gas/liquid separator by a stream of argon, through a membrane dryer, and to the AFS detector. Frequency of apurinic/apyrimidinic sites (AP sites) Genomic DNA was isolated by a method based on that developed by Edwards, Johnstone & Thompson (1991), with some modifications. Fresh cap tissue (approx. 5 g) was cut into pea-sized pieces with a scalpel and homogenized in three times its volume 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). Using an ice bath extreme care was taken to avoid overheating of homogenates. Following centrifugation (12 000 g, 15 min, 4 x), aliquots (1.5 ml) of supernatant were stored (<2 h, 4 x). Supernatants (125 mL) were mixed with 1000 ml of extraction buffer (200 mM Tris, 250 mM NaCl, 25 mM EDTA, 0.5 % w/v SDS, pH 7.6) and the samples were vortexed (5 s) and finally stored (<2 hr, 4 x). Samples were centrifuged (12 000 g, 1 min, 4 x), and supernatants (750 ml) transferred to fresh Eppendorf tubes containing isopropanol (750 ml, 20 x), and handshaken. Following incubation (2 min, 20 x) and centrifugation (12 000 g, 5 min, 20 x), supernatants were removed. Pellets were vacuum dried and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5, 100 ml). Concentrations of DNA in all samples were determined by measuring the optical density (OD) at 260 nm, whereas DNA purities (i.e. degree of contamination of DNA with proteins or RNA) were deduced from the OD260/OD280 ratio. Only samples with a OD260/OD280 ratio of o1.75 were accepted. Samples were diluted with TE buffer to yield final solutions of 100 mg/ml DNA. An assay kit utilizing a biotinylated aldehyde-reactive probe (bio-ARP) was used to determine the frequency of AP sites in purified DNA (K-Assay DNA Oxidative Damage Kit, Kamiya Biomedical, Seattle, WA). The predominant bio-ARP reactive group at the AP site was an open-chain aldehyde derived from DNA’s deoxyribose (Manoharan et al. 1988, Doetsch & Cunningham 1990). A standard curve was prepared based on dilutions of a DNA standard with 40 AP sites per 1r105 base pairs. Analysis of randomly selected samples spiked with standard DNA facilitated computation of recoveries, which fell within the range 92–99 % (80 % of the samples o95% recovery). Concentration of lipid hydroperoxides Lipid hydroperoxides were determined by a specific assay (K-Assay LPO-CC kit, Kamiya Biomedical, Seattle, WA) which utilizes the observation (Ohishi et al. 1985) that hemoglobin catalyzes the reaction of

1389 lipid hydroperoxides with the methylene blue derivative MCDP (10-N-methylcarbamoyl-3,7-dimethylamino10-H-phenothiazine), forming an equimolar concentration of methylene blue with maximum absorbance at 675 nm. A standard curve was made from 0 to 50 nmol mlx1 cumene hydroperoxide. According to Walther, Jobe & Ikegami (1998) this assay kit has a linear range between 2–300 nmol mlx1. Randomly selected samples spiked with known amounts of cumene hydroperoxide standard were analyzed, and recoveries calculated. Recoveries fell within the range of 94–103 % (80 % of the samples o98% recovery). Concentrations of lipid hydroperoxides were expressed as nmol mlx1 undiluted tissue extract. Normalization of data All measurements were carried out in duplicate. Prior to statistical analyses, data of fungal metal concentrations were normalized to a fresh weight basis using the water content data. Frequencies of AP sites, concentrations of lipid hydroperoxides, and soil metal concentrations were not normalized. Statistical treatment of data Distribution analysis of the data revealed that metal concentrations in fruit bodies were not generally normally distributed. Values for normalized metal concentrations were transformed logarithmically for analysis, although logarithmic transformation failed to normalize the distribution of the concentrations of Cd and Zn. These distributions were, however, similar in their dispersions and distributional shape. Consequently, 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. Furthermore, since the bivariate correlations could not necessarily be assumed to be linear, nonparametric Spearman’s correlation analysis was chosen to test the degree of monotonic, but not necessarily linear, correlation between metal concentrations in fruit bodies (caps) and oxidative damage to DNA or lipids. P<0.05 was chosen as the level of statistical significance throughout.

RESULTS Metal concentrations in caps of Boletus edulis varied within wide limits between the exposed area at Odda and the four reference areas, especially for the nonessential metals Cd and Hg. Caps of B. edulis differing 319-fold in Cd concentration (308 vs 0.965 mg gx1, D.W. basis), 7.91-fold in Zn (741 vs 93.7 mg gx1), 7.81-fold in Cu (139 vs 17.8 mg gx1), and 185-fold in Hg (54.1 vs 0.292 mg gx1) were obtained in the present study. DNA and lipids are among the cellular macromolecules of finite chemical stability that are susceptible to

Damage to DNA and lipids in Boletus edulis

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Table 1. Comparisons of size, water content, metal concentration data, frequencies of AP sites, and concentrations of lipid hydroperoxides between fruiting bodies (caps) of metal exposed group and reference group (Mann–Whitney U-test). (Arithmetic mean¡arithmetic standard deviation unless otherwise indicated). Exposed group Reference group (n=16) (n=15) P-value Mass of fruit body (g) 457¡101 Cap diam (cm) 11.1¡3.1 Water content (%) 92.0¡0.2 Cd (mg gx1)a, b 148.4¡66.6 580¡139 Zn (mg gx1)a, b Cu (mg gx1)a, b 97.8¡23.6 Hg (mg gx1)a, b 14.1¡12.4 AP sitesc 9.7¡1.8 8.5¡2.0 Lipid hydroperoxidesd

390¡140 10.5¡2.2 92.0¡0.2 2.85¡2.00 128¡21.2 31.2¡16.3 2.10¡2.58 3.2¡1.8 2.8¡1.6

NS NS NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

NS, Not significant. a D.W. basis. b Geometric mean¡geometric standard deviation. c Number of AP sites per 100 000 DNA base pairs. d nmol mlx1 undiluted tissue extract.

oxidative damage by ROS (Halliwell 1992, Halliwell & Gutteridge 1999). The results of the investigations for the exposed group and the reference group are summarized in Table 1. There were no significant differences in mass of the entire fruiting body, cap diameter or water content between the groups. In the exposed group, concentrations of Cd, Zn, Cu, and Hg, frequencies of apurinic/apyrimidinic (AP) sites in DNA (a pre-mutagenic modification of bases in DNA), and concentrations of lipid hydroperoxides were significantly elevated (Mann–Whitney U-test, P<0.001). When the entire study selection (n=31) is considered, several significant positive correlations were observed between metal concentrations and AP site frequency (Fig. 1, Table 2) or lipid hydroperoxide concentrations (Fig. 2, Table 2). The correlation between the frequency of AP sites and concentration of liquid peroxides was also positively and highly significant (Fig. 3). However, for the samples in the reference group (n=15), corresponding correlations are predominantly negative (Table 3), although a significantly negative correlation was observed only between Hg and lipid hydroperoxides. DISCUSSION Despite the accumulation of certain toxic metals in mushrooms such as the edible Boletus edulis has been known for decades, few attempts have been made to measure possible harmful effects at the molecular level. Based on the ecological importance of mycorrhizal fungi and their continuous exposure in the soil, the responses of these organisms to long-term heavy metal contamination could give important information for assessing environmental risks of contamination. Our results showed the following four points : (1) fruit bodies of B. edulis growing in soils polluted with a mixture of transition metals (exposed group), experience increased frequency of apurinic/apyrimidinic (AP)

sites in DNA and concentration of lipid hydroperoxides relative to specimens from relatively unpolluted areas (reference group) ; (2) there were significant positive correlations between concentrations of Cd, Zn or Cu and the degree of oxidative damage to DNA and lipids for the whole study selection (n=31), and for Hg, there was a significant positive correlation with the frequency of AP sites, whereas the correlation between Hg and lipid hydroperoxides was insignificant; (3) a good agreement was established between oxidative damage to DNA and lipids; and (4) a significant negative relationship was noted between Hg and lipid hydroperoxides in the reference group. Negative insignificant trends were noted for the reference group between concentrations of each of the four metals and frequency of AP sites or concentration of lipid hydroperoxides, with one exception for the association for the latter with Zn. These findings suggest that in B. edulis low concentrations of Hg, possibly also other of the metals determined in the present study, may induce ‘J-shaped’ dose-response curves. To our knowledge, this is the first study to indicate that oxidative damage of nucleic acids and lipids is involved in the toxicity of metals to wild-growing macromycetes. Recent studies of mammalian cells have demonstrated that increased intracellular concentrations of Cd and certain other transition metals may give rise to nonspecific oxidative damage to biomolecules, including nucleic acids, lipids and proteins (Fatur et al. 2002, Hartwig et al. 2002a, b). Our results bring to light the relevance of these previous findings to macrofungi living in metal-contaminated areas. The concentrations of Cd and Hg reported here for the exposed area are very high compared with earlier published data for B. edulis from polluted areas. Cd concentrations in this species seem to rarely exceed 100 mg gx1, even in specimens sampled from sites highly polluted by this metal (Kalacˇ Burda & Staskova 1991, Collin-Hansen et al. 2002). Concentrations of Hg in the exposed group were also very high compared with previous findings from areas heavily polluted with Hg (Kalacˇ, Svoboda & Havlı´ cˇkova´ 2004). The majority of previous studies on uptake of metals in macromycetes have been performed in the context of human exposure via consumption. From the human perspective, Cd is generally regarded as the most deleterious heavy metal in mushrooms, due to the potential of some of the popular edible macromycetes to accumulate Cd to high concentrations in their fruit bodies (Kalacˇ et al. 2004). Contaminated food is the major source of Cd for nonsmokers and non-occupationally exposed individuals. Conflicting results have been reported for the uptake of metals from mushrooms by humans. Whereas early studies by Schellmann, Hilz & Opitz (1980) and Diehl & Schlemmer (1984) reported that a relatively small proportion (f10 %) of ingested Cd is absorbed in the gastrointestinal tract, further reports have observed a comparable or higher absorption from mushrooms

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(A)

100 [Zn], µg g–1, fresh weight basis

[Cd], µg g–1, fresh weight basis

100

10

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12

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0

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(D) [Cu], µg g–1, fresh weight basis

10 [Hg], µg g–1, fresh weight basis

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100

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4 6 8 10 AP sites per 100 000 b.p.

12

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Fig. 1. Relationships between frequency of apurinic/apyrimidinic (AP) sites in DNA and concentrations of Cd (A), Zn (B), Hg (C) or Cu (D) in 31 wild-growing fruiting bodies (caps) of Boletus edulis. 16 samples (#) were collected near a Zn smelter, the remaining 15 ($) from relatively unpolluted areas. See Table 2 for Spearman’s rSp and P values. Note logarithmic scale on ordinate axis. Table 2. Spearman correlation factors (rSp) for the degree of association between metal concentrations in fruit bodies (wet weight basis) and oxidative damage to DNA (frequency of AP sites) or lipids (concentration of lipid hydroperoxides). n=31.

AP sites Lipid hydroperoxides

Cd

Zn

Cu

Hg

0.646** 0.444*

0.788** 0.642**

0.753** 0.615**

0.416* 0.248

* P<0.05. ** P<0.01.

than from inorganic cadmium salts (Seeger et al. 1986, Lind et al. 1995, Mitra et al. 1995). Hopefully, future work will bring some clarification to the question of metal availability from mushrooms. Regardless of the uncertainties surrounding the question of bioavailability of metals from mushroom tissues, concentrations of the toxic metals Cd and Hg in caps of B. edulis close to the Outokumpu Norzinc at Odda smelter of up to 308 mg gx1 Cd and 54.1 mg gx1 Hg (D.W. basis) call for a general warning to be made against ingestion of even small amounts of this species collected near sources of atmospheric metal emissions.

Concentrations of Cd, Zn, and Cu in fruit bodies of B. edulis near this smelter reported in the present study are significantly higher than corresponding data in our previous study (Collin-Hansen et al. 2002), although the sampling sites were collected from the same or similar sites in the two studies. Clearly, local, topographic differences could be the reason for the discrepancies. It should be noted that this difference is not likely to emerge from differing analytical conditions, since the same equipment for sampling, sample preparation, and metal determinations was used, and the same flasks of standard reference material were used in the two studies, yielding satisfactory results for analytical precision in both studies. To test the validity of the results from the present study, selected samples and standard reference material from our previous study were re-analysed with respect to Cd, Zn, and Cu together with a selection of samples from the present study. This failed to reveal any significant deviations in analytical conditions between the two studies (data not shown). One interesting observation in this respect is that the density of B. edulis fruit bodies was markedly higher

Damage to DNA and lipids in Boletus edulis

1392 (B)

(A)

100 [Zn], µg g–1, fresh weight basis

[Cd], µg g–1, fresh weight basis

100

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8 10 6 [LPO] (nmol ml–1)

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100 [Cu], µg g–1, fresh weight basis

[Hg], µg g–1, fresh weight basis

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Fig. 2. Relationships between concentration of lipid hydroperoxides (expressed in terms of nmol mlx1 of undiluted tissue extract) and concentrations of Cd (A), Zn (B), Hg (C) or Cu (D) in 31 wild-growing fruiting bodies (caps) of Boletus edulis. 16 samples (#) were collected near a Zn smelter, the remaining 15 ($) from relatively unpolluted areas. See Table 2 for Spearman’s rSp and P values. Note logarithmic scale on ordinate axis. Table 3. Spearman correlation factors (rSp) for the reference group quantifying the degree of association between metal concentrations in fruit bodies (wet weight basis) and oxidative damage to DNA (frequency of AP sites) or lipids (concentration of lipid hydroperoxides). n=15.

AP sites Lipid hydroperoxides

Cd

Zn

Cu

Hg

x0.354 x0.332

x0.211 0.131

x0.264 x0.446

x0.289 x0.532*

* P<0.05.

when samples were collected for the present study in 2002 than in 1998, the time of collection of samples for our previous study (Collin-Hansen et al. 2002). According to Kalacˇ et al. (2004), uptake of metals in fruiting bodies seem to be affected by the age of the mycelium (i.e. that older mycelium is capable of transferring more metals to fruit bodies than is younger mycelium), although this suggestion suffers from a lack of supportive experimental evidence. It might be speculated that climatic differences between years can explain the observed discrepancies, e.g. that the high temperature during the summer of 2002 induced the production of fruit bodies at Odda by old mycelia

which had not produced fruit bodies for several years. Another factor is that the high temperature would be expected to increase the flow of water through fruit bodies, thereby creating an increased potential of metal transfer from mycelium to fruit bodies. One may also speculate that mycelium containing high concentrations of toxic metals such as Cd and Hg (e.g. old mycelium) may require better conditions for fructification to be stimulated. The exact reasons for the discrepancies in metal concentrations between our two studies, however, remain elusive. Mutagenic effects of Cd are generally weak in bacterial and in standard mammalian cell mutation assays. Consequently, the carcinogenic mechanism(s) of cadmium remain(s) largely unknown. However, Filipicˇ & Hei (2004) demonstrated that Cd carcinogenicity in mammalian cells is, at least in part, due to mutagenic effects mediated by ROS generated DNA damage, and to inhibition of DNA repair mechanisms. Notably, pre-treatment with buthionine sulfoximine (which depletes intracellular glutathione levels) increased the cytotoxicity and mutagenicity of Cd, whereas concurrent treatment with Cd and the ROS scavenger dimethyl sulfoxide markedly lowered the number of

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14

[Lipid hydroperoxides] (nmol ml–1)

12 10 8 6 4 2 0 0

2

4

6 8 10 AP sites per 100 000 b.p.

12

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16

Fig. 3. Relationship between frequency of apurinic/apyrimidinic (AP) sites in DNA and concentration of lipid hydroperoxides (rSp = 0.843, P<0.001) in Boletus edulis collected near a Zn smelter (#) or from relatively unpolluted areas ($).

cadmium-induced mutations relative to treatment with Cd alone. Oxidative damage to antioxidant enzymes has recently been suggested as a plausible mechanism underlying the apoptotic effects of Cd (Wa¨tjen & Beyersmann 2004). Decreased expressions of such enzymes due to interference of metals such as Cd (Ovelgo¨nne et al. 1995) and Hg (Sa´nchez Urı´ a & SanzMedel 1998) with proteins involved in DNA and protein synthesis are other possible mechanisms for the increased levels of oxidative damage at high levels of metal exposure in the present study. Furthermore, high intracellular concentrations of Hg may result in precipitation of selenium (Se) as insoluble mercury selenide (HgSe), thus removing Se which could normally act as a co-factor for scavenging of H2O2 and lipid peroxides by glutathione peroxidase (Imura & Naganuma 1991). Mercuric ion (Hg2+), one of the strongest thiol-binding agents known, may also complex glutathione and hamper its function as an important cellular antioxidant (Stohs & Bagchi 1995). Based on the results from these previous studies, it would not be surprising if the elevated oxidative damage to DNA and lipids seen in metal-exposed B. edulis in the present study is an effect of a combination of the increased activity of ROS and reduced activity of proteins involved in the breakdown of ROS species or repair of ROS-induced damage. Turning to the perspective of effects of potentially toxic metals to macromycetes, there are far too few studies so far to enable any firm conclusions regarding dose-response characteristics or real hypotheses concerning the broader ecological consequences to these organisms of metals in the environment. However, the significant negative relationship between Hg and lipid hydroperoxides in the reference group (Table 3) suggests a ‘J-shaped’ (biphasic) dose-response curve. Such

dose-response relationships are indicative of a hormetic effect, i.e. that low doses of this metal have a protective effect against lipid peroxidation. One possible explanation for hormesis lies in a modest overcompensation to a disruption in homeostasis. This overcompensation might result from the induction of molecular defence and (or) repair systems by low doses of the toxicant (Southam & Ehrlich 1943, Luckey 1959). When the toxic effect is lipid peroxidation, these defence systems include phospholipase and glutathione peroxidase. Negative, insignificant trends were noticed in the reference group for the rest of the associations between metal exposure and response, except from the very weak (insignificant) positive association between Zn and lipid hydroperoxides. Hormetic dose-response relationships are not at all unusual in toxicology, indeed a recent article by Calabrese & Baldwin (2003) argues that the dose response of peptides generally conforms to the hormetic model. There are strong indications that the hormetic dose-response curve is more fundamental than the threshold or linear models, and that this model often provides more reliable estimates of low-dose risk (Calabrese & Baldwin 2003). Although Spearman’s non-parametric correlation analysis is generally more suited for non-linear relationships than are parametric tests, it will, like any parametric or non-parametric test for correlation, perform poorly in cases such as relationships described by J- or U-shaped curves (Bhattacharyya & Johnson 1977: 406). However, since the association between Hg and lipid hydroperoxides was the only one of the doseresponse relationships that was significant when the reference group was treated alone, Spearman’s correlation analysis was found appropriate. During sample preparation in the laboratory, all fruiting bodies were thoroughly examined for insect

Damage to DNA and lipids in Boletus edulis larvae, since incorporation of animal DNA or lipids was likely to significantly affect the results. It should be noted that only one sample (5.0 %) of the exposed group from Odda were rejected of this reason, whereas eight samples (35 %) from the reference group were rejected due to the presence of insect larvae. This difference might represent a protective effect of high concentrations of potentially toxic metals towards parasitism, calling for studies on the insecticidal potential of tissue from metal-accumulating edible mushrooms from areas contaminated with such metals. Although several of the species of macromycetes that are toxic to insects (and indeed have been used to kill or anaesthetize insects or keep them away from clothing) are reported as being accumulators of metals (e.g. Amanita sp., Lycoperdon sp., Lepista sp.), some insecticidal mushrooms are not metal accumulators (e.g. Cantharellus cibarius). Wang et al. (2002) point to proteins (e.g. lectins and hemolysins) as being important in the defence of fruit bodies of macromycetes against parasitism by insects. Metals were not determined in their study, nor is it stated whether fruit bodies were collected from ‘background ’ or metalcontaminated areas. It appears likely, however, that the results of Wang et al. (2002) are representative for background areas. Notably in this respect, Mier et al. (1996) studied the insecticidal effect of fruit bodies of 175 species of macromycetes, presumably from noncontaminated areas, and noticed that the minimal toxic dose of B. edulis to Drosophila melanogaster differed >3-fold between fruit bodies, indicating an effect of substrate conditions upon the insecticidal properties. To our knowledge no studies have compared the insecticidal effects of tissues from one fungal species grown on media containing different concentrations of metals. The above suggests an effort to estimate the possible involvement of metals in protective effect against parasitism by insects in metal-accumulating species that are not generally regarded as insecticidal. Several plant species seem to utilize hyperaccumulation of metals as a defence mechanism against herbivores (Boyd & Martens 1992, Shier 1994). Among several hypotheses regarding the evolutionary ‘ raison d’eˆtre ’ of metal hyperaccumulation in plants reviewed by Boyd & Martens (1992), the metal defence hypothesis has received the most supporting evidence (Boyd 1998, Boyd & Martens 1998). In an elegant set of experiments, leaves of the nickel (Ni) hyperaccumulating plant Thlaspi montanum var. montanum grown on Ni-rich soil were administered to larvae of Pieris rapae, a generalist folivore. The larvae were not able to cocoon and consequently died, whereas larvae fed with Thlaspi leaves from plants grown on soil with normal Ni concentrations developed into adults (Boyd & Martens 1994). Fruit bodies are organs specialized for spore production. Earlier studies have demonstrated that concentrations of metals such as Cd and Hg are generally higher in cap tissue than in the stipe, and often highest

1394 in the spore-forming (sporophore) part of the cap (Melgar et al. 1998, Falandysz et al. 2003a, b, own data not shown). Based on these observations, the results presented here call for studies on the integrity of DNA in spores from fruiting bodies of metal-accumulating macromycetes from areas contaminated with metals. Such studies could give answers to the question of whether metal-induced DNA alterations in these organisms under such conditions may be passed on to the next generation. In rats, Cd has been shown to inhibit lipogenesis by binding to the thiol group of coenzyme A, thereby reducing the serum levels of free fatty acids and, consequently, of lipid peroxides (Fujita 1992). The total lipid concentration was not determined in the present study, thus the possibility of such an inhibitory effect cannot be eliminated. We suggest that future studies should take this into account by including determinations of total lipid concentration. Lipid peroxidation has been regarded as a more reasonable mechanism for the acute cytotoxicity of several transition metals than the damaging effects of metal ions upon proteins (Yamada et al. 1992, Ogihara et al. 1995). Nevertheless, oxidation of proteins in organisms subject to metal stress is an emerging field of study (Stadtman & Levine 2000), and to fully assess the oxidative stress induced by a mixture of metals in B. edulis, the topic of protein oxidation in this species warrants further investigation. In summary, our study suggests that the elevated concentrations of Cd, Zn, Cu, and Hg found in wildgrowing B. edulis living near a Zn smelter result in structural damage to lipids and DNA in fruit bodies. Future studies are needed to clarify the potential roles of ROS formation and inhibition of proteins involved in the repair of oxidative damage to DNA and lipids in metal-induced toxicity in B. edulis, and of the ecological relevance of the high metal concentrations reported in the present study.

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