Mushrooms from a Cd and Zn contaminated spruce forest:

Occurence of heavy metals and heavy-metal binding proteins

K. E. Yttri^1#), R. A. Andersen²⁾, B. O. Berthelsen¹⁾, C. Collin-Hansen¹⁾, E. Steinnes¹⁾.

¹⁾ Norwegian University of Science and Technology, Department of Chemistry, 7491 Trondheim, Norway

²⁾ Norwegian University of Science and Technology, Department of Zoology, 7491 Trondheim, Norway

^# e-mail of corresp. author: karly@stud.ntnu.no

Abstract

Cap and stalk of wild growing macrofungi and corresponding topsoil were collected in the vicinity (range 0.7 – 20 km) of a zinc smelter in western Norway in order to study concentrations of Cd, Cu and Zn and to provide knowledge about the heavy-metal binding properties of the fungi. The topsoil was found to be heavily contaminated, with Cd and Zn levels within the range of 5-30 ppm and 1100-7600 ppm respectively. Particularly Cd, but also Zn, appeared to be accumulated to high levels in the species collected. Concentrations as high as 125 ppm and 400 ppm d.wt. respectively were found in the edible species Boletus edulis. Detectable levels of Cd/Zn-metallothioneins were observed in all fungal samples analysed using the Cd-Chelex assay, a finding matching the AAS-analysis of heavy-metal binding protein fractions separated by gel permeation chromatography. The amino acid composition of heavy-metal binding protein fractions from the species Amanita muscaria, isolated by anion exchange chromatography, excludes the presence of proteins of the metallothionein family in this species.

Introduction

Elevated concentrations of heavy metals often occur in fruiting bodies of macrofungi growing in polluted areas (Stijve and Besson, 1976). It is therefore plausible to suggest that fungi may act as an important intermediate link for some toxic elements from soil to humans, either directly by consumption or indirectly via animals. The fact that Cd is quite mobile in soil and toxic to humans even in very low concentrations, makes accumulation of this element of special concern.

Fungi have several ways of dealing with elevated levels of heavy metals in their surroundings, amongst these are induction and binding of metals such as Cd, Cu and Zn to cystein rich proteins and polypeptides of the metallothionein (MT) family (Münger and Lerch, 1985; Rauser, 1990; Gadd, 1993). MT is a class of low molecular weight (< 10 kDa) proteins and polypeptides known to bind considerable amounts of d¹⁰ metal ions in metal thiolate clusters (Hamer, 1986). There has repeatedly been reported a high correlation between elevated concentrations of MT and reduced toxicity of free heavy-metal ions, including both in vivo and in vitro studies (Webb, 1987). However, little is known about the speciation and effects of heavy metals in the macrofungal tissue and the role of MT in such organisms.

The main purpose of the present study was to look for correlations between heavy-metal and MT concentrations in the fungal tissue and to some extent to identify the characteristics of the heavy-metal binding protein as well as giving a brief insight in the pollution status in the area of concern.

Methods

Wild growing macrofungi and corresponding topsoil were collected near a zinc smelter located in western Norway. The following species were included: Amanita muscaria, Boletus edulis, Leccinum rufescens, Leccinum scaber, and Xerocomus subtomentosus. Among these species only A. muscaria is not edible. The topsoil was sampled by cutting a 10´10 cm² area around the stalk which included the humic layer. Cd, Cu and Zn concentrations in mushrooms and topsoil were determined by AAS (flame mode) after wet ashing. Fungal samples spiked with ¹⁰⁹Cd tracer were separated on a Sephadex G-75 column (3 – 80 kDa) and the eluate monitored at 254 nm. Cd activity in the eluted protein fractions was measured by gamma spectrometry to detect the fractions with the highest metal-binding capacity. AAS determinations for Cd, Cu and Zn were also performed on protein fractions not spiked with radioactivity. ¹⁰⁹Cd containing protein fractions from Sephadex G-75 separation were further separated on a DEAE Sepharose anionic exchange column and eluted by a linear gradient of Tris-HCl. The eluate was monitored at 254 nm and Cd activity was measured by gamma spectrometry. Amino acid composition was determined in those protein fractions showing high correlation between UV absorbance and ¹⁰⁹Cd activity. Concentrations of Cd/Zn-MT and total-MT were quantified by the Cd-Chelex assay (Klein et al., 1990) and the thiomolybdate-Chelex assay (Bartsch et al., 1990) respectively, Cu-MT-concentrations being the difference between the two.

Results and Discussion

Heavy metals in wild growing mushrooms and in topsoil collected near the Zn-smelter (contaminated area) clearly show elevated concentrations of Cd and Zn compared to samples collected farther away from the smelter (little contaminated area) (see Table 1). 125 ppm Cd d.wt. in the cap of the edible and very common species Boletus edulis is of concern related to human exposure. An intake of only 4 g of this specimen exceeds the predicted weekly tolerable intake stipulated by the WHO. Although rather high concentrations is being reported for the species in this survey, there are strong indications that the uptake of certain heavy metals are strongly species dependent (Melgar et al., 1998) (see Figure 1).

Detectable levels of Cd,Zn metallothioneins were observed in all fungal samples analysed using the Cd-Chelex assay. Cu-MT was detected in only 54% of the samples. No significant correlations were found between concentrations of Cd/Zn-MT or Cu-MT and Cd, Cu or Zn concentrations in the mushrooms. These observations could indicate that the proteins responsible for binding heavy metals in these species are not of the MT family, although they possess high metal binding affinity.

AAS determinations for Cd, Cu and Zn, performed on protein fractions separated on a Sephadex G-75 column, indicated the presence of low molecular weight proteins with an elevated affinity for the examined heavy metals (see Figure 3) in the species Amanita muscaria and Boletus edulis.

These observations are supported by the findings of ¹⁰⁹Cd binding to selected protein fractions (see Figure 2) in the two species mentioned.

Amino acid composition of heavy-metal binding protein fractions from the species Amanita muscaria, isolated by anion exchange chromatography, excludes the presence of proteins from the MT family in this species. This may be concluded by the almost total absence of cystein. The amino acid composition shows a certain similarity to that of cadmium-mycophosphatin isolated by Meisch et al. (1983) from Agaricus bisporus.

Further studies need to be performed in order to identify the heavy-metal binding proteins of Amanita muscaria and other wild growing macrofungi.

Table 1: Concentrations of Cd, Cu and Zn in topsoil and in fruiting bodies (cap) of 5 species of wild growing

macrofungi and estimated levels of Cd/Zn-MT and Cu-MT in these species of macrofungi. Samples are

collected in two areas: "Contaminated area" and "Little contaminated area" respectively 0.7 – 2.0 km and

20 km from the smelter.

Species	Area	Soil^1)			Fruiting body^1) (cap)			Cd/Zn-MT^1)	Cu-MT^1)
		Cd	Cu	Zn	Cd	Cu	Zn
A. muscaria	Contaminated Little cont.	24.9 22.7 21.5 10.5 9.61 6.73 7.44 6.82 <0.825 <0.825 <0.825	57.6 198 177 60.0 55.6 42.5 24.2 18.6 42.9 8.80 8.41	6950 7620 5150 1330 1590 1590 1690 1380 65.7 160 99.2	40.7 21.6 11.1 35.8 26.8 24.4 52.9 48.7 19.9 8.00 9.46	49.2 47.3 28.4 46.3 46.0 27.3 52.7 46.2 44.8 18.3 29.6	311 209 100 221 93.9 195 292 231 111 90.0 74.8	48.7 33.4 525 51.4 79.6 51,4 1020 351 78.9 62.2 70.8	371 264 287 88.1 53.5 n.d.^2) n.d. n.d. n.d. 1.10 127
B. edulis	Contaminated Little cont.	29.2 19.8 4.39 10.3 1.03 <0.825	106 89.2 25.0 54.4 22.6 6.78	5340 5240 1080 2270 63.4 60.3	52.1 126 34,4 8.70 4.04 5.79	89.0 78.4 48.5 24.8 38.6 33.3	288 388 342 183 81.5 127	645 2270 294 13.9 257 394	n.d. n.d. 41.4 111 325 189
L. rufescens	Contaminated	8.00 10.7	33.8 54.4	1760 2010	3.00 41.0	21.9 94.5	115 238	160 213	n.d. n.d.
L. scaber	Little cont.	2.08 <0.825	10.4 39.0	315 98.2	7.74 2.63	19.6 26.9	173 58.9	140 121	n.d. 379
X. subomentosus	Contaminated	31.0 21.5 4.61	142 105 21.8	7450 6080 1240	41.7 18.7 26.0	72.9 55.4 64.5	396 210 211	37.6 65.8 102	251 n.d. n.d.

1) (mg g^-1)

2) n.d. = not detected.

Figure 1: [Cd] in B. edulis (D) (…) (n = 6) and Figure 2: Elution profile (¾) (254 nm) of extract

A. muscaria (") (¾) (n = 11) (cap) as a B. edulis (cap) spiked with ¹⁰⁹Cd (¼).

function of [Cd]in soil.

Figure 3: Elution profile (¾) (254 nm) of extract of B. edulis (cap) including AAS values for Cd (D), Cu (à) and Zn (O).

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