MUSHROOMS FROM A
COPPER CONTAMINATED BIRCH FOREST: CONCENTRATIONS OF HEAVY METALS AND
METALLOTHIONEINS AND SUPEROXIDE DISMUTASE ACTIVITY
C.
Collin-Hansen*#, R. A. Andersen**,
B. O. Berthelsen*, E. Steinnes*, K. E. Yttri*
*: Department of Chemistry, Norwegian
University of Science and Technology, N-7491 Trondheim, Norway.
**: Department of Zoology, Norwegian
University of Science and Technology, N-7491 Trondheim, Norway.
#e-mail address of corresp.
author: chrico@postkassa.no
ABSTRACT
Concentrations of Cu, Zn and Cd in samples of surface
soil and five species of ectomycorrhizal fungi (cap and stalk) from an area
near a copper smelter in northern Norway are reported. Concentrations of
Cu-binding, Cd/Zn-binding and total metallothionin-like proteins (Cu-MT,
Cd,Zn-MT and total MTs, respectively) were determined using the
thiomolybdate-Chelex and cadmium-Chelex method. Relative activity of superoxide
dismutases (SODs) was determined by the riboflavine-NBT method. Different
species showed large variations in Cu, Zn and Cd uptake from the same soils.
However, no significant correlations were found, either between concentrations
of heavy metals and MTs, or between heavy metal concentrations and relative SOD
activity. Explanations to these observations are suggested.
INTRODUCTION
Most plant species participate in mycorrhizal
symbiosis under natural conditions. It thus seems reasonable to suggest that
fungi may be of major importance in the transport of at least some elements
from soil to animals and human beings (Lepp et
al., 1987), either indirectly (via plants) or directly. Numerous studies
have indicated that mycorrhizal fungi can be of importance in ameliorating the
phytotoxicity of a number of heavy metals (e.g.
Gildon and Tinker, 1983; Brown and Wilkins, 1985). However, little is known
about the mechanisms behind such detoxification. Knowledge about these
mechanisms could clearly be of importance in the agricultural sciences.
Metallothioneins (MTs) are low molecular weight
metal-binding proteins that have become widely used as indicators of prior
metal exposure. However, extrapolation of findings of strong correlations
between exposure and MT levels in laboratory experiments to wild-living
organisms has been questioned.
The superoxide radical (×O2-) is
synthesized during normal metabolism in all aerobs, but increased exposure of
several heavy metals may lead to increased levels of ×O2- in the organism. ×O2- can cause damage by
oxidizing [4Fe-4S] clusters in dehydratases, which leads to inactivation of the
enzyme and release of Fe(II). Fe(II) then may convert H2O2
to OH- and the hydroxyl radical (×OH). Alternatively, Fe(II) may
bind to the DNA and give rise to potent oxidants in its close vicinity,
increasing the possibility of mutagenesis (Fridovich, 1995). Induction of
superoxide dismutase (SOD) as a response to heavy metal exposure has been well
studied in yeasts under laboratory conditions, but little is known about heavy
metal induced oxygen radical damage in macrofungi. The main objective of this
study was to look for simple correlations between heavy metal exposure on one
side and concentration and activity of MT and SOD, respectively, on the other.
METHODS
Soil and mushroom samples were collected along a
deposition gradient in the western direction from the former Sulitjelma copper
smelter situated in northern Norway (see figure 1). Emphasis was put on edible
ectomycorrhizal fungi, but some toxic ectomycorrhizal species known to be
potent accumulators of heavy metals were included as well. The material
included the species Boletus edulis, Amanita muscaria, Russula xerampelina, Leccinum
rufescens and Lactarius torminosus.
Surface soil was sampled as a 20´20cm2 square around
the stalk, in order to provide a measure of heavy metals available to each
fruit body. Heavy metal concentrations in soil and mushroom samples were
determined by flame AAS after nitric acid digestion. Concentrations of total MT
and Cd,Zn-MT were determined by the thiomolybdate-Chelex (Bartsch et al., 1990) and Cadmium-Chelex method
(Klein et al., 1990), respectively.
Cu-MT concentrations were calculated as the difference between the two.
Relative activity of SOD in caps of Amanita
muscaria was measured by the riboflavine-NBT method, according to Beauchamp
and Fridovich (1971).
RESULTS AND DISCUSSION
Although by the time of sample collection eleven years
had passed since the smelter was shut down, effects of the smelter emissions
were still obvious (e.g. low soil
organic matter, low plant species diversity and density). The results indicate
that Cu, Zn and Cd in topsoil and mushrooms collected near the smelter are
still elevated relative to samples collected farther away in the prevailing
wind direction (see table 1). Deviations from this pattern are attributed to
low metal-binding ability in topsoil close to the smelter because of reduced
organic matter content. There are strong indications that fungal concentrations
of Cu, Zn and Cd are more dependent on species dependent factors than on HNO3-soluble
topsoil concentrations.
One individual of each of the species Amanita muscaria (not consumed by human
beings) and the edible mushroom Boletus
edulis showed Cd levels in cap as high as about 30 ppm dry weight. This may
lead to appreciable exposure of humans and grazing animals, as well as of
animals in forest food chains.
Figure 1. Map of Norway showing study area and sampling sites.
Sites 1 to 4 represent a deposition gradient, whereas site 5 was chosen as a
reference site.
Table 1. Heavy metal concentrations in topsoil samples and concentrations of
heavy metals, Cu-metallothionein (Cu-MT) and Cd,Zn-metallothionein (Cd,Zn-MT)
in fruit bodies (caps) (all concentrations are mg g-1, dry wt; n.d.= not
detected).
|
|
|
Soil |
Cap |
||||||
|
Species |
Site |
Cu |
Zn |
Cd |
Cu |
Zn |
Cd |
Cu-MT |
Cd,Zn-MT |
|
B. edulis |
1 |
893 |
67 |
<0.83 |
313 |
158 |
12 |
81 |
20 |
|
2 |
2839 |
220 |
0.98 |
216 |
217 |
26 |
n.d. |
482 |
|
|
3 |
1793 |
54 |
2.1 |
427 |
150 |
11 |
340 |
191 |
|
|
R. xerampe-lina |
1 |
3711 |
398 |
3.1 |
--- |
--- |
--- |
n.d. |
681 |
|
2 |
1274 |
378 |
0.87 |
170 |
215 |
3.4 |
63 |
121 |
|
|
3 |
56 |
109 |
<0.83 |
25 |
156 |
2.2 |
178 |
152 |
|
|
4 |
42 |
74 |
1.0 |
64 |
279 |
3.1 |
n.d. |
81 |
|
|
5 |
20 |
39 |
<0.83 |
73 |
179 |
<1.9 |
103 |
30 |
|
|
L. rufescens |
1 |
486 |
62 |
<0.83 |
83 |
136 |
2.4 |
59 |
30 |
|
2 |
2813 |
249 |
1.4 |
135 |
313 |
16 |
n.d. |
39 |
|
|
3 |
990 |
88 |
1.2 |
73 |
188 |
4.9 |
147 |
n.d. |
|
|
4 |
397 |
75 |
1.0 |
56 |
116 |
3.9 |
31 |
18 |
|
|
5 |
14 |
58 |
<0.83 |
66 |
122 |
<1.9 |
n.d. |
274 |
|
|
A. muscaria |
1 |
3202 |
225 |
<0.83 |
66 |
166 |
13 |
143 |
55 |
|
2 |
1788 |
178 |
<0.83 |
79 |
180 |
36 |
194 |
2.7 |
|
|
3 |
2178 |
327 |
3.0 |
76 |
139 |
8.7 |
n.d. |
246 |
|
|
4 |
163 |
46 |
<0.83 |
37 |
187 |
11 |
n.d. |
268 |
|
|
L. tormi-nosus |
3 |
3797 |
395 |
2.7 |
19 |
194 |
<1.9 |
n.d. |
49 |
|
4 |
193 |
78 |
<0.83 |
21 |
188 |
<1.9 |
131 |
59 |
|
|
5 |
22 |
64 |
<0.83 |
7.8 |
96 |
<1.9 |
n.d. |
19 |
|
The results shown in figure 2 indicate the presence of
Cu-MT and Cd,Zn-MT in most samples, but no significant correlations were found
between concentrations of these proteins (or total MT) and heavy metal exposure
(data not shown). The lack of significant correlations may be explained at
least in part by the low sample number (n£5 for each species). It should
be stressed, however, that it remains to investigate closer whether these
proteins should be classified as MT, according to the definitions given by Fowler
et al. (1987).
Weak and insignificant correlations were found between
SOD activity and concentrations of Cu (r=0.45), Zn (r=0.43) and Cd (r=0.34) in
14 caps of Amanita muscaria (data not
shown).
These findings may be accounted for by the fact that fungal
defense against heavy metal toxicity has been shown to include a number of
different tolerance and resistance mechanisms, including extracellular
precipitation and complexation and binding to cell walls (review: Gadd, 1993).
In addition, previous research has shown that MT may act as scavengers of free
radicals (Abel and de Ruiter, 1989), suggesting a rather complex relation
between induction of MT and SOD as a result of heavy metal exposure.
Figure 2. Concentrations of Cu-MT and
Cd,Zn-MT in caps from five species of ectomycorrhizal fungi.
REFERENCES
Abel, J, de Ruiter, N (1989) Toxicol. Lett. 47:
191-195.
Bartsch, R, Klein, D, Summer, KH (1990) Arch. Toxicol.
64: 177-180.
Beauchamp, C, Fridovich, I (1971) Anal. Biochem. 44:
276-287.
Brown, MT, Wilkins, DA (1985) Trans. Brit. Mycol. Soc.
84: 367-369.
Fowler, BA, Hildebrand, CE, Kojima, Y, Webb, M (1987)
In: Metallothionein II (JHR Kägi, Y Kojima, Editors), Basel, Birkhäuser Verlag,
pp. 19-22.
Fridovich (1995) Annu. Rev. Biochem. 64: 97-112.
Gadd, GM (1993) New Phytol. 124: 25-60.
Gildon, A, Tinker, PB (1983) New Phytol. 95: 247-261.
Klein, D, Bartsch, R, Summer, KH (1990) Analyt.
Biochem. 189: 35-39.
Lepp, NW, Harrison, SCS, Morrell, BG (1987) Env. Geochem.
Health 9: 61-64.