ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 66 (2007) 36–43

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Effects of heavy metals on the production of thiol compounds by the

aquatic fungi Fontanospora fusiramosa and Flagellospora curta
Luı́s Guimarães-Soares, Cláudia Pascoal, Fernanda Cássio
Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Received 18 January 2005; received in revised form 26 August 2005; accepted 11 October 2005
Available online 29 November 2005

Abstract

The aquatic hyphomycetes Fontanospora fusiramosa and Flagellospora curta isolated from a clean and a metal-contaminated site,
respectively, were tested for the production of thiol compounds when exposed to Cd, Zn, and Cu for short- and long-term periods. After
8 days, control cultures of F. curta had a total thiol (T-SH) concentration in mycelia of 3.4670.37 mmol g
1 dry mass, which was 2.4
times greater than that of F. fusiramosa. In both species, nonprotein (NP-SH) and protein-bound (PB-SH) thiols accounted for 30% and
70% of T-SH, respectively. F. curta increased the production of thiol compounds, namely NP-SH, more rapidly than F. fusiramosa when
exposed to Cd or Zn. The greater increases in either NP-SH or PB-SH occurred in F. fusiramosa after long-term exposure to all metals; in
this case, the increases of PB-SH overwhelmed those of NP-SH. Long-term exposure to metals also increased the mycelial protein
concentration.
r 2005 Elsevier Inc. All rights reserved.

Keywords: Aquatic hyphomycetes; Fungi; Heavy metals; Thiol compounds

1. Introduction precipitation of metal ions (Baldrian, 2003; Gadd, 1993;

Heavy metals are continuously being mobilized and
dispersed into the biosphere by natural processes and
human activities, such as agriculture, mining, and industry,
and can constitute a serious environmental hazard (Ayres,
1992). While some heavy metals (e.g., Zn and Cu) are
essential to living organisms, others (e.g., Cd) have no
apparent biological function. However, both essential and
nonessential heavy metals can be toxic above a critical
concentration that depends on the organism, the physico-
chemical properties of the metal, and the environmental
factors (Blaudez et al., 2000b; Gadd, 1993; Gimmler et al.,
2001).
Fungi, like all living organisms, have evolved a set of
mechanisms that control and respond to the uptake and
accumulation of heavy metals. Possible interactions be-
tween toxic metals and fungi include (a) production and
secretion of organic acids, polysaccharides, melanins, or
proteins and subsequent binding/complexation and/or
Corresponding author. Fax.: +351 253678980.
E-mail address: fcassio@bio.uminho.pt (F. Cássio).
Martino et al., 2002; Sayer and Gadd, 2001), (b) metal
binding to cell walls (Blaudez et al., 2000a), (c) transport of
metal cations (Blaudez et al., 2000a; Clemens et al., 2002;
Li et al., 1997), (d) chemical transformation of metals
(Gadd, 1993), (e) organellar compartmentation (Blaudez et
al., 2000a; Clemens et al., 2002; Li et al., 1997), and (f)
synthesis of thiol-containing compounds, such as the
nonproteinaceous glutathione and phytochelatins and the
metallothionein proteins of families 8–13 (fungi I–VI
MTs), which can sequester metal ions (Binz and Kägi,
1999; Cervantes and Gutierrez-Corona, 1994; Cobbett and
Goldsbrough, 2002; Gadd, 1993; Miersch et al., 2001).
Aquatic hyphomycetes are a group of freshwater fungi
that play a key role as decomposers of plant detritus in
streams (Bärlocher, 1992). Owing to their enzymatic
capabilities they ensure the transformation of organic
matter into a food source more accessible to higher trophic
levels, primarily to macroinvertebrate detritivores (Sub-
erkropp, 1998). Few studies have examined the effects of
heavy metals on aquatic hyphomycetes, including the
effects of Fe and Mn (Bermingham et al., 1996b) and Cd
(Abel and Bärlocher, 1984) on the growth and sporulation

0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2005.10.005
 ARTICLE IN PRESS
L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43
and the effects of Cd, Cu and Zn on the growth and thiol
compound production (Miersch et al., 1997, 2001) in a few
species of this group of fungi. Heavy metal exposure has
been reported to depress leaf litter decomposition in
streams (Bermingham et al., 1996a; Duarte et al., 2004)
and to decrease the activity of aquatic fungi (Duarte et al.,
2004). Although fungal diversity has been recognized to
decrease due to heavy metal exposure (Bermingham et al.,
1996a), several species of aquatic hyphomycetes have been
found in metal-polluted streams (Krauss et al., 2001;
Pascoal et al., 2005a).
Flagellospora curta is an aquatic hyphomycete species
which has been consistently found on decomposing leaves
in polluted streams in northwest Portugal (Pascoal and
Cássio, 2004; Pascoal et al., 2003, 2005a). The presence of
this species at metal-polluted sites, where other widely
distributed species fail to appear, suggests that it should
have mechanisms to overcome metal toxicity. In contrast,
Fontanospora fusiramosa, a species which has been
restricted to clean streams (Pascoal et al., 2005b), may be
absent from metal-polluted sites due to a low metal
tolerance. Because metal detoxification and homeostasis
require intracellular complexation of metals, we expect that
increased fungal metal tolerance may be related to the pool
of thiol-containing compounds. This could mean higher
constitutive levels, higher capacity of production, and/or a
more rapid response to metal exposure. In this work, the
production of thiol compounds after short- and long-term
exposure to the heavy metals Cd, Zn, and Cu was
examined in F. fusiramosa and F. curta isolated from a
clean and a metal-polluted site of the same river,
respectively. The production of both nonprotein and
protein-bound thiols was analyzed to discriminate their
relative contribution to metal tolerance in these aquatic
fungi.
2. Materials and methods
2.1. Fungal species and culture maintenance
F. fusiramosa Marvanová, Peter J. Fisher et Descals (UMB-25.01) was
isolated from foam at the source of the Este River (northwest Portugal)
and F. curta J. Webster (UMB-39.01) was isolated from alder leaves
collected 10 km downstream, near the industrial park of the town of
Braga. In contrast to the clean site, the polluted site had high nutrient
+
loads (e.g., N-NO

1
3 4.97 mg L , N-NH4 0.25 mg L
1, and P-PO3
4

1
0.18 mg L ; Pascoal et al., 2005a) from domestic sewage and agriculture
and increased levels of heavy metals in the water column (e.g., Zn
2.02 mg L
1, Cu 5.87 mg L
1, Cd 0.02 mg L
1, Pb 5.30 mg L
1, and Cr
0.20 mg L
1; Gonc- alves, 2001) and sediments (e.g., Cu 518.1 g kg
1, Cd
0.14 g kg
1, and Pb 16.4 g kg
1; Soares et al., 1999). Details on the fungal
species are presented in Marvanová et al. (2003) and Pascoal et al. (2005b).
Fungi were maintained on medium containing 2% (w/v) malt extract
and 2% (w/v) agar, at 18 1C and under permanent artificial light.
2.2. Growth in liquid medium, harvesting, and transfer of mycelia
Fungi were grown in liquid medium containing 1% (w/v) malt extract,
with pH adjusted to 5.0 with HCl prior to autoclaving. Sterilized
(Filtropur S, 0.2 mm; Sarstedt) stock solutions of Cd, Zn, and Cu (chloride
37
salts; Sigma) were used to obtain concentrations of metals that inhibited
biomass production for 8 days by 50% (EC50) and 80% (EC80).
Concentration values for F. fusiramosa were Cd 1.2 and 3 mM, Zn 78
and 100 mM, and Cu 35 and 50 mM. Values for F. curta were Cd 20 and
28 mM, Zn 78 and 130 mM, and Cu 68 and 90 mM. Culture flasks were
incubated on a shaker (160 rpm, Certomat BS 3; B. Braun Biotech Int.) at
18 1C and under permanent artificial light.
To evaluate the effects of heavy metals on the production of thiol
compounds, the aquatic hyphomycetes were grown for 8 days in liquid
medium with or without heavy metals added at the concentrations
indicated above. At this time, cultures of both fungi were at the same
growth phase, with biomass increasing linearly with time (not shown).
Growth was achieved by inoculating 1-L Erlenmeyer flasks containing
400 mL of medium, in the case of F. fusiramosa, and 2-L flasks containing
1 L of medium, in the case of F. curta, with 6 conidia mL
1 (final
concentration). Conidial suspensions were obtained through 3 days
aeration of agar slices of 14–21-day-old cultures in sterile deionized water.
To test short-term effects of heavy metals on the production of thiol
compounds, mycelia grown 8 days in liquid medium without added heavy
metals were aseptically harvested by filtration (0.5 mm mesh), washed with
cold sterile deionized water, and then transferred to fresh medium
(0.15–0.20 mg dry mass mL
1) with or without added heavy metals at the
concentrations indicated above. Mycelia were incubated in 500-mL
Erlenmeyer flasks containing 200 mL of medium in the case of F.
fusiramosa and in 1-L flasks containing 500 mL of medium in the case of F.
curta, for periods from 14 to 158 h during which both fungi were actively
growing (not shown).
Different flask volumes were used because F. curta produces less
biomass than F. fusiramosa. However, the medium/air volume ratios used
in this work did not significantly affect the biomass production per 1 L of
medium (not shown).
2.3. Preparation of cell-free extracts
Mycelia were harvested as described above, rinsed twice with cold
deionized water, and blotted dry between layers of filter paper. Mycelia
were then quick-frozen in liquid nitrogen and ground with 2 g of purified
sea sand (Merck) per 1 g of mycelium wet mass in a precooled pestle and
mortar. The resultant paste was suspended in icecold 20 mM Tris(hy-
droxymethyl)aminomethane (Tris; Merck), pH 7.5, containing 1 mM
ethylenediaminetetraacetic acid disodium salt (Sigma), previously gassed
with a nitrogen stream, at a concentration of 3.5 mL per 1 g of mycelium
wet mass. The homogenate was centrifuged at 6200g for 10 min (4 1C) and
the pellet discarded. The supernatant was further centrifuged at 18,000g
consecutively for 50 and 15 min (4 1C), and the pellets were discarded in
both cases. The final supernatant was filtered (Filtropur S, 0.45 mm;
Sarstedt) and kept on ice until used. Under these conditions oxidation of
thiol compounds was minimized, as indicated by the absence of significant
differences in the measured thiol content of a glutathione solution
(reduced GSH) submitted or not to the steps of extraction/clarification
(not shown). Portions of mycelium were dried to constant mass at 85 1C to
convert wet to dry mass.
2.4. Estimation of total (T-SH), nonprotein (NP-SH), and
protein-bound (PB-SH) thiols
The concentration of T-SH and NP-SH was determined with 5,50 -
dithio-bis(2-nitrobenzoic acid) (DTNB; Sigma) according to Sedlak and
Lindsay (1968). To determine T-SH, 50-mL aliquots of the cell-free
extracts were mixed with 150 mL of 0.2 M Tris, pH 8.2, and 10 mL of
0.01 M DTNB, and the mixtures were brought to 1.0 mL with absolute
methanol (Riedel-de Haën). After 15 min, the mixtures were centrifuged
(3000g, 15 min) and the absorbance was measured at 412 nm (Perkin
Elmer Lambda 2 UV/VIS Spectrometer) against a reagent blank (without
sample), using reduced glutathione (Sigma) as standard.
To determine NP-SH, aliquots of 500 mL of the cell-free extracts were
mixed with 400 mL of milli-Q water and 100 mL of 50% trichloroacetic acid
 ARTICLE IN PRESS
38 L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43

(Riedel-de Haën). The mixtures were gently shaken for 12 min before PB-SH (Table 1). Mycelial protein concentration was
being centrifuged (3000g, 15 min). Then 200 mL of the supernatant was
51.1475.57 mg g
1 dry mass (Table 1).
mixed with 400 mL of 0.4 M Tris, pH 8.9, and 10 mL of 0.01 M DTNB, and
absorbance was measured within 3 min of DTNB addition as indicated The effects of heavy metal addition on the production of
above. SH compounds and on protein concentration in mycelia of
The concentration of PB-SH was calculated by subtracting the NP-SH F. curta exposed to metal concentrations that inhibited
from T-SH concentrations. fungal biomass in 50% or 80% are shown in Fig. 1. The
Since samples might contain compounds absorbing in the absence of exposure to the highest Cd concentration significantly
DTNB, sample blanks (without DTNB) were used to correct the
absorbance values. Buffers and solutions were gassed 1–2 min with a increased the NP-SH concentration in mycelia (one-way
nitrogen stream before use. ANOVA, Tukey, Po0:05). Both Zn concentrations
significantly increased the levels of protein and SH
2.5. Determination of protein concentration
compounds, except for NP-SH (one-way ANOVA, Tukey,
Po0:05, for all cases but NP-SH). The exposure to Cu did
The protein concentration was determined by the Lowry method not significantly affect the production of any SH com-
(Lowry et al., 1951) with bovine serum albumin (Sigma) as standard. pounds in F. curta (one-way ANOVA, PX0:07). Protein
concentration in mycelia exposed to the lowest Cu
2.6. Data analysis concentration was significantly higher than that in control
(one-way ANOVA, Tukey, Po0:05).
Data on the effects of heavy metals on the production of thiol F. fusiramosa mycelia grown 8 days in liquid medium
compounds by aquatic hyphomycetes and on the mycelial protein without added heavy metals had lower concentrations of T-
concentration were expressed as percentage of control. Data were then SH and protein (2.4 and 3 times, respectively) than those
divided by 1000 and arcsine square root transformed to achieve normal
observed in F. curta (Table 1). The effects of heavy metal
distribution (Kolmogorov–Smirnov test, P40:1; Zar, 1996).
Data on the effects of metal concentration on the production of thiol addition on the production of SH compounds (T-SH, NP-
compounds and protein after 8 days of growth were analyzed with one-
way ANOVA followed by Tukey’s test (Zar, 1996). Data on short-term
effects of metals on the production of thiol compounds and protein were
analyzed with two-way ANOVA, with exposure time and metal
concentration as factors, and further analyzed with Tukey’s test (Zar,
1996). Each data category (T-SH, NP-SH, PB-SH, and protein) was
analyzed independently for each fungal species and metal.
Data analysis was performed using GraphPad Prism 3.02 for Windows
(GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Effects of heavy metals on the production of thiol

compounds
When F. curta was grown 8 days in liquid medium
without added heavy metals T-SH concentration in the
mycelia, expressed as mmol g
1 dry mass, was 3.4670.37,
of which 1.1670.09 was NP-SH and 2.3070.29 was
Fig. 1. Effects of Cd, Zn, and Cu on the concentration of total (black),
nonprotein (gray) and protein-bound (dashed) thiols, and protein (white)
in 8-day-grown mycelia of F. curta. Data are percentage of the control
(control ¼ 100%). Mean7SE of three independent experiments. Actual
values of controls are in Table 1.

Table 1
Concentration of total (T-SH), nonprotein (NP-SH), and protein-bound (PB-SH) thiols and protein in mycelia of aquatic hyphomycetes grown with no
addition of heavy metals for 8 days and then transferred to fresh medium for different incubation periods

Species Incubation period (h) Thiol compounds (mmol g
1 dry mass) Protein (mg g
1 dry mass)

T-SH NP-SH PB-SH

F. curta 0 3.4670.37 1.1670.09 2.3070.29 51.1475.57

14 3.8870.40 1.3970.20 2.4970.29 68.2474.69
62 2.6570.11 1.0370.10 1.6170.11 38.6572.83
110 2.5470.12 1.1670.05 1.3870.10 27.8772.20
158 2.1470.27 1.1670.03 0.9870.27 17.5272.42
F. fusiramosa 0 1.4270.08 0.4270.02 1.0070.08 16.8171.43
14 1.7370.08 0.5170.04 1.2270.07 28.8872.09
62 1.7570.09 0.4370.02 1.3370.10 24.6671.13
110 1.3070.07 0.3570.02 0.9570.08 17.0071.40

Mean7SE of at least 3 independent experiments.

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L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43 39

(A)

Fig. 2. Effects of Cd, Zn, and Cu on the concentration of total (black),

nonprotein (gray) and protein-bound (dashed) thiols, and protein (white)
in 8-day-grown mycelia of F. fusiramosa. Data are percentage of the
control (control ¼ 100%). Mean7SE of three independent experiments.
Actual values of controls are in Table 1.
(B)

SH, and PB-SH) and on the protein concentration in F.

fusiramosa mycelia are shown in Fig. 2. At the highest Cd
concentration, the levels of SH compounds were signifi-
cantly higher than those of control and the lowest Cd
concentration, while protein concentration was signifi-
cantly higher than control only (one-way ANOVA, Tukey,
Po0:05). The addition of both Zn concentrations to the
culture medium led to significant increases in the concen-
trations of SH compounds and protein (one-way ANOVA,
Tukey, Po0:05). The exposure to the highest Cu concen-
tration significantly increased the levels of SH compounds
and protein in F. fusiramosa (one-way ANOVA, Tukey,
Po0:05).
3.2. Short-term effects of heavy metals on the production of
thiol compounds
The effects of heavy metals on the production of SH-
compounds and on the protein concentration in mycelia of
F. curta were examined in 8-day-old cultures exposed to
metals for 14–158 h (Fig. 3). The production of T-SH and
NP-SH was significantly affected by Cd concentration but
not by exposure time (two-way ANOVA, Pp0:03 and
PX0:34, respectively; Fig. 3A). The T-SH level in mycelia
exposed to the highest Cd concentration was significantly
higher than that in control after 14 h of exposure (Tukey,
Po0:05). At this time, the level of NP-SH was significantly
higher in both Cd concentrations and increased with the
increase of Cd concentration (Tukey, Po0:001, for all
cases).
Zinc concentration and exposure time significantly
affected the production of T-SH and NP-SH in F. curta
(two-way ANOVA, Pp0:006; Fig. 3B). The highest Zn
concentration increased the levels of T-SH after 14 and 62 h
(Tukey, Po0:05). Both Zn concentrations increased
NP-SH levels (Tukey, Po0:01), despite that after 62 h the
increase was significant only at the highest Zn concentra-
tion (Tukey, Po0:05).
(C)
Fig. 3. Short-term effects of Cd (A), Zn (B), and Cu (C) on the
concentration of total (black), nonprotein (gray) and protein-bound
(dashed) thiols, and protein (white) in F. curta. Mycelia were grown 8 days
with no addition of metal ions and then transferred to fresh medium with
either no addition or addition of the metals for different periods
(14–158 h). Data are percentage of the control (control ¼ 100%).
Mean7SE of three independent experiments. Actual values of controls
are in Table 1. nd, not determined; nm, not measurable.
In F. curta the production of T-SH and NP-SH was
significantly affected by Cu concentration but not by
exposure time (two-way ANOVA, Pp0:008 and PX0:25,
respectively; Fig. 3C). NP-SH level in mycelia was
decreased by exposure to the highest Cu concentration
after 62 h (Tukey, Po0:05) and by both Cu concentrations
at longer times (Tukey, Po0:01). Mycelial protein
concentration was significantly increased by exposure to
the highest Cu concentration for 158 h (Tukey, Po0:05).
When 8-day-old mycelia of F. fusiramosa were exposed
to Cd for 14–110 h, the concentrations of T-SH, PB-SH,
and protein were not affected by either Cd concentration or
exposure time (two-way ANOVA, PX0:17; Fig. 4A). On
the contrary, exposure to the highest Cd concentration for
62 h significantly increased the level of NP-SH compared
with the control and the lowest Cd concentration (Tukey,
Po0:05).
 ARTICLE IN PRESS
40 L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43
(A)
(B)
(C)
Fig. 4. Short-term effects of Cd (A), Zn (B), and Cu (C) on the
concentration of total (black), nonprotein (gray) and protein-bound
(dashed) thiols, and protein (white) in F. fusiramosa. Mycelia were grown
8 days with no addition of metal ions and then transferred to fresh
medium with either no addition or addition of the metals for different
periods (14–110 h). Data are percentage of the control (control ¼ 100%).
Mean7SE of three independent experiments. Actual values of controls
are in Table 1.
The concentrations of T-SH, PB-SH, and protein in F.
fusiramosa were not significantly affected by either Zn
concentration or exposure time (two-way ANOVA,
PX0:054; Fig. 4B). The production of NP-SH was affected
by exposure time and time–concentration interaction (two-
way ANOVA, Pp0:01). Lower levels of NP-SH were
observed after 62 h of exposure to the highest Zn
concentration compared with either the control or other
exposure times (Tukey, Po0:05).
Concentrations of SH compounds and protein in F.
fusiramosa were significantly affected by exposure time but
not by Cu concentration (two-way ANOVA, Pp0:005 and
PX0:173, respectively; Fig. 4C). Mycelia exposed to the
highest Cu concentration had significantly higher levels of
all SH compounds and protein after 62 h than after 14 h
(Tukey, Po0:05). After 62 h, mycelia exposed to the lowest
Cu concentration had significantly higher level of protein
(Tukey, Po0:05), whereas exposure to the highest Cu
concentration significantly increased the levels of T-SH,
PB-SH, and protein in relation to those of control (Tukey,
Po0:05).
4. Discussion
Data from the literature indicate a greater tolerance to
heavy metals in fungi isolated from metal-polluted sites
(e.g., Colpaert et al., 2000; Martino et al., 2000; Miersch et
al., 1997), but this is not always the case (e.g., Baldrian and
Gabriel, 2002; Blaudez et al., 2000b; Colpaert et al., 2000;
Miersch et al., 1997). In this study, the effects of Cd, Zn,
and Cu on biomass production were less pronounced in F.
curta than in F. fusiramosa, as indicated by higher EC50
and EC80 values estimated for the former species. The EC50
values were 17 times higher for Cd and 2 times higher for
Cu in F. curta than in F. fusiramosa, increasing the
importance of elucidating the mechanisms underlying the
tolerance of these aquatic fungi to heavy metals. Taking
into account that (i) F. curta was isolated from a metal-
contaminated site and F. fusiramosa from a clean site and
(ii) F. curta is a dominant species in polluted streams from
northwest Portugal at sites where F. fusiramosa was never
found (Pascoal and Cássio, 2004; Pascoal et al., 2003,
2005b), species identity might be an important feature
when heavy metal tolerance is considered. The isolation
and characterization of other strains of these aquatic
hyphomycete species would be helpful to clarify this point,
since some studies show high fungal intraspecific variability
in metal tolerance (Baldrian and Gabriel, 2002; Blaudez et
al., 2000b).
F. fusiramosa grown in medium without added heavy
metals had a T-SH concentration 2.4 times lower than that
of F. curta, the species isolated from the metal contami-
nated site, suggesting that high levels of constitutive thiol
compounds might be relevant for metal detoxification.
Because streams can be either occasionally or chronically
exposed to effluents containing heavy metals, consideration
of both short- and long-term effects of heavy metals on
aquatic hyphomycetes is important. Responses of thiol
compounds to heavy metals were observed later in F.
fusiramosa (after 62 h) than in F. curta (after 14 h). The
more rapid response of F. curta to metal exposure may also
contribute to its presence at metal-contaminated sites. The
magnitude of thiol compound responses to long-term
exposure in F. fusiramosa was generally greater than that
in F. curta, suggesting that the inductive production of
thiol compounds might be particularly important to face
heavy metal toxicity in species from clean sites. Similarly, a
strain of the aquatic hyphomycete Articulospora tetracladia
isolated from a clean stream, in contrast to a strain isolated
from a metal-polluted stream, increased the production of
thiol compounds when grown in the presence of Cd
(Miersch et al., 2001).
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In both fungal species, NP-SH and PB-SH accounted for
approximately 30% and 70% of T-SH, respectively, when
grown without added heavy metals. Short- and long-term
exposure to Cd led to an increased production of NP-SH in
both F. curta and F. fusiramosa. Miersch et al. (1997, 2001)
also found increased synthesis of NP-SH by exposure to Cd
in aquatic hyphomycete species isolated from either a
nonpolluted stream (Heliscus lugdunensis and A. tetracla-
dia) or a stream occasionally receiving effluents with Cd
(Varicosporium elodeae). A linear increase in glutathione
levels with increasing Cd concentration was found in a
strain of A. tetracladia isolated from a nonpolluted stream
(Miersch et al., 2001), and glutathione is reported to
participate in the complexation of Cd in Saccharomyces
cerevisiae (Li et al., 1997). The increase of NP-SH in
response to Cd exposure occurs in various organisms, e.g.,
the yeast Schizosaccharomyces pombe (Al-Lahham et al.,
1999), the ascomycete Neurospora crassa (Kneer et al.,
1992), the zygomycete Mucor racemosus (Miersch et al.,
2001), the ectomycorrhizal fungus Paxillus involutus
(Courbot et al., 2004), the marine microalga Tetraselmis
suecica (Pérez-Rama et al., 2001), and the water moss
Fontinalis antipyretica (Bruns et al., 2001). The production
of PB-SH was also stimulated in Cd-adapted mycelia of F.
fusiramosa, suggesting that protein compounds may also be
involved in Cd detoxification in aquatic hyphomycetes.
Indeed, in the aquatic hyphomycete H. lugdunensis Cd
exposure induced the production of proteins with
5–15 kDa, some of which reacted with antibodies against
metallothionein (Miersch et al., 2003). The production of
metallothioneins was reported to protect cells against Cd
toxicity in P. involutus (Courbot et al., 2004), in Yarrowia
lipolytica (Strouhal et al., 2003) and in a Cd-resistant strain
of S. cerevisiae (Macreadie et al., 1994).
In contrast to F. curta, short-term exposure to Zn did
not increase SH compound levels in F. fusiramosa.
However, both species exhibited increased production of
PB-SH in Zn-adapted mycelia, and in the case of F.
fusiramosa NP-SH production was also increased. Zinc is
known to induce the production of phytochelatins and/or
phytochelatin-related peptides in S. cerevisiae (Kneer et al.,
1992) and S. pombe (Hayashi and Mutoh, 1994). In
addition to phytochelatins, metallothioneins are also
produced after Zn exposure in S. pombe (Borrelly et al.,
2002).
The increased level of T-SH in Cu-adapted mycelia of F.
fusiramosa was due to the enhanced production of both
NP-SH and PB-SH. Copper induces the synthesis of
phytochelatins in S. pombe (Hayashi and Mutoh, 1994)
and in S. cerevisiae (Kneer et al., 1992). In the latter
(Macreadie et al., 1994) and in Candida glabrata (Mehra et
al., 1989), Agaricus bisporus (Münger and Lerch, 1985),
and N. crassa (Münger et al., 1987), production of
metallothioneins is also observed after Cu exposure. On
the contrary, Cu did not lead to increased levels of any of
the thiol compounds in F. curta, and a decrease of NP-SH
levels at shorter exposure times was observed. Miersch et
41
al. (2001) reported diminished levels of glutathione in Cu-
exposed A. tetracladia, which were not accompanied by the
synthesis of phytochelatins. The decrease in the measured
levels of NP-SH may indicate that these compounds were
oxidized in Cu-exposed mycelia, probably due to Cu
participation in Fenton-type reactions (see Bai et al.
(2003)). Not independently from this, an imbalance in the
synthesis/recycling and consumption/degradation of NP-
SH may have occurred due to an excess of Cu ions.
Contrarily to that observed in F. fusiramosa, the protein
concentration in mycelia of F. curta decreased at later times
after transference to fresh medium (62–158 h; Table 1).
Considering that both fungi were at the same growth phase
before transference and continued to grow, we have no
straightforward explanation for the decrease in protein
concentration in F. curta. After metal exposure, the protein
concentration in mycelia of both aquatic hyphomycete
species increased whenever increased levels of PB-SH were
observed. This finding can be due, at least partially, to an
increased production of metal-chelating proteins including
metallothioneins, as suggested for the freshwater proto-
zoon Euglena gracilis after Cu and Zn exposure (Einicker-
Lamas et al., 2002) and for the frog Rana ridibunda after
Cd exposure (Vogiatzis and Loumbourdis, 1998, 1999).
5. Conclusions
F. curta had higher levels of thiol compounds and
increased their production more rapidly than F. fusiramosa
when exposed to Cd or Zn, suggesting that both the
constitutive levels of thiol compounds and their rapid
increases after metal exposure may account for the
presence of F. curta in metal-polluted streams. The species
F. fusiramosa, consistently found at clean sites, showed a
greater increase in the production of thiol compounds in
mycelia adapted to all metals. However, the delayed thiol
compound response of F. fusiramosa may have compro-
mised its survival in metal-polluted streams. In addition,
both NP-SH and PB-SH appear to be involved in metal
detoxification in aquatic hyphomycetes. Nevertheless,
further studies using strains of these species isolated from
different sites would be helpful to clarify whether the
response of thiol compounds to metals is species and/or
strain dependent.
Acknowledgments
This work was supported by the Portuguese project
POCTI/34024/BSE/2000. L. Guimarães-Soares was sup-
ported by the Portuguese Foundation for the Science and
the Technology (SFRH/BD/3193/2000).
References
Abel, T.H., Bärlocher, F., 1984. Effects of cadmium on aquatic
hyphomycetes. Appl. Environ. Microbiol. 48, 245–251.
 ARTICLE IN PRESS
42 L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43
Al-Lahham, A., Rohde, V., Heim, P., Leuchter, R., Veeck, J., Wunderlich,
C., Wolf, K., Zimmermann, M., 1999. Biosynthesis of phytochelatins in
the fission yeast. Phytochelatin synthesis: a second role for the glutathione
synthetase gene of Schizosaccharomyces pombe. Yeast 15, 385–396.
Ayres, R.U., 1992. Toxic heavy metals: materials cycle optimization. Proc.
Natl. Acad. Sci. USA 89, 815–820.
Bai, Z., Harvey, L.M., McNeil, B., 2003. Oxidative stress in submerged
cultures of fungi. Crit. Rev. Biotechnol. 23, 267–302.
Baldrian, P., 2003. Interactions of heavy metals with white-rot fungi.
Enzyme Microb. Technol. 32, 78–91.
Baldrian, P., Gabriel, J., 2002. Intraspecific variability in growth response
to cadmium of the wood-rotting fungus Piptoporus betulinus.
Mycologia 94, 428–436.
Bärlocher, F., 1992. The Ecology of Aquatic Hyphomycetes. Springer,
Berlin.
Bermingham, S., Maltby, L., Cooke, R.C., 1996a. Effects of a coal mine
effluent on aquatic hyphomycetes. I. Field study. J. Appl. Ecol. 33,
1311–1321.
Bermingham, S., Maltby, L., Cooke, R.C., 1996b. Effects of a coal mine
effluent on aquatic hyphomycetes. II. Laboratory toxicity experiments.
J. Appl. Ecol. 33, 1322–1328.
Binz, P.-A., Kägi, J.H.R., 1999. Metallothionein: molecular evolution and
classification. In: Klaassen, C. (Ed.), Metallothionein IV. Birkhäuser
Verlag, Basel, pp. 7–13.
Blaudez, D., Botton, B., Chalot, M., 2000a. Cadmium uptake and
subcellular compartmentation in the ectomycorrhizal fungus Paxillus
involutus. Microbiology 146, 1109–1117.
Blaudez, D., Jacob, C., Turnau, K., Colpaert, J.V., Ahonen-Jonnarth, U.,
Finlay, R., Botton, B., Chalot, M., 2000b. Differential responses of
ectomycorrhizal fungi to heavy metals in vitro. Mycol. Res. 104,
1366–1371.
Borrelly, G.P.M., Harrison, M.D., Robinson, A.K., Cox, S.G., Robinson,
N.J., Whitehall, S.K., 2002. Surplus zinc is handled by Zym1
metallothionein and Zhf endoplasmic reticulum transporter in
Schizosaccharomyces pombe. J. Biol. Chem. 277, 30394–30400.
Bruns, I., Sutter, K., Menge, S., Neumann, D., Krauss, G.-J., 2001.
Cadmium lets increase the glutathione pool in bryophytes. J. Plant
Physiol. 158, 79–89.
Cervantes, C., Gutierrez-Corona, F., 1994. Copper resistance mechanisms
in bacteria and fungi. FEMS Microbiol. Rev. 14, 121–137.
Clemens, S., Bloss, T., Vess, C., Neumann, D., Nies, D.H., zur Nieden,
U., 2002. A transporter in the endoplasmic reticulum of Schizosac-
charomyces pombe cells mediates zinc storage and differentially affects
transition metal tolerance. J. Biol. Chem. 277, 18215–18221.
Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins:
roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant
Biol. 53, 159–182.
Colpaert, J.V., Vandenkoornhuyse, P., Adriaensen, K., Vangronsveld, J.,
2000. Genetic variation and heavy metal tolerance in the ectomycor-
rhizal basidiomycete Suillus luteus. New Phytol. 147, 367–379.
Courbot, M., Diez, L., Ruotolo, R., Chalot, M., Leroy, P., 2004.
Cadmium-responsive thiols in the ectomycorrhizal fungus Paxillus
involutus. Appl. Environ. Microbiol. 70, 7413–7417.
Duarte, S., Pascoal, C., Cássio, F., 2004. Effects of zinc on leaf
decomposition by fungi in streams: studies in microcosms. Microb.
Ecol. 48, 366–374.
Einicker-Lamas, M., Mezian, G.A., Fernandes, T.B., Silva, F.L.S.,
Guerra, F., Miranda, K., Attias, M., Oliveira, M.M., 2002. Euglena
gracilis as a model for the study of Cu2+ and Zn2+ toxicity and
accumulation in eukaryotic cells. Environ. Pollut. 120, 779–786.
Gadd, G.M., 1993. Interactions of fungi with toxic metals. New Phytol.
124, 25–60.
Gimmler, H., de Jesus, J., Greiser, A., 2001. Heavy metal resistance of the
extreme acidotolerant filamentous fungus Bispora sp. Microb. Ecol. 42,
87–98.
Gonc- alves, M.A.P., 2001. Determinac- ão de metais pesados em águas
superficiais recolhidas no rio Este. M.Sc. Thesis, University of Minho,
Braga, Portugal.
Hayashi, Y., Mutoh, N., 1994. Cadystin (phytochelatin) in fungi. In:
Winkelmann, G., Winge, D.R. (Eds.), Metal Ions in Fungi. Marcel
Dekker, New York, pp. 311–337.
Kneer, R., Kutchan, T.M., Hochberger, A., Zenk, M.H., 1992.
Saccharomyces cerevisiae and Neurospora crassa contain heavy metal
sequestering phytochelatin. Arch. Microbiol. 157, 305–310.
Krauss, G., Bärlocher, F., Schreck, P., Wennrich, R., Glässer, W., Krauss,
G.-J., 2001. Aquatic hyphomycetes occur in hyperpolluted waters in
Central Germany. Nova Hedwigia 72, 419–428.
Li, Z.-S., Lu, Y.-P., Zhen, R.-G., Szczypka, M., Thiele, D.J., Rea, P.A.,
1997. A new pathway for vacuolar cadmium sequestration in
Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathio-
nato)cadmium. Proc. Natl. Acad. Sci. USA 94, 42–47.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193,
265–275.
Macreadie, I.G., Sewell, A.K., Winge, D.R., 1994. Metal ion resistance
and the role of metallothionein in yeast. In: Winkelmann, G., Winge,
D.R. (Eds.), Metal Ions in Fungi. Marcel Dekker, New York, pp.
279–310.
Martino, E., Franco, B., Piccoli, G., Stocchi, V., Perotto, S., 2002.
Influence of zinc ions on protein secretion in a heavy metal tolerant
strain of the ericoid mycorrhizal fungus Oidiodendron maius. Mol. Cell.
Biochem. 231, 179–185.
Martino, E., Turnau, K., Girlanda, M., Bonfante, P., Perotto, S., 2000.
Ericoid mycorrhizal fungi from heavy metal polluted soils: their
identification and growth in the presence of zinc ions. Mycol. Res. 104,
338–344.
Marvanová, L., Pascoal, C., Cássio, F., 2003. New and rare hyphomycetes
from streams of northwest Portugal. Part I. Cryptogam. Mycol. 24,
339–358.
Mehra, R.K., Garey, J.R., Butt, T.R., Gray, W.R., Winge, D.R.,
1989. Candida glabrata metallothioneins. Cloning and sequence of
the genes and characterization of proteins. J. Biol. Chem. 264,
19747–19753.
Miersch, J., Bärlocher, F., Bruns, I., Krauss, G.-J., 1997. Effects of
cadmium, copper, and zinc on growth and thiol content of aquatic
hyphomycetes. Hydrobiologia 346, 77–84.
Miersch, J., Friedenberger, M., Augustin, R., Wohlfromm, S., Rother,
M., Menge, S., Schierhorn, A., Rücknagel, P., Krauss, G.-J., 2003.
Heavy metal-induced peptides and proteins in the aquatic fungus
Heliscus lugdunensis Sacc. et Therry. Eighth International Congress on
Amino Acids and Proteins (Abstracts), Rome, Italy, September 5–9
(Amino Acids 25, 182).
Miersch, J., Tschimedbalshir, M., Bärlocher, F., Grams, Y., Pierau, B.,
Schierhorn, A., Krauss, G.-J., 2001. Heavy metals and thiol
compounds in Mucor racemosus and Articulospora tetracladia. Mycol.
Res. 105, 883–889.
Münger, K., Lerch, K., 1985. Copper metallothionein from the fungus
Agaricus bisporus: chemical and spectroscopic properties. Biochemistry
24, 6751–6756.
Münger, K., Germann, U.A., Lerch, K., 1987. The Neurospora crassa
metallothionein gene. Regulation of expression and chromosomal
location. J. Biol. Chem. 262, 7363–7367.
Pascoal, C., Cássio, F., 2004. Contribution of fungi and bacteria to leaf
litter decomposition in a polluted river. Appl. Environ. Microbiol. 70,
5266–5273.
Pascoal, C., Cássio, F., Marvanová, L., 2005a. Anthropogenic stress may
affect aquatic hyphomycete diversity more than leaf decomposition in
a low-order stream. Arch. Hydrobiol. 162, 481–496.
Pascoal, C., Marvanová, L., Cássio, F., 2005b. Aquatic hyphomycete
diversity in streams of Northwest Portugal. Fungal Divers. 19,
109–128.
Pascoal, C., Pinho, M., Cássio, F., Gomes, P., 2003. Assessing structural
and functional ecosystem condition using leaf breakdown: studies on a
polluted river. Freshwater Biol. 48, 2033–2044.
Pérez-Rama, M., López, C.H., Alonso, J.A., Vaamonde, E.T., 2001. Class
III metallothioneins in response to cadmium toxicity in the marine
 ARTICLE IN PRESS
L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43
microalga Tetraselmis suecica (Kylin) Butch. Environ. Toxicol. Chem.
20, 2061–2066.
Sayer, J.A., Gadd, G.M., 2001. Binding of cobalt and zinc by organic
acids and culture filtrates of Aspergillus niger grown in the absence or
presence of insoluble cobalt or zinc phosphate. Mycol. Res. 105,
1261–1267.
Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound, and
nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal.
Biochem. 25, 192–205.
Soares, H.M.V.M., Boaventura, R.A.R., Machado, A.A.S.C., Esteves da
Silva, J.C.G., 1999. Sediments as monitors of heavy metal contamina-
tion in the Ave river basin (Portugal): multivariate analysis of data.
Environ. Pollut. 105, 311–323.
Strouhal, M., Kizek, R., Vacek, J., Trnková, L., Němec, M., 2003.
Electrochemical study of heavy metals and metallothionein in yeast
Yarrowia lipolytica. Bioelectrochemistry 60, 29–36.
43
Suberkropp, K.F., 1998. Microorganisms and organic matter decomposi-
tion. In: Naiman, R.J., Bilby, R.E. (Eds.), River Ecology and
Management: Lessons from the Pacific Coastal Ecoregion. Springer,
New York, pp. 120–143.
Vogiatzis, A.K., Loumbourdis, N.S., 1998. Cadmium accumulation in
liver and kidneys and hepatic metallothionein and glutathione levels in
Rana ridibunda, after exposure to CdCl2. Arch. Environ. Contam.
Toxicol. 34, 64–68.
Vogiatzis, A.K., Loumbourdis, N.S., 1999. A study of glycogen, lactate,
total fats, protein, and glucose concentration in the liver of the frog
Rana ridibunda, after exposure to cadmium for 30 days. Environ.
Pollut. 104, 335–340.
Zar, J.H., 1996. Biostatistical Analysis, third ed. Prentice-Hall, Englewood
Cliffs, NJ.