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ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 66 (2007) 36–43
www.elsevier.com/locate/ecoenv

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 g1 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.
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Keywords: Aquatic hyphomycetes; Fungi; Heavy metals; Thiol compounds

1. Introduction precipitation of metal ions (Baldrian, 2003; Gadd, 1993;
Martino et al., 2002; Sayer and Gadd, 2001), (b) metal
Heavy metals are continuously being mobilized and binding to cell walls (Blaudez et al., 2000a), (c) transport of
dispersed into the biosphere by natural processes and metal cations (Blaudez et al., 2000a; Clemens et al., 2002;
human activities, such as agriculture, mining, and industry, Li et al., 1997), (d) chemical transformation of metals
and can constitute a serious environmental hazard (Ayres, (Gadd, 1993), (e) organellar compartmentation (Blaudez et
1992). While some heavy metals (e.g., Zn and Cu) are al., 2000a; Clemens et al., 2002; Li et al., 1997), and (f)
essential to living organisms, others (e.g., Cd) have no synthesis of thiol-containing compounds, such as the
apparent biological function. However, both essential and nonproteinaceous glutathione and phytochelatins and the
nonessential heavy metals can be toxic above a critical metallothionein proteins of families 8–13 (fungi I–VI
concentration that depends on the organism, the physico- MTs), which can sequester metal ions (Binz and Kägi,
chemical properties of the metal, and the environmental 1999; Cervantes and Gutierrez-Corona, 1994; Cobbett and
factors (Blaudez et al., 2000b; Gadd, 1993; Gimmler et al., Goldsbrough, 2002; Gadd, 1993; Miersch et al., 2001).
2001). Aquatic hyphomycetes are a group of freshwater fungi
Fungi, like all living organisms, have evolved a set of that play a key role as decomposers of plant detritus in
mechanisms that control and respond to the uptake and streams (Bärlocher, 1992). Owing to their enzymatic
accumulation of heavy metals. Possible interactions be- capabilities they ensure the transformation of organic
tween toxic metals and fungi include (a) production and matter into a food source more accessible to higher trophic
secretion of organic acids, polysaccharides, melanins, or levels, primarily to macroinvertebrate detritivores (Sub-
proteins and subsequent binding/complexation and/or erkropp, 1998). Few studies have examined the effects of
heavy metals on aquatic hyphomycetes, including the
Corresponding author. Fax.: +351 253678980. effects of Fe and Mn (Bermingham et al., 1996b) and Cd
E-mail address: fcassio@bio.uminho.pt (F. Cássio). (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
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L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43 37

and the effects of Cd, Cu and Zn on the growth and thiol salts; Sigma) were used to obtain concentrations of metals that inhibited
biomass production for 8 days by 50% (EC50) and 80% (EC80).
compound production (Miersch et al., 1997, 2001) in a few
Concentration values for F. fusiramosa were Cd 1.2 and 3 mM, Zn 78
species of this group of fungi. Heavy metal exposure has and 100 mM, and Cu 35 and 50 mM. Values for F. curta were Cd 20 and
been reported to depress leaf litter decomposition in 28 mM, Zn 78 and 130 mM, and Cu 68 and 90 mM. Culture flasks were
streams (Bermingham et al., 1996a; Duarte et al., 2004) incubated on a shaker (160 rpm, Certomat BS 3; B. Braun Biotech Int.) at
and to decrease the activity of aquatic fungi (Duarte et al., 18 1C and under permanent artificial light.
2004). Although fungal diversity has been recognized to To evaluate the effects of heavy metals on the production of thiol
compounds, the aquatic hyphomycetes were grown for 8 days in liquid
decrease due to heavy metal exposure (Bermingham et al., medium with or without heavy metals added at the concentrations
1996a), several species of aquatic hyphomycetes have been indicated above. At this time, cultures of both fungi were at the same
found in metal-polluted streams (Krauss et al., 2001; growth phase, with biomass increasing linearly with time (not shown).
Pascoal et al., 2005a). 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
Flagellospora curta is an aquatic hyphomycete species
1 L of medium, in the case of F. curta, with 6 conidia mL1 (final
which has been consistently found on decomposing leaves concentration). Conidial suspensions were obtained through 3 days
in polluted streams in northwest Portugal (Pascoal and aeration of agar slices of 14–21-day-old cultures in sterile deionized water.
Cássio, 2004; Pascoal et al., 2003, 2005a). The presence of To test short-term effects of heavy metals on the production of thiol
this species at metal-polluted sites, where other widely compounds, mycelia grown 8 days in liquid medium without added heavy
distributed species fail to appear, suggests that it should metals were aseptically harvested by filtration (0.5 mm mesh), washed with
cold sterile deionized water, and then transferred to fresh medium
have mechanisms to overcome metal toxicity. In contrast, (0.15–0.20 mg dry mass mL1) with or without added heavy metals at the
Fontanospora fusiramosa, a species which has been concentrations indicated above. Mycelia were incubated in 500-mL
restricted to clean streams (Pascoal et al., 2005b), may be Erlenmeyer flasks containing 200 mL of medium in the case of F.
absent from metal-polluted sites due to a low metal fusiramosa and in 1-L flasks containing 500 mL of medium in the case of F.
tolerance. Because metal detoxification and homeostasis curta, for periods from 14 to 158 h during which both fungi were actively
growing (not shown).
require intracellular complexation of metals, we expect that Different flask volumes were used because F. curta produces less
increased fungal metal tolerance may be related to the pool biomass than F. fusiramosa. However, the medium/air volume ratios used
of thiol-containing compounds. This could mean higher in this work did not significantly affect the biomass production per 1 L of
constitutive levels, higher capacity of production, and/or a medium (not shown).
more rapid response to metal exposure. In this work, the
production of thiol compounds after short- and long-term 2.3. Preparation of cell-free extracts
exposure to the heavy metals Cd, Zn, and Cu was
examined in F. fusiramosa and F. curta isolated from a Mycelia were harvested as described above, rinsed twice with cold
deionized water, and blotted dry between layers of filter paper. Mycelia
clean and a metal-polluted site of the same river, were then quick-frozen in liquid nitrogen and ground with 2 g of purified
respectively. The production of both nonprotein and sea sand (Merck) per 1 g of mycelium wet mass in a precooled pestle and
protein-bound thiols was analyzed to discriminate their mortar. The resultant paste was suspended in icecold 20 mM Tris(hy-
relative contribution to metal tolerance in these aquatic droxymethyl)aminomethane (Tris; Merck), pH 7.5, containing 1 mM
fungi. 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
2. Materials and methods 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
2.1. Fungal species and culture maintenance both cases. The final supernatant was filtered (Filtropur S, 0.45 mm;
Sarstedt) and kept on ice until used. Under these conditions oxidation of
F. fusiramosa Marvanová, Peter J. Fisher et Descals (UMB-25.01) was thiol compounds was minimized, as indicated by the absence of significant
isolated from foam at the source of the Este River (northwest Portugal) differences in the measured thiol content of a glutathione solution
and F. curta J. Webster (UMB-39.01) was isolated from alder leaves (reduced GSH) submitted or not to the steps of extraction/clarification
collected 10 km downstream, near the industrial park of the town of (not shown). Portions of mycelium were dried to constant mass at 85 1C to
Braga. In contrast to the clean site, the polluted site had high nutrient convert wet to dry mass.
+
loads (e.g., N-NO 1
3 4.97 mg L , N-NH4 0.25 mg L1, and P-PO3 4
1
0.18 mg L ; Pascoal et al., 2005a) from domestic sewage and agriculture 2.4. Estimation of total (T-SH), nonprotein (NP-SH), and
and increased levels of heavy metals in the water column (e.g., Zn protein-bound (PB-SH) thiols
2.02 mg L1, Cu 5.87 mg L1, Cd 0.02 mg L1, Pb 5.30 mg L1, and Cr
0.20 mg L1; Gonc- alves, 2001) and sediments (e.g., Cu 518.1 g kg1, Cd
The concentration of T-SH and NP-SH was determined with 5,50 -
0.14 g kg1, and Pb 16.4 g kg1; Soares et al., 1999). Details on the fungal
dithio-bis(2-nitrobenzoic acid) (DTNB; Sigma) according to Sedlak and
species are presented in Marvanová et al. (2003) and Pascoal et al. (2005b).
Lindsay (1968). To determine T-SH, 50-mL aliquots of the cell-free
Fungi were maintained on medium containing 2% (w/v) malt extract
extracts were mixed with 150 mL of 0.2 M Tris, pH 8.2, and 10 mL of
and 2% (w/v) agar, at 18 1C and under permanent artificial light.
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
2.2. Growth in liquid medium, harvesting, and transfer of mycelia (3000g, 15 min) and the absorbance was measured at 412 nm (Perkin
Elmer Lambda 2 UV/VIS Spectrometer) against a reagent blank (without
Fungi were grown in liquid medium containing 1% (w/v) malt extract, sample), using reduced glutathione (Sigma) as standard.
with pH adjusted to 5.0 with HCl prior to autoclaving. Sterilized To determine NP-SH, aliquots of 500 mL of the cell-free extracts were
(Filtropur S, 0.2 mm; Sarstedt) stock solutions of Cd, Zn, and Cu (chloride mixed with 400 mL of milli-Q water and 100 mL of 50% trichloroacetic acid
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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 g1 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
Fig. 1. Effects of Cd, Zn, and Cu on the concentration of total (black),
When F. curta was grown 8 days in liquid medium
nonprotein (gray) and protein-bound (dashed) thiols, and protein (white)
without added heavy metals T-SH concentration in the in 8-day-grown mycelia of F. curta. Data are percentage of the control
mycelia, expressed as mmol g1 dry mass, was 3.4670.37, (control ¼ 100%). Mean7SE of three independent experiments. Actual
of which 1.1670.09 was NP-SH and 2.3070.29 was 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 g1 dry mass) Protein (mg g1 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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(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 (C)
and protein in F. fusiramosa (one-way ANOVA, Tukey,
Fig. 3. Short-term effects of Cd (A), Zn (B), and Cu (C) on the
Po0:05). concentration of total (black), nonprotein (gray) and protein-bound
(dashed) thiols, and protein (white) in F. curta. Mycelia were grown 8 days
3.2. Short-term effects of heavy metals on the production of with no addition of metal ions and then transferred to fresh medium with
thiol compounds 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
The effects of heavy metals on the production of SH- are in Table 1. nd, not determined; nm, not measurable.
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 In F. curta the production of T-SH and NP-SH was
not by exposure time (two-way ANOVA, Pp0:03 and significantly affected by Cu concentration but not by
PX0:34, respectively; Fig. 3A). The T-SH level in mycelia exposure time (two-way ANOVA, Pp0:008 and PX0:25,
exposed to the highest Cd concentration was significantly respectively; Fig. 3C). NP-SH level in mycelia was
higher than that in control after 14 h of exposure (Tukey, decreased by exposure to the highest Cu concentration
Po0:05). At this time, the level of NP-SH was significantly after 62 h (Tukey, Po0:05) and by both Cu concentrations
higher in both Cd concentrations and increased with the at longer times (Tukey, Po0:01). Mycelial protein
increase of Cd concentration (Tukey, Po0:001, for all concentration was significantly increased by exposure to
cases). the highest Cu concentration for 158 h (Tukey, Po0:05).
Zinc concentration and exposure time significantly When 8-day-old mycelia of F. fusiramosa were exposed
affected the production of T-SH and NP-SH in F. curta to Cd for 14–110 h, the concentrations of T-SH, PB-SH,
(two-way ANOVA, Pp0:006; Fig. 3B). The highest Zn and protein were not affected by either Cd concentration or
concentration increased the levels of T-SH after 14 and 62 h exposure time (two-way ANOVA, PX0:17; Fig. 4A). On
(Tukey, Po0:05). Both Zn concentrations increased the contrary, exposure to the highest Cd concentration for
NP-SH levels (Tukey, Po0:01), despite that after 62 h the 62 h significantly increased the level of NP-SH compared
increase was significant only at the highest Zn concentra- with the control and the lowest Cd concentration (Tukey,
tion (Tukey, Po0:05). Po0:05).
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40 L. Guimarães-Soares et al. / Ecotoxicology and Environmental Safety 66 (2007) 36–43

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
(A)

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
(B) 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
(C)
hyphomycete species would be helpful to clarify this point,
Fig. 4. Short-term effects of Cd (A), Zn (B), and Cu (C) on the since some studies show high fungal intraspecific variability
concentration of total (black), nonprotein (gray) and protein-bound in metal tolerance (Baldrian and Gabriel, 2002; Blaudez et
(dashed) thiols, and protein (white) in F. fusiramosa. Mycelia were grown al., 2000b).
8 days with no addition of metal ions and then transferred to fresh
F. fusiramosa grown in medium without added heavy
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%). metals had a T-SH concentration 2.4 times lower than that
Mean7SE of three independent experiments. Actual values of controls of F. curta, the species isolated from the metal contami-
are in Table 1. 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
The concentrations of T-SH, PB-SH, and protein in F. of both short- and long-term effects of heavy metals on
fusiramosa were not significantly affected by either Zn aquatic hyphomycetes is important. Responses of thiol
concentration or exposure time (two-way ANOVA, compounds to heavy metals were observed later in F.
PX0:054; Fig. 4B). The production of NP-SH was affected fusiramosa (after 62 h) than in F. curta (after 14 h). The
by exposure time and time–concentration interaction (two- more rapid response of F. curta to metal exposure may also
way ANOVA, Pp0:01). Lower levels of NP-SH were contribute to its presence at metal-contaminated sites. The
observed after 62 h of exposure to the highest Zn magnitude of thiol compound responses to long-term
concentration compared with either the control or other exposure in F. fusiramosa was generally greater than that
exposure times (Tukey, Po0:05). in F. curta, suggesting that the inductive production of
Concentrations of SH compounds and protein in F. thiol compounds might be particularly important to face
fusiramosa were significantly affected by exposure time but heavy metal toxicity in species from clean sites. Similarly, a
not by Cu concentration (two-way ANOVA, Pp0:005 and strain of the aquatic hyphomycete Articulospora tetracladia
PX0:173, respectively; Fig. 4C). Mycelia exposed to the isolated from a clean stream, in contrast to a strain isolated
highest Cu concentration had significantly higher levels of from a metal-polluted stream, increased the production of
all SH compounds and protein after 62 h than after 14 h thiol compounds when grown in the presence of Cd
(Tukey, Po0:05). After 62 h, mycelia exposed to the lowest (Miersch et al., 2001).
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In both fungal species, NP-SH and PB-SH accounted for al. (2001) reported diminished levels of glutathione in Cu-
approximately 30% and 70% of T-SH, respectively, when exposed A. tetracladia, which were not accompanied by the
grown without added heavy metals. Short- and long-term synthesis of phytochelatins. The decrease in the measured
exposure to Cd led to an increased production of NP-SH in levels of NP-SH may indicate that these compounds were
both F. curta and F. fusiramosa. Miersch et al. (1997, 2001) oxidized in Cu-exposed mycelia, probably due to Cu
also found increased synthesis of NP-SH by exposure to Cd participation in Fenton-type reactions (see Bai et al.
in aquatic hyphomycete species isolated from either a (2003)). Not independently from this, an imbalance in the
nonpolluted stream (Heliscus lugdunensis and A. tetracla- synthesis/recycling and consumption/degradation of NP-
dia) or a stream occasionally receiving effluents with Cd SH may have occurred due to an excess of Cu ions.
(Varicosporium elodeae). A linear increase in glutathione Contrarily to that observed in F. fusiramosa, the protein
levels with increasing Cd concentration was found in a concentration in mycelia of F. curta decreased at later times
strain of A. tetracladia isolated from a nonpolluted stream after transference to fresh medium (62–158 h; Table 1).
(Miersch et al., 2001), and glutathione is reported to Considering that both fungi were at the same growth phase
participate in the complexation of Cd in Saccharomyces before transference and continued to grow, we have no
cerevisiae (Li et al., 1997). The increase of NP-SH in straightforward explanation for the decrease in protein
response to Cd exposure occurs in various organisms, e.g., concentration in F. curta. After metal exposure, the protein
the yeast Schizosaccharomyces pombe (Al-Lahham et al., concentration in mycelia of both aquatic hyphomycete
1999), the ascomycete Neurospora crassa (Kneer et al., species increased whenever increased levels of PB-SH were
1992), the zygomycete Mucor racemosus (Miersch et al., observed. This finding can be due, at least partially, to an
2001), the ectomycorrhizal fungus Paxillus involutus increased production of metal-chelating proteins including
(Courbot et al., 2004), the marine microalga Tetraselmis metallothioneins, as suggested for the freshwater proto-
suecica (Pérez-Rama et al., 2001), and the water moss zoon Euglena gracilis after Cu and Zn exposure (Einicker-
Fontinalis antipyretica (Bruns et al., 2001). The production Lamas et al., 2002) and for the frog Rana ridibunda after
of PB-SH was also stimulated in Cd-adapted mycelia of F. Cd exposure (Vogiatzis and Loumbourdis, 1998, 1999).
fusiramosa, suggesting that protein compounds may also be
involved in Cd detoxification in aquatic hyphomycetes.
5. Conclusions
Indeed, in the aquatic hyphomycete H. lugdunensis Cd
exposure induced the production of proteins with
F. curta had higher levels of thiol compounds and
5–15 kDa, some of which reacted with antibodies against
increased their production more rapidly than F. fusiramosa
metallothionein (Miersch et al., 2003). The production of
when exposed to Cd or Zn, suggesting that both the
metallothioneins was reported to protect cells against Cd
constitutive levels of thiol compounds and their rapid
toxicity in P. involutus (Courbot et al., 2004), in Yarrowia
increases after metal exposure may account for the
lipolytica (Strouhal et al., 2003) and in a Cd-resistant strain
presence of F. curta in metal-polluted streams. The species
of S. cerevisiae (Macreadie et al., 1994).
F. fusiramosa, consistently found at clean sites, showed a
In contrast to F. curta, short-term exposure to Zn did
greater increase in the production of thiol compounds in
not increase SH compound levels in F. fusiramosa.
mycelia adapted to all metals. However, the delayed thiol
However, both species exhibited increased production of
compound response of F. fusiramosa may have compro-
PB-SH in Zn-adapted mycelia, and in the case of F.
mised its survival in metal-polluted streams. In addition,
fusiramosa NP-SH production was also increased. Zinc is
both NP-SH and PB-SH appear to be involved in metal
known to induce the production of phytochelatins and/or
detoxification in aquatic hyphomycetes. Nevertheless,
phytochelatin-related peptides in S. cerevisiae (Kneer et al.,
further studies using strains of these species isolated from
1992) and S. pombe (Hayashi and Mutoh, 1994). In
different sites would be helpful to clarify whether the
addition to phytochelatins, metallothioneins are also
response of thiol compounds to metals is species and/or
produced after Zn exposure in S. pombe (Borrelly et al.,
strain dependent.
2002).
The increased level of T-SH in Cu-adapted mycelia of F.
fusiramosa was due to the enhanced production of both Acknowledgments
NP-SH and PB-SH. Copper induces the synthesis of
phytochelatins in S. pombe (Hayashi and Mutoh, 1994) This work was supported by the Portuguese project
and in S. cerevisiae (Kneer et al., 1992). In the latter POCTI/34024/BSE/2000. L. Guimarães-Soares was sup-
(Macreadie et al., 1994) and in Candida glabrata (Mehra et ported by the Portuguese Foundation for the Science and
al., 1989), Agaricus bisporus (Münger and Lerch, 1985), the Technology (SFRH/BD/3193/2000).
and N. crassa (Münger et al., 1987), production of
metallothioneins is also observed after Cu exposure. On References
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