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Process Biochemistry 44 (2009) 288–295

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Biomarkers of oxidative stress in the fungal strain Humicola lutea under copper exposure Ekaterina Z. Krumova a, Svetlana B. Pashova a, Pavlina A. Dolashka-Angelova b, Tzvetanka Stefanova a, Maria B. Angelova a,* a b

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bonchev str., 1113 Sofia, Bulgaria Institute of Organic Chemistry, Bulgarian Academy of Sciences, 9, Acad. G. Bonchev str., 26, 1113 Sofia, Bulgaria

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 January 2008 Received in revised form 3 October 2008 Accepted 31 October 2008

The fungal strain Humicola lutea 103 was used as a model organism to examine the relationship between copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Spores or submerged cultures were treated with different copper concentrations and the oxidative stress-inducing agent paraquat (PQ). Oxidative stress biomarkers such as reactive oxygen species (ROS), cyanide-resistant respiration, protein carbonyls, reserve carbohydrates, and antioxidant defence were identified in cells treated or not treated with either copper ions or PQ. Copper inhibited the growth and conidiospore formation of H. lutea 103 in a concentration-dependent manner. This treatment also resulted in increased superoxide anion radical formation. Copper stress was furthermore accompanied by transient accumulation of trehalose and glycogen, as well as increased protein carbonyl content. Compared to control cultures, copper-treated mycelia demonstrated a marked increase in the activity of protective enzymes (superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase). These increased antioxidant enzyme activities were blocked by inhibitors of protein synthesis, suggesting that de novo enzyme formation was involved. Biomarker response to the heavy metal was similar to treatment with known ROS generators such as PQ. The observed hyper-oxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells. ß 2008 Elsevier Ltd. All rights reserved.

Keywords: Filamentous fungi Oxidative stress biomarkers Copper toxicity ROS generation Carbonyl groups Antioxidant enzymes

1. Introduction Reactive oxygen species (ROS) such as superoxide (O2), hydrogen peroxide (H2O2), and the hydroxyl radical (OH) are produced intracellularly in all aerobic organisms and are normally in balance with antioxidant molecules. Oxidative stress occurs when this critical balance is disrupted due to depletion of antioxidants or excess accumulation of ROS [1]. ROS generation can occur via several mechanisms [2]. Regardless of how or where they are generated, increased levels of ROS can damage all types of biological molecules, including DNA, lipids, proteins, and carbohydrates. Such damage influences numerous cellular processes, leading to compromised cell function or to cell death [3]. In response to harmful ROS, aerobically growing organisms (eukaryotic and prokaryotic) have evolved multiple defence mechanisms for protection of their cellular components [4]. The

* Corresponding author. E-mail addresses: [email protected], [email protected] (M.B. Angelova). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.10.023

first line of defence against ROS is prevention of their formation. There are also protective proteins that remove ROS, and secondary defences consist of enzymes that remove and repair the products of oxidatively damaged components [5]. Despite the existence of many nonenzymatic anti-oxidant compounds (glutathione, atocopherol, ascorbate), the most efficient way to eliminate ROS is through catalysis by antioxidant enzymes such as superoxide dismutase [EC 1.15.1.1.] (SOD), catalase [EC 1.11.1.6.] (CAT), and peroxidases [EC 1.11.1.7.] (POXs) [6]. Environmental stresses such as exposure to heavy metals are known to promote ROS formation in cells, potentially overwhelming antioxidant defences [7]. This is particularly true of redox-active metals like copper (Cu) that catalyze the Fenton reaction and can accelerate generation of highly damaging OH radicals from O2 and H2O2 substrates [8,2]. At the same time, Cu serves an essential role in biological processes because of its catalytic and structural properties. Thus, the maintenance of Cu homeostasis at the cellular level is crucial for aerobic organisms [9]. The phenomenon of Cu activation of ROS production is described as oxidative stress in different models [10,11]. Exposure to this heavy metal provokes a pronounced response of antioxidant systems [11,12].

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In filamentous fungi, defence mechanisms against Cu stress are currently under investigation [13]. Attention has been paid mainly to alterations in the antioxidant defence system in the presence of Cu ions [14,15]. However, the evaluation of events such as ROS generation, oxidative damage to proteins, and biosynthesis of reserve carbohydrates that are typical of both copper and oxidative stress have been seldom performed. Our previous studies revealed that the fungal strain Humicola lutea 103 produces naturally glycosylated Cu/Zn-superoxide dismutase (Cu/Zn-SOD) at high levels [16,17,18]. Such glycosylated SODs could only be isolated in limited cases. H. lutea enzymes protected against myeloid Graffi tumours in hamsters and experimental influenza virus infection in mice [16,17]. Our preliminary study showed that addition of Cu ions to productive medium resulted in increased enzyme activity. Thus, the Cu stress response in H. lutea cultures might be of industrial interest. The aim of the present investigation was to compare the fungal cell response against copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Specifically, we investigated the effect of increased concentrations of Cu ions on biomarkers of cyanide-resistant respiration (CNRR), ROS, carbonyl groups, and reserve carbohydrates, as well as on the growth of H. lutea cultures. We present evidence indicating that responses to Cu treatment resemble those caused by oxidative stress-inducing agents. This report also suggests a relevant role for antioxidant enzymes in Cu resistance. 2. Materials and methods 2.1. Materials Paraquat (PQ), nitro blue tetrazolium (NBT), cytochrome c, horseradish peroxidase type VI-A, NADPH, a-amyloglucosidase from Aspergillus niger, trehalase, 2,4-dinitrophenylhydrazine (DNPH), hydroxyurea (HU), actinomycin D (AD), 8azaguanine (8-AG), and cycloheximide (CHI) were obtained from Sigma–Aldrich (Deisenhofen, Germany). All other chemicals used in this study were of the highest analytical grade. 2.2. Fungal strain and culture conditions The fungal strain H. lutea 103 from the Mycological Collection at the Institute of Microbiology, Sofia, was used throughout and maintained at 4 8C on beer agar, pH 6.3. To monitor the Cu-resistance of the fungal strain, conidiospores were cultivated in Petri dishes (d = 10 mm) with beer agar supplemented with various concentrations of CuSO4 for 10 days at a temperature of 30 8C. For submerged cultivation, both seed and productive media were used [19]. For the inoculum, 80 ml of seed medium was inoculated with 5 ml of spore suspension at a concentration of 2  108 spores ml1 in 500 ml Erlenmeyer flasks. Cultivation was performed on a shaker (220 rpm) at 30 8C for 24 h. Then, 6 ml of seed culture were transferred to 500 ml Erlenmeyer flasks containing 74 ml of production medium. The cultures were grown at 30 8C for 96 h. For investigation of the effects of different Cu concentrations, cultures were incubated with 3 mM PQ or various concentrations of CuSO4 in order to achieve 40, 70, 100, 150, and 300 mg/ml Cu ions. Results were evaluated from repeated experiments using three or five parallel runs. For some experiments, non-growing mycelium was used. In these cases cells were cultivated for 24 h (mid-logarithmic growth phase) in the seed medium as described above. Then, 1 g of wet mycelium was added to 40 ml of medium III (KH2PO4: 5 g/l and MgSO47H2O: 2.5 g/l, pH 7.8) with or without stress-inducing agents (3 mM PQ or 40, 70, 100, 150, and 300 mg/ml Cu ions) in 500 ml Erlenmeyer flasks, followed by incubation at 30 8C on a shaker (220 rev/min) for 30, 60, and 120 min. For experiments concerning inhibition of protein synthesis, non-growing cells were cultivated in the presence of 180 mg/ml actinomycin D, 500 mg/ml hydroxyurea, 150 mg/ml 8-azaguanine, or 180 mg/ml cycloheximide for 1 h precultivation. This time was necessary for penetration of the inhibitors into the fungal cells. After pre-cultivation, the mycelium was treated with Cu ions or PQ for 3 h. 2.3. Cell-free extract preparation and isolation of mitochondria All steps during the isolation of mitochondria were performed at 0–4 8C. Mycelial disruption and mitochondrial isolation were performed according to Lambowitz [20] with some modifications. Cells from 24 h cultures were harvested using a filter paper-covered funnel (Whatman No. 4 filter, Clifton, USA) with a sieve connected to a vacuum pump, and washed repeatedly on the filter with distilled water and then once with isolation buffer (0.25 M sucrose, 5 mM EDTA, 0.15% bovine serum

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albumin, pH 7.5). For every 10 g of wet weight hyphae, 5 g of quartz sand and 20 ml of the same buffer containing 0.3 mM phenylmethylsulfonyl fluoride (PMSF) were added to the mortar and ground together for 1–2 min at 4 8C. After addition of another 10 ml of the same buffer, the slurry was centrifuged 3 times at 1500  g for 15 min to remove the quartz sand and undisrupted cells. The resulting supernatant was further centrifuged at 15,000  g for 30 min and the liquid fraction was used as the cell-free extract (CFE). The 15,000  g precipitate was resuspended in the same volume of isolation buffer and centrifuged for 40 min at 30,590  g. Then, the supernatant was discarded and the crude mitochondrial pellet (CMP) was carefully washed with SEM buffer (0.25 M sucrose; 5 mM EDTA; 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS)/KOH, pH 7.5). This procedure was repeated three times, and the collected mitochondria were resuspended in SEM buffer. For measurement of ROS generation, the mitochondria were used immediately following their isolation. 2.4. Enzyme activity determination SOD activity was measured in CFE by NBT reduction [21]. One unit of SOD activity was defined as the amount of SOD required for inhibition of the reduction of NBT by 50% (A560) and was expressed as units per mg protein (U/mg protein). Cyanide (5 mM) was used to distinguish between the cyanide-sensitive isoenzyme Cu/ZnSOD and cyanide-resistant Mn SOD. Cu/Zn-SOD activity was obtained as total activity minus the activity in the presence of 5 mM cyanide. Catalase was assayed by the method of Beers and Sizer [22], in which the decomposition of H2O2 was analysed spectrophotometrically at 240 nm. One unit of catalase activity was defined as the amount of enzyme that decomposes 1 mmol H2O2/min at an initial H2O2 concentration of 30 mM at pH 7.0 and 25 8C. Glucose 6-phosphate dehydrogenase (G6PD) activity was measured by glucose 6-phosphate-dependent reduction of NADP+ [23]. One unit was equivalent to 1 mmol of substrate reduced per min. The specific activity is given as U/mg protein. 2.5. Measurement of total and cyanide-resistant respiration Oxygen uptake by fungal cell suspensions treated or not treated with Cu or PQ was measured at 30 8C in the absence and presence of 10 mM cyanide by using an oxygen meter, type 5221-ELWRO (Poland), and expressed as milligrams O2 per gram dry weight per minute. The measurement was carried out as previously described [24]. 2.6. Determination of ROS For measurement of O2 production rate, the method of superoxide dismutaseinhibitable reduction of cytochrome c was used [25] with some modifications. Briefly, cell suspensions or crude mitochondrial pellets taken from 24 h cultures grown in the presence of 3 mM PQ or various Cu2+ concentrations, were incubated for 60 min at 30 8C on a water bath rotary shaker at 150 rpm. The reaction mixtures contained 50 mM cytochrome c, 2% non-autoclaved glucose, and 20 mM NADPH in the presence and absence of 50 mg ml1 of superoxide dismutase from bovine erythrocytes in 0.05 M potassium phosphate buffer at pH 7.8. The reaction was stopped by cooling in an ice-cold water bath. The cells were removed by centrifugation before reading the absorbance at 550 nm to determine the extent of cytochrome c reduction. A molar extinction coefficient of 2.11  104 was used to calculate the concentration of reduced cytochrome c. For measurement of hydrogen peroxide production, the method of Pick and Mizel [26] was used. Briefly, fungal cells or CMP were suspended in 0.05 M potassium phosphate buffer at pH 7.8 containing 50 mg ml1 horseradish peroxidase type VI-A. After incubation at 30 8C for 45 min, the reaction was stopped by addition of 1N NaOH, and the absorbance was read at 620 nm. For calculations, a standard curve with H2O2 concentrations (from 5 to 50 mmol) was used. 2.7. Measurement of protein carbonyl content Protein oxidative damage was measured spectrophotometrically as protein carbonyl content using the DNPH binding assay [27], slightly modified by Adachi and Ishii [28]. Following Cu treatment, the cell-free extracts were incubated with DNPH for 1 h at 37 8C; proteins were precipitated in 10% cold TCA and washed with ethanol:ethylacetate (1:1), to remove excess DNPH and finally dissolved in 6 M guanidine chloride, pH 2. The optimal density was measured at 380 nm, and the carbonyl content was calculated using a molar extinction coefficient of 21 mM1 cm1, resulting in final measurement of nanomoles of DNPH incorporated (protein carbonyls) per mg of protein. 2.8. Determination of reserve carbohydrates In order to determine glycogen and trehalose content, a procedure previously described by Becker [29] and Vandecamen et al. [30] and then modified by Parrou et al. [31] was used. Soluble reducing sugars were determined by the SomogyNelson method [32].

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Fig. 1. Growth of H. lutea spores on the agar medium in presence of 0–500 mg/ml Cu2+. The fungal spore from 7 days culture were grown on the beer agar with addition of CuSO4 in concentration to achieve above mentioned Cu2+ content. 1: control; 2: 70; 3: 100; 4: 200; 5: 300; 6: 500 mg/ml Cu2+, respectively. 2.9. PAGE electrophoresis

CuZn-SOD antibody preparation and immunoblot analysis were performed according to Towbin et al. [33] as previously described by us [17].

Reduction of the dry weight to 84, 76, 69 and 29% of the control occurred at 70, 100, 150 and 300 mg/ml. PQ was used for comparison to the heavy metal treatment. This herbicide (methyl viologen, 1,10 -dimethyl-4,40 -bipyridinium dichloride) is a classical agent that induces oxidative stress in aerobic cells by participating in redox cycling with cellular enzymes. Fig. 2 shows that exposure to 3 mM PQ resulted in almost 50% reduction of biomass in comparison to the control.

2.11. Other analytical methods

3.2. Cu exposure affects CNRR

Protein was estimated by the Lowry procedure [34] using crystalline bovine albumin as a standard. Dry weight determination was performed on samples of mycelia harvested throughout the culture period. The culture fluid was filtered through a Whatman (Clifton, USA) No. 4 filter. The separated mycelia were washed twice with distilled water and dried to a constant weight at 105 8C.

Measurement of the inhibition of cytochrome-dependent respiration (i.e. cyanide-sensitive respiration, CNSR) and CNRR allowed estimation of the influence of different Cu2+ contents and

The SOD isoenzyme profile was determined using polyacrylamide gels. Forty micrograms total protein were applied to 10% nondenaturing PAGE and stained for superoxide dismutase activity as described by Beauchamp and Fridovich [21]. 2.10. Western blot analysis

2.12. Statistical evaluation of the results The results obtained in this investigation were evaluated from at least three repeated experiments using three or five parallel runs. Statistical comparisons between controls and treated cultures were made using Student’s t-test for MIE (mean interval estimation) or one-away analysis of variance (ANOVA) followed by Dunnet’s post-test, with a significance level of 0.05.

3. Results To determine the relationship between oxidative stress and copper toxicity, we first evaluated the copper resistance of the model strain and then measured several oxidative stress biomarkers and the antioxidant status of the fungus. 3.1. Effect of Cu ions on growth and cell differentiation The growth of fungal spores on agar medium supplemented with Cu2+ from 70 to 500 mg/ml is demonstrated in Fig. 1; exposure to CuSO4 affected the growth of H. lutea spores at all concentrations used in a concentration-dependent manner. Moreover, heavy metal content inhibited the formation of conidiospores. Furthermore, growth of H. lutea 103 was studied in relation to 40–300 mg/ml copper exposure under submerged conditions. Fig. 2 demonstrates the maximum biomass content for the different variants. Treatment of fungal cells with 40 mg/ml Cu2+ induced a slight increase (about 18%) in mycelia weight while at above 40 mg/ml, the biomass content decreased continuously.

Fig. 2. Effect of enhanced Cu2+ concentrations and PQ treatment on biomass production by H. lutea, cultivated under submerged conditions. The fungal strain was grown in productive medium supplemented with 0–300 mg/ml Cu2+ or 3 mM PQ during 72 h. The results are representative of five independent determinations in triplicate. Bars represent SD of means. The effect of treatment was significant for the Cu and PQ (p  0.05).

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3.3. Effect of Cu stress on ROS generation

Fig. 3. Stimulation of cyanide-resistant respiration in fungal cultures treated by enhanced Cu ions concentrations or 3 mM PQ. The results are representative of five independent determinations in triplicate. Bars represent SD of means. The effect of treatment was significant for the Cu and PQ (p  0.05).

3 mM PQ on both respiratory pathways in H. lutea. Fig. 3 shows an increase of 50–100% in CNRR in the presence of stress-inducing agents as compared to the control. Copper treatment had no effect on the cyanide-sensitive oxygen uptake rate (CNSR; 264–287 mg O2  106/min per mg dry weight for control samples and cells treated with 40–300 mg/ml Cu2+ and 3 mM PQ), whereas it accelerated cyanide-resistant oxygen consumption. Oxidative stress induced by 3 mM PQ in H. lutea cells showed increased CNRR similar to that caused by high copper concentrations.

To further characterize the potential involvement of oxidative stress on Cu ion stress, the generation of ROS was determined in both intact cells and mitochondrial fractions. Fig. 4 shows the effect of Cu2+ on superoxide anion and H2O2 production in H. lutea cells taken from 24 h culture. Elevated O2 content was found at all Cu concentrations tested. Fig. 4A shows that 70 and 100 mg/ml copper markedly increased the O2 level (by about 133 and 166%, respectively) as compared to the control. Cells treated with 150 and 300 mg/ml showed extremely high O2 levels (475 and 750% of the control, respectively). At the same time, exposure to the same Cu2+ concentrations had no effect on H2O2 levels as compared to the control (Fig. 4B). This assay was previously used to detect a 5-fold increase in H2O2 levels upon heat-shock in A. niger cells [35]. Fig. 4 also depicts a more active process of ROS generation in mitochondrial fractions than in intact cells. The dependence of  O2 generation on Cu content of up to 100 mg/ml is clearly illustrated in Fig. 4C; a plateau in the generation of O2 appears at Cu ion concentrations between 100 and 300 mg/ml. Again, Cu treatment did not result in significant changes in H2O2 production (Fig. 4D). The effects of the redox-cycling compound PQ were compared to those of copper exposure. Oxidative stress induced by 3 mM PQ resulted in increased ROS generation similar to that caused by copper treatment. 3.4. Cu exposure caused protein oxidation Carbonyl formation is a marker of protein oxidation. Increased protein damage upon exposure of exponential phase cultures to 40, 70 and 150 mg/ml Cu2+ was evident after 30, 60 and 120 min for H.

Fig. 4. Dose dependent increase in ROS generation (superoxide anion radicals—A, C, and H2O2—B, D) in the whole cells (A, B) and mitochondria (C, D) treated with Cu2+ or PQ. The results are representative of five independent determinations in triplicate. Bars represent SD of means. The effect of treatment was significant for the Cu and PQ for the superoxide anion content (p  0.05).

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Fig. 5. Time-dependent protein oxidation induced by copper- or PQ treatment of H. lutea cells. Fungal mycelia taken from 24 h culture were cultivated with or without stress-agents (3 mM PQ or 40, 70, 100, 150, and 300 mg/ml Cu ions, respectively) for 30, 60, and 120 min, respectively. The results are representative of five independent determinations in triplicate. Bars represent SD of means. Statistical differences (compared with control) are determined where p < 0.05 as calculated by ANOVA followed by Dunnet’s post-test.

lutea treated cells as compared to the control (Fig. 5). A remarkable increase in carbonyls was detected after exposure to 40 and 70 mg/ ml Cu2+ (1.3–2.4-fold), indicating a hyper-oxidative state. Similar stress conditions were observed at a copper concentration of 100 mg/ml, which increased carbonyl contents from 1.5- to 2.7fold as compared to the control. A higher metal concentration resulted in faster carbonyl group production; while 70 and 100 mg/ ml copper ions caused significantly increased oxidation after 60 and 120 min, Cu concentrations of 150 and 300 mg/ml showed this effect (128 and 236% increase) after 30 min. A similar trend was demonstrated in experiments with PQ. 3.5. Effect of stress on glycogen and trehalose content Another physiological consequence of copper exposure in H. lutea is the accumulation of glycogen and trehalose through stress. Fig. 6A shows that all Cu2+ concentrations tested caused a sizeable increase in glycogen content. Two hours after the treatment with 40, 70 and 150 mg/ml copper ions, the glycogen levels increased from 2011 to 3461, 3856 and 4227 mg glucose/g, respectively. Non-treated control cells showed a 2-fold increase in trehalose level after 60 min of incubation, followed by a sharp decrease to near the basal level (Fig. 6B). Fig. 6B shows that the observed increase was transient; for the first 60 min, trehalose levels were 2.5–3-fold higher than the basal level. Although stress continued for the next 60 min, the trehalose content fell to the control level. Increases in reserve carbohydrates were commensurate with PQ treatment. 3.6. Cu effect on SOD, CAT and G6PD activity To compare the antioxidant response of the fungal cells to Cu toxicity and oxidative stress, altered cellular antioxidant enzyme defence was investigated in the presence of elevated copper concentrations and 3 mM PQ (Fig. 7). Activities of SOD, CAT and G6PD were simultaneously assayed in Cu- and PQ-treated nongrowing cells (see Section 2) over a period of 2 h. During that time, SOD activity increased in a concentration-dependent manner when treated with up to 150 mg/ml Cu2+ (Fig. 7A). The observed

Fig. 6. Changes in the glycogen (A) and trehalose (B) accumulation in H. lutea cells treated with copper or PQ. The results are representative of five independent determinations in triplicate. Bars represent SD of means. Statistical differences (compared with control) are determined where p < 0.05 as calculated by ANOVA followed by Dunnet’s post-test.

increase was from 2.5- to 4.0-fold over the activity of the control. Higher copper concentrations (300 mg/ml) resulted in some attenuation of the response, but SOD activity was still increased in comparison to untreated cells. Some increase in CAT activity (from 12 to 40% as compared to the control) was also observed in cells treated with Cu+2 (Fig. 7B). In contrast, no significant changes in G6PDH activity were noticed at up to 100 mg/ml metal content (Fig. 7C). On the other hand, elevated Cu concentrations (150 and 300 mg/ml) increased enzyme activity by 2.6–4.1-fold after 2 h treatment. These results indicate that G6PDH was only induced at higher Cu levels. The increased total SOD activity (Fig. 7A) was largely due to the Cu/Zn-SOD isoform, which showed a 2.5–4.4-fold increase as compared to the control culture; we found no change in the activity of Mn-SOD (Fig. 8A). To confirm the levels of MnSOD and Cu/ZnSOD activity in the fungal cultures, we used the native gel technique. Fig. 8B shows that Mn-SOD activity did not change with increased copper content. In contrast, Cu/Zn-SOD activity was significantly increased in a concentration-dependent manner. Protein expression of Cu/Zn-SOD was also assessed by Western blotting (Fig. 8C). Consistent with the activity assay, increased Cu/ Zn-SOD immunoreactivity was seen in lysates of copper-treated cells as compared to the control. 3.7. Effect of protein synthesis inhibitors on Cu-induced elevation of SOD To determine whether Cu treatment stimulated H. lutea cells to synthesize more SOD and Cat de novo, protein synthesis was inhibited at different stages. Results with 1 h pre-cultivation of

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Fig. 7. Antioxidant response of H. lutea cells against copper- and PQ-induced stress. (A) SOD, (B) CAT, (C) G6PD activities in cell-free extract from H. lutea cells cultivated in the presence of enhanced Cu2+ concentrations or 3 mM PQ. The results are representative of tree independent determinations in triplicate. Bars represent SD of means. The effect of treatment was significant for the Cu and PQ (p  0.05).

mycelium in the presence of the inhibitors are shown in Table 1 (see Section 2). Hydroxyurea, which is known to interfere with DNA replication but not with subsequent stages of protein synthesis [36], did not prevent the increase of SOD and CAT by Cu and PQ. Actinomycin D inhibits RNA polymerase and thus inhibits transcription of DNA to RNA [37]. This antibiotic completely inhibited the accumulation of the tested enzymes. Treatment with CHI (inhibitor of translation [38]) and antimetabolite 8-azaguanine (inhibitor of translation initiation [39]) also prevented the induction of SOD and CAT under copper treatment and oxidative stress. 4. Discussion Copper is an essential for the normal growth and development of all organisms, including filamentous fungi, as it serves as a cofactor for many physiological processes. However, it can be highly toxic at excessive levels. The studies presented here show that increased concentrations of copper inhibited growth and conidiospore formation of H. lutea 103 in a concentrationdependent manner. Simultaneously, these results confirm our previous indication of high resistance of H. lutea cells to copper treatment [17]. The critical toxicity level of Cu2+ toward fungal spores is above 500 mg/ml (7.88 mM) and is thus much larger than for other microorganisms [40]. A similar trend of copper tolerance

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Fig. 8. Effect of copper treatment on isoenzyme profiles of SOD in H. lutea cells. (A) Mn- and Cu/Zn-SOD activity in free-cell extracts from fungal cultures grown under normophysiological and Cu2+-stress conditions evaluated by NBT reduction. (B) Mn- and Cu/Zn-SOD activity evaluated by polyacrylamide gel electrophoresis (10% gel) lane 1, standard Cu/Zn-SOD from bovine erythrocytes; lane 2, standard MnSOD from E. coli; lane 3, SOD in the control cells; lanes 4 and 5, SOD in the cells cultivated in presence of 70 and 150 mg/ml Cu2+, respectively. (C) Western blot analysis of extracts from identical cultures: lane 1, purified Cu/Zn-SOD from H. lutea cells is shown as the positive control; lane 2, cells grown in the productive medium without Cu2+; lanes 3, and 4 cells grown in presence of 70 and 150 mg/ml Cu2+, respectively. The separated proteins were transferred to nintrocellulose membranes and probed with anti-H. lutea Cu/Zn-SOD polyclonal antibodies.

has been established for the model strain under conditions of submerged cultivation. Copper resistance has been demonstrated in a number of microorganisms including fungi such as A. niger [41], Podospora anserina [14], brown rot fungi (Antrodia vaillantii, Leucogyrophana pinastri) [42] and several others [43]. The main finding of this paper is that copper treatment seems to induce oxidative stress events in H. lutea cells. First, our results indicate a correlation between increased copper concentrations and increased ROS levels. Furthermore, even unstressed fungal cells seemed to produce O2 and H2O2, presumably due to single electron reduction of 2% of the consumed oxygen as previously suggested [44]. Our direct assay showed significantly increased  O2 levels in the presence 40–300 mg/ml copper ions. This increase was more notable in isolated mitochondria than intact cells. In contrast to O2, copper did not affect the production of H2O2. Similar direct analyses of ROS content in fungal cells have not often been reported. According to Osiewacz and Stumpferl

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Table 1 Inhibition of replication, transcription and translation in H. lutea cells, cultivated in presence of 70 mg/ml Cu ions or 3 mM PQ. Inhibitor

Enzyme activity [U/mg protein] With Cu2+

Without stress factor

Control Hydroxyurea Actinomycin D 8-Azaguanine Cyclohexamide

With PQ

SOD

CAT

SOD

CAT

SOD

CAT

11.2  1.12 12.8  1.09 13.1  1.34 11.8  0.98 11.4  1.15

3.2  0.47 3.1  0.65 3.9  0.73 4.1  0.59 3.1  0.44

41.5  3.12 39.6  2.89 16.2  2.04 13.5  1.27 14.1  1.14

5.2  0.78 5.8  0.69 2.6  0.49 2.1  0.54 2.6  0.37

54.2  6.11 48.2  6.09 17.8  2.45 12.1  2.09 13.5  1.67

6.7  1.32 5.9  1.28 2.2  0.76 3.1  0.49 2.6  0.55

The results are representative of five independent determinations in triplicate. The treatment with actinomycin D, 8-azaguanine and cycloheximide are significant for Cu and PQ (p  0.05).

[10], cellular copper levels play a significant role in generation of oxygen metabolites by the fungus P. anserina. Other studies have examined copper-induced ROS in higher eukaryotes [45,46]. Existing evidence suggests that copper increases both O2 and H2O2 levels [47,48]. Our data show that copper stress probably imposes an oxidative burden, of which O2 would be a major component. Qian et al. [49] suggested the superoxide anion to be the major ROS component in Cu-exposed human cells, since Cumediated decrease of Cu/Zn-SOD in astrocytoma cells resulted in increased DCF-DA-reactive ROS without concomitant changes in Mn-SOD or catalase. Possible explanations for the lack of H2O2 accumulation could be that the capacity of cellular H2O2 defences (e.g., catalase) was not exceeded [25], or that the Haber-Weiss cycle was enhanced by increased intracellular copper [50]. The current study with H. lutea indicated that Cu treatment increased oxygen consumption that was not sensitive to 10 mM cyanide. CNRR represents the presence of an electron acceptor different from the cyanide-inhibited cytochrome c oxidase (COX). In plants, fungi and protists, an alternative oxidase (AOX) protein exists [51]. Many studies have shown that AOX gene expression is induced by respiratory inhibitors and ROS such as H2O2 and superoxide radical anions [52,53]. Induction of AOX gene expression by respiratory inhibitors such as cyanide is also thought to be mediated by ROS arising from inhibition of the cytochrome respiratory pathway [54]. In P. anserina, AOX seems to be activated upon COX depletion [14]. Furthermore, it seems to be repressed by Cu [15]. In Escherichia coli, CNRR has also been used to identify electron-shunting activities of redox-cycling drugs such as PQ [30]. This may result in increased oxy-intermediate generation [25], but in P. anserina the activation of AOX is known to reduce ROS levels [14]. Cu-mediated increases in CNRR levels might thus represent the activation of AOX or increased electron-shunting. Compared to the control variants, copper-treated cells displayed increased protein carbonyl content, a biomarker of oxidative stress. Thus, heavy metal stress caused oxidation of intracellular proteins. ROS can modify macromolecules such as proteins by formation of carbonyl groups via oxidation of specific amino acid residues or the protein backbone [55]. The increase in oxidative protein damage by heavy metal treatment was similar to that observed upon treatment with known ROS generators such as PQ. Increased protein carbonylation has also been observed in hyper-oxygenated cultures of white-rot fungus Phanerochaete chrysosporium [56]. The present study also showed that copper stress in H. lutea was accompanied by accumulation of reserve carbohydrates such as trehalose and glycogen. These compounds have different physiological roles: trehalose might be a more general stress protectant and assist chaperones in controlling protein denaturation and renaturation, whereas glycogen is a storage carbohydrate [57]. Previous reports have linked glycogen to microbial viability, suggesting that it is involved in the stress response [58]. Our findings of enhanced trehalose and glycogen levels after copper

treatment agree with earlier studies about the microbial response against heavy metal stress including copper [59,60]. It is noteworthy that increases in trehalose were followed by a sharp decrease that was probably related to fast consumption of this compound. Expression of the genes encoding the enzymes in production and breakdown of trehalose is remarkably co-regulated and enhanced under essentially all stress conditions in bacteria, yeasts, fungi, and invertebrates [61]. Finally, copper-induced oxidative stress was indicated by enhanced antioxidant enzyme activities. Our results demonstrate that treatment with 0–300 mg/ml copper mainly activates SOD. Of the SOD isoforms present in H. lutea, Cu/Zn-SOD was induced by heavy metal, whereas Mn-SOD was not significantly affected (Fig. 8A). PAGE and blot analysis further suggested that Cu/Zn-SOD is involved in the fungal defence against copper stress. Cu/Zn-SOD induction by Cu has also been reported in filamentous fungi [15], methylotrophic yeasts [62] and plants [48,11]. Induction of CAT was detected under the same conditions, but to a lesser degree. Contradictionary data have been reported regarding CAT activity during copper stress. In most cases, CAT activity did not change or was even suppressed [11,48]. Conversely, some studies included CAT in the cellular response to copper treatment [62]. In the present experiments, cells treated with Cu2+ above 100 mg/ml appeared to show reduced CAT activity as compared to cells treated with 40–100 mg/ml Cu (Fig. 7). The O2 level was also the highest with high Cu2+ (500% of control; Fig. 4); reduced CAT activity may thus reflect inhibition by O2 as previously suggested [11]. Pre-treatment with inhibitors of protein synthesis (AD, 8-AG and CHI) followed by copper treatment led to significantly decreased SOD and CAT activities as compared to treatment with metal alone, suggesting de novo synthesis of antioxidant enzymes (Table 1). We also found that oxidative stress caused by high concentrations of CuSO4 (above 150–300 mg/ml) could stimulate the expression of G6PD in H. lutea cells. As previously reported, G6PD is the key enzyme for NADPH generation in the cytosol. Thus, this enzyme plays an important role in the fungal response against Cu2+ stress by regulating intracellular redox status [63]. In summary, our results provide additional evidence for oxidative stress-mediated copper toxicity in filamentous fungi. The observed hyperoxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells. Despite the significant induction of antioxidant enzymes, copper exposure still has deleterious effects, probably mediated by the overloading of antioxidant defences. Acknowledgements This work was supported by grant No. LST.CLG980520/2003 from NATO and grant K-11302/03 from the National Scientific

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