- Email: [email protected]
Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium
Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium Jennifer Dorts a,⁎, Aurélie Bauwin a, Patrick Kestemont a, Sabrina Jolly b, Wilfried Sanchez b, Frédéric Silvestre a a b
Research Unit in Environmental and Evolutionary Biology (URBE), The University of Namur (FUNDP), Rue de Bruxelles 61, B-5000, Namur, Belgium Institut National de l'Environnement Industriel et des Risques (INERIS), Unité d'Evaluation des Risques Ecotoxicologiques, BP2, 60550, Verneuil-en-Halatte, France
a r t i c l e
i n f o
Article history: Received 12 July 2011 Received in revised form 27 September 2011 Accepted 29 September 2011 Available online 19 October 2011 Keywords: Antioxidant enzymes Cadmium Fish Heat stress Oxidative stress Proteasomal activity Temperature
a b s t r a c t Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl2 at nominal concentrations of 0.01 and 1 mg/L) for 4 days at either 15 °C or 21 °C. First, exposure to 1 mg Cd/L induced a high mortality rate (67%) in fish held at 21 °C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21 °C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15 °C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Pollution of aquatic environments by heavy metals including cadmium is a world-wide problem due to the persistency and continuing accumulation of these elements. Cadmium (Cd) is a non-essential toxic metal commonly detected in aquatic and terrestrial environments where it is released from both natural and anthropogenic sources, including industrial, agricultural, and mining activities (Burger, 2008). Although the average Cd content in European rivers roughly varies from 10 to 100 ng/L (OSPAR, 2002), concentrations up to 30 μg/L have been reported in the Molse Nete river (Scheldt basin, Belgium) (Knapen et al., 2004) and levels up to 86 μg/L have been reported in the Meuse River (Eijsden, Belgium) in 2006 (RIWA, 2007). Within cells, Cd has multiple pathways of toxicity due to its high affinity for sulfhydryl groups (SH) that play an important role in the redox balance of the cell and in the structure and function of many enzymes (Valko et al.,
⁎ Corresponding author. Tel.: + 32 81 724287; fax: + 32 81 724362. E-mail addresses: [email protected] (J. Dorts), [email protected] (A. Bauwin), [email protected] (P. Kestemont), [email protected] (S. Jolly), [email protected] (W. Sanchez), [email protected] (F. Silvestre). 1532-0456/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2011.10.003
2005). This resulting change in the redox state of the cell is believed to be associated with oxidation of macromolecules, altered calcium homeostasis, as well as disturbances in the antioxidant defense system (Stohs and Bagchi, 1995). The susceptibility of ectotherms to metal pollution can be strongly affected by environmental temperature due to its direct influence on all physiological and biochemical reactions. The current increases in average temperatures and fluctuations in temperature extremes due to global climate change are considered as one of the new and important threats to aquatic ecosystems (Brönmark and Hansson, 2002). Previous studies have shown that elevated temperatures tend to enhance toxic effects of metals on organisms that may be partially explained by the higher uptake rates of metals and a higher intrinsic sensitivity of organisms (Cairns et al., 1975; Heugens et al., 2001; Sokolova and Lannig, 2008). According to Sokolova and Lannig (2008), interference with aerobic metabolism, including energy demand, oxygen supply, and mitochondrial function, forms a physiological basis for interactions between environmental temperature and metal pollution. In addition to energy disturbance, tissues of Cdexposed oysters (Crassostrea virginica) accumulate elevated levels of lipid peroxidation products at higher temperatures indicating the onset of oxidative stress (Lannig et al., 2006). This view is further
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
supported by the fact that Cd-induced oxidative stress in oyster mitochondria was particularly pronounced at elevated temperature as indicated by increased production of reactive oxygen species (ROS) and decreased activity of mitochondrial aconitase (Cherkasov et al., 2007). Elevated temperature and metal exposure, such as to Cd, are known to induce oxidative stress due to the excessive ROS production and/or interferences with the antioxidant defense system (Valko et al., 2005; Abele et al., 2007). The effects of temperature and Cd exposure as single environmental factors on oxidative stress have been extensively studied in aquatic organisms, including fish (Almeida et al., 2002; Lushchak and Bagnyukova, 2006a, 2006b). However, to our knowledge there are no studies that address interactive effects of these two potentially pro-oxidant stressors on oxidative stress status in fish. Oxygen free radicals can elicit widespread damage to the vital cellular components, including lipids, proteins and DNA, which can eventually lead to cell death. For instance, oxidative stress contributes to structural changes or misfolding of cellular proteins (Stadtman, 1992). Cells possess a protective mechanism to overcome the potentially toxic accumulation of oxidatively modified proteins, namely an increase in proteolysis (Grune et al., 1995). Previous studies have suggested that oxidized proteins are degraded in an ATP- and ubiquitin-independent pathway by the 20S proteasome (Davies, 2001; Shringarpure et al., 2003). Additionally several studies demonstrated that oxidative stressors can alter the activity of the ubiquitin– proteasome system (UPS) (Shang and Taylor, 1995; FigueiredoPereira et al., 1998). Changes in ubiquitination in response to temperature (Hofmann and Somero, 1995; Buckley et al., 2001) and Cd exposure (McDonagh and Sheehan, 2006) have been previously studied in bivalves. Downs et al. (2001) also reported in grass shrimp Palaeomonetes pugio that ubiquitin levels increase in response to high temperature, Cd, bunker fuel, and diesel fuel. However the relationship between potentially pro-oxidant stressors (such as temperature and metal exposure) and the proteasome complex remains poorly understood, especially in fish. The aim of the present study was to investigate the combined effects of temperature and Cd exposure on the oxidative stress status of the European bullhead, Cottus gobio, a small bottom-dwelling freshwater cottid fish widespread throughout Europe. Bullhead typically lives in well oxygenated stream waters from 2 to 16.5 °C (Andreasson, 1971), whereas Elliott and Elliott (1995) found the critical thermal limits of bullhead to be − 4.2 and 27.7 °C. Furthermore, this species has become endangered in several parts of its distribution area as a result of pollution and habitat destruction and is known for its sensitivity to temperature changes (Utzinger et al., 1998; Dorts et al., 2011a). To address this question, biochemical markers of oxidative stress including antioxidant enzyme activity and lipid peroxidation level, as a consequence of oxidative deterioration of membrane lipids, were assessed in liver and gill tissues of bullhead subjected to different Cd concentrations at either 15 °C or 21 °C for 4 days. In addition, the effects of these two stressors on the 20S proteasome activity (chymotrypsin-like) were investigated for the first time in a fish species. 2. Material and methods 2.1. Animals and exposure condition Investigations and animal care were conducted according to the guidelines for the use and care of laboratory animals and in compliance with Belgian and European regulations on animal welfare. Adult European bullhead of both genders weighing 10.4 ± 2.8 g were caught by electrofishing in the Samson River (Belgium) in May 2010. Fish were acclimated to laboratory conditions in dechlorinated tap water at 15.8 ± 1.4 °C under a 14:10 h (light/dark) photoperiod for 4 weeks before the experiment. During the acclimation period,
319
fish were fed daily to apparent satiation with chironomid larvae (Chironomus sp.). After acclimation, 144 fish were randomly distributed over 18 tanks filled with 12 L dechlorinated tap water. Fish were exposed to CdCl2 (Sigma C2544) at nominal concentrations of 0.01 and 1 mg/L for 4 days at either 15 °C or 21 °C. The temperature was increased by 2 °C per day from 15 to 21 °C. The tested concentrations were chosen according to LC50 values reported for related fish species in the literature (Mebane, 2006; Besser et al., 2007) and to a previous study which evaluated the toxicity of short-term Cd exposure in liver and gills of C. gobio by monitoring the response of several enzymes involved in energy metabolism, and by undertaking a proteomic analysis using 2D-DIGE technique (Dorts et al., 2011b). Each treatment included three replicate tanks, with 8 fish per tank. After 4 days of exposure, each fish was weighed, and liver and gills were collected on ice, directly snap-frozen in liquid nitrogen and stored at − 80 °C until homogenization. Animals were not fed during exposure, and half-water was gently siphoned out, replaced, and recontaminated every day. Total Cd concentrations in the exposure water were monitored every other day using a Sector Field Inductively Coupled Plasma Mass Spectrometer (Thermo Finnigan Element 2). Certified reference water samples (riverine water certified reference material SLRS-4, National Research Council Canada) were also analyzed for Cd during each analytical run; measured Cd concentrations were consistently within the certified range. Cd water concentrations were stable over the course of the experiment; the mean concentrations and standard deviations in the 0.01 and 1 mg/L treatment were 0.009 ± 0.001 and 0.834 ± 0.064 mg/L, respectively. 2.2. Oxidative stress markers 2.2.1. Sample preparation Oxidative stress markers were assessed in liver and gills from 2 individual fish per replicate tank. Tissues were homogenized in ice-cold phosphate buffer (100 mM, pH 7.5) containing 20% glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor. The homogenates were centrifuged at 10,000 g for 15 min at 4 °C and the post-mitochondrial fractions were kept at − 80 °C for biochemical assays. Protein contents were determined using the method of Bradford (1976) with bovine serum albumin as a standard. 2.2.2. Lipid peroxidation level LPO results in the production of various compounds including the malondialdehyde (MDA) consequently to free radicals production, and was assessed by the thiobarbituric acid reactive substances assay TBARS (Ohkawa et al., 1979). LPO products react with thiobarbituric acid (TBA) and the product is quantified by fluorimetry (excitation wavelength 515 nm, emission wavelength 553 nm). A calibration curve with increasing MDA concentrations allowed the calculation of LPO expressed as nmol MDA equiv./g protein. 2.2.3. Antioxidant enzyme activities Prior to biochemical analysis, assays were optimized for the bullhead to assume optimal conditions including saturating conditions, linearity of enzyme rates and homogenate amount. All assays were performed in microtiter plates and experimental temperature was 20 °C. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined using the method of Paoletti et al. (1986). The decrease of the rate of NADH oxidation was measured spectrophotometrically at 340 nm for 6 min. One unit of activity is defined as the amount of enzyme causing 50% inhibition of NADH oxidation and the results were expressed as unit per g protein. Catalase (CAT, EC 1.11.1.6) activity was estimated using the method previously described by Babo and Vasseur (1992). The destruction of hydrogen peroxide (H2O2) was
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
2.3. Chymotrypsin-like proteasome activity Chymotrypsin-like activity of 20S proteasome was assessed in liver and gills from 4 pooled fish per replicate tank using a luminescent assay (Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay, Promega). Tissues were homogenized in a hypotonic buffer (10 mM Tris–HCl pH 7.5, 5 mM MgCl2, 0.5 mM DTT, and 5 mM ATP) according to the manufacturer's recommendation. The homogenates were centrifuged at 1000 ×g for 10 min at 4 °C and the supernatants were kept at − 80 °C for enzyme activity assay. Proteasome activity was normalized to total protein contents determined by a bicinchoninic acid (BCA) assay (Pierce) with bovine serum albumin as a standard. 2.4. Statistical analysis Data were expressed as mean ± S.D. Normality analysis of data was assessed by the Shapiro–Wilk W test. Homogeneity of variances was tested by the Bartlett test. Effects of temperature, Cd exposure and their interaction on the studied variables were tested using the analysis of variance (ANOVA). Fisher LSD tests were used for posthoc comparisons at a 5% significant level. All tests were performed using the Statistica 5.5 software (StatSoft, Tulsa, OK, USA). 3. Results No mortality was observed except for fish exposed to 1 mg Cd/L at 21 °C for 4 days (67% mortality). Due to this high mortality, the biochemical assays on surviving fish exposed to 1 mg Cd/L were not undertaken. 3.1. Lipid peroxidation TBARS levels measured in liver and gill tissues of bullhead in response to temperature and Cd exposure are presented in Fig. 1. In both tissues, no interaction effects of temperature and Cd exposure in TBARS levels were observed. However, TBARS levels were significantly influenced by temperature in liver (P = 0.0023), independently of Cd exposure. Exposure to 21 °C resulted in a significant 29% decrease of TBARS levels in liver.
A
Lipid peroxidation (nmol MDA equiv./g protein)
measured spectrophotometrically at 240 nm for 2 min. The enzyme activity was expressed as μmol of H2O2 consumed per min per mg protein. Glutathione peroxidase (GPx, EC 1.11.1.9) activity was determined using the method of Paglia and Valentine (1967). Oxidation of NADPH was recorded spectrophotometrically at 340 nm for 1 min. The enzyme activity was calculated as μmol NADPH oxidized per min per g protein. Glutathione reductase (GR, EC 1.8.1.7) activity was estimated using the method of Carlberg and Mannervik (1985). The GR activity is evaluated by following NADPH concentration at 340 nm during 6 min. The enzyme activity was expressed in μmol of NADPH oxidized per min per g protein. Glutathione transferase (GST, EC 2.5.1.18) activity assay was conducted according to Habig et al. (1974). The conjugation of reduced glutathione (GSH) with 1chloro-2,-dinitrobenzene (CDNB) via GST activity was recorded spectrophotometrically at 340 nm for 5 min. The activity was expressed as μmol of CDNB conjugate formed per min per g protein.
B
Lipid peroxidation (nmol MDA equiv./g protein)
320
**
8000
6000
4000
2000
0 1200 1000 800 600 400 200 0 0 mg Cd/L
0.01 mg Cd/L
Fig. 1. LPO level (nmol MDA equiv./g protein) in liver (A) and gills (B) of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. Data are represented as mean ± S.D. (n = 6). Statistically significant differences in LPO levels between treatments are denoted by stars (**P b 0.01).
glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST). Nevertheless, the hepatic activities of SOD and GR were significantly affected by Cd exposure (P = 0.0012 and P = 0.0007, respectively), regardless of heat stress. Exposure to 0.01 mg Cd/L significantly increased SOD activity by 54%, while a 45% significant decrease occurred in GR activity. In contrast, the activities of GPx and GST were significantly influenced by temperature in gills (P = 0.005 and P = 0.042, respectively), independently of Cd exposure. Exposure to 21 °C resulted in a significant 24% increase of GPx activity, whereas the activity of GST was significantly reduced by 38% as compared to fish exposed to 15 °C. 3.3. Chymotrypsin-like proteasome activity The activity of chymotrypsin-like 20S proteasome in liver and gill tissues of bullhead in response to temperature and Cd exposure is presented in Fig. 4. In both tissues, the proteasome activity was significantly affected by temperature (P = 0.013 and P = 0.008 in liver and gills, respectively), whereas Cd exposure and the combination of both stressors did not influence its activity. Within groups exposed to 21 °C we observed a significant increase in the activity of proteasome by approximately 25% in both liver and gill tissues (1188.2 ± 204.2 and 3650.2 ± 716.6 RLU/mg protein at 15 °C compared to 1611.5 ±249.8 and 4896.8 ± 464.9 RLU/mg protein at 21 °C, in liver and gills, respectively). 4. Discussion
3.2. Antioxidant enzyme activities The activities of antioxidant enzymes in liver and gill tissues in response to temperature and Cd exposure are depicted in Figs. 2 and 3, respectively. Due to small quantities of tissues, the activities of all antioxidant enzymes could not have been assessed in fish gills. Regardless of tissues, we did not observe any interaction between temperature and Cd exposure for the activities of studied antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT),
To the best of our knowledge, the present study was the first attempt to address the interactive effects of elevated temperature and Cd stress on oxidative stress status in the fish C. gobio. Overall, elevated temperature and Cd exposure as single stressor strongly influenced the antioxidant defense system in bullhead with clear tissuespecific responses. In addition, a temperature rise of 6 °C affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues.
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
A
20
GPx activity (U/g protein)
SOD activity (U/g protein)
A
**
15 10 5 0
100 80 60 40 20 0
1000
GST activity (U/g protein)
1000
CAT activity (U/mg protein)
B 1200
600 400 200 0
**
120
B 1200 800
*
800 600 400 200 0
15°C 0 mg Cd/L
800 600 400 200 0
15°C 0 mg Cd/L
21°C 0.01 mg Cd/L
Sokolova and Lannig, 2008). In the present study, exposure to 1 mg Cd/L for 4 days induced a high mortality rate for bullhead at 21 °C. The influence of temperature on metal toxicity is a complex matter and can be attributed to the relative changes in the rate of metal uptake, elimination, diffusion and biotransformation of an organism.
25
A
20 ***
15 10 5 0 600 500
Chymotrypsin-like proteasome activity (RLU/mg protein)
GR activity (U/g protein)
GST activity (U/g protein)
E
21°C 0.01 mg Cd/L
Fig. 3. Activities of antioxidant enzymes in gills of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. (A) GPx — glutathione peroxidase and (B) GST — glutathione S-transferase. Data are represented as mean ± S.D. (n = 6). Statistically significant differences in enzyme activities between treatments are denoted by stars (*P b 0.05; **P b 0.01).
1000
B
400 300 200 100 0 15°C 0 mg Cd/L
21°C 0.01 mg Cd/L
Fig. 2. Activities of antioxidant enzymes in liver of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. (A) SOD — superoxide dismutase, (B) CAT — catalase, (C) GPx — glutathione peroxidase, (D) GR — glutathione reductase, and (E) GST — glutathione S-transferase. Data are represented as mean ± S.D. (n = 6). Statistically significant differences in enzyme activities between treatments are denoted by stars (**P b 0.01; ***P b 0.001).
Experimental evidence indicates that an increase in environmental temperature results in elevated mortality rates in metal-exposed ectotherms (reviewed in Cairns et al., 1975; Heugens et al., 2001;
Chymotrypsin-like proteasome activity (RLU/mg protein)
GPx activity (U/g protein)
C 1200
D
321
*
2000
1000
0
**
6000
4000
2000
0
15°C 0 mg Cd/L
21°C 0.01 mg Cd/L
Fig. 4. Chymotrypsin-like 20S proteasome activity (RLU/mg protein) in liver (A) and gills (B) of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. Data are represented as mean ± S.D. (n = 3). Statistically significant differences in proteasome activity between treatments are denoted by stars (*P b 0.05; **P b 0.01).
322
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
Results from acute toxicity tests evidenced that a temperature increase reduced the 96 h LC50 values for different aquatic species exposed to Cd (Rathore and Khangarot, 2002; Heugens et al., 2003; Prato et al., 2009). Oxidative stress is an important component of the stress response in aquatic organisms exposed to a variety of insults as a result of changes in environmental conditions such as thermal stress or exposure to pollution (Lesser, 2006). To minimize the damaging effects of reactive oxygen species (ROS), cells possess both enzymatic and nonenzymatic antioxidant defenses. The main antioxidant enzyme systems consist of superoxide dismutase (SOD), which detoxifies O2•−, catalase (CAT), which reduces H2O2 to water and oxygen, and glutathione peroxidase (GPx), which reduces both H2O2 and organic peroxides (Halliwell and Gutteridge, 1989). In the present study, the changes observed in liver and gills of C. gobio suggested a tissue-specific antioxidant response following exposure to enhanced temperature and Cd. Several studies dealing with the responses of antioxidant enzymes to a variety of laboratory exposures have been reported but the responses are transient and variable depending on the intensity and duration of the stress applied to the organism in addition to the susceptibility of the exposed species and organs (Ballesteros et al., 2009). Several studies have demonstrated that Cd-induced toxic effects involve lipid peroxidation and variations in the level of antioxidant enzymes culminating in oxidative stress (Ait-Aissa et al., 2003; Liu et al., 2009). Cd is not a redox-active metal and does not directly generate free radicals (Ercal et al., 2001). Its pro-oxidant properties might be due to: (1) displacement of Fe 2+, which induces ROS via Fentonlike reaction (Casalino et al., 1997); (2) inhibition of antioxidant enzyme activities (Casalino et al., 2002); (3) depletion of GSH content (Figueiredo-Pereira et al., 1998); (4) inhibition of the respiratory chain in mitochondria (Wang et al., 2004). With respect to hepatic antioxidant enzymes, the present study highlighted effects of Cd exposure, regardless of heat stress. This could be related to the fact that the liver is one of the major sites of Cd accumulation in fish (Kim et al., 2004) and is the most critical organ for detoxification process. First, our results showed an increase in SOD activity following cadmium stress, suggesting that a rise in the O2•− generation rate might have taken place and the protective role of this enzyme against metal-induced oxidative stress. The reported increase in SOD activity in Cd-exposed bullhead would result in a higher generation of H2O2. However, the activities of the two H2O2-scavenging enzymes, i.e. CAT and GPx, were not affected by Cd exposure in liver of C. gobio. Previously, Jia et al. (2011) showed increased SOD activities in liver of carp Cyprinus carpio var. color following exposure to 0.4, 0.5 and 0.7 mg Cd/L for 7 days, while SOD activity was inhibited at higher Cd concentrations of 1 and 2 mg/L. The present study also reported a decrease in GR activity in response to Cd exposure. GR enzyme, which catalyzes the reduction of oxidized glutathione (GSSG), is not involved in antioxidant defense in the same way as the SOD activity. However it is important in maintaining the GSH/GSSG homeostasis under stress conditions (Winston and Digiulio, 1991). A decline in GR activity could be due to the change in the availability of NADPH in the cell and may result in GSH depletion if extra synthesis of GSH cannot take place to preserve its redox status, as a result of prooxidative effects (Zhang et al., 2004). Cirillo et al. (2011) observed that GSH concentrations and the antioxidant enzyme activities of GPx and GR showed an overall decreasing trend in liver of the gilthead sea bream Sparus aurata exposed to 0.1 mg Cd/L for 11 days. The metabolic rate of ectotherms and, hence, oxygen consumption are proportional to environmental temperature (Hochachka and Somero, 2002). Furthermore, the rate of ROS generation is related to oxygen consumption (Halliwell and Gutteridge, 1989). Therefore, it can be expected that the intensification of respiration at higher temperature may result in enhanced ROS production, oxidation of cellular constituents and a response of antioxidant and associated
enzyme systems (Lushchak and Bagnyukova, 2006a). In the present study, a water temperature rise of 6 °C increased the activity of GPx in gills of bullhead, while the branchial GST activity was inhibited in comparison to control groups. The effects of heat stress observed herein on gills of C. gobio may be related to their physiological role in respiration. Additionally, gills have a large exchange area that is in continuous contact with surrounding water, and slight environmental changes could be transferred easily to cellular defense systems. GSH and enzymes associated with its metabolism provide a major defense against ROS induced cellular damage. It functions mainly as a sulfhydryl buffer, but GSH also serves to detoxify compound either via conjugation reactions catalyzed by GST or directly, as it is the case with hydroperoxides in the GPx catalyzed reaction (Nordberg and Arner, 2001). In the present study, an increased activity of GPx suggests an excess H2O2 production as a result of heat stress. This enhanced activity might be of particular importance in the reduction of lipid hydroperoxides thereby preventing the chain propagation reaction leading to the deleterious effects of lipid peroxidation (Parihar et al., 1997). Several studies dealing with the responses of antioxidant enzymes to enhanced environmental temperature have been reported in various fish species (Parihar et al., 1997; Heise et al., 2006; Lushchak and Bagnyukova, 2006b; Bagnyukova et al., 2007a, 2007b). However, these reports were inconclusive and showed wide individual differences. Exceeding the defensive ability of antioxidant systems can result in damage to cellular constituents, including lipids, proteins and DNA, and a state of oxidative stress ensues (Livingstone, 2001). Previous studies have suggested that exposure to environmental contaminants (Livingstone, 2001; Shi et al., 2005; Cirillo et al., 2011) or other environmental stresses (e.g. temperature) (Parihar and Dubey, 1995; Lushchak and Bagnyukova, 2006a; Bagnyukova et al., 2007a, 2007b) can affect the peroxidation of lipids in different fish tissues. In the present study, neither cadmium nor temperature was able to stimulate the lipid peroxidation process (based on TBARS levels) in gills of C. gobio, supposedly due to effective antioxidant responses. However, a slight decrease of lipid peroxidation was observed in liver of bullhead in response to heat stress. In the present study, the lipid peroxidation process could have occurred earlier during applied stresses as suggested by previous studies (Parihar and Dubey, 1995; Bagnyukova et al., 2007b). Bullhead could also have sufficient antioxidant systems such as non-enzymatic antioxidant defenses that were not assayed in our study. Additionally, although lipid peroxidation has been considered to be the primary process responsible for cadmium toxicity, several authors do not agree on cadmium-induced lipid peroxidation as a basis for cell damage, as many toxic events occur prior to enhanced lipid peroxidation (Casalino et al., 1997, 2000). Apart from lipid peroxidation, oxidative stress is one of the mechanisms that contribute to structural changes or misfolding of proteins. ROS can lead to a range of reversible and irreversible covalent modifications of amino acid side-chain of proteins (reviewed by Ghezzi and Bonetto, 2003). According to Grune (2000), the degradation of non-functional, oxidized proteins is an essential part of the antioxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic proteins is the proteasomal system, which consists of the 20S core proteasome and a multitude of various regulators. To our knowledge, the present study is the first devoted to measure the activity of chymotrypsin-like 20S proteasome in response to heat and cadmium stress in a fish species. As seen here, exposure to elevated temperature resulted in an increased activity of proteasome in liver and gill tissues of C. gobio, regardless of Cd exposure. Similarly, Lamarre et al. (2010) have observed an increased 20S proteasome activity in liver of the wolffish Anarhichas minor acclimated to high temperature. From our results, it may be thought that the proteasomal activity was enhanced to overcome the potentially toxic accumulation of damaged proteins in response to heat stress. Interestingly, an increased expression of a subunit of the 19S
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
proteasome particle (PSMD2) has been observed in gills of C. gobio either exposed to Cd (Dorts et al., 2011b) or acclimated to high temperature (Dorts et al., 2011c). Overall, the induction of proteasomal activity as observed in the present study and the aforementioned increased expression of a subunit of the proteasome activator 19S, suggest that the protein quality control could be a possible target of environmental stressors, such as heat stress, in fish. Combination of exposure to elevated temperatures and trace metals is an environmentally relevant scenario that is expected to become more widespread in the future with global warming. Although no interaction effect on the oxidative stress status of bullhead was observed in the present report, earlier studies have shown interactive effects of enhanced temperature and toxic metals on oxidative stress status of aquatic ectotherms (Lannig et al., 2006; Cherkasov et al., 2007; Verlecar et al., 2007; Lapointe et al., 2011). For instance, oysters C. virginica exposed to both temperature and Cd stress suffered high mortality accompanied by elevated lipid peroxidation products (Lannig et al., 2006), and displayed elevated ROS production and oxidative damage as indicated by aconitase inactivation (Cherkasov et al., 2007). As a corollary, the current study demonstrates that the concomitant exposure to heat and high Cd concentration resulted in a high mortality rate of C. gobio. Furthermore, the present results did not highlight any interactive effects but rather indicated the importance of each abiotic factor, i.e., temperature and Cd, on several aspects of the oxidative stress response. We showed clear tissue-specific and stress-specific antioxidant responses and the induction of proteasomal activity in fish in response to heat stress. This work provides insights into the biochemical events involved in the response to heat and Cd in fish and suggests that further studies on additional endpoints could provide crucial information to better understand the interactive effects of elevated temperature and metal exposure in fish.
Acknowledgments The authors thank A. Evrad and M.-C. Forget from URBE and J. Navez from Musée Royal de l'Afrique Centrale (Tervuren, Belgium) for valuable help during animal husbandry, and biochemical, and chemical analyses, respectively. W. Sanchez and S. Jolly were supported by the French Ministry for Ecology (P190-AP08/09). This study was supported by a FNRS PhD fellowship to J. Dorts.
References Abele, D., Philipp, E., Gonzalez, P.M., Puntarulo, S., 2007. Marine invertebrate mitochondria and oxidative stress. Front. Biosci. 12, 933–946. Ait-Aissa, S., Ausseil, O., Palluel, O., Vindimian, E., Garnier-Laplace, J., Porcher, J.M., 2003. Biomarker responses in juvenile rainbow trout (Oncorhynchus mykiss) after single and combined exposure to low doses of cadmium, zinc, PCB77 and 17beta-oestradiol. Biomarkers 8, 491–508. Almeida, J.A., Diniz, Y.S., Marques, S.F.G., Faine, L.A., Ribas, B.O., Burneiko, R.C., Novelli, E.L.B., 2002. The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environ. Int. 27, 673–679. Andreasson, S., 1971. Feeding Habits of a Sculpin (Cottus gobio L. Pisces) Population. The Institute of Freshwater Research, Drottningholm, Sweden. Rep. No. 51. Babo, S., Vasseur, P., 1992. In vitro effects of thiram on liver antioxidant enzymeactivities of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 22, 61–68. Bagnyukova, T.V., Danyliv, S.I., Zin'ko, O.S., Lushchak, V.I., 2007a. Heat shock induces oxidative stress in rotan Perccottus glenii tissues. J. Therm. Biol. 32, 255–260. Bagnyukova, T.V., Lushchak, O.V., Storey, K.B., Lushchak, V.I., 2007b. Oxidative stress and antioxidant defense responses by goldfish tissues to acute change of temperature from 3 to 23 degrees C. J. Therm. Biol. 32, 227–234. Ballesteros, M.L., Wunderlin, D.A., Bistoni, M.A., 2009. Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan. Ecotoxicol. Environ. Saf. 72, 199–205. Besser, J.M., Mebane, C.A., Mount, D.R., Ivey, C.D., Kunz, J.L., Greer, I.E., May, T.W., Ingersoll, C.G., 2007. Sensitivity of mottled sculpins (Cottus bairdi) and rainbow trout (Onchorhynchus mykiss) to acute and chronic toxicity of cadmium, copper, and zinc. Environ. Toxicol. Chem. 26, 1657–1665.
323
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brönmark, C., Hansson, L.-H., 2002. Environmental issues in lakes and ponds: current state and perspectives. Environ. Conserv. 29, 290–306. Buckley, B.A., Owen, M.E., Hofmann, G.E., 2001. Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J. Exp. Biol. 204, 3571–3579. Burger, J., 2008. Assessment and management of risk to wildlife from cadmium. Sci. Total Environ. 389, 37–45. Cairns Jr., J., Heath, A.G., Parker, B.C., 1975. The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologica 47, 135–171. Carlberg, I., Mannervik, B., 1985. Glutathione reductase. Methods Enzymol. 113, 484–490. Casalino, E., Sblano, C., Landriscina, C., 1997. Enzyme activity alteration by cadmium administration to rats: the possibility of iron involvement in lipid peroxidation. Arch. Biochem. Biophys. 346, 171–179. Casalino, E., Calzaretti, G., Sblano, C., Landriscina, C., 2000. Cadmium-dependent enzyme activity alteration is not imputable to lipid peroxidation. Arch. Biochem. Biophys. 383, 288–295. Casalino, E., Calzaretti, G., Sblano, C., Landriscina, C., 2002. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 179, 37–50. Cherkasov, A.A., Overton Jr., R.A., Sokolov, E.P., Sokolova, I.M., 2007. Temperaturedependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation. J. Exp. Biol. 210, 46–55. Cirillo, T., Amodio Cocchieri, R., Fasano, E., Lucisano, A., Tafuri, S., Ferrante, M.C., Carpene, E., Andreani, G., Isani, G., 2011. Cadmium accumulation and antioxidant responses in Sparus aurata exposed to waterborne cadmium. Arch. Environ. Contam. Toxicol. doi:10.1007/s00244-011-9676-9. Davies, K.J., 2001. Degradation of oxidized proteins by the 20S proteasome. Biochimie 83, 301–310. Dorts, J., Grenouillet, G., Douxfils, J., Mandiki, S.N.M., Milla, S., Silvestre, F., Kestemont, P., 2011a. Evidence that elevated water temperature affects the reproductive physiology of the European bullhead Cottus gobio. Fish Physiol. Biochem. doi:10.1007/ s10695-011-9515-y. Dorts, J., Kestemont, P., Dieu, M., Raes, M., Silvestre, F., 2011b. Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio. J. Proteome Res. 10, 470–478. Dorts, J., Kestemont, P., Thézenas, M.-L., Raes, M., Silvestre, F., 2011c. Heat Acclimation of Cottus gobio Interacts with Subsequent Cadmium Exposure Resulting in a Specific Proteome Response. Downs, C., Fauth, J., Woodley, C., 2001. Assessing the health of grass shrimp (Palaeomonetes pugio) exposed to natural and anthropogenic stressors: a molecular biomarker system. Mar. Biotechnol. 3, 380–397. Elliott, J.M., Elliott, J.A., 1995. The critical thermal limits for the bullhead, Cottus gobio, from 3 populations in North-West England. Freshw. Biol. 33, 411–418. Ercal, N., Gurer-Orhan, H., Aykin-Burns, N., 2001. Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539. Figueiredo-Pereira, M.E., Yakushin, S., Cohen, G., 1998. Disruption of the intracellular sulfhydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquitination in neuronal cells. J. Biol. Chem. 273, 12703–12709. Ghezzi, P., Bonetto, V., 2003. Redox proteomics: identification of oxidatively modified proteins. Proteomics 3, 1145–1153. Grune, T., 2000. Oxidative stress, aging and the proteasomal system. Biogerontology 1, 31–40. Grune, T., Reinheckel, T., Joshi, M., Davies, K.J., 1995. Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. J. Biol. Chem. 270, 2344–2351. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine. Clarendon Press, Oxford, UK. Heise, K., Puntarulo, S., Nikinmaa, M., Abele, D., Portner, H.O., 2006. Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout Zoarces viviparus L. J. Exp. Biol. 209, 353–363. Heugens, E.H., Hendriks, A.J., Dekker, T., van Straalen, N.M., Admiraal, W., 2001. A review of the effects of multiple stressors on aquatic organisms and analysis of uncertainty factors for use in risk assessment. Crit. Rev. Toxicol. 31, 247–284. Heugens, E.H., Jager, T., Creyghton, R., Kraak, M.H., Hendriks, A.J., Van Straalen, N.M., Admiraal, W., 2003. Temperature-dependent effects of cadmium on Daphnia magna: accumulation versus sensitivity. Environ. Sci. Technol. 37, 2145–2151. Hochachka, P.W., Somero, G.N., 2002. Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, Oxford, UK. Hofmann, G., Somero, G., 1995. Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198, 1509–1518. Jia, X., Zhang, H., Liu, X., 2011. Low levels of cadmium exposure induce DNA damage and oxidative stress in the liver of Oujiang colored common carp Cyprinus carpio var. color. Fish Physiol. Biochem. 37, 97–103. Kim, S.G., Jee, J.H., Kang, J.C., 2004. Cadmium accumulation and elimination in tissues of juvenile olive flounder, Paralichthys olivaceus after sub-chronic cadmium exposure. Environ. Pollut. 127, 117–123. Knapen, D., Bervoets, L., Verheyen, E., Blust, R., 2004. Resistance to water pollution in natural gudgeon (Gobio gobio) populations may be due to genetic adaptation. Aquat. Toxicol. 67, 155–165.
324
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
Lamarre, S., Blier, P., Driedzic, W., Le François, N., 2010. White muscle 20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor. J. Fish Biol. 76, 1565–1575. Lannig, G., Flores, J.F., Sokolova, I.M., 2006. Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat. Toxicol. 79, 278–287. Lapointe, D., Pierron, F., Couture, P., 2011. Individual and combined effects of heat stress and aqueous or dietary copper exposure in fathead minnows (Pimephales promelas). Aquat. Toxicol. 104, 80–85. Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiology ecology. Annu. Rev. Physiol. 68, 253–278. Liu, J., Qu, W., Kadiiska, M.B., 2009. Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol. Appl. Pharmacol. 238, 209–214. Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656–666. Lushchak, V.I., Bagnyukova, T.V., 2006a. Temperature increase results in oxidative stress in goldfish tissues. 1. Indices of oxidative stress. Comp. Biochem. Physiol. 143C, 30–35. Lushchak, V.I., Bagnyukova, T.V., 2006b. Temperature increase results in oxidative stress in goldfish tissues. 2. Antioxidant and associated enzymes. Comp. Biochem. Physiol. 143C, 36–41. McDonagh, B., Sheehan, D., 2006. Redox proteomics in the blue mussel Mytilus edulis: carbonylation is not a pre-requisite for ubiquitination in acute free radicalmediated oxidative stress. Aquat. Toxicol. 79, 325–333. Mebane, C.A., 2006. Cadmium Risks to Freshwater Life: Derivation and Validation of Low-effect Criteria Values Using Laboratory and Field Studies (Version 1.1). U.S. Geological Survey Scientific Investigations Report 2006–5245. Nordberg, J., Arner, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287–1312. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. OSPAR, 2002. Cadmium. Hazardous Substances Series. OSPAR Commission. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Paoletti, F., Aldinucci, D., Mocali, A., Caparrini, A., 1986. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal. Biochem. 154, 536–541. Parihar, M., Dubey, A., 1995. Lipid peroxidation and ascorbic acid status in respiratory organs of male and female freshwater catfish Heteropneustes fossilis exposed to temperature increase. Comp. Biochem. Physiol. 112C, 109–113. Parihar, M.S., Javeri, T., Hemnani, T., Dubey, A.K., Prakash, P., 1997. Responses of superoxide dismutase, glutathione peroxidase and reduced glutathione antioxidant
defenses in gills of the freshwater catfish (Heteropneustes fossilis) to short-term elevated temperature. J. Therm. Biol. 22, 151–156. Prato, E., Biandolino, F., Scardicchio, C., 2009. Effects of temperature on the sensitivity of Gammarus aequicauda (Martynov, 1931) to cadmium. Bull. Environ. Contam. Toxicol. 83, 469–473. Rathore, R.S., Khangarot, B.S., 2002. Effects of temperature on the sensitivity of sludge worm Tubifex tubifex Muller to selected heavy metals. Ecotoxicol. Environ. Saf. 53, 27–36. RIWA - Maas/Meuse, 2007. Communiqué de presse électronique 14 mars 2007 [online]. Available from http://www.riwa-maas.org/download/fr/nieuws/39.pdf. Shang, F., Taylor, A., 1995. Oxidative stress and recovery from oxidative stress are associated with altered ubiquitin conjugating and proteolytic activities in bovine lens epithelial cells. Biochem. J. 307, 297–303. Shi, H., Sui, Y., Wang, X., Luo, Y., Ji, L., 2005. Hydroxyl radical production and oxidative damage induced by cadmium and naphthalene in liver of Carassius auratus. Comp. Biochem. Physiol. C 140C, 115–121. Shringarpure, R., Grune, T., Mehlhase, J., Davies, K.J., 2003. Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J. Biol. Chem. 278, 311–318. Sokolova, I.M., Lannig, G., 2008. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Clim. Res. 37, 181–201. Stadtman, E.R., 1992. Protein oxidation and aging. Science 257, 1220–1224. Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. Utzinger, J., Roth, C., Peter, A., 1998. Effects of environmental parameters on the distribution of bullhead Cottus gobio with particular consideration of the effects of obstructions. J. Appl. Ecol. 35, 882–892. Valko, M., Morris, H., Cronin, M.T., 2005. Metals, toxicity and oxidative stress. Curr. Med. Chem. 12, 1161–1208. Verlecar, X., Jena, K., Chainy, G., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chem. Biol. Interact. 167, 219–226. Wang, Y., Fang, J., Leonard, S.S., Rao, K.M., 2004. Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic. Biol. Med. 36, 1434–1443. Winston, G.W., Digiulio, R.T., 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137–161. Zhang, J.F., Wang, X.R., Guo, H.Y., Wu, J.C., Xue, Y.Q., 2004. Effects of water-soluble fractions of diesel oil on the antioxidant defenses of the goldfish, Carassius auratus. Ecotoxicol. Environ. Saf. 58, 110–116.
Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium Jennifer Dorts a,⁎, Aurélie Bauwin a, Patrick Kestemont a, Sabrina Jolly b, Wilfried Sanchez b, Frédéric Silvestre a a b
Research Unit in Environmental and Evolutionary Biology (URBE), The University of Namur (FUNDP), Rue de Bruxelles 61, B-5000, Namur, Belgium Institut National de l'Environnement Industriel et des Risques (INERIS), Unité d'Evaluation des Risques Ecotoxicologiques, BP2, 60550, Verneuil-en-Halatte, France
a r t i c l e
i n f o
Article history: Received 12 July 2011 Received in revised form 27 September 2011 Accepted 29 September 2011 Available online 19 October 2011 Keywords: Antioxidant enzymes Cadmium Fish Heat stress Oxidative stress Proteasomal activity Temperature
a b s t r a c t Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl2 at nominal concentrations of 0.01 and 1 mg/L) for 4 days at either 15 °C or 21 °C. First, exposure to 1 mg Cd/L induced a high mortality rate (67%) in fish held at 21 °C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21 °C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15 °C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Pollution of aquatic environments by heavy metals including cadmium is a world-wide problem due to the persistency and continuing accumulation of these elements. Cadmium (Cd) is a non-essential toxic metal commonly detected in aquatic and terrestrial environments where it is released from both natural and anthropogenic sources, including industrial, agricultural, and mining activities (Burger, 2008). Although the average Cd content in European rivers roughly varies from 10 to 100 ng/L (OSPAR, 2002), concentrations up to 30 μg/L have been reported in the Molse Nete river (Scheldt basin, Belgium) (Knapen et al., 2004) and levels up to 86 μg/L have been reported in the Meuse River (Eijsden, Belgium) in 2006 (RIWA, 2007). Within cells, Cd has multiple pathways of toxicity due to its high affinity for sulfhydryl groups (SH) that play an important role in the redox balance of the cell and in the structure and function of many enzymes (Valko et al.,
⁎ Corresponding author. Tel.: + 32 81 724287; fax: + 32 81 724362. E-mail addresses: [email protected] (J. Dorts), [email protected] (A. Bauwin), [email protected] (P. Kestemont), [email protected] (S. Jolly), [email protected] (W. Sanchez), [email protected] (F. Silvestre). 1532-0456/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2011.10.003
2005). This resulting change in the redox state of the cell is believed to be associated with oxidation of macromolecules, altered calcium homeostasis, as well as disturbances in the antioxidant defense system (Stohs and Bagchi, 1995). The susceptibility of ectotherms to metal pollution can be strongly affected by environmental temperature due to its direct influence on all physiological and biochemical reactions. The current increases in average temperatures and fluctuations in temperature extremes due to global climate change are considered as one of the new and important threats to aquatic ecosystems (Brönmark and Hansson, 2002). Previous studies have shown that elevated temperatures tend to enhance toxic effects of metals on organisms that may be partially explained by the higher uptake rates of metals and a higher intrinsic sensitivity of organisms (Cairns et al., 1975; Heugens et al., 2001; Sokolova and Lannig, 2008). According to Sokolova and Lannig (2008), interference with aerobic metabolism, including energy demand, oxygen supply, and mitochondrial function, forms a physiological basis for interactions between environmental temperature and metal pollution. In addition to energy disturbance, tissues of Cdexposed oysters (Crassostrea virginica) accumulate elevated levels of lipid peroxidation products at higher temperatures indicating the onset of oxidative stress (Lannig et al., 2006). This view is further
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
supported by the fact that Cd-induced oxidative stress in oyster mitochondria was particularly pronounced at elevated temperature as indicated by increased production of reactive oxygen species (ROS) and decreased activity of mitochondrial aconitase (Cherkasov et al., 2007). Elevated temperature and metal exposure, such as to Cd, are known to induce oxidative stress due to the excessive ROS production and/or interferences with the antioxidant defense system (Valko et al., 2005; Abele et al., 2007). The effects of temperature and Cd exposure as single environmental factors on oxidative stress have been extensively studied in aquatic organisms, including fish (Almeida et al., 2002; Lushchak and Bagnyukova, 2006a, 2006b). However, to our knowledge there are no studies that address interactive effects of these two potentially pro-oxidant stressors on oxidative stress status in fish. Oxygen free radicals can elicit widespread damage to the vital cellular components, including lipids, proteins and DNA, which can eventually lead to cell death. For instance, oxidative stress contributes to structural changes or misfolding of cellular proteins (Stadtman, 1992). Cells possess a protective mechanism to overcome the potentially toxic accumulation of oxidatively modified proteins, namely an increase in proteolysis (Grune et al., 1995). Previous studies have suggested that oxidized proteins are degraded in an ATP- and ubiquitin-independent pathway by the 20S proteasome (Davies, 2001; Shringarpure et al., 2003). Additionally several studies demonstrated that oxidative stressors can alter the activity of the ubiquitin– proteasome system (UPS) (Shang and Taylor, 1995; FigueiredoPereira et al., 1998). Changes in ubiquitination in response to temperature (Hofmann and Somero, 1995; Buckley et al., 2001) and Cd exposure (McDonagh and Sheehan, 2006) have been previously studied in bivalves. Downs et al. (2001) also reported in grass shrimp Palaeomonetes pugio that ubiquitin levels increase in response to high temperature, Cd, bunker fuel, and diesel fuel. However the relationship between potentially pro-oxidant stressors (such as temperature and metal exposure) and the proteasome complex remains poorly understood, especially in fish. The aim of the present study was to investigate the combined effects of temperature and Cd exposure on the oxidative stress status of the European bullhead, Cottus gobio, a small bottom-dwelling freshwater cottid fish widespread throughout Europe. Bullhead typically lives in well oxygenated stream waters from 2 to 16.5 °C (Andreasson, 1971), whereas Elliott and Elliott (1995) found the critical thermal limits of bullhead to be − 4.2 and 27.7 °C. Furthermore, this species has become endangered in several parts of its distribution area as a result of pollution and habitat destruction and is known for its sensitivity to temperature changes (Utzinger et al., 1998; Dorts et al., 2011a). To address this question, biochemical markers of oxidative stress including antioxidant enzyme activity and lipid peroxidation level, as a consequence of oxidative deterioration of membrane lipids, were assessed in liver and gill tissues of bullhead subjected to different Cd concentrations at either 15 °C or 21 °C for 4 days. In addition, the effects of these two stressors on the 20S proteasome activity (chymotrypsin-like) were investigated for the first time in a fish species. 2. Material and methods 2.1. Animals and exposure condition Investigations and animal care were conducted according to the guidelines for the use and care of laboratory animals and in compliance with Belgian and European regulations on animal welfare. Adult European bullhead of both genders weighing 10.4 ± 2.8 g were caught by electrofishing in the Samson River (Belgium) in May 2010. Fish were acclimated to laboratory conditions in dechlorinated tap water at 15.8 ± 1.4 °C under a 14:10 h (light/dark) photoperiod for 4 weeks before the experiment. During the acclimation period,
319
fish were fed daily to apparent satiation with chironomid larvae (Chironomus sp.). After acclimation, 144 fish were randomly distributed over 18 tanks filled with 12 L dechlorinated tap water. Fish were exposed to CdCl2 (Sigma C2544) at nominal concentrations of 0.01 and 1 mg/L for 4 days at either 15 °C or 21 °C. The temperature was increased by 2 °C per day from 15 to 21 °C. The tested concentrations were chosen according to LC50 values reported for related fish species in the literature (Mebane, 2006; Besser et al., 2007) and to a previous study which evaluated the toxicity of short-term Cd exposure in liver and gills of C. gobio by monitoring the response of several enzymes involved in energy metabolism, and by undertaking a proteomic analysis using 2D-DIGE technique (Dorts et al., 2011b). Each treatment included three replicate tanks, with 8 fish per tank. After 4 days of exposure, each fish was weighed, and liver and gills were collected on ice, directly snap-frozen in liquid nitrogen and stored at − 80 °C until homogenization. Animals were not fed during exposure, and half-water was gently siphoned out, replaced, and recontaminated every day. Total Cd concentrations in the exposure water were monitored every other day using a Sector Field Inductively Coupled Plasma Mass Spectrometer (Thermo Finnigan Element 2). Certified reference water samples (riverine water certified reference material SLRS-4, National Research Council Canada) were also analyzed for Cd during each analytical run; measured Cd concentrations were consistently within the certified range. Cd water concentrations were stable over the course of the experiment; the mean concentrations and standard deviations in the 0.01 and 1 mg/L treatment were 0.009 ± 0.001 and 0.834 ± 0.064 mg/L, respectively. 2.2. Oxidative stress markers 2.2.1. Sample preparation Oxidative stress markers were assessed in liver and gills from 2 individual fish per replicate tank. Tissues were homogenized in ice-cold phosphate buffer (100 mM, pH 7.5) containing 20% glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor. The homogenates were centrifuged at 10,000 g for 15 min at 4 °C and the post-mitochondrial fractions were kept at − 80 °C for biochemical assays. Protein contents were determined using the method of Bradford (1976) with bovine serum albumin as a standard. 2.2.2. Lipid peroxidation level LPO results in the production of various compounds including the malondialdehyde (MDA) consequently to free radicals production, and was assessed by the thiobarbituric acid reactive substances assay TBARS (Ohkawa et al., 1979). LPO products react with thiobarbituric acid (TBA) and the product is quantified by fluorimetry (excitation wavelength 515 nm, emission wavelength 553 nm). A calibration curve with increasing MDA concentrations allowed the calculation of LPO expressed as nmol MDA equiv./g protein. 2.2.3. Antioxidant enzyme activities Prior to biochemical analysis, assays were optimized for the bullhead to assume optimal conditions including saturating conditions, linearity of enzyme rates and homogenate amount. All assays were performed in microtiter plates and experimental temperature was 20 °C. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined using the method of Paoletti et al. (1986). The decrease of the rate of NADH oxidation was measured spectrophotometrically at 340 nm for 6 min. One unit of activity is defined as the amount of enzyme causing 50% inhibition of NADH oxidation and the results were expressed as unit per g protein. Catalase (CAT, EC 1.11.1.6) activity was estimated using the method previously described by Babo and Vasseur (1992). The destruction of hydrogen peroxide (H2O2) was
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
2.3. Chymotrypsin-like proteasome activity Chymotrypsin-like activity of 20S proteasome was assessed in liver and gills from 4 pooled fish per replicate tank using a luminescent assay (Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay, Promega). Tissues were homogenized in a hypotonic buffer (10 mM Tris–HCl pH 7.5, 5 mM MgCl2, 0.5 mM DTT, and 5 mM ATP) according to the manufacturer's recommendation. The homogenates were centrifuged at 1000 ×g for 10 min at 4 °C and the supernatants were kept at − 80 °C for enzyme activity assay. Proteasome activity was normalized to total protein contents determined by a bicinchoninic acid (BCA) assay (Pierce) with bovine serum albumin as a standard. 2.4. Statistical analysis Data were expressed as mean ± S.D. Normality analysis of data was assessed by the Shapiro–Wilk W test. Homogeneity of variances was tested by the Bartlett test. Effects of temperature, Cd exposure and their interaction on the studied variables were tested using the analysis of variance (ANOVA). Fisher LSD tests were used for posthoc comparisons at a 5% significant level. All tests were performed using the Statistica 5.5 software (StatSoft, Tulsa, OK, USA). 3. Results No mortality was observed except for fish exposed to 1 mg Cd/L at 21 °C for 4 days (67% mortality). Due to this high mortality, the biochemical assays on surviving fish exposed to 1 mg Cd/L were not undertaken. 3.1. Lipid peroxidation TBARS levels measured in liver and gill tissues of bullhead in response to temperature and Cd exposure are presented in Fig. 1. In both tissues, no interaction effects of temperature and Cd exposure in TBARS levels were observed. However, TBARS levels were significantly influenced by temperature in liver (P = 0.0023), independently of Cd exposure. Exposure to 21 °C resulted in a significant 29% decrease of TBARS levels in liver.
A
Lipid peroxidation (nmol MDA equiv./g protein)
measured spectrophotometrically at 240 nm for 2 min. The enzyme activity was expressed as μmol of H2O2 consumed per min per mg protein. Glutathione peroxidase (GPx, EC 1.11.1.9) activity was determined using the method of Paglia and Valentine (1967). Oxidation of NADPH was recorded spectrophotometrically at 340 nm for 1 min. The enzyme activity was calculated as μmol NADPH oxidized per min per g protein. Glutathione reductase (GR, EC 1.8.1.7) activity was estimated using the method of Carlberg and Mannervik (1985). The GR activity is evaluated by following NADPH concentration at 340 nm during 6 min. The enzyme activity was expressed in μmol of NADPH oxidized per min per g protein. Glutathione transferase (GST, EC 2.5.1.18) activity assay was conducted according to Habig et al. (1974). The conjugation of reduced glutathione (GSH) with 1chloro-2,-dinitrobenzene (CDNB) via GST activity was recorded spectrophotometrically at 340 nm for 5 min. The activity was expressed as μmol of CDNB conjugate formed per min per g protein.
B
Lipid peroxidation (nmol MDA equiv./g protein)
320
**
8000
6000
4000
2000
0 1200 1000 800 600 400 200 0 0 mg Cd/L
0.01 mg Cd/L
Fig. 1. LPO level (nmol MDA equiv./g protein) in liver (A) and gills (B) of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. Data are represented as mean ± S.D. (n = 6). Statistically significant differences in LPO levels between treatments are denoted by stars (**P b 0.01).
glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST). Nevertheless, the hepatic activities of SOD and GR were significantly affected by Cd exposure (P = 0.0012 and P = 0.0007, respectively), regardless of heat stress. Exposure to 0.01 mg Cd/L significantly increased SOD activity by 54%, while a 45% significant decrease occurred in GR activity. In contrast, the activities of GPx and GST were significantly influenced by temperature in gills (P = 0.005 and P = 0.042, respectively), independently of Cd exposure. Exposure to 21 °C resulted in a significant 24% increase of GPx activity, whereas the activity of GST was significantly reduced by 38% as compared to fish exposed to 15 °C. 3.3. Chymotrypsin-like proteasome activity The activity of chymotrypsin-like 20S proteasome in liver and gill tissues of bullhead in response to temperature and Cd exposure is presented in Fig. 4. In both tissues, the proteasome activity was significantly affected by temperature (P = 0.013 and P = 0.008 in liver and gills, respectively), whereas Cd exposure and the combination of both stressors did not influence its activity. Within groups exposed to 21 °C we observed a significant increase in the activity of proteasome by approximately 25% in both liver and gill tissues (1188.2 ± 204.2 and 3650.2 ± 716.6 RLU/mg protein at 15 °C compared to 1611.5 ±249.8 and 4896.8 ± 464.9 RLU/mg protein at 21 °C, in liver and gills, respectively). 4. Discussion
3.2. Antioxidant enzyme activities The activities of antioxidant enzymes in liver and gill tissues in response to temperature and Cd exposure are depicted in Figs. 2 and 3, respectively. Due to small quantities of tissues, the activities of all antioxidant enzymes could not have been assessed in fish gills. Regardless of tissues, we did not observe any interaction between temperature and Cd exposure for the activities of studied antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT),
To the best of our knowledge, the present study was the first attempt to address the interactive effects of elevated temperature and Cd stress on oxidative stress status in the fish C. gobio. Overall, elevated temperature and Cd exposure as single stressor strongly influenced the antioxidant defense system in bullhead with clear tissuespecific responses. In addition, a temperature rise of 6 °C affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues.
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
A
20
GPx activity (U/g protein)
SOD activity (U/g protein)
A
**
15 10 5 0
100 80 60 40 20 0
1000
GST activity (U/g protein)
1000
CAT activity (U/mg protein)
B 1200
600 400 200 0
**
120
B 1200 800
*
800 600 400 200 0
15°C 0 mg Cd/L
800 600 400 200 0
15°C 0 mg Cd/L
21°C 0.01 mg Cd/L
Sokolova and Lannig, 2008). In the present study, exposure to 1 mg Cd/L for 4 days induced a high mortality rate for bullhead at 21 °C. The influence of temperature on metal toxicity is a complex matter and can be attributed to the relative changes in the rate of metal uptake, elimination, diffusion and biotransformation of an organism.
25
A
20 ***
15 10 5 0 600 500
Chymotrypsin-like proteasome activity (RLU/mg protein)
GR activity (U/g protein)
GST activity (U/g protein)
E
21°C 0.01 mg Cd/L
Fig. 3. Activities of antioxidant enzymes in gills of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. (A) GPx — glutathione peroxidase and (B) GST — glutathione S-transferase. Data are represented as mean ± S.D. (n = 6). Statistically significant differences in enzyme activities between treatments are denoted by stars (*P b 0.05; **P b 0.01).
1000
B
400 300 200 100 0 15°C 0 mg Cd/L
21°C 0.01 mg Cd/L
Fig. 2. Activities of antioxidant enzymes in liver of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. (A) SOD — superoxide dismutase, (B) CAT — catalase, (C) GPx — glutathione peroxidase, (D) GR — glutathione reductase, and (E) GST — glutathione S-transferase. Data are represented as mean ± S.D. (n = 6). Statistically significant differences in enzyme activities between treatments are denoted by stars (**P b 0.01; ***P b 0.001).
Experimental evidence indicates that an increase in environmental temperature results in elevated mortality rates in metal-exposed ectotherms (reviewed in Cairns et al., 1975; Heugens et al., 2001;
Chymotrypsin-like proteasome activity (RLU/mg protein)
GPx activity (U/g protein)
C 1200
D
321
*
2000
1000
0
**
6000
4000
2000
0
15°C 0 mg Cd/L
21°C 0.01 mg Cd/L
Fig. 4. Chymotrypsin-like 20S proteasome activity (RLU/mg protein) in liver (A) and gills (B) of C. gobio exposed to 0.01 mg Cd/L for 4 days either at 15 °C or 21 °C. Data are represented as mean ± S.D. (n = 3). Statistically significant differences in proteasome activity between treatments are denoted by stars (*P b 0.05; **P b 0.01).
322
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
Results from acute toxicity tests evidenced that a temperature increase reduced the 96 h LC50 values for different aquatic species exposed to Cd (Rathore and Khangarot, 2002; Heugens et al., 2003; Prato et al., 2009). Oxidative stress is an important component of the stress response in aquatic organisms exposed to a variety of insults as a result of changes in environmental conditions such as thermal stress or exposure to pollution (Lesser, 2006). To minimize the damaging effects of reactive oxygen species (ROS), cells possess both enzymatic and nonenzymatic antioxidant defenses. The main antioxidant enzyme systems consist of superoxide dismutase (SOD), which detoxifies O2•−, catalase (CAT), which reduces H2O2 to water and oxygen, and glutathione peroxidase (GPx), which reduces both H2O2 and organic peroxides (Halliwell and Gutteridge, 1989). In the present study, the changes observed in liver and gills of C. gobio suggested a tissue-specific antioxidant response following exposure to enhanced temperature and Cd. Several studies dealing with the responses of antioxidant enzymes to a variety of laboratory exposures have been reported but the responses are transient and variable depending on the intensity and duration of the stress applied to the organism in addition to the susceptibility of the exposed species and organs (Ballesteros et al., 2009). Several studies have demonstrated that Cd-induced toxic effects involve lipid peroxidation and variations in the level of antioxidant enzymes culminating in oxidative stress (Ait-Aissa et al., 2003; Liu et al., 2009). Cd is not a redox-active metal and does not directly generate free radicals (Ercal et al., 2001). Its pro-oxidant properties might be due to: (1) displacement of Fe 2+, which induces ROS via Fentonlike reaction (Casalino et al., 1997); (2) inhibition of antioxidant enzyme activities (Casalino et al., 2002); (3) depletion of GSH content (Figueiredo-Pereira et al., 1998); (4) inhibition of the respiratory chain in mitochondria (Wang et al., 2004). With respect to hepatic antioxidant enzymes, the present study highlighted effects of Cd exposure, regardless of heat stress. This could be related to the fact that the liver is one of the major sites of Cd accumulation in fish (Kim et al., 2004) and is the most critical organ for detoxification process. First, our results showed an increase in SOD activity following cadmium stress, suggesting that a rise in the O2•− generation rate might have taken place and the protective role of this enzyme against metal-induced oxidative stress. The reported increase in SOD activity in Cd-exposed bullhead would result in a higher generation of H2O2. However, the activities of the two H2O2-scavenging enzymes, i.e. CAT and GPx, were not affected by Cd exposure in liver of C. gobio. Previously, Jia et al. (2011) showed increased SOD activities in liver of carp Cyprinus carpio var. color following exposure to 0.4, 0.5 and 0.7 mg Cd/L for 7 days, while SOD activity was inhibited at higher Cd concentrations of 1 and 2 mg/L. The present study also reported a decrease in GR activity in response to Cd exposure. GR enzyme, which catalyzes the reduction of oxidized glutathione (GSSG), is not involved in antioxidant defense in the same way as the SOD activity. However it is important in maintaining the GSH/GSSG homeostasis under stress conditions (Winston and Digiulio, 1991). A decline in GR activity could be due to the change in the availability of NADPH in the cell and may result in GSH depletion if extra synthesis of GSH cannot take place to preserve its redox status, as a result of prooxidative effects (Zhang et al., 2004). Cirillo et al. (2011) observed that GSH concentrations and the antioxidant enzyme activities of GPx and GR showed an overall decreasing trend in liver of the gilthead sea bream Sparus aurata exposed to 0.1 mg Cd/L for 11 days. The metabolic rate of ectotherms and, hence, oxygen consumption are proportional to environmental temperature (Hochachka and Somero, 2002). Furthermore, the rate of ROS generation is related to oxygen consumption (Halliwell and Gutteridge, 1989). Therefore, it can be expected that the intensification of respiration at higher temperature may result in enhanced ROS production, oxidation of cellular constituents and a response of antioxidant and associated
enzyme systems (Lushchak and Bagnyukova, 2006a). In the present study, a water temperature rise of 6 °C increased the activity of GPx in gills of bullhead, while the branchial GST activity was inhibited in comparison to control groups. The effects of heat stress observed herein on gills of C. gobio may be related to their physiological role in respiration. Additionally, gills have a large exchange area that is in continuous contact with surrounding water, and slight environmental changes could be transferred easily to cellular defense systems. GSH and enzymes associated with its metabolism provide a major defense against ROS induced cellular damage. It functions mainly as a sulfhydryl buffer, but GSH also serves to detoxify compound either via conjugation reactions catalyzed by GST or directly, as it is the case with hydroperoxides in the GPx catalyzed reaction (Nordberg and Arner, 2001). In the present study, an increased activity of GPx suggests an excess H2O2 production as a result of heat stress. This enhanced activity might be of particular importance in the reduction of lipid hydroperoxides thereby preventing the chain propagation reaction leading to the deleterious effects of lipid peroxidation (Parihar et al., 1997). Several studies dealing with the responses of antioxidant enzymes to enhanced environmental temperature have been reported in various fish species (Parihar et al., 1997; Heise et al., 2006; Lushchak and Bagnyukova, 2006b; Bagnyukova et al., 2007a, 2007b). However, these reports were inconclusive and showed wide individual differences. Exceeding the defensive ability of antioxidant systems can result in damage to cellular constituents, including lipids, proteins and DNA, and a state of oxidative stress ensues (Livingstone, 2001). Previous studies have suggested that exposure to environmental contaminants (Livingstone, 2001; Shi et al., 2005; Cirillo et al., 2011) or other environmental stresses (e.g. temperature) (Parihar and Dubey, 1995; Lushchak and Bagnyukova, 2006a; Bagnyukova et al., 2007a, 2007b) can affect the peroxidation of lipids in different fish tissues. In the present study, neither cadmium nor temperature was able to stimulate the lipid peroxidation process (based on TBARS levels) in gills of C. gobio, supposedly due to effective antioxidant responses. However, a slight decrease of lipid peroxidation was observed in liver of bullhead in response to heat stress. In the present study, the lipid peroxidation process could have occurred earlier during applied stresses as suggested by previous studies (Parihar and Dubey, 1995; Bagnyukova et al., 2007b). Bullhead could also have sufficient antioxidant systems such as non-enzymatic antioxidant defenses that were not assayed in our study. Additionally, although lipid peroxidation has been considered to be the primary process responsible for cadmium toxicity, several authors do not agree on cadmium-induced lipid peroxidation as a basis for cell damage, as many toxic events occur prior to enhanced lipid peroxidation (Casalino et al., 1997, 2000). Apart from lipid peroxidation, oxidative stress is one of the mechanisms that contribute to structural changes or misfolding of proteins. ROS can lead to a range of reversible and irreversible covalent modifications of amino acid side-chain of proteins (reviewed by Ghezzi and Bonetto, 2003). According to Grune (2000), the degradation of non-functional, oxidized proteins is an essential part of the antioxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic proteins is the proteasomal system, which consists of the 20S core proteasome and a multitude of various regulators. To our knowledge, the present study is the first devoted to measure the activity of chymotrypsin-like 20S proteasome in response to heat and cadmium stress in a fish species. As seen here, exposure to elevated temperature resulted in an increased activity of proteasome in liver and gill tissues of C. gobio, regardless of Cd exposure. Similarly, Lamarre et al. (2010) have observed an increased 20S proteasome activity in liver of the wolffish Anarhichas minor acclimated to high temperature. From our results, it may be thought that the proteasomal activity was enhanced to overcome the potentially toxic accumulation of damaged proteins in response to heat stress. Interestingly, an increased expression of a subunit of the 19S
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
proteasome particle (PSMD2) has been observed in gills of C. gobio either exposed to Cd (Dorts et al., 2011b) or acclimated to high temperature (Dorts et al., 2011c). Overall, the induction of proteasomal activity as observed in the present study and the aforementioned increased expression of a subunit of the proteasome activator 19S, suggest that the protein quality control could be a possible target of environmental stressors, such as heat stress, in fish. Combination of exposure to elevated temperatures and trace metals is an environmentally relevant scenario that is expected to become more widespread in the future with global warming. Although no interaction effect on the oxidative stress status of bullhead was observed in the present report, earlier studies have shown interactive effects of enhanced temperature and toxic metals on oxidative stress status of aquatic ectotherms (Lannig et al., 2006; Cherkasov et al., 2007; Verlecar et al., 2007; Lapointe et al., 2011). For instance, oysters C. virginica exposed to both temperature and Cd stress suffered high mortality accompanied by elevated lipid peroxidation products (Lannig et al., 2006), and displayed elevated ROS production and oxidative damage as indicated by aconitase inactivation (Cherkasov et al., 2007). As a corollary, the current study demonstrates that the concomitant exposure to heat and high Cd concentration resulted in a high mortality rate of C. gobio. Furthermore, the present results did not highlight any interactive effects but rather indicated the importance of each abiotic factor, i.e., temperature and Cd, on several aspects of the oxidative stress response. We showed clear tissue-specific and stress-specific antioxidant responses and the induction of proteasomal activity in fish in response to heat stress. This work provides insights into the biochemical events involved in the response to heat and Cd in fish and suggests that further studies on additional endpoints could provide crucial information to better understand the interactive effects of elevated temperature and metal exposure in fish.
Acknowledgments The authors thank A. Evrad and M.-C. Forget from URBE and J. Navez from Musée Royal de l'Afrique Centrale (Tervuren, Belgium) for valuable help during animal husbandry, and biochemical, and chemical analyses, respectively. W. Sanchez and S. Jolly were supported by the French Ministry for Ecology (P190-AP08/09). This study was supported by a FNRS PhD fellowship to J. Dorts.
References Abele, D., Philipp, E., Gonzalez, P.M., Puntarulo, S., 2007. Marine invertebrate mitochondria and oxidative stress. Front. Biosci. 12, 933–946. Ait-Aissa, S., Ausseil, O., Palluel, O., Vindimian, E., Garnier-Laplace, J., Porcher, J.M., 2003. Biomarker responses in juvenile rainbow trout (Oncorhynchus mykiss) after single and combined exposure to low doses of cadmium, zinc, PCB77 and 17beta-oestradiol. Biomarkers 8, 491–508. Almeida, J.A., Diniz, Y.S., Marques, S.F.G., Faine, L.A., Ribas, B.O., Burneiko, R.C., Novelli, E.L.B., 2002. The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environ. Int. 27, 673–679. Andreasson, S., 1971. Feeding Habits of a Sculpin (Cottus gobio L. Pisces) Population. The Institute of Freshwater Research, Drottningholm, Sweden. Rep. No. 51. Babo, S., Vasseur, P., 1992. In vitro effects of thiram on liver antioxidant enzymeactivities of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 22, 61–68. Bagnyukova, T.V., Danyliv, S.I., Zin'ko, O.S., Lushchak, V.I., 2007a. Heat shock induces oxidative stress in rotan Perccottus glenii tissues. J. Therm. Biol. 32, 255–260. Bagnyukova, T.V., Lushchak, O.V., Storey, K.B., Lushchak, V.I., 2007b. Oxidative stress and antioxidant defense responses by goldfish tissues to acute change of temperature from 3 to 23 degrees C. J. Therm. Biol. 32, 227–234. Ballesteros, M.L., Wunderlin, D.A., Bistoni, M.A., 2009. Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan. Ecotoxicol. Environ. Saf. 72, 199–205. Besser, J.M., Mebane, C.A., Mount, D.R., Ivey, C.D., Kunz, J.L., Greer, I.E., May, T.W., Ingersoll, C.G., 2007. Sensitivity of mottled sculpins (Cottus bairdi) and rainbow trout (Onchorhynchus mykiss) to acute and chronic toxicity of cadmium, copper, and zinc. Environ. Toxicol. Chem. 26, 1657–1665.
323
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brönmark, C., Hansson, L.-H., 2002. Environmental issues in lakes and ponds: current state and perspectives. Environ. Conserv. 29, 290–306. Buckley, B.A., Owen, M.E., Hofmann, G.E., 2001. Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J. Exp. Biol. 204, 3571–3579. Burger, J., 2008. Assessment and management of risk to wildlife from cadmium. Sci. Total Environ. 389, 37–45. Cairns Jr., J., Heath, A.G., Parker, B.C., 1975. The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologica 47, 135–171. Carlberg, I., Mannervik, B., 1985. Glutathione reductase. Methods Enzymol. 113, 484–490. Casalino, E., Sblano, C., Landriscina, C., 1997. Enzyme activity alteration by cadmium administration to rats: the possibility of iron involvement in lipid peroxidation. Arch. Biochem. Biophys. 346, 171–179. Casalino, E., Calzaretti, G., Sblano, C., Landriscina, C., 2000. Cadmium-dependent enzyme activity alteration is not imputable to lipid peroxidation. Arch. Biochem. Biophys. 383, 288–295. Casalino, E., Calzaretti, G., Sblano, C., Landriscina, C., 2002. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 179, 37–50. Cherkasov, A.A., Overton Jr., R.A., Sokolov, E.P., Sokolova, I.M., 2007. Temperaturedependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation. J. Exp. Biol. 210, 46–55. Cirillo, T., Amodio Cocchieri, R., Fasano, E., Lucisano, A., Tafuri, S., Ferrante, M.C., Carpene, E., Andreani, G., Isani, G., 2011. Cadmium accumulation and antioxidant responses in Sparus aurata exposed to waterborne cadmium. Arch. Environ. Contam. Toxicol. doi:10.1007/s00244-011-9676-9. Davies, K.J., 2001. Degradation of oxidized proteins by the 20S proteasome. Biochimie 83, 301–310. Dorts, J., Grenouillet, G., Douxfils, J., Mandiki, S.N.M., Milla, S., Silvestre, F., Kestemont, P., 2011a. Evidence that elevated water temperature affects the reproductive physiology of the European bullhead Cottus gobio. Fish Physiol. Biochem. doi:10.1007/ s10695-011-9515-y. Dorts, J., Kestemont, P., Dieu, M., Raes, M., Silvestre, F., 2011b. Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio. J. Proteome Res. 10, 470–478. Dorts, J., Kestemont, P., Thézenas, M.-L., Raes, M., Silvestre, F., 2011c. Heat Acclimation of Cottus gobio Interacts with Subsequent Cadmium Exposure Resulting in a Specific Proteome Response. Downs, C., Fauth, J., Woodley, C., 2001. Assessing the health of grass shrimp (Palaeomonetes pugio) exposed to natural and anthropogenic stressors: a molecular biomarker system. Mar. Biotechnol. 3, 380–397. Elliott, J.M., Elliott, J.A., 1995. The critical thermal limits for the bullhead, Cottus gobio, from 3 populations in North-West England. Freshw. Biol. 33, 411–418. Ercal, N., Gurer-Orhan, H., Aykin-Burns, N., 2001. Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539. Figueiredo-Pereira, M.E., Yakushin, S., Cohen, G., 1998. Disruption of the intracellular sulfhydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquitination in neuronal cells. J. Biol. Chem. 273, 12703–12709. Ghezzi, P., Bonetto, V., 2003. Redox proteomics: identification of oxidatively modified proteins. Proteomics 3, 1145–1153. Grune, T., 2000. Oxidative stress, aging and the proteasomal system. Biogerontology 1, 31–40. Grune, T., Reinheckel, T., Joshi, M., Davies, K.J., 1995. Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. J. Biol. Chem. 270, 2344–2351. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine. Clarendon Press, Oxford, UK. Heise, K., Puntarulo, S., Nikinmaa, M., Abele, D., Portner, H.O., 2006. Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout Zoarces viviparus L. J. Exp. Biol. 209, 353–363. Heugens, E.H., Hendriks, A.J., Dekker, T., van Straalen, N.M., Admiraal, W., 2001. A review of the effects of multiple stressors on aquatic organisms and analysis of uncertainty factors for use in risk assessment. Crit. Rev. Toxicol. 31, 247–284. Heugens, E.H., Jager, T., Creyghton, R., Kraak, M.H., Hendriks, A.J., Van Straalen, N.M., Admiraal, W., 2003. Temperature-dependent effects of cadmium on Daphnia magna: accumulation versus sensitivity. Environ. Sci. Technol. 37, 2145–2151. Hochachka, P.W., Somero, G.N., 2002. Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, Oxford, UK. Hofmann, G., Somero, G., 1995. Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198, 1509–1518. Jia, X., Zhang, H., Liu, X., 2011. Low levels of cadmium exposure induce DNA damage and oxidative stress in the liver of Oujiang colored common carp Cyprinus carpio var. color. Fish Physiol. Biochem. 37, 97–103. Kim, S.G., Jee, J.H., Kang, J.C., 2004. Cadmium accumulation and elimination in tissues of juvenile olive flounder, Paralichthys olivaceus after sub-chronic cadmium exposure. Environ. Pollut. 127, 117–123. Knapen, D., Bervoets, L., Verheyen, E., Blust, R., 2004. Resistance to water pollution in natural gudgeon (Gobio gobio) populations may be due to genetic adaptation. Aquat. Toxicol. 67, 155–165.
324
J. Dorts et al. / Comparative Biochemistry and Physiology, Part C 155 (2012) 318–324
Lamarre, S., Blier, P., Driedzic, W., Le François, N., 2010. White muscle 20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor. J. Fish Biol. 76, 1565–1575. Lannig, G., Flores, J.F., Sokolova, I.M., 2006. Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat. Toxicol. 79, 278–287. Lapointe, D., Pierron, F., Couture, P., 2011. Individual and combined effects of heat stress and aqueous or dietary copper exposure in fathead minnows (Pimephales promelas). Aquat. Toxicol. 104, 80–85. Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiology ecology. Annu. Rev. Physiol. 68, 253–278. Liu, J., Qu, W., Kadiiska, M.B., 2009. Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol. Appl. Pharmacol. 238, 209–214. Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656–666. Lushchak, V.I., Bagnyukova, T.V., 2006a. Temperature increase results in oxidative stress in goldfish tissues. 1. Indices of oxidative stress. Comp. Biochem. Physiol. 143C, 30–35. Lushchak, V.I., Bagnyukova, T.V., 2006b. Temperature increase results in oxidative stress in goldfish tissues. 2. Antioxidant and associated enzymes. Comp. Biochem. Physiol. 143C, 36–41. McDonagh, B., Sheehan, D., 2006. Redox proteomics in the blue mussel Mytilus edulis: carbonylation is not a pre-requisite for ubiquitination in acute free radicalmediated oxidative stress. Aquat. Toxicol. 79, 325–333. Mebane, C.A., 2006. Cadmium Risks to Freshwater Life: Derivation and Validation of Low-effect Criteria Values Using Laboratory and Field Studies (Version 1.1). U.S. Geological Survey Scientific Investigations Report 2006–5245. Nordberg, J., Arner, E.S., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31, 1287–1312. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. OSPAR, 2002. Cadmium. Hazardous Substances Series. OSPAR Commission. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Paoletti, F., Aldinucci, D., Mocali, A., Caparrini, A., 1986. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal. Biochem. 154, 536–541. Parihar, M., Dubey, A., 1995. Lipid peroxidation and ascorbic acid status in respiratory organs of male and female freshwater catfish Heteropneustes fossilis exposed to temperature increase. Comp. Biochem. Physiol. 112C, 109–113. Parihar, M.S., Javeri, T., Hemnani, T., Dubey, A.K., Prakash, P., 1997. Responses of superoxide dismutase, glutathione peroxidase and reduced glutathione antioxidant
defenses in gills of the freshwater catfish (Heteropneustes fossilis) to short-term elevated temperature. J. Therm. Biol. 22, 151–156. Prato, E., Biandolino, F., Scardicchio, C., 2009. Effects of temperature on the sensitivity of Gammarus aequicauda (Martynov, 1931) to cadmium. Bull. Environ. Contam. Toxicol. 83, 469–473. Rathore, R.S., Khangarot, B.S., 2002. Effects of temperature on the sensitivity of sludge worm Tubifex tubifex Muller to selected heavy metals. Ecotoxicol. Environ. Saf. 53, 27–36. RIWA - Maas/Meuse, 2007. Communiqué de presse électronique 14 mars 2007 [online]. Available from http://www.riwa-maas.org/download/fr/nieuws/39.pdf. Shang, F., Taylor, A., 1995. Oxidative stress and recovery from oxidative stress are associated with altered ubiquitin conjugating and proteolytic activities in bovine lens epithelial cells. Biochem. J. 307, 297–303. Shi, H., Sui, Y., Wang, X., Luo, Y., Ji, L., 2005. Hydroxyl radical production and oxidative damage induced by cadmium and naphthalene in liver of Carassius auratus. Comp. Biochem. Physiol. C 140C, 115–121. Shringarpure, R., Grune, T., Mehlhase, J., Davies, K.J., 2003. Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J. Biol. Chem. 278, 311–318. Sokolova, I.M., Lannig, G., 2008. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Clim. Res. 37, 181–201. Stadtman, E.R., 1992. Protein oxidation and aging. Science 257, 1220–1224. Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. Utzinger, J., Roth, C., Peter, A., 1998. Effects of environmental parameters on the distribution of bullhead Cottus gobio with particular consideration of the effects of obstructions. J. Appl. Ecol. 35, 882–892. Valko, M., Morris, H., Cronin, M.T., 2005. Metals, toxicity and oxidative stress. Curr. Med. Chem. 12, 1161–1208. Verlecar, X., Jena, K., Chainy, G., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chem. Biol. Interact. 167, 219–226. Wang, Y., Fang, J., Leonard, S.S., Rao, K.M., 2004. Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic. Biol. Med. 36, 1434–1443. Winston, G.W., Digiulio, R.T., 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137–161. Zhang, J.F., Wang, X.R., Guo, H.Y., Wu, J.C., Xue, Y.Q., 2004. Effects of water-soluble fractions of diesel oil on the antioxidant defenses of the goldfish, Carassius auratus. Ecotoxicol. Environ. Saf. 58, 110–116.