Impact of season on liver mitochondrial oxidative stress and the expression of HSP70 in grey mullets from contaminated estuary
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- Padmini, E. & Vijaya Geetha, B. Ecotoxicology (2009) 18: 304. doi:10.1007/s10646-008-0282-1
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Heat shock proteins (HSPs) are the ubiquitous feature of cells in which these proteins cope with stress induced denaturation of other proteins. HSP70 is found to play a primary role in cellular defense under stress condition. In the present investigation, the seasonal impact on environmental stress induced mitochondrial HSP70 (mtHSP70) expression in the liver mitochondria was examined in grey mullets, Mugil cephalus living in the Ennore estuary (polluted site) was compared with the Kovalam estuary (unpolluted site) over the course of two seasons viz monsoon and summer from April 2006 to March 2008. Oxidative stress was determined along with mtHSP70 expression studies in fish liver mitochondria collected from these two estuaries for both the seasons. The liver mitochondria of grey mullet fish collected from polluted Ennore estuary showed increased levels of lipid peroxide and mtHSP70 expression along with decrease in total antioxidant capacity and glutathione redox ratio levels (cP < 0.05) when compared to unpolluted Kovalam estuary fish. In the fish liver mitochondria of Ennore estuary, there was significant seasonal variation (bP < 0.05) in both oxidative stress marker levels (34% increase) and mitochondrial HSP70 expression (33% increase) with increased level during summer season but the Kovalam estuary fish did not show any significant seasonal variation. In the Ennore estuary fish that are exposed to chronic environmental stress, the overexpression of liver mtHSP70 particularly during summer season may confer differential effects on the cell survival by protection against oxidative stress induced changes.
KeywordsAntioxidantsEstuarymtHSP70Mugil cephalusMitochondriaOxidative stressPollutantsSeasonal influences
Toxic contaminants may enter estuarine ecosystems through a variety of pathways. When sediment contaminant levels become sufficiently high, they may impact resident biota (Fulton et al. 2006). Many industrial and agricultural processes have contributed to the contamination of fresh water systems thereby causing adverse effects on aquatic biota and human health (Dautremepuits et al. 2004). Estuaries, the main contributor of fisheries in India suffer from severe damage due to increased industrialization and urbanization along coastal areas (Padmini et al. 2004). Due to the rapid industrialization, metal contamination is one of the serious problems that is faced by the Ennore estuary located in North Chennai, Tamilnadu state of India. Heavy metals induce oxidative stress in fish by generating ROS by their participation in Fenton reaction (Padmini et al. 2008c). Fish inhabiting the highly polluted Ennore estuary developed an enhanced state of oxidative stress characterized by increased levels of lipid peroxides (LPO), lipid hydroperoxides (LHP), and conjugated diene (CD) when compared to the Kovalam estuary fish in the isolated liver mitochondrial fraction (Padmini and Vijaya Geetha 2007a, b, c).
Environmental stress is especially important at many levels of biological organization as it induces damage at the molecular, cellular, and the organismal level and plays a key role in the genesis of biological novelty (Hoffmann and Hercus 2000). It also induces an adaptive response that usually compensates the noxa and helps the organisms to attain stress tolerance (Calabrese 2006). HSPs are a group of universal proteins that are rapidly induced in response to events that cause physiological stress including exposures to elevated temperature, metals, drugs, hypoxia, and conditions that leads to oxidative stress (Jolly and Morimoto 2000). Mitochondrial HSP70 (MtHSP70) is involved in several different functions and acts as a part of the mitochondrial protein import machinery (Rassow and Pfanner 1995). It is also necessary to maintain sufficient mitochondrial membrane potential necessary for efficient protein import (Moczko et al. 1995).
Emerging evidence shows that the increased oxidative stress or nitrative stress and consequent radical mediated damage observed in cells under any conditions begins in the mitochondria, which are the major sites of free radical species (ROS) production (Duchen 2004). Pollutants like heavy metals, polycyclic aromatic hydrocarbons (PAH) are found to be the main contaminants of waters of Ennore estuary which contributes to oxidative stress due to their reactive oxygen species generating power (Padmini et al. 2008c; Padmini and Vijaya Geetha 2008). Seasonal variation, one of several biological factors have been reported to have a significant influence on estuaries (Wilhelm Filho et al. 2001). Our recent laboratory investigations show that there is seasonal variation in the HSP90α and HSP70 expression in fish hepatocytes from Ennore estuary (Padmini et al. 2008a; Padmini and Usharani 2008).
It has been hypothesized that stress proteins, being integrators of diverse aspects of protein damage, may provide added value to biomonitoring programs. The present work is the first attempt to explore the influence of environmental stress on oxidative stress associated mitochondrial HSP70 expression in grey mullets inhabiting the polluted estuary which is challenged by effluents from several industries surrounding this site. The study also reports for the first time about the seasonal impact on mitochondrial HSP70 expressions under polluted condition in natural environment.
Materials and methods
Malondialdehyde, thiobarbituric acid, 2, 2′-azinobis 3-ethylbenzothiaziline-6-sulphonate (ABTS), trolox, β-aminocaproic acid, phenylmethane sulfonyl fluoride, and N-ethylmaleimide (NEM) were purchased from Sigma–Aldrich, USA. O-phthalaldehyde (OPT), hydrogen peroxide, and ethylenediamine tetraacetic acid (EDTA) were from Sisco research laboratories, Mumbai, India. Mouse monoclonal anti-HSP70 antibody (SPS-810) and Rabbit polyclonal anti-β-Actin (CSA-400) was obtained from Stressgen Bioreagents, Columbia, Victoria Canada and immunohistochemical staining kit was from Piercenet, USA.
Two estuaries were chosen as the experimental sites for the present study. Kovalam estuary (12°49′N, 80°5′E) is situated on the east coast of India and is about 35 km south of Chennai. It runs parallel to the sea coast and extends to a distance of 20 km. The temperature, salinity and pH of this estuary ranged between 27–29°C, 26.7–28.4 ppt and pH 7.18–7.33, respectively. It was chosen as the unpolluted site for the present investigation as it is surrounded by high vegetation and it is free from industrial or urban pollution. Ennore estuary (13°14′N, 80°20′E) also situated on the east coast of India, is about 15 km north of Chennai. It runs parallel to the sea coast and extends over a distance of 36 km. The temperature, salinity, and pH of this estuary ranged between 30–37°C, 32.8–38.6 ppt and pH 7.64–8.73, respectively. This estuary was chosen as the polluted site as in its immediate coastal neighborhood, a number of industries are situated which include petrochemicals, fertilizers, pesticides, oil refineries, rubber factory, and thermal power stations, etc., that discharge their effluents into this estuary. Contamination of Ennore estuary by heavy metals like lead, cadmium, mercury, zinc, iron, etc., to a significant extent compared to Kovalam estuary has been confirmed by previous studies (Raghunathan and Srinivasan 1983; Padmini and Vijaya Geetha 2007b). Also the Ennore estuary significantly differs from Kovalam estuary in their physical, chemical, and biological factors due to pollution (Padmini and Vijaya Geetha 2007a).
M. cephalus (grey mullets), a natural inhabitant of the estuaries, identified by the use of FAO species identification sheets was chosen as the experimental animal for the study (Fischer and Bianchi 1984). Grey mullets, M. cephalus with average length of 20–30 cm were collected freshly every month for two seasons viz summer and monsoon (October–March for monsoon season and April–September for summer season). The samples were collected from both the estuaries during the period of April 2006 to March 2008 to study the effect of seasons. The fish samples were collected with baited minnow traps in a shallow estuary and were placed immediately into insulated containers filled with aerated estuarine water at ambient temperature. Fish was sacrificed by severing their spinal cord and the liver was removed immediately for the isolation of mitochondria.
Isolation of fish liver mitochondria
The procedure of Johnson and Lardy (1967) with slight modifications was employed to isolate mitochondria. About 1 g of tissue was weighed, washed twice with ice cold buffer, and 5 ml of Kohler’s homogenizing buffer was added. The homogenate was centrifuged in the refrigerated centrifuge at 500g for 10 min. The supernatant was recentrifuged at 8,000g for 30 min. The resulting pellet containing the mitochondrial fraction was resuspended and recentrifuged under same experimental conditions, to obtain the pure fraction of mitochondria. We purified the crude mitochondrial fraction by resuspending such fraction in three packed cell volumes of mitochondrial suspension buffer (10 mM Tris–HCl, pH 6.7, 0.15 mM MgCl2, 0.25 M sucrose, 1 mM PMSF, 1 mM DTT) and centrifuging at 9,500g for 5 min for repelleting the pure mitochondria. It was suspended in 0.25 M sucrose solution (pH 7.4) and homogenized for 1 min, which was then used for further studies. The presence of mitochondria in the pellet fraction was confirmed by the assay of succinate dehydrogenase enzyme (marker enzyme). Proteins were estimated by the method of Bradford (1976) with the use of bovine serum albumin as the standard.
Measurement of lipid peroxidation, glutathione redox ratio and total antioxidant capacity
Lipid peroxidation was measured by high performance liquid chromatography (HPLC) according to the Lykkesfeldt (2001) method that was described previously (Padmini et al. 2008a), which involves measurement of the malondialdehyde (MDA) adduct extracted from mitochondrial samples using thiobarbituric acid (TBA) reagent. The results were expressed as nanomoles of MDA formed/mg of mitochondrial protein.
The thiol status was assessed spectrofluorimetrically based on the reaction of O-phthalaldehyde (OPT) as a fluorescent reagent with GSH at pH 8.0 and GSSG with N-ethylmaleimide (NEM) at pH 12.0 using the method of Hissin and Hilf (1976) that was described previously (Padmini et al. 2008a). The fluorescence and excitation were determined at 420 and 350 nm, respectively, and the values were expressed as nanomoles of GSH or GSSG/mg protein.
Total antioxidant capacity (TAC) was determined according to the Erel (2004) method using ABTS reagent that was described previously (Padmini et al. 2008a). The degree of quenching of radical generation in individual samples, indicative of the presence of antioxidant activity, was quantified by comparison with a traditional standard trolox and the assay results were expressed in terms of mmol trolox equivalent/L.
Western blotting of HSP70
Western blotting was performed according to the method of Towbin et al. (1979). The fish liver mitochondrial samples (100 μg of protein) along with HSP70 standards were run using 10% SDS-polyacrylamide gel electrophoresis in Dual electrophoretic apparatus according to the method of Laemmli (1970). The separated proteins were electrotransferred from the gel slabs onto 0.45 μM PVDF membrane (Biotrace, Germany). The membranes were then blocked with 5% Blotto overnight at room temperature with agitation. One of the membranes was probed with mouse alkaline phosphatase conjugated monoclonal antibody raised against HSP70 diluted 1:3000 (SPA-810; Stressgen Bioreagents, Columbia, Canada) for 3 h. The 3-bromo-4-chloro-indolylphosphate-nitroblue tetrazolium (BCIP-NBT) substrate (Sigma, St Louis, MO, USA) system was used to detect the alkaline phosphatase conjugate as described by the manufacturer. The band intensities were scanned with the HP Scan Imager and quantified using the TotalLab Software, GELS, USA. Using a standard graph obtained from the HSP70 standards, HSP70 levels in fish liver mitochondrial samples were calculated. Similar treatment for the other blot sheet was performed by incubating it in rabbit polyclonal anti β-Actin antibody diluted 1:2000 (CSA-400; Stressgen Bioreagents, Columbia, Canada). Following this the blot sheet was incubated with appropriate alkaline phosphatase-labeled secondary antibody prepared in PBS-Tween-20 and the immunoreactive proteins were detected using the BCIP-NBT substrate system and subsequent quantification of the bands were made using Gel documentation system (TotalLab GELS, USA).
Immunofluorescence studies of HSP70
Indirect Immunofluorescence microscopy was performed according to the method of Pringle et al. (1989) with some modifications. Deparaffinized and rehydrated sections were treated with 3% hydrogen peroxide in absolute methanol for 5 min in order to inhibit endogenous peroxidase activity. Antigen retrieval was performed by heat treating sections in citrate buffer at pH 6 in a microwave oven for 5 min (3 cycles). To reduce non specific binding, slides were incubated in 10% normal goat serum for 10 min at room temperature. After that the slides were incubated for 1 h with primary antibody HSP70 (SPA-810; Stressgen, Canada) in a humidified chamber at 4°C. After three washes with 0.05 M TBS, the primary antibody was detected with antimouse IgG Alexa fluor 594 (1:400 dilution) in PBS-0.1% BSA by incubating at room temperature for 30 min. After that slides were rinsed with 0.05 M TBS buffer dehydrated and mounted with coverslips. Then the fluorescence of HSP70 was detected using confocal microscope (Leica TCS Sp-2 XL) in 40× magnification which was seen as green fluor.
Data were analysed using commercially available statistical software package (SPSS for windows 7.5 version). Two way ANOVA test was performed to find out the significance of variations between the polluted site and unpolluted site fish samples and also the seasonal variation in oxidative stress levels and HSP70 expression levels. Results were presented as mean ± SD. A P value < 0.05 was considered to be statistically significant.
Oxidative stress status in response to environmental pollutants
Levels of lipid peroxide, glutathione redox ratio and total antioxidant capacity in liver mitochondria of M. cephalus inhabiting unpolluted and polluted sites during monsoon and summer seasons
Fish from unpolluted site
Fish from polluted site
Lipid peroxide (nanomoles of MDA/mg protein)
1.23 ± 0.04
1.45 ± 0.05a
1.9 ± 0.08c
2.74 ± 0.12b,c
Glutathione redox ratio (in percentage)
41.49 ± 2.8
40.13 ± 2.4a
35.2 ± 1.48c
24.47 ± 1.2b,c
Total antioxidant capacity (millimoles of trolox equivalent/liter)
1.1 ± 0.06
0.96 ± 0.055a
0.72 ± 0.04c
0.51 ± 0.045b,c
In the polluted site samples, the comparison of LPO and TAC levels (Table 1) showed that as the LPO level increases, the TAC level decreases proportionately reflecting the defective antioxidant status when compared to the unpolluted site samples.
Environmental stress induced expression of mtHSP 70
Immunofluorescent expression of HSP70
The comparison of oxidative stress marker levels against mtHSP70 levels (Table 1; Fig. 1) demonstrated that LPO level increases as the TAC level decreases and however to overcome and protect the cell from increased oxidative stress and damage, HSP70 increases proportionately with the LPO levels and protect the cell from such situation which would probably aid in cellular survival.
Mitochondria plays a crucial role in the cellular metabolism by performing many, in part essential, biosynthetic reactions (Lill and Kispal 2000). It also participates in important regulative processes like apoptosis (Jiang and Wang 2004). Hence the maintenance of mitochondrial activity by preserving protein content and function is the key aspect of cellular survival particularly under stressed conditions.
In case of moderate stress, the inner balance is usually restored but under severe or prolonged stress conditions, the compensatory abilities of the organism may be exhausted which results in physiological disturbance or even death. It was already reported that the grey mullets, Mugil cephalus surviving in the polluted Ennore estuary are subjected to prolonged oxidative stress that leads to considerable DNA fragmentation which may decrease the cellular survivality (Padmini et al. 2008b).
Regulation of mitochondrial function plays a crucial role in adaptation and environmental stress tolerance. Recent studies on mitochondrial function under a variety of physiological and stress conditions led to the realization that the mitochondria is highly tuned and prone to malfunction in response to even moderate stress, rather than operating reliably in the background (Nicholls 2002). To investigate the known mechanisms behind adaptation and responses to stress and to identify new mechanisms an integrative approach is especially important. A better understanding is needed to improve our knowledge about adaptation to the environmental contamination changes.
In the present study, during both the seasons, the lipid peroxidation product MDA was significantly increased and the GRR and TAC levels were significantly decreased in polluted site samples than unpolluted site samples, the condition representing a typical association with acute and chronic stress in fish exposed to pollutants. During monsoon and summer seasons, in the Ennore estuary inhabiting fish, the percentage increase in the MDA level was significant (54 and 75%, respectively) and similarly the TAC and GRR levels showed a significant decrease (35 and 47% for TAC; 15 and 38% for GRR) that was inversely proportional to the MDA levels when compared with the Kovalam estuary during monsoon and summer season.
The above results indicate significant increase in the LPO levels and significant decrease in TAC and GRR levels in Ennore estuary sample when compared to Kovalam estuary sample during summer and monsoon seasons. But the percentage difference between the two sites is comparatively high in the samples collected during summer season. On further seasonal variation analysis of the results obtained, the fish liver mitochondrial sample collected from the Ennore estuary showed a significant seasonal difference (34% increase in LPO values; 29 and 31% decrease in TAC and GRR levels) when compared to the Kovalam estuary fish which showed an insignificant seasonal variation (17% increase in LPO values; only 13 and 4% decrease in TAC and GRR levels) of oxidative stress and antioxidant status parameters.
The observed increase in lipid peroxide levels is usually suggestive of enhanced oxidative stress (Lykkesfeldt 2007). The increased lipid radical formation would decrease glutathione redox ratio and total antioxidant capacity in liver mitochondria of polluted site samples revealed that the protective effect was still insufficient to mop up the bulk of the free radicals produced necessitating the condition that these fish must have an effective counter mechanism to overcome the buildup of oxidative stress over a critical level.
Earlier studies have assessed the impact of stressors/contaminants on fish oxidative stress markers and HSPs response in in vitro condition by exposing them to a single anthropogenic stress under unpolluted site led conditions, neglecting to consider the cumulative effect of all stresses to which the organisms are subjected within their environment (Nadeau et al. 2001; Molina et al. 2002). In various fish tissues, HSPs respond to a wide range of stressors and so these proteins are reported to be indicators of stressed states in fish (Iwama et al. 2003). The mitochondrial matrix inhabiting mtHSP70, a member of the HSP70 family mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature and also responsible for the maintenance of the morphology of mitochondria (Liu et al. 2001).
In Ennore estuary, the results of mtHSP70 expression levels showed significant elevation (44 and 53% increase) when compared to Kovalam estuary during monsoon and summer seasons, respectively, which shows that the percentage of elevation is more during summer season. The observed result was supported by a previous study which reported that the contaminant release was associated with the altered HSP70 expression levels in the liver and gill of the cray fish living in the polluted stream system (Triebskorn et al. 2002). On further analysis of the results obtained, the mtHSP70 level in the Ennore estuary fish showed a significant seasonal variation (33% increase) when compared to Kovalam estuary sample which shows only 24% increase during summer season. This led to a conclusion that during summer season, the Ennore estuary fish are at the higher level of oxidative stress and need to have increased levels of HSP70 to adapt themselves and survive in the polluted stress condition and hence significant increase in the mtHSP70 levels.
Analysis of the fish liver mitochondria collected at the unpolluted site showed lower LPO, higher GRR and TAC, and normal HSP70 expressions with less significant differences between summer and monsoon seasons whereas the fish collected at the polluted site showed a significant increase in LPO and HSP70 expression and decrease in GRR and TAC levels during summer season when compared to its respective monsoon season. The increased LPO and decreased GRR and TAC levels, shown by polluted site fish coincided with the increase in the concentration of pollutants due to water evaporation during summer. By immunofluorescence studies, it was confirmed that the HSP70 gets over expressed in the liver tissue samples collected from the polluted site in comparison with the unpolluted site. The over expression of HSP70 observed in polluted site fish liver sections may be due to the environmental pollution induced oxidative stress condition.
Seasonal variations have a potential to affect estuarine organism due to changes in physiochemical variables and some biological factors (Tait 1981) which may be the likely reason for the observed results with regard to the levels of LPO, GRR, TAC and mtHSP70 expression. Its consideration is essential during biomonitoring program and when sampling is done at different periods of the year. Seasonal variation in the hydrographic parameters and metal contamination status in the Ennore estuary have been already reported (Padmini and Vijaya Geetha 2007a, b).
Seasonal adjustments in the antioxidant defense of this fish suggest that this mechanism is a common adaptation in thermoconformer vertebrates and invertebrates. The results have also indirectly indicated the seasonal variations in the activity of several enzymes which participate in the cellular defense system (represented by total antioxidant levels) that is involved in the adaptive response of organisms to pollution. Together, our data corroborate previous study that shows a positive correlation between antioxidant defenses and seasonality (Wilhelm Filho et al. 2001). In Ennore estuary fish, the decrease in the activity of antioxidant defense system with respect to GRR and TAC levels, occurring during summer may be directly responsible for the enhanced susceptibility of grey mullets to oxidative stress during this period.
In the Kovalam estuary inhabiting fish, HSP levels showed little change with seasons indicating that these species did not experience pollution induced stress. But in the Ennore estuary, the higher levels of HSP70 during summer seasons inhabiting fish may be adaptive in maintaining native protein structures under environmental stress. There must be sufficient concentrations of HSPs to cope satisfactorily with the levels of aberrant proteins that are generated at any given environmental stress condition. Higher constitutive levels of HSPs in eurythermal ectotherms during summer could provide cells with an adequate ability to process partially unfolded proteins. In monsoon, due to dilution of environmental pollutants caused by greater inflow of rainwater, protein denaturation may occur less frequently, and low concentration of HSPs may be adequate (Dietz and Somero 1992). But in Ennore estuary fish, that are exposed to chronic environmental stress, the overexpression of liver mtHSP70 particularly during summer season may confer differential effects on the cell survival by protection against oxidative stress induced changes.
In conclusion, we report for the first time that mtHSP70 overexpression may play a key role in environmental adaptation processes in grey mullets protecting the cells from oxidative stress in natural field conditions. Additionally, our studies also show the seasonal variations in mitochondrial HSP70 expression under polluted conditions. So, the seasonal influences should be better understood in order to make any inferences regarding cellular homeostasis in fish liver cell.
This study is a part of the DST Project Ref. No DST: SP/SO/AS-10/2003 funded by the Ministry of Science and Technology, Government of India.