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Response of glutathione system and carotenoids to sublethal copper in the postlarvae of Penaeus indicus
Ecotoxicology and Environmental Safety 75 (2012) 127–133
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Response of glutathione system and carotenoids to sublethal copper in the postlarvae of Penaeus indicus Rupa Vani Paila n, Prabhakara Rao Yallapragada, Samuel David Raj Thatipaka Division of Physiology and Toxicology, Department of Zoology, Andhra University, Visakhapatnam 530003, India
a r t i c l e i n f o
abstract
Article history: Received 5 June 2011 Received in revised form 22 August 2011 Accepted 29 August 2011 Available online 25 September 2011
The objective of this study is to determine the effect of sublethal copper on the glutathione system and carotenoids of Penaeus indicus postlarvae (PL) when subjected to short- and long-term exposure in the laboratory. The PL were exposed to 0.1641 ppm (sublethal) copper for a period of 30 days with sampling intervals of 24, 48, 96 h and 10, 20, 30 days. Variations in the activity of Glutathione reductase (GR), Glutathione peroxidase (GPx) and Glutathione-S-transferase (GSTase) were measured as biomarkers of toxicity. A significant (P o0.05) increase in the GR and GPx activity of the exposed PL till 20 days of exposure and thereafter a significant decrease indicates susceptibility of the PL to oxidative stress upon chronic exposure. Similarly, a significant decrease in GSTase activity during long-term exposures in the exposed PL reflects possible failure of the detoxification system. A possible role of carotenoids to combat oxidative stress against copper toxicity is also discussed. & 2011 Elsevier Inc. All rights reserved.
Keywords: Glutathione reductase Glutathione peroxidase Glutathione-S-transferase Carotenoids Copper toxicity Penaeus indicus Postlarvae
1. Introduction While the vast majority of complex life on Earth requires oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by producing reactive oxygen species (ROS) (Davies, 1995). Consequently, organisms contain a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids (Sies, 1997; Vertuani et al., 2004). In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell (Davies, 1995; Sies, 1997). However, since ROS do have useful functions in cells, such as redox signaling, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level (Rhee, 2006). When the production of ROS overwhelms the antioxidant system, an imbalance between the formation and removal of ROS occurs thereby rendering the cells more susceptible to oxidative damage and results in simultaneous decline in the antioxidant defense mechanism. This leads to damage of cellular organelles and changes in certain enzymatic activities, cumulating to a situation known as oxidative stress n
Corresponding author. Fax: þ91 891 2525611/2755324. E-mail addresses: [email protected] (R.V. Paila), [email protected] (P.R. Yallapragada), [email protected] (S.D.R. Thatipaka). 0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.08.023
(Zhang et al., 2008). The relevance of glutathione system in defense against oxidative stress is being increasingly studied, because of its potential utility to provide biochemical biomarkers that could be used in environmental monitoring system. The key enzymes for the detoxication of ROS in all organisms are superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) as well as glutathione-S-transferase (GSTase). Glutathione-S-transferase (GSTase) catalyzes the conjugation of GSH with a variety of electrophilic metabolites. This enzyme participates in defense against oxidative stress by detoxifying endogenous harmful compounds like hydroxyalkenals and base propenals or DNA hydroperoxides and electrophilic xenobiotics (Cnubben et al., 2001). Thus, induction of GSTase is considered beneficial to handle environmental stress (van der Oost et al., 2003). Glutathione peroxidase (GPx) with peroxidase activity protects the organism from oxidative damage by reducing lipid hydroperoxides to their corresponding alcohols. It also reduces free hydrogen peroxide to water and thereby catalyzes the conversion of reduced glutathione (GSH) to an oxidized form (GSSG) (Nordberg and Arner, 2001). Glutathione reductase (GR) reduces glutathione disulfide (GSSG) to GSH, which is an important cellular antioxidant (Mannervik, 1987). For every mole of oxidized glutathione (GSSG) one mole of NADPH is required to reduce GSSG to GSH from which, two reduced GSH molecules are gained that act as antioxidants, scavenging ROS in the cell.
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Carotenoids are a class of natural, fat-soluble, organic pigments belonging to the category of tetraterpenoids and produces characteristic yellow, orange or red colors. They play a potential protective role as biological antioxidants by two general mechanisms: (i) quenching of singlet oxygen and dissipating the energy as heat, and (ii) scavenging of radicals to prevent or terminate chain reactions (Miki, 1991). Metal toxicity results from nonspecific metal binding, which can inactivate important regulatory enzymes by displacing essential metal ions from catalytic sites, by altering catalytic cysteine groups or by allosterically altering the functional conformation of proteins (Mason and Jenkins, 1996). The basis for copper cytotoxicity is that both cupric and cuprous ions can participate in oxidation and reduction reactions i.e. capable of redox cycling. In the presence of superoxide (O2 ) or reducing agents such as ascorbic acid or GSH, Cu2 þ can be reduced to Cu þ , which is capable of catalyzing the formation of hydroxyl radicals (OH) from hydrogen peroxide (H2O2) via the Haber–Weiss reaction (Bremner, 1998). Thus, copper is capable of producing ROS and inducing DNA strand breaks and oxidation of bases (Kawanishi et al., 1989). The white shrimp Penaeus indicus widely distributed along the east coast of India is well known for its palatability, high market value and is a commercially important crustacean for the Indian fishing industry. The present investigation was intended to determine the effect of short- and long-term sublethal copper exposure on glutathione system of Penaeus indicus postlarvae (PL) as well as to evaluate the role of carotenoids against copper toxicity.
2. Materials and methods This study was conducted in accordance with the institutional (Andhra University) guidelines for animal experiments. 2.1. Chemicals All chemicals and reagents were obtained from Sigma Chemical Co. (St Louis, Missouri, USA). 2.2. Animals Equal-sized PL (16–23 mm) of Penaeus indicus were obtained from the wild catch of Gosthani estuary located near Bheemunipatnam (17.51N 83.31E), 30 km from Visakhapatnam in Andhra Pradesh, on the east coast of India. The PL were carefully transported to laboratory and were acclimatized to laboratory conditions for three days in 50 L tanks containing seawater (salinity 10%, temperature 29 71 1C, dissolved oxygen 8.0 and pH 7.8). During this period, constant aeration was provided and PL were fed with commercial diet, having no residual traces of copper (EPAC-XL, INVE, Belgium) twice a day. Excess feed was removed daily by siphoning and ambient water in the tank was renewed daily. 2.3. Sublethal exposure and sampling The 96 h LC50 value for same size group previously obtained by Paila and Yallapragada (2010) was used for the present study (96 h LC50 for P. indicus PL was 0.8204 mg/L of copper toxicity). Therefore, a concentration of 1/5 of 96 h LC50 value, i.e., 164.1 mg/L was considered as sublethal (Paila and Yallapragada, 2010) and further experiments were carried out on exposure to this nominal concentration. A stock solution of copper (10 g/L) was prepared from CuSO4 5H2O (AR) in distilled water. An appropriate volume of the freshly prepared stock solution was added to the seawater to obtain the final desired concentration. The PL (about 600 in number) were then exposed to this toxicant for a period of 30 days in plastic tanks of 50 L capacity. A parallel control was maintained without the metal toxicant. During the course of the study, salinity (10%), temperature (297 1 1C), dissolved oxygen (8.0) and pH (7.8) were regularly measured and adjusted using a portable auto analyzer (WTW Multiline P4, Merck). Seawater was renewed every 24 h and copper concentration was re-established in the exposure tanks after the daily water change. Excess feed was removed daily by siphoning and the tanks were washed daily. Feeding was stopped six hours prior to sampling. Whole PL
samples were collected both from control and exposed tanks at six different time intervals i.e., 24, 48 and 96 h (short-term), 10, 20 and 30 days (long-term) and were kept at 70 1C until analysis, which was carried out within one week. Five replicates were maintained at each interval.
2.4. Sample preparation After the exposure periods, the control and exposed PL were sacrificed and soft tissues were isolated. Whole body of ten individuals for one sample was taken for each biochemical assay. A 10% homogenate for each sample was prepared in Tris HCl buffer 0.026 M (pH 8.0) for GSTase activity. For GPx and GR assays, the homogenate (10%) was similarly made in potassium phosphate buffer of 0.05 M (pH 7.0) and 0.5 M (pH 7.0) concentration, respectively. The homogenate was centrifuged differentially at 4000 rpm for 15 min at 4 1C for GSTase activity and at 16000g for 45 min at 4 1C for GPx and GR activity. The supernatant was further centrifuged at 16,000 rpm for 60 min at 4 1C for GSTase activity and at 30,000 rpm for 1 h at 4 1C for GPx and GR activity. The resultant supernatant was then used as an enzyme source.
2.5. Enzymatic assays 2.5.1. Estimation of glutathione-S-transferase activity Glutathione-S-tranferase (GSTase) activity was estimated by following the method of Habig et al. (1974). The reaction mixture was prepared by adding 2.4 ml of 0.026 M potassium phosphate buffer, 100 ml of 0.33 M 1-chloro-2,4-dinitrobenzene (CDNB), 400 ml of distilled water. An amount of 100 ml of homogenate containing 50 mg of protein was taken in the reaction mixture followed by addition of 100 ml 0.33 M of GSH. The change in absorbance was monitored at 340 nm for one minute in spectrophotometer. One unit of activity is equal to micromole of thioester formed/mg protein/min.
2.5.2. Estimation of glutathione peroxidase activity Glutathione peroxidase (GPx) was determined by following the modified version of Flohe and Gunzler (1984). The reaction mixture was prepared by adding 600 ml of potassium phosphate buffer (0.05 M, pH 7.0, 0.1 mM EDTA), 100 ml of 10 mM GSH (reduced form), 100 ml of 1.5 mM NADPH and 100 ml of GR (0.24 units). An amount of 100 ml of whole PL homogenate supernatant fraction was added to the reaction mixture and incubated at 37 1C for 10 min. To this, 50 ml of 12 mM t-butyl hydroperoxide was added. The change in the absorbance was measured at 340 nm for one minute. One unit of activity is equal to millimole of NADPH oxidized/mg protein/min.
2.5.3. Estimation of glutathione reductase activity Glutathione reductase (GR) was determined by following the method of Calberg and Mannervik (1985). An amount of 50 ml of NADPH (2 mM) in 10 mM Tris HCl buffer (pH 7.0) was added in a cuvette containing 50 ml of GSSG (20 mM) in potassium phosphate buffer (0.5 M, pH 7.0, 0.2 mM EDTA) and 850 ml of potassium phosphate buffer. An amount of 50 ml of whole PL homogenate supernatant was added to the NADPH-GSSG buffered solution. The absorbance was read at 340 nm using spectrophotometer for minute. One unit of activity is equal to millimole of NADPH oxidized/mg protein/min. Total soluble protein contents for the above enzymatic activities were determined according to Lowry et al. (1951), using bovine serum albumin (BSA) as a standard.
2.5.4. Estimation of total carotenoids The extraction of total carotenoid content from whole PL samples was estimated by the method of Susan and Damodaran (1997). Uniform-sized live PL were collected from both the control and exposed tanks at each exposure period, thoroughly washed with double distilled water, blotted on filter paper and their wet weights were taken. Total carotenoids were extracted by homogenizing the PL with acetone in glass homogenizer. The colored supernatant was collected and fresh acetone was added again to the pellet. Homogenization was continued until the acetone extract turned colorless. This acetone extract was then filtered under reduced pressure in a scintered glass funnel. The total volume of the extract was measured and its optical density was recorded at 455 nm using spectrophotometer against an acetone blank. The concentration of total carotenoids was calculated by using the following formula (Susan and Damodaran, 1997): Total carotenoid concentration ðmg=100 mg tissueÞ ¼ 0:4 DV=W where D ¼ optical density of the extract measured at the wavelength of carotenoid absorption maxima i.e. 455 nm, V ¼total volume of carotenoid extract (ml), W¼ total wet weight of the tissue (mg) from which the carotenoids were extracted. The results were expressed as mg/g wet weight tissue.
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2.6. Statistical analysis All assays were repeated five times. The data were analyzed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test, using Graphpad Prism 5 software package to study the significant differences between the means (n¼10, five samples in duplicate) of control and exposed samples for the six time intervals as well as to study changes over time.
3. Results 3.1. Glutathione-S-transferase activity The result of GSTase activity in the PL of P. indicus exposed to sublethal copper along with their respective controls for different time intervals is presented in Fig. 1. The figure shows that though there is an increase in the GSTase activity in the exposed PL over its respective control during short-term exposure i.e. from 24 h to 96 h, the increase is not significant at 5% level. However, longterm exposure to sublethal copper has resulted in a significant (F: 159.8; Po0.0001) decrease in the GSTase activity of the exposed PL over its respective control. The decrease in the activity was 11.62%, 30.62% and 33.49% for 10, 20 and 30 days, respectively. Significant (F: 412.7; P o0.0001) time-dependent decrease in GSTase activity of the exposed PL was noticed from 10 days onwards when compared to short-term exposures as shown in Fig. 2 and Table 1.
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Table 1 Levels of significance for GSTase activity of the exposed Penaeus indicus PL tested over time. GSTase activity Exposure period 24 h 48 h 96 h 10 days 20 days 30 days
24 h
96 h
10 days
20 days
30 days
nnn
nnn
nnn
nnn
nnn
– ns ns
ns – ns
ns ns –
nnn
nnn
nnn
nnn
nnn
nnn
nnn
-
nnn
nnn
nnn
nnn
nnn
nnn
–
nnn
nnn
nnn
nnn
nnn
nnn
Note: ns¼ not significant. 0.001% level. nnn
48 h
n
¼ significant at 0.01% level.
– nn
¼ significant at
¼significant at 0.0001% level.
3.2. Glutathione peroxidase activity Levels of GPx activity in the PL of P. indicus exposed to sublethal copper for different periods of exposure along with
Fig. 3. Glutathione peroxidase activity in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper along with their respective controls for different experimental periods. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD. ***¼ Po 0.0001 over its respective control.
Fig. 1. Glutathione-S-transferase activity in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper along with their respective controls for different experimental periods. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD. **¼ Po 0.001 and *** ¼P o0.0001 over their respective control.
their respective controls are presented in Fig. 3. The results show a significant (F: 47.14; Po0.0001) increase in the GPx activity in the exposed PL over its respective control only for short-term exposures i.e 24 h to 96 h. Though long-term exposures showed an increase in the levels of GPx activity of the exposed PL over their respective controls upon 10 and 20 days of exposure, the increase was not significant. However, long-term exposure for 30 days showed a significant (Po0.0001) decrease in the GPx activity in the exposed PL over its respective control. The percent increase in the GPx activity for different time intervals was 29.27%, 17.73%, 37.02%, 16.18% and 6.49% for 24 h, 48 h, 96 h, 10 days and 20 days, respectively. However, 30 days exposure showed 20.47% decrease in GPx activity in the exposed PL over its respective control. Fig. 4 and Table 2 shows a gradual, timedependent and significant (F: 71.56; P o0.0001) decrease in GPx activity of the exposed PL from 96 h onwards and a complete reversal of the activity was observed on 30 days of exposure. 3.3. Glutathione reductase activity
Fig. 2. Time-dependent changes in the GSTase activity of the exposed Penaeus indicus PL. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD.
Fig. 5 indicates GR activity in P. indicus PL subjected to sublethal copper exposures for different time intervals along with their respective controls. Though, an increase in the GR activity of the exposed PL over its respective control was noticed from 24 h till 20 days of exposure, a significant (F: 17.64; Po0.0001) increase in the activity was noticed only from 10 days onwards. There was a gradual increase in the GR activity from 12.93% (24 h)
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GR-Exposed
GR activity
(nmoles of NADPH oxidised /mg protein/min.)
1.5 1.0 0.5 0.0 -0.5
24hr
48hr
96hr 10d 20d Exposure period
30d
-1.0 -1.5
Fig. 4. Time-dependent changes in the GPx activity of the exposed Penaeus indicus PL. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD.
Table 2 Levels of significance for GPx activity of the exposed Penaeus indicus PL tested over time. GPx activity Exposure period 24 h 48 h 96 h 10 days 20 days 30 days
24 h
48 h
96 h
10 days
20 days
30 days
nn
n
nnn
nnn
nnn
ns
nnn
nnn
nnn
–
nnn
nnn
nnn
n
ns
nnn
nnn
nnn
nnn
– ns
ns -
nnn
nnn
nnn
nnn
nnn
nnn
–
– ns
ns –
nn
Fig. 6. Time-dependent changes in the GR activity of the exposed Penaeus indicus PL. Bars represent means (n ¼10, five samples in duplicate) and vertical lines SD.
Table 3 Levels of significance for GR activity of the exposed Penaeus indicus PL tested over time. GR activity Exposure period
24 h
48 h
96 h
10 days
20 days
30 days
24 hrs 48 h 96 h 10 days 20 days 30 days
– ns ns
ns – ns
ns ns –
nnn
n
nnn
nn
nnn
nnn
nn
n
n
ns
ns
– ns
ns ns ns –
nnn
nnn
nnn
nnn
nnn
–
n
nnn nnn nnn
nnn
Note: ns ¼not significant. n
Note: ns¼not significant. n
¼ significant at 0.01% level. ¼ significant at 0.001% level. nnn ¼significant at 0.0001% level.
nn
¼ significant at 0.01% level. ¼significant at 0.001% level. ¼significant at 0.0001% level
nnn
nn
Fig. 7. Total carotenoid content in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper for different experimental periods. Bars represent means (n ¼10, five samples in duplicate) and vertical lines SD. ***¼ Po 0.0001, **¼ Po 0.001 and * ¼P o 0.01 over their respective control.
Fig. 5. Glutathione reductase activity in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper for different experimental periods. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD. ***¼ P o0.0001, ** ¼P o0.001 and * ¼ Po 0.01 over their respective control.
to 25.88% (20 days). The percent increase for other time intervals was 14.1, 16.99 and 22.25 for 48 h, 96 h and 10 days exposure, respectively. However, long-term exposure for 30 days showed a significant (Po0.0001) decrease of about 19.68% in the GR activity of the exposed PL over its respective control. Fig. 6 and Table 3 shows that when tested against time, the exposed samples showed a significant (F: 66.4; Po0.0001) and gradual decrease in the enzyme activity from 10 days onwards with a complete reversal of the activity at 30 days exposure similar to GPx activity.
3.4. Total carotenoid content The results of total carotenoid content in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper are shown in Fig. 7 for both short- and long-term exposures along with their respective controls. The data clearly show an increase in the total carotenoid content of the exposed PL compared to their respective controls for all time intervals. Though the increase was not significant for 24 and 48 h exposure, thereafter a significant (F: 409.6; Po0.0001) increase was noticed from 96 h onwards till 30 days of sublethal copper exposure over their respective controls. The percent increase in the carotenoid content recorded for the exposed PL over its respective control was 1.61, 3.04, 10.6, 23.35, 45.44 and 46.83 for 24, 48, 96 h, 10, 20 and 30 days of sublethal copper exposure, respectively. There was a gradual
R.V. Paila et al. / Ecotoxicology and Environmental Safety 75 (2012) 127–133
Total Carotenoids - Exposed 0.20 (µg/gm wet wt.)
Total Carotenoids
0.25
0.15 0.10 0.05 0.00 24hr
48hr
96hr
10d
20d
30d
Exposure period Fig. 8. Time-dependent changes in the total carotenoid content of the exposed Penaeus indicus PL. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD.
Table 4 Levels of significance for total carotenoid content of the exposed Penaeus indicus PL tested over time. Total carotenoids Exposure period
24 h
48 h
96 h
10 days
20 days
30 days
24 h 48 h 96 h 10 days 20 days 30 days
– ns
ns –
nn
nnn
nnn
nnn
n
nnn
nnn
nn
nn
n
nnn
nnn
nnn
nnn
nnn
nn
– ns ns ns
ns – ns ns
ns ns – ns
ns ns ns –
Note: ns¼not significant. n nn
¼significant at 0.01% level. ¼ significant at 0.001% level. ¼ significant at 0.0001% level.
nnn
decrease in total carotenoid content from 15.93 mg/g wet weight at 24 h to 3.03 mg/g at 30 days in the control PL. Similarly, a gradual decrease from 16.19 mg/g wet weight at 24 h to 4.45 mg/g wet weight at 30 days in the exposed PL was noticed. When tested over time, a gradual, time-dependent and significant (F: 13.49; Po0.0001) increase in the carotenoid content of the exposed PL was noticed from 48 h onwards (Fig. 8 and Table 4).
4. Discussion The decrease in GSTase activity of the exposed Penaeus indicus PL upon long-term exposure (Figs. 1 and 2) could be due to the increased oxidative stress as evidenced by higher levels of lipid peroxidation products (LPP) in the exposed PL over time, which was reported in our previous study (Paila and Yallapragada, in press). Different hypotheses explain the inhibition of GSTase activity. First, biotransformation steps by cytochrome P450 enzymes may produce a cocktail of different metabolites competing with GSTase substrates for the active sites on the GSTase enzyme (Egaas et al., 1999). Second, Gallangher and Sheehy (2000) postulated that lower activities may be caused by a decrease in the synthesis of GSTase proteins at a molecular level. Therefore, decreased activity of the detoxification enzyme GSTase in P. indicus PL exposed to sublethal copper explains its failure to detoxify under chronic exposure thus leading to higher level of LPP in the exposed PL. This test result is in good agreement with Li et al. (2008) who reported significant decrease in GSTase activity in giant freshwater prawn Macrobrachium rosenbergii
131
exposed to 0.01 mg/L of copper and suggested that GSTase is a suitable biomarker of environmental copper stress in M. rosenbergii. Effect of copper on GSTase activity in crustaceans has been reported by some investigators mostly in crabs like Oziotelphusa senex senex (Reddy, 1999); Scylla serrata (Reddy, 1997) and reported higher GSTase activity suggesting activation of the detoxification enzyme under conditions of stress. However, our results suggest complete failure of the detoxification enzyme in P. indicus PL upon long-term exposure to 0.164 mg/L of copper and therefore, GSTase can be used as a suitable biomarker of environmental copper stress in P. indicus PL. CAT and GPx have complementary roles in H2O2 detoxication (Barata et al., 2005). The initial significant increase in GPx activity of the exposed PL (Fig. 3) suggests the development of an adaptive response for protection against oxidative stress for short-term exposures only. Pan and Zhang (2006) have similarly reported an initial increase and then a decrease in GPx activity in gills and hepatopancreas of cadmium exposed marine crab Charybdis japonica. Our previous study (Paila and Yallapragada, in press) showed gradual increasing levels of LPP from 24 h to 30 days suggesting gradual increase in oxidative stress and a significant increase in catalase activity of the exposed PL from 96 h till 30 days whereas 24 and 48 h showed insignificant (P40.05) change. Therefore, it would be important to mention here that, our results are in good agreement with Yan and Harding (1997) who suggested that when cellular H2O2 is less, GPx is more functional than catalase in the removal of H2O2 and GPx is reported to protect cells against low level oxidant stress. Hence, GPx activity was significantly higher from 24 h till 96 h when cellular H2O2 was less and thereafter CAT becomes functional from 96 h till 30 days when cellular H2O2 is more. Thus, the increase in GPx activity of the exposed PL over its respective control was much lower on longer exposures i.e. 16.18% and 6.49% for 10 and 20 days of exposure, respectively, whereas a decrease by 20.47% on 30 days of exposure (Figs. 3 and 4). Geret et al. (2003) has similarly reported increased catalase and decreased GPx activity in the gills of clam Ruditapes decussates obtained from sites of metal pollution. Monteiro et al. (2006) pointed out that GPx activity can decrease by negative feedback either from excess of substrate or damage induced by oxidative modification. Ballesteros et al. (2009) stated that reduced GPx activity in an exposed tissue could indicate that its antioxidant capacity was exceeded by the amount of hydroperoxide products. Thus, inhibition of GPx activity might reflect its reduced capacity to scavenge H2O2 and lipid hydroperoxides produced in the tissues and therefore a possible failure of the antioxidant system upon longterm copper exposures. The role of GPx as sensitive biomarkers for monitoring environmental pollution in invertebrates has been suggested by Cossu et al. (2000). The role of GR is to maintain the cytosolic concentration of GSH in the cells at the expense of NADPH. GSH is substrate for GSTase and cofactor for GPx (van der Oost et al., 2003). The increase in the GR activity from 12.93% (24 h) to 25.88% (20 days) in the exposed PL over their respective controls indicate the possible development of an adaptive response of the exposed animals gradually, to overcome the increasing stress against copper toxicity. According to Zhang et al. (2004), the inhibition of GR activity could be due to the change in the availability of NADPH in the cell. Decreased activity of GPx on 30 days exposure could have therefore triggered a decrease in GR activity too at the same exposure period, possibly to maintain the cytosolic concentration of GSH as also mentioned by Ballesteros et al. (2009). Hence, when tested against time, a gradual decrease in the enzyme activity was noticed from 10 days onwards, with a complete reversal of the activity at 30 days exposure similar to GPx activity (Fig. 6). Therefore, there has been almost an equal
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reduction by 19.68% in the GR activity and by 20.47% in GPx activity of the exposed PL over their respective controls. The aim of the present investigation was also to evaluate the role of carotenoids in combating heavy metal stress in Penaeus indicus PL when exposed to 164.1 mg/L of copper for a period of 30 days. A gradual, time-dependent and significant (F: 409.6; Po0.0001) increase in the total carotenoid content of the exposed PL from 10.6% (96 h) to 46.83% (30 days) over their respective controls (Fig. 7) can be attributed to three important factors as per the existing literature. Firstly, carotenoids are supposed to play an important role during hypoxic conditions (Karnaukhov, 1971, 1973), secondly the role of carotenoid as an antioxidant under conditions of oxidative stress (Miki et al., 1994) and finally, the role of carotenoids as a biomarker of environmental pollution (Karnaukhov et al., 1977). In our previous investigation (Paila and Yallapragada, 2010), we have reported a gradual and time-dependent significant (Po0.05) decrease in the oxygen consumption rates of the exposed PL ranging from 13.33% (24 h) to 34.25% (30 days) of sublethal copper exposure compared to their respective controls. It is therefore suggested that the exposed PL were under extreme hypoxic conditions for all the exposure periods. Therefore, in the present study, a significant (Po0.0001) increase in the total carotenoid content of the exposed PL over their respective controls from 96 h onwards could be an adaptive measure to give protection to cells during hypoxic conditions as suggested by Karnaukhov (1973). Karnaukhov (1971) reported that during oxidative metabolism in the animal cells, carotenoids provide necessary energy requirements to the cells under the conditions of acute low rate of oxygen penetration into the tissues and can form the system of oxygen reserve in special intracellular organoid, which was named as carotenoxysome. Proposed role of carotenoids as lipids antioxidants, which are able to protect against oxidation and other destructive process mediated by free radicals was stated by Miki et al. (1994). Astaxanthin is a strong inhibitor of lipid peroxidation and has been proposed as ‘‘super vitamin E’’ by Miki (1991). Shimidzu et al. (1996) reported that carotenoids are singlet oxygen quenchers in marine organisms. Astaxanthin has been reported by Chien et al. (2003) to increase antioxidant defense capability in P. monodon. The role of marine carotenoids as antioxidant defense has been reviewed by Miyashita (2009). Therefore, it can be suggested that the exposed P. indicus PL in our present investigation might have contributed to the rise in carotenoid levels to protect the cell membrane from oxidative damage against metalinduced toxicity. Karnaukhov et al. (1977) investigated and stated that the higher carotenoid concentration in the molluscan bodies can be used as a biomarker, which was probably because of the animal’s adaptation to the condition of higher degree of environmental pollution. We can therefore conclude from our findings that the significant increase in the total carotenoid content of the exposed P. indicus PL is an adaptation of the animal to combat metalinduced lipid peroxidation, increased oxidative stress and decreased oxygen consumption rate. Carotenoids can therefore be used as a biomarker of environmental pollution caused by heavy metal copper.
5. Conclusion The present investigation therefore, proves that, though copper is an essential element, even sublethal doses can induce significantly high levels of oxidative stress in P. indicus PL, which cannot be overcome by glutathione system upon chronic exposures. Enzymes of glutathione system can therefore serve as
biomarkers for copper toxicity. Carotenoids serve to protect P. indicus PL from oxidative damage against metal-induced toxicity and can be used as a biomarker and postlarvae of P. indicus as a bioindicator of copper pollution. Therefore, longterm copper exposures along coastal ecosystems should receive great attention and further investigations should be carried out.
Acknowledgments This work was supported by a fellowship grant from Council for Scientific and Industrial Research (CSIR), New Delhi, India. The authors are grateful to Department of Zoology, Andhra University for providing the research facilities. References 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. Barata, C., Lekumberri, I., Vila-Escale, M., Prat, N., Porte, C., 2005. Trace metal concentration, antioxidant enzyme activities and susceptibility to oxidative stress in the tricoptera larvae Hydropsyche exocellata from the Llobregat river basin (NE Spain). Aquat. Toxicol. 74, 3–19. Bremner, I., 1998. Manifestations of copper. Am. J. Clin. Nutr. 36, 551–561. Calberg, I., Mannervik, B., 1985. Glutathione reductase. Methods Enzymol. 113, 484–490. Chien, Y.H., Pan, C.H., Hunter, B., 2003. The resistance to physical stresses by Penaeus monodon juveniles fed diets supplemented with astaxanthin. Aquaculture 216, 177–191. Cnubben, N.H.P., Rietjens, I.M.C.M., Wortelboer, H., van Zanden, J., van Bladeren, P.J., 2001. The interplay of glutathione-related processes in antioxidant defense. Environ. Toxicol. Pharmacol. 10, 41–152. Cossu, C., Doyotte, A., Babut, M., Exinger, A., Vasseur, P., 2000. Antioxidant biomarkers in freshwater bivalves, Unio tumidus, in response to different contamination profiles of aquatic sediments. Ecotoxicol. Environ. Saf. 45, 106–121. Davies, K.J., 1995. Oxidative stress: the paradox of aerobic life. Biochem. Soc. Symp. 61, 1–31. Egaas, E., Sandvik, M., Fjeld, E., Kallqvist, T., Goksoyr, A., Svensen, A., 1999. Some effects of the fungicide propiconazole on cytochrome P450 and glutathioneStransferase in brown trout Salmo trutta. Comp. Biochem. Physiol. 122, 337–344. Flohe, L., Gunzler, W.A., 1984. Assays of glutathione peroxidase. Methods Enzymol. 105, 114–121. Gallangher, E.P., Sheehy, K.M., 2000. Altered glutathione-S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake. Mar. Environ. Res. 50, 399–403. Geret, F., Serafim, A., Bebianno, M.J., 2003. Antioxidant enzyme activities, metallothioneins and lipid peroxidation as biomarkers in Ruditapes decussatus? Ecotoxicology 12, 417–426. Habig, W.H., Pabat, M.J., Jakoby, W.B., 1974. Glutathione-S-transferase: the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7193. Karnaukhov, V.N., 1971. Role of carotenoids in intracellular oxygen deposition. Dokl. Akad. Nauk SSSR 196, 1221–1224. Karnaukhov, V.N., 1973. The role of carotenoids in the formation of lipofuscin and the adaptation of animal cells to oxygen insufficiency. Tsitologia 15 (5), 538–542. Karnaukhov, V.N., Milovidova, N.Y., Kargopolova, I.N., 1977. On a role of carotenoids in tolerance of sea molluscs to environmental pollution. Comp. Biochem. Physiol. 56A, 189–193. Kawanishi, S., Inoe, S., Yamamoto, K., 1989. Hydroxyl radical and singlet oxygen production and DNA damage induced by carcinogenic metal compounds and hydrogen peroxide. Biol. Trace Elem. Res. 21, 367–372. Li, N., Zhao, Y., Yang, J., 2008. Effects of water-borne copper on digestive and metabolic enzymes of the giant freshwater prawn, Macrobrachium rosenbergii. Arch. Environ. Contam. Toxicol. 55, 86–93. Lowry, O.M., Rosebrough, N.J., Farr, A.C., Landall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 195, 265–275. Mannervik, B., 1987. The enzymes of glutathione metabolism: an overview. Biochem. Soc. Trans. 15, 717–718. Mason, A.Z., Jenkins, K.D., 1996. Metal detoxification in aquatic organisms. in: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. IUPAC Press, London, pp. 479–608. Miki, W., 1991. Biological functions and activities of animal carotenoids. Pure Appl. Chem. 63, 141–146. Miki, W., Otaki, N., Shimidzu, N., Yokoyama, A., 1994. Carotenoids as free radical scavengers in marine animals. J. Mar. Biotechnol. 2, 35–37. Miyashita, K., 2009. Function of marine carotenoids. In: Yoshikawa, T., (Ed.), Food Factors for Health Promotion, vol. 61. Forum Nutrition, Basel, Karger, pp. 136–146.
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Monteiro, D.A., Almeida, J.A., Rantin, F.T., Kalinin, A.L., 2006. Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion). Comp. Biochem. Physiol. 143C, 141–149. Nordberg, J., Arner, E.S.J., 2001. Reactive oxygen species, antioxidants and the mammalian thioredoxin system. Free Radical Biol. Med. 31 (11), 1287–1312. Paila, R.V., Yallapragada, P.R., 2010. Bioaccumulation and toxic effects of copper on growth, oxygen consumption by the postlarvae of Penaeus indicus. Chem. Ecol. 26, 209–221. Paila, R.V., Yallapragada, P.R. Antioxidant responses and lipid peroxidation of Penaeus indicus postlarvae subjected to sublethal copper exposure. Crustaceana, in press. doi:10.1163/156854011X587496. Pan, L., Zhang, H., 2006. Metallothionein, antioxidant enzymes and DNA strand breaks as biomarkers of Cd exposure in a marine crab, Charybdis japonica. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 144, 67–75. Reddy, P.S., 1997. Modulations in antioxidant enzymes in the gill and hepatopancreas of the edible crab Scylla serrata during exposure to cadmium and copper. Fresenius Environ. Bull. 6, 589–597. Reddy, P.S., 1999. Modulations in antioxidant enzymes in the hepatopancreas and gill of the freshwater crab Oziotelphysa senex senex during exposure to cadmium and copper. Fresenius Environ. Bull. 8, 445–452.
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Response of glutathione system and carotenoids to sublethal copper in the postlarvae of Penaeus indicus Rupa Vani Paila n, Prabhakara Rao Yallapragada, Samuel David Raj Thatipaka Division of Physiology and Toxicology, Department of Zoology, Andhra University, Visakhapatnam 530003, India
a r t i c l e i n f o
abstract
Article history: Received 5 June 2011 Received in revised form 22 August 2011 Accepted 29 August 2011 Available online 25 September 2011
The objective of this study is to determine the effect of sublethal copper on the glutathione system and carotenoids of Penaeus indicus postlarvae (PL) when subjected to short- and long-term exposure in the laboratory. The PL were exposed to 0.1641 ppm (sublethal) copper for a period of 30 days with sampling intervals of 24, 48, 96 h and 10, 20, 30 days. Variations in the activity of Glutathione reductase (GR), Glutathione peroxidase (GPx) and Glutathione-S-transferase (GSTase) were measured as biomarkers of toxicity. A significant (P o0.05) increase in the GR and GPx activity of the exposed PL till 20 days of exposure and thereafter a significant decrease indicates susceptibility of the PL to oxidative stress upon chronic exposure. Similarly, a significant decrease in GSTase activity during long-term exposures in the exposed PL reflects possible failure of the detoxification system. A possible role of carotenoids to combat oxidative stress against copper toxicity is also discussed. & 2011 Elsevier Inc. All rights reserved.
Keywords: Glutathione reductase Glutathione peroxidase Glutathione-S-transferase Carotenoids Copper toxicity Penaeus indicus Postlarvae
1. Introduction While the vast majority of complex life on Earth requires oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by producing reactive oxygen species (ROS) (Davies, 1995). Consequently, organisms contain a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids (Sies, 1997; Vertuani et al., 2004). In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell (Davies, 1995; Sies, 1997). However, since ROS do have useful functions in cells, such as redox signaling, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level (Rhee, 2006). When the production of ROS overwhelms the antioxidant system, an imbalance between the formation and removal of ROS occurs thereby rendering the cells more susceptible to oxidative damage and results in simultaneous decline in the antioxidant defense mechanism. This leads to damage of cellular organelles and changes in certain enzymatic activities, cumulating to a situation known as oxidative stress n
Corresponding author. Fax: þ91 891 2525611/2755324. E-mail addresses: [email protected] (R.V. Paila), [email protected] (P.R. Yallapragada), [email protected] (S.D.R. Thatipaka). 0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.08.023
(Zhang et al., 2008). The relevance of glutathione system in defense against oxidative stress is being increasingly studied, because of its potential utility to provide biochemical biomarkers that could be used in environmental monitoring system. The key enzymes for the detoxication of ROS in all organisms are superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) as well as glutathione-S-transferase (GSTase). Glutathione-S-transferase (GSTase) catalyzes the conjugation of GSH with a variety of electrophilic metabolites. This enzyme participates in defense against oxidative stress by detoxifying endogenous harmful compounds like hydroxyalkenals and base propenals or DNA hydroperoxides and electrophilic xenobiotics (Cnubben et al., 2001). Thus, induction of GSTase is considered beneficial to handle environmental stress (van der Oost et al., 2003). Glutathione peroxidase (GPx) with peroxidase activity protects the organism from oxidative damage by reducing lipid hydroperoxides to their corresponding alcohols. It also reduces free hydrogen peroxide to water and thereby catalyzes the conversion of reduced glutathione (GSH) to an oxidized form (GSSG) (Nordberg and Arner, 2001). Glutathione reductase (GR) reduces glutathione disulfide (GSSG) to GSH, which is an important cellular antioxidant (Mannervik, 1987). For every mole of oxidized glutathione (GSSG) one mole of NADPH is required to reduce GSSG to GSH from which, two reduced GSH molecules are gained that act as antioxidants, scavenging ROS in the cell.
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Carotenoids are a class of natural, fat-soluble, organic pigments belonging to the category of tetraterpenoids and produces characteristic yellow, orange or red colors. They play a potential protective role as biological antioxidants by two general mechanisms: (i) quenching of singlet oxygen and dissipating the energy as heat, and (ii) scavenging of radicals to prevent or terminate chain reactions (Miki, 1991). Metal toxicity results from nonspecific metal binding, which can inactivate important regulatory enzymes by displacing essential metal ions from catalytic sites, by altering catalytic cysteine groups or by allosterically altering the functional conformation of proteins (Mason and Jenkins, 1996). The basis for copper cytotoxicity is that both cupric and cuprous ions can participate in oxidation and reduction reactions i.e. capable of redox cycling. In the presence of superoxide (O2 ) or reducing agents such as ascorbic acid or GSH, Cu2 þ can be reduced to Cu þ , which is capable of catalyzing the formation of hydroxyl radicals (OH) from hydrogen peroxide (H2O2) via the Haber–Weiss reaction (Bremner, 1998). Thus, copper is capable of producing ROS and inducing DNA strand breaks and oxidation of bases (Kawanishi et al., 1989). The white shrimp Penaeus indicus widely distributed along the east coast of India is well known for its palatability, high market value and is a commercially important crustacean for the Indian fishing industry. The present investigation was intended to determine the effect of short- and long-term sublethal copper exposure on glutathione system of Penaeus indicus postlarvae (PL) as well as to evaluate the role of carotenoids against copper toxicity.
2. Materials and methods This study was conducted in accordance with the institutional (Andhra University) guidelines for animal experiments. 2.1. Chemicals All chemicals and reagents were obtained from Sigma Chemical Co. (St Louis, Missouri, USA). 2.2. Animals Equal-sized PL (16–23 mm) of Penaeus indicus were obtained from the wild catch of Gosthani estuary located near Bheemunipatnam (17.51N 83.31E), 30 km from Visakhapatnam in Andhra Pradesh, on the east coast of India. The PL were carefully transported to laboratory and were acclimatized to laboratory conditions for three days in 50 L tanks containing seawater (salinity 10%, temperature 29 71 1C, dissolved oxygen 8.0 and pH 7.8). During this period, constant aeration was provided and PL were fed with commercial diet, having no residual traces of copper (EPAC-XL, INVE, Belgium) twice a day. Excess feed was removed daily by siphoning and ambient water in the tank was renewed daily. 2.3. Sublethal exposure and sampling The 96 h LC50 value for same size group previously obtained by Paila and Yallapragada (2010) was used for the present study (96 h LC50 for P. indicus PL was 0.8204 mg/L of copper toxicity). Therefore, a concentration of 1/5 of 96 h LC50 value, i.e., 164.1 mg/L was considered as sublethal (Paila and Yallapragada, 2010) and further experiments were carried out on exposure to this nominal concentration. A stock solution of copper (10 g/L) was prepared from CuSO4 5H2O (AR) in distilled water. An appropriate volume of the freshly prepared stock solution was added to the seawater to obtain the final desired concentration. The PL (about 600 in number) were then exposed to this toxicant for a period of 30 days in plastic tanks of 50 L capacity. A parallel control was maintained without the metal toxicant. During the course of the study, salinity (10%), temperature (297 1 1C), dissolved oxygen (8.0) and pH (7.8) were regularly measured and adjusted using a portable auto analyzer (WTW Multiline P4, Merck). Seawater was renewed every 24 h and copper concentration was re-established in the exposure tanks after the daily water change. Excess feed was removed daily by siphoning and the tanks were washed daily. Feeding was stopped six hours prior to sampling. Whole PL
samples were collected both from control and exposed tanks at six different time intervals i.e., 24, 48 and 96 h (short-term), 10, 20 and 30 days (long-term) and were kept at 70 1C until analysis, which was carried out within one week. Five replicates were maintained at each interval.
2.4. Sample preparation After the exposure periods, the control and exposed PL were sacrificed and soft tissues were isolated. Whole body of ten individuals for one sample was taken for each biochemical assay. A 10% homogenate for each sample was prepared in Tris HCl buffer 0.026 M (pH 8.0) for GSTase activity. For GPx and GR assays, the homogenate (10%) was similarly made in potassium phosphate buffer of 0.05 M (pH 7.0) and 0.5 M (pH 7.0) concentration, respectively. The homogenate was centrifuged differentially at 4000 rpm for 15 min at 4 1C for GSTase activity and at 16000g for 45 min at 4 1C for GPx and GR activity. The supernatant was further centrifuged at 16,000 rpm for 60 min at 4 1C for GSTase activity and at 30,000 rpm for 1 h at 4 1C for GPx and GR activity. The resultant supernatant was then used as an enzyme source.
2.5. Enzymatic assays 2.5.1. Estimation of glutathione-S-transferase activity Glutathione-S-tranferase (GSTase) activity was estimated by following the method of Habig et al. (1974). The reaction mixture was prepared by adding 2.4 ml of 0.026 M potassium phosphate buffer, 100 ml of 0.33 M 1-chloro-2,4-dinitrobenzene (CDNB), 400 ml of distilled water. An amount of 100 ml of homogenate containing 50 mg of protein was taken in the reaction mixture followed by addition of 100 ml 0.33 M of GSH. The change in absorbance was monitored at 340 nm for one minute in spectrophotometer. One unit of activity is equal to micromole of thioester formed/mg protein/min.
2.5.2. Estimation of glutathione peroxidase activity Glutathione peroxidase (GPx) was determined by following the modified version of Flohe and Gunzler (1984). The reaction mixture was prepared by adding 600 ml of potassium phosphate buffer (0.05 M, pH 7.0, 0.1 mM EDTA), 100 ml of 10 mM GSH (reduced form), 100 ml of 1.5 mM NADPH and 100 ml of GR (0.24 units). An amount of 100 ml of whole PL homogenate supernatant fraction was added to the reaction mixture and incubated at 37 1C for 10 min. To this, 50 ml of 12 mM t-butyl hydroperoxide was added. The change in the absorbance was measured at 340 nm for one minute. One unit of activity is equal to millimole of NADPH oxidized/mg protein/min.
2.5.3. Estimation of glutathione reductase activity Glutathione reductase (GR) was determined by following the method of Calberg and Mannervik (1985). An amount of 50 ml of NADPH (2 mM) in 10 mM Tris HCl buffer (pH 7.0) was added in a cuvette containing 50 ml of GSSG (20 mM) in potassium phosphate buffer (0.5 M, pH 7.0, 0.2 mM EDTA) and 850 ml of potassium phosphate buffer. An amount of 50 ml of whole PL homogenate supernatant was added to the NADPH-GSSG buffered solution. The absorbance was read at 340 nm using spectrophotometer for minute. One unit of activity is equal to millimole of NADPH oxidized/mg protein/min. Total soluble protein contents for the above enzymatic activities were determined according to Lowry et al. (1951), using bovine serum albumin (BSA) as a standard.
2.5.4. Estimation of total carotenoids The extraction of total carotenoid content from whole PL samples was estimated by the method of Susan and Damodaran (1997). Uniform-sized live PL were collected from both the control and exposed tanks at each exposure period, thoroughly washed with double distilled water, blotted on filter paper and their wet weights were taken. Total carotenoids were extracted by homogenizing the PL with acetone in glass homogenizer. The colored supernatant was collected and fresh acetone was added again to the pellet. Homogenization was continued until the acetone extract turned colorless. This acetone extract was then filtered under reduced pressure in a scintered glass funnel. The total volume of the extract was measured and its optical density was recorded at 455 nm using spectrophotometer against an acetone blank. The concentration of total carotenoids was calculated by using the following formula (Susan and Damodaran, 1997): Total carotenoid concentration ðmg=100 mg tissueÞ ¼ 0:4 DV=W where D ¼ optical density of the extract measured at the wavelength of carotenoid absorption maxima i.e. 455 nm, V ¼total volume of carotenoid extract (ml), W¼ total wet weight of the tissue (mg) from which the carotenoids were extracted. The results were expressed as mg/g wet weight tissue.
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2.6. Statistical analysis All assays were repeated five times. The data were analyzed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test, using Graphpad Prism 5 software package to study the significant differences between the means (n¼10, five samples in duplicate) of control and exposed samples for the six time intervals as well as to study changes over time.
3. Results 3.1. Glutathione-S-transferase activity The result of GSTase activity in the PL of P. indicus exposed to sublethal copper along with their respective controls for different time intervals is presented in Fig. 1. The figure shows that though there is an increase in the GSTase activity in the exposed PL over its respective control during short-term exposure i.e. from 24 h to 96 h, the increase is not significant at 5% level. However, longterm exposure to sublethal copper has resulted in a significant (F: 159.8; Po0.0001) decrease in the GSTase activity of the exposed PL over its respective control. The decrease in the activity was 11.62%, 30.62% and 33.49% for 10, 20 and 30 days, respectively. Significant (F: 412.7; P o0.0001) time-dependent decrease in GSTase activity of the exposed PL was noticed from 10 days onwards when compared to short-term exposures as shown in Fig. 2 and Table 1.
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Table 1 Levels of significance for GSTase activity of the exposed Penaeus indicus PL tested over time. GSTase activity Exposure period 24 h 48 h 96 h 10 days 20 days 30 days
24 h
96 h
10 days
20 days
30 days
nnn
nnn
nnn
nnn
nnn
– ns ns
ns – ns
ns ns –
nnn
nnn
nnn
nnn
nnn
nnn
nnn
-
nnn
nnn
nnn
nnn
nnn
nnn
–
nnn
nnn
nnn
nnn
nnn
nnn
Note: ns¼ not significant. 0.001% level. nnn
48 h
n
¼ significant at 0.01% level.
– nn
¼ significant at
¼significant at 0.0001% level.
3.2. Glutathione peroxidase activity Levels of GPx activity in the PL of P. indicus exposed to sublethal copper for different periods of exposure along with
Fig. 3. Glutathione peroxidase activity in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper along with their respective controls for different experimental periods. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD. ***¼ Po 0.0001 over its respective control.
Fig. 1. Glutathione-S-transferase activity in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper along with their respective controls for different experimental periods. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD. **¼ Po 0.001 and *** ¼P o0.0001 over their respective control.
their respective controls are presented in Fig. 3. The results show a significant (F: 47.14; Po0.0001) increase in the GPx activity in the exposed PL over its respective control only for short-term exposures i.e 24 h to 96 h. Though long-term exposures showed an increase in the levels of GPx activity of the exposed PL over their respective controls upon 10 and 20 days of exposure, the increase was not significant. However, long-term exposure for 30 days showed a significant (Po0.0001) decrease in the GPx activity in the exposed PL over its respective control. The percent increase in the GPx activity for different time intervals was 29.27%, 17.73%, 37.02%, 16.18% and 6.49% for 24 h, 48 h, 96 h, 10 days and 20 days, respectively. However, 30 days exposure showed 20.47% decrease in GPx activity in the exposed PL over its respective control. Fig. 4 and Table 2 shows a gradual, timedependent and significant (F: 71.56; P o0.0001) decrease in GPx activity of the exposed PL from 96 h onwards and a complete reversal of the activity was observed on 30 days of exposure. 3.3. Glutathione reductase activity
Fig. 2. Time-dependent changes in the GSTase activity of the exposed Penaeus indicus PL. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD.
Fig. 5 indicates GR activity in P. indicus PL subjected to sublethal copper exposures for different time intervals along with their respective controls. Though, an increase in the GR activity of the exposed PL over its respective control was noticed from 24 h till 20 days of exposure, a significant (F: 17.64; Po0.0001) increase in the activity was noticed only from 10 days onwards. There was a gradual increase in the GR activity from 12.93% (24 h)
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GR-Exposed
GR activity
(nmoles of NADPH oxidised /mg protein/min.)
1.5 1.0 0.5 0.0 -0.5
24hr
48hr
96hr 10d 20d Exposure period
30d
-1.0 -1.5
Fig. 4. Time-dependent changes in the GPx activity of the exposed Penaeus indicus PL. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD.
Table 2 Levels of significance for GPx activity of the exposed Penaeus indicus PL tested over time. GPx activity Exposure period 24 h 48 h 96 h 10 days 20 days 30 days
24 h
48 h
96 h
10 days
20 days
30 days
nn
n
nnn
nnn
nnn
ns
nnn
nnn
nnn
–
nnn
nnn
nnn
n
ns
nnn
nnn
nnn
nnn
– ns
ns -
nnn
nnn
nnn
nnn
nnn
nnn
–
– ns
ns –
nn
Fig. 6. Time-dependent changes in the GR activity of the exposed Penaeus indicus PL. Bars represent means (n ¼10, five samples in duplicate) and vertical lines SD.
Table 3 Levels of significance for GR activity of the exposed Penaeus indicus PL tested over time. GR activity Exposure period
24 h
48 h
96 h
10 days
20 days
30 days
24 hrs 48 h 96 h 10 days 20 days 30 days
– ns ns
ns – ns
ns ns –
nnn
n
nnn
nn
nnn
nnn
nn
n
n
ns
ns
– ns
ns ns ns –
nnn
nnn
nnn
nnn
nnn
–
n
nnn nnn nnn
nnn
Note: ns ¼not significant. n
Note: ns¼not significant. n
¼ significant at 0.01% level. ¼ significant at 0.001% level. nnn ¼significant at 0.0001% level.
nn
¼ significant at 0.01% level. ¼significant at 0.001% level. ¼significant at 0.0001% level
nnn
nn
Fig. 7. Total carotenoid content in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper for different experimental periods. Bars represent means (n ¼10, five samples in duplicate) and vertical lines SD. ***¼ Po 0.0001, **¼ Po 0.001 and * ¼P o 0.01 over their respective control.
Fig. 5. Glutathione reductase activity in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper for different experimental periods. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD. ***¼ P o0.0001, ** ¼P o0.001 and * ¼ Po 0.01 over their respective control.
to 25.88% (20 days). The percent increase for other time intervals was 14.1, 16.99 and 22.25 for 48 h, 96 h and 10 days exposure, respectively. However, long-term exposure for 30 days showed a significant (Po0.0001) decrease of about 19.68% in the GR activity of the exposed PL over its respective control. Fig. 6 and Table 3 shows that when tested against time, the exposed samples showed a significant (F: 66.4; Po0.0001) and gradual decrease in the enzyme activity from 10 days onwards with a complete reversal of the activity at 30 days exposure similar to GPx activity.
3.4. Total carotenoid content The results of total carotenoid content in Penaeus indicus PL exposed to sublethal (164.1 mg/L) copper are shown in Fig. 7 for both short- and long-term exposures along with their respective controls. The data clearly show an increase in the total carotenoid content of the exposed PL compared to their respective controls for all time intervals. Though the increase was not significant for 24 and 48 h exposure, thereafter a significant (F: 409.6; Po0.0001) increase was noticed from 96 h onwards till 30 days of sublethal copper exposure over their respective controls. The percent increase in the carotenoid content recorded for the exposed PL over its respective control was 1.61, 3.04, 10.6, 23.35, 45.44 and 46.83 for 24, 48, 96 h, 10, 20 and 30 days of sublethal copper exposure, respectively. There was a gradual
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Total Carotenoids - Exposed 0.20 (µg/gm wet wt.)
Total Carotenoids
0.25
0.15 0.10 0.05 0.00 24hr
48hr
96hr
10d
20d
30d
Exposure period Fig. 8. Time-dependent changes in the total carotenoid content of the exposed Penaeus indicus PL. Bars represent means (n¼ 10, five samples in duplicate) and vertical lines SD.
Table 4 Levels of significance for total carotenoid content of the exposed Penaeus indicus PL tested over time. Total carotenoids Exposure period
24 h
48 h
96 h
10 days
20 days
30 days
24 h 48 h 96 h 10 days 20 days 30 days
– ns
ns –
nn
nnn
nnn
nnn
n
nnn
nnn
nn
nn
n
nnn
nnn
nnn
nnn
nnn
nn
– ns ns ns
ns – ns ns
ns ns – ns
ns ns ns –
Note: ns¼not significant. n nn
¼significant at 0.01% level. ¼ significant at 0.001% level. ¼ significant at 0.0001% level.
nnn
decrease in total carotenoid content from 15.93 mg/g wet weight at 24 h to 3.03 mg/g at 30 days in the control PL. Similarly, a gradual decrease from 16.19 mg/g wet weight at 24 h to 4.45 mg/g wet weight at 30 days in the exposed PL was noticed. When tested over time, a gradual, time-dependent and significant (F: 13.49; Po0.0001) increase in the carotenoid content of the exposed PL was noticed from 48 h onwards (Fig. 8 and Table 4).
4. Discussion The decrease in GSTase activity of the exposed Penaeus indicus PL upon long-term exposure (Figs. 1 and 2) could be due to the increased oxidative stress as evidenced by higher levels of lipid peroxidation products (LPP) in the exposed PL over time, which was reported in our previous study (Paila and Yallapragada, in press). Different hypotheses explain the inhibition of GSTase activity. First, biotransformation steps by cytochrome P450 enzymes may produce a cocktail of different metabolites competing with GSTase substrates for the active sites on the GSTase enzyme (Egaas et al., 1999). Second, Gallangher and Sheehy (2000) postulated that lower activities may be caused by a decrease in the synthesis of GSTase proteins at a molecular level. Therefore, decreased activity of the detoxification enzyme GSTase in P. indicus PL exposed to sublethal copper explains its failure to detoxify under chronic exposure thus leading to higher level of LPP in the exposed PL. This test result is in good agreement with Li et al. (2008) who reported significant decrease in GSTase activity in giant freshwater prawn Macrobrachium rosenbergii
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exposed to 0.01 mg/L of copper and suggested that GSTase is a suitable biomarker of environmental copper stress in M. rosenbergii. Effect of copper on GSTase activity in crustaceans has been reported by some investigators mostly in crabs like Oziotelphusa senex senex (Reddy, 1999); Scylla serrata (Reddy, 1997) and reported higher GSTase activity suggesting activation of the detoxification enzyme under conditions of stress. However, our results suggest complete failure of the detoxification enzyme in P. indicus PL upon long-term exposure to 0.164 mg/L of copper and therefore, GSTase can be used as a suitable biomarker of environmental copper stress in P. indicus PL. CAT and GPx have complementary roles in H2O2 detoxication (Barata et al., 2005). The initial significant increase in GPx activity of the exposed PL (Fig. 3) suggests the development of an adaptive response for protection against oxidative stress for short-term exposures only. Pan and Zhang (2006) have similarly reported an initial increase and then a decrease in GPx activity in gills and hepatopancreas of cadmium exposed marine crab Charybdis japonica. Our previous study (Paila and Yallapragada, in press) showed gradual increasing levels of LPP from 24 h to 30 days suggesting gradual increase in oxidative stress and a significant increase in catalase activity of the exposed PL from 96 h till 30 days whereas 24 and 48 h showed insignificant (P40.05) change. Therefore, it would be important to mention here that, our results are in good agreement with Yan and Harding (1997) who suggested that when cellular H2O2 is less, GPx is more functional than catalase in the removal of H2O2 and GPx is reported to protect cells against low level oxidant stress. Hence, GPx activity was significantly higher from 24 h till 96 h when cellular H2O2 was less and thereafter CAT becomes functional from 96 h till 30 days when cellular H2O2 is more. Thus, the increase in GPx activity of the exposed PL over its respective control was much lower on longer exposures i.e. 16.18% and 6.49% for 10 and 20 days of exposure, respectively, whereas a decrease by 20.47% on 30 days of exposure (Figs. 3 and 4). Geret et al. (2003) has similarly reported increased catalase and decreased GPx activity in the gills of clam Ruditapes decussates obtained from sites of metal pollution. Monteiro et al. (2006) pointed out that GPx activity can decrease by negative feedback either from excess of substrate or damage induced by oxidative modification. Ballesteros et al. (2009) stated that reduced GPx activity in an exposed tissue could indicate that its antioxidant capacity was exceeded by the amount of hydroperoxide products. Thus, inhibition of GPx activity might reflect its reduced capacity to scavenge H2O2 and lipid hydroperoxides produced in the tissues and therefore a possible failure of the antioxidant system upon longterm copper exposures. The role of GPx as sensitive biomarkers for monitoring environmental pollution in invertebrates has been suggested by Cossu et al. (2000). The role of GR is to maintain the cytosolic concentration of GSH in the cells at the expense of NADPH. GSH is substrate for GSTase and cofactor for GPx (van der Oost et al., 2003). The increase in the GR activity from 12.93% (24 h) to 25.88% (20 days) in the exposed PL over their respective controls indicate the possible development of an adaptive response of the exposed animals gradually, to overcome the increasing stress against copper toxicity. According to Zhang et al. (2004), the inhibition of GR activity could be due to the change in the availability of NADPH in the cell. Decreased activity of GPx on 30 days exposure could have therefore triggered a decrease in GR activity too at the same exposure period, possibly to maintain the cytosolic concentration of GSH as also mentioned by Ballesteros et al. (2009). Hence, when tested against time, a gradual decrease in the enzyme activity was noticed from 10 days onwards, with a complete reversal of the activity at 30 days exposure similar to GPx activity (Fig. 6). Therefore, there has been almost an equal
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reduction by 19.68% in the GR activity and by 20.47% in GPx activity of the exposed PL over their respective controls. The aim of the present investigation was also to evaluate the role of carotenoids in combating heavy metal stress in Penaeus indicus PL when exposed to 164.1 mg/L of copper for a period of 30 days. A gradual, time-dependent and significant (F: 409.6; Po0.0001) increase in the total carotenoid content of the exposed PL from 10.6% (96 h) to 46.83% (30 days) over their respective controls (Fig. 7) can be attributed to three important factors as per the existing literature. Firstly, carotenoids are supposed to play an important role during hypoxic conditions (Karnaukhov, 1971, 1973), secondly the role of carotenoid as an antioxidant under conditions of oxidative stress (Miki et al., 1994) and finally, the role of carotenoids as a biomarker of environmental pollution (Karnaukhov et al., 1977). In our previous investigation (Paila and Yallapragada, 2010), we have reported a gradual and time-dependent significant (Po0.05) decrease in the oxygen consumption rates of the exposed PL ranging from 13.33% (24 h) to 34.25% (30 days) of sublethal copper exposure compared to their respective controls. It is therefore suggested that the exposed PL were under extreme hypoxic conditions for all the exposure periods. Therefore, in the present study, a significant (Po0.0001) increase in the total carotenoid content of the exposed PL over their respective controls from 96 h onwards could be an adaptive measure to give protection to cells during hypoxic conditions as suggested by Karnaukhov (1973). Karnaukhov (1971) reported that during oxidative metabolism in the animal cells, carotenoids provide necessary energy requirements to the cells under the conditions of acute low rate of oxygen penetration into the tissues and can form the system of oxygen reserve in special intracellular organoid, which was named as carotenoxysome. Proposed role of carotenoids as lipids antioxidants, which are able to protect against oxidation and other destructive process mediated by free radicals was stated by Miki et al. (1994). Astaxanthin is a strong inhibitor of lipid peroxidation and has been proposed as ‘‘super vitamin E’’ by Miki (1991). Shimidzu et al. (1996) reported that carotenoids are singlet oxygen quenchers in marine organisms. Astaxanthin has been reported by Chien et al. (2003) to increase antioxidant defense capability in P. monodon. The role of marine carotenoids as antioxidant defense has been reviewed by Miyashita (2009). Therefore, it can be suggested that the exposed P. indicus PL in our present investigation might have contributed to the rise in carotenoid levels to protect the cell membrane from oxidative damage against metalinduced toxicity. Karnaukhov et al. (1977) investigated and stated that the higher carotenoid concentration in the molluscan bodies can be used as a biomarker, which was probably because of the animal’s adaptation to the condition of higher degree of environmental pollution. We can therefore conclude from our findings that the significant increase in the total carotenoid content of the exposed P. indicus PL is an adaptation of the animal to combat metalinduced lipid peroxidation, increased oxidative stress and decreased oxygen consumption rate. Carotenoids can therefore be used as a biomarker of environmental pollution caused by heavy metal copper.
5. Conclusion The present investigation therefore, proves that, though copper is an essential element, even sublethal doses can induce significantly high levels of oxidative stress in P. indicus PL, which cannot be overcome by glutathione system upon chronic exposures. Enzymes of glutathione system can therefore serve as
biomarkers for copper toxicity. Carotenoids serve to protect P. indicus PL from oxidative damage against metal-induced toxicity and can be used as a biomarker and postlarvae of P. indicus as a bioindicator of copper pollution. Therefore, longterm copper exposures along coastal ecosystems should receive great attention and further investigations should be carried out.
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