CHITRA, K. C. AND SAJITHA, R.

Transcription

1 Journal of Cell and Tissue Research Vol. 14(2) (214) (Available online at ISSN: ; E-ISSN: Original Article EFFECT OF BISPHENOL-A ON THE ANTIOXIDANT DEFENSE SYSTEM AND ITS IMPACT ON THE ACTIVITY OF SUCCINATE DEHYDROGENASE IN THE GILL OF FRESHWATER FISH, OREOCHROMIS MOSSAMBICUS? CHITRA, K. C. AND SAJITHA, R. Department of Zoology, University of Calicut, Malappuram , Kerala E. mail: Cell: Received: March 1, 214; Accepted: March 25, 214 Abstract: The study evaluated that bisphenol A induces oxidative stress to the exposed fishes by time-dependent reduction in the antioxidant enzymes and concomitant increase in the lipid peroxidation in the gill of Oreochromis. A significant reduction in the marker enzyme, succinate dehydrogenase (SDH) was also observed and this could be due to the impairment of aerobic metabolism in bisphenol- A treated fishes. Exposure to bisphenol- A for 2 days caused principal alterations in the gills of treated fishes as destruction of primary and secondary lamellae, upliftment of gill epithelium, hyperplasia and vasodialation in the lamellar epithelium. Thus the present study summarizes that the increased production of oxygen free radicals due to the exposure of bisphenol A inhibited the activities of antioxidant enzymes and succinate dehydrogenase thereby inducing oxidative stress and also destruct the normal architecture of gill in the fish. Key words: Bisphenol A, Oxidative stress, Succinate dehydrogenase INTRODUCTION Bisphenol A (C 15 H 16 ), an organic compound having two phenol functional groups is used to make polycarbonate polymers and epoxy resins, along with other materials employed to make plastics. The final products of bisphenol A has been found in some of the domestic materials such as, adhesives, protective coatings, powder paints, automotive lenses, protective window glazing, building materials, compact disks, optical lenses, thermal paper, paper coatings, as a developer in dyes, encapsulation of electrical and electronic parts [1]. The chemical structure of bisphenol A is similar to the estrogenic compound diethylstilbestrol thus raising concerns in recent years. The United States Food and Drug Administration (FDA) in 21 raised concerns regarding exposure of fetuses, infants and young children to bisphenol A. In September 21, Canada became the first country to declare bisphenol A as a toxic substance. Recently, in India the usage of plastic beverage containers and the release of bisphenol A-containing industrial wastewater have increased comparatively resulting to the human exposure. A large amount of bisphenol A has been liberated from polycarbonate plastics subjected to high temperature [2] or from incompletely polymerized epoxy resins [3]. Indeed, significant quantities of bisphenol A have been detected in liquids from canned vegetables (2 mg/ can) and foods that are exposed to high temperature during autoclaving [4,5] and in the saliva (2-3 mg/ ml) of dental patients fitted with restorative material [6]. In the European Union and Canada the use of bisphenol A is banned even in baby bottles as it has been found in infant liquid formula in concentrations varying from.48 to 11 ng/ g and rarely found in infant powder formula [7]. Bisphenol A has also been 4219

2 J. Cell Tissue Research reported to be found in both the placenta and the amniotic fluid of pregnant mice. A study has detected an average of 2.8 ng/ ml bisphenol A in the blood of 9 out of the 1 umbilical cords tested. After the baby is born, maternal exposure can continue to affect the infant through transfer of bisphenol A to the infant via breast milk [8]. Such environmental contaminants are generally known to cause an increase in peroxidative processes within cells, causing oxidative stress [9]. Aquatic organisms have developed a comprehensive antioxidant defense system, comprising both molecular and enzymatic defenses, against the dangers of oxygen radicals, thereby preventing excess oxidation and damage. There are several evidences reporting the toxicity effect of bisphenol A on freshwater fishes, but limited information is available on the effect of this compound on the antioxidant system of Oreochromis mossambicus. The mitochondrial respiratory stress marker enzyme succinate dehydrogenase is widely used as an indicator of osmoregulatory activity. The activity of the enzyme was also evaluated in order to determine the impact of bisphenol A on aerobic metabolism. Based on the above context the present work has been carried out to study the effect of bisphenol A on the reactive oxygen species generation in the gill of the fresh water fish, Oreochromis mossambicus. MATERIALS AND METHODS Collection and maintenance of animal: Fresh water fish, Oreochromis mossambicus weighing 1 ± 2 g and length 7.5 ± 1 cm were collected from a fish farm, Kaloos Aquarium, Kotakkal, Malappuram District, Kerala, India. Fishes were acclimatized to the laboratory conditions for four weeks with constant supply of water and good lighting system. They were maintained in well-aerated tubs (4 L capacity), which was dechlorinated and sustained with fresh water flow and waste water discharge. Bath was changed every 24 h, which was dechlorinated, respectively. Preliminary tests: The physico-chemical features of the tap water were estimated as per APHA [1]. Water temperature in the test ranged from 28 ± 2 C during the experiment, oxygen saturation of water ranged between 7 and 1 %, ph is 6.5 to 7.5 which were monitored using a standardized procedures. The LC 5 values in the respective time intervals were determined by probit analysis, with a confident limit of 5 % level [11]. Preliminary tests were conducted to provide guidance on range of concentration of bisphenol A to use in the bioassay. Five specimens were placed in each tub of replicates so that ten fishes were maintained in each test and aerated using tubed motorized pumps. Monofilament netting was used to cover the tanks to prevent the specimens from jumping out of test solutions. The behaviour of specimens was observed and death was also recorded throughout the study. Evaluation of median lethal concentration (LC 5 ): The concentration of the pollutant at which 5 percentage of the test animals dies during a specific period or the concentration lethal to one half of the test population is referred to as median lethal concentration (LC 5 ) or median tolerance limit. For determining LC 5 concentration separate circular plastic tubs of 4 L of water capacity were taken and different concentrations of bisphenol A were added. Then, 1 fish were introduced into each tub. A control tub with 4 L of water and 1 fish were also maintained (no toxicant). The lethal concentration for 5 % killing (LC 5 ) values was computed on the basis of probit analysis [11] for 96 h, which was 1 mg/ L. One-tenth of the dosage (1 mg/ L) of bisphenol A was chosen in the present study. Chemicals: Bisphenol A (4, 4-Isopropylidenediphenol) of 97% purity was obtained from SISCO Research Laboratories Pvt. Ltd., Mumbai, India. Malondialdehyde, NADPH, glutathione oxidized, thiobarbituric acid and pyrogallol were obtained from Himedia Laboratories, Mumbai, India. All other chemicals were of analytical grade and obtained from local commercial sources. Treatments: There were three groups, two tanks each with toxicant doses maintained for 1 and 2 days, respectively and two tanks with control fishes. Single dose with double durations were used in present study. Ten fish specimens were used for every test and also in control groups. The first group of fishes was maintained in toxicant-free water and was used as control and the second group was treated with bisphenol A at 1 mg/ L for 1 days. The third group was treated with bisphenol A at 1 mg/ L for 2 days. Biochemical estimation of gill was performed at the end of 1 and 2 days and the histology of gill was done at the end of 2 days of bisphenol A treatment, at the same time the control group was also maintained. 422

3 Chitra and Sajitha Body weight (g) nmol pyrogallol oxidized/ min/ mg protein Fig. 1: Effect of bisphenol A on the body weight of Oreochromis mossambicus Fig. 2: Effect of bisphenol A on the activity of superoxide dismutase in the gill of Oreochromis mossambicus µmol H2O2 consumed/ min/ mg protein nmol NADPH oxidized/ min/ mg protein Fig. 3: Effect of bisphenol A on the activity of catalase in the gill of Oreochromis mossambicus Fig. 4: Effect of bisphenol A on the activity of glutathione reductase in the gill of Oreochromis mossambicus µmol malondialdehyde produced/ min/ mg protein mg formazan formed/ min/ mg protein Fig. 5: Effect of bisphenol A on the level of lipid peroxidation in the gill of Oreochromis mossambicus Fig. 6: Effect of bisphenol A on the activity of succinate dehydrogenase in the gill of Oreochromis mossambicus 4221

4 J. Cell Tissue Research Killing of animals: The fish was caught very gently using a small dip net, one at a time with least disturbance. At the end of each exposure time, fishes were weighed and decapitated. Gill of both control and treated groups were dissected and stored at 4ºC until the analyses were performed. Tissue processing and biochemical analysis: A 1% (w/ v) homogenate of gill was prepared in icecold normal saline with the help of a motor-driven glass Teflon homogenizer on crushed ice for a minute. The homogenate was centrifuged at 8 g for 15 min at 4 C to obtain the supernatant, which was then used for the analyses. Protein was estimated by the method of Lowry et al [12] with BSA as the standard. Activity of superoxide dismutase was estimated by the method of Marklund and Marklund [13]. The activity of catalase was measured by the method of Claiborne [14]. Glutathione reductase was assayed by the method of Carlberg and Mannervik [15]. Level of lipid peroxidation was measured by the method of Ohkawa et al [16]. Succinate dehydrogenase was measured as described by Slater and Bonner [17]. Histology of gill tissues: Gill tissue was collected by sacrificing the fish and it was fixed in 1 % buffered formalin for 24 hours. Tissue was dehydrated in ascending grades of alcohol and was cleared in xylene until they became translucent. Tissue was transferred to molten paraffin wax for 1 hour to remove xylene completely and then impregnated with wax. Then the blocks were cut in a rotary microtome to prepare sections of thickness 4 to 6 microns. The sections were stained with haematoxylin and eosin and mounted in DPx. The structural alteration was observed under light microscope in the sections of gill of fish and was compared with those of control tissues. Photomicrographs were taken using Cannon shot camera fitted to the Carl Zeiss Axioscope 2 Plus Trinocular Research Microscope. Statistical analyses:statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Duncan s Multiple Range test using statistical package SPSS 17.. Differences were considered to be significant at p<.5 against control group. Data are presented as mean ± SD for ten animals per group. All biochemical estimations were carried out in duplicate. RESULTS Body weights:administration of bisphenol A at the sub lethal dose of 1 mg/ L showed a significant reduction in the body weight after 2 days of treatment whereas no changes were observed after 1 days (Fig. 1). Biochemical parameters: Administration of bisphenol A at sub lethal dose of 1mg/ L for 1 days and 2 days significantly (p<.5) decreased the activities of superoxide dismutase, catalase and glutathione reductase in the gills of fishes in concomitant manner as compared with the corresponding group of control animals (Figs. 2-4). The level of lipid peroxidation was increased significantly (p<.5) at 1 and 2 days of bisphenol A treatment in gills of the test animal when compared with the corresponding group of control animals (Fig. 5). There was a significant (p<.5) decrease in the activity of SDH in the gill of bisphenol A-treated fishes after 1 and 2 days of exposure as compared with the control group (Fig. 6). Gill histopathology: The fishes from the bisphenol A treated group presented morphological alterations when compared with the control groups. The analysis of the control group showed that the gill lamellae are separated from each other (Fig. 7), but in the treated group, after 2 days of bisphenol A exposure, presented three principal alterations: upliftment of gill epithelium (Fig. 8), gill lamellar fusion (Fig.9) and hypertrophy and vasodialation in secondary lamellae (Fig. 1). DISCUSSION Bisphenol A, one of the estrogenic environmental contaminants, get released into the environment during handling, unloading, heating, as well as accidental spills at the time of manufacture of polycarbonate and epoxy resins, flame retardants, and other plastic products. In the present study freshwater fish, Oreochromis mossambicus was used as an experimental model, and bisphenol A was exposed in a time-dependent manner for 1 and 2 days to study its antioxidant status and the histopathological changes in the gills of Oreochromis, which proposes their potential role as early-warning bio-indicator. Fishes constitute good test-organisms to monitor the water quality, especially small freshwater fishes, since 4222

5 Chitra and Sajitha Fig. 7 Fig. 8 Fig. 9 Fig. 1 Fig. 7: Photomicrograph showing histology of gill of the control fish (X 4 magnification) Fig. 8: Photomicrograph of gill histology in bisphenol A-treated fish showing upliftment of gill epithelium (X 4 magnification) Fig. 9: Photomicrograph showing gill histology of bisphenol A-treated fish showing primary and secondary gill lamellar fusion (X 4 magnification) Fig. 1: Photomicrograph showing gill histology of bisphenol A-treated fish showing hypertrophy and vasodialation (X 4 magnification) 4223

6 J. Cell Tissue Research they can be maintained in laboratory and can be easily exposed to toxic substances in a similar way of the other high vertebrates, and in this way, could be used to assess the presence of substances that have the potential to cause toxic effects in humans. O. mossambicus is a very important fresh water fish species in south India. Fish gills are the main target of several aquatic pollutants, being its epithelium, an excellent model to examine the effects of dissolved substances in the tissues; besides this, gills are the most seriously affected organs due to their constant contact with the water [18]. In the present study bisphenol A showed a significant reduction in the body weight after 2 days of treatment whereas no changes were observed after 1 days. Depression in body weight or reduction in the body weight gain may reflect a variety of responses, including internal and external environmental factors such as rejection of feed, treatment induced anorexia or systemic toxicity. However, the present study was designed to test the hypothesis that elevated reactive oxygen species activity and decreased antioxidant enzyme activity may contribute to the toxic effect of bisphenol A in gills of fishes and that could be supplemented with histopathological study of the gill tissue. Fish gills are constituted by primary filaments and secondary lamellae, which are formed, basically, by three different cell types: pillar cells, respiratory cells and erythrocytes that circulate in the lamellae interior. Gills are the first target of waterborne pollutants since it is constantly in contact with the water, accomplishing exchanges; besides this, it presents a high adaptation capacity. It is well known that changes in fish gill are among the most commonly recognized responses to environmental pollutants [19]. Gills also participate in many important functions in fish, such as respiration, osmoregulation and excretion, remain in close contact with the external environment, and particularly sensitive to changes in the quality of the water, are thus considered as the primary target of the contaminants [2]. Alterations like epithelial lifting, hyperplasia and hypertrophy of the epithelial cells, besides partial fusion of some secondary lamellae are examples of defense mechanisms, in general, these results in the increase of the distance between the external environment and the blood and thus serve as a barrier to the entrance of contaminants [19]. Histological alterations observed in fish gills are acknowledged as a fast and valid method to determine the damages caused by exposure to different pollutants in fishes [21]. However, fish exposed to bisphenol A shows several histological alterations, namely upliftment of gill epithelium, fusion of secondary gill lamellae and lamellar axis vasodilation. The lifting of respiratory epithelium is the histological change observed, probably induced by the incidence of severe edema [22]. Edema with lifting of lamellar epithelium could serve as a mechanism of defense, because separation of epithelial lamellae increases the distance across which waterborne pollutants must diffuse to reach the bloodstream [23]. However, these results also were found in fish exposed to other pollutants. The edema, epithelial lifting as well as lamellar fusion also are defensive mechanisms that reduce the branchial superficial area in contact with the external milieu. Lesions in the gill morphology could lead to functional alterations and interference in fundamental process such as maintenance of osmoregulation and antioxidant defense of gills. The present study was thus designed to test the hypothesis that elevated reactive oxygen species activity and decreased antioxidant enzyme activity may contribute to the toxic effect of bisphenol A in gills of fishes. In the present study bisphenol A exposure for 1 and 2 days significantly (p<.5) decreased the activities of superoxide dismutase, catalase and glutathione reductase in a timedependent manner in the gills of fishes as compared with the corresponding group of control animals. Aerobic organisms, which derive their energy from the reduction of oxygen, are susceptible to the damaging actions of the small amounts of superoxide anion ( - ), hydroxyl radical ( OH) and hydrogen peroxide (H 2 ) that inevitably form during the metabolism of oxygen, especially in the reduction of oxygen by the electron transfer system of mitochondria. These three species, together with unstable intermediates in the peroxidation of lipids, are referred to as Reactive Oxygen Species (ROS). Damage from ROS as a result of an imbalance between radical-generating and radical-scavenging systems results in a condition called oxidative stress. Free radicals/ ROS generated in tissues and subcellular compartments are effectively scavenged by the antioxidant defence system, which constitutes 4224

7 antioxidant enzymes such as, superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. Superoxide dismutase (SOD) catalysis the dismutation of superoxide to hydrogen peroxide (H 2 ) and oxygen ( ). The conversion of H 2 to 2H 2 O is by the enzyme glutathione peroxidase and the conversion of H 2 to and H 2 O is by the enzyme catalase. Since the reaction catalyzed by glutathione peroxidase requires glutathione (GSH) as substrate and depends in part on the ratio of glutathione disulfide (GSSG): GSH, the concentrations of these reactants and their ratio, which is a reflection of the redox state of the cell, are important to ROS detoxification. Glutathione peroxidase/ reductase directly act as antioxidant enzymes to inhibit lipid peroxidation [24]. In the present study a decrease in the activity of superoxide dismutase has been shown to increase the level of superoxide anion, which is known to inactivate catalase activity [25]. Similarly, catalase or glutathione peroxidase has been shown to eliminate hydrogen peroxide from the cell leading to the inactivation of superoxide dismutase and generation of lipid peroxides [26]. In the present study bisphenol A exposure concomitantly increased (p<.5) the level of lipid peroxidation after 1 and 2 days in gills of the test animal when compared with the corresponding group of control animals. Increased lipid peroxidation may indicate an increased oxygen free radical generation [27]. Accumulation of hydrogen peroxide at low concentration is not reactive, but at a higher concentration promotes the formation of highly toxic hydroxyl radicals in the presence of Fe 2+, which attack almost all biomolecules including membrane lipids causing formation of lipid peroxides [28]. The mitochondrial respiratory stress marker enzyme succinate dehydrogenase is primarily involved in the oxidative catabolism of sugars, which is used as an indicator of osmoregulatory activity. This enzyme is concentrated in the chloride cells within fish gills. In the present study exposure of adult Oreochromis at sub lethal concentration of bisphenol A resulted in a significant decrease in succinate dehydrogenase activity in gill after 1 and 2 days, which may be due to the impairment of aerobic metabolism in bisphenol A-treated fishes [29]. Inhibition of stress marker enzyme could be considered as an important marker to indicate the state of fish health and their physiological conditions. The result shows that fish exposed to bisphenol A is unable to bear the stress Chitra and Sajitha 4225 caused due to the pollutant and resulted in a shift towards anaerobiosis at organ level during sub lethal intoxication. CONCLUSION Thus the adverse effect of bisphenol A on gill may be due to induction of oxidative stress and the results of the investigation show that sub lethal dose of bisphenol A caused imbalance in the pro-oxidant and antioxidant status in the gills of fish. The decreased activity of succinate dehydrogenase is due to impairment of aerobic metabolism and stress-related shift towards anaerobiosis. ACKNOWLEDGMENT The authors acknowledge the availability of fund from UGC-SAP/ BSR, Government of India. REFERENCES [1] US Environmental Protection Agency. Bisphenol A: final test rule. Federal register, 51(181): , OTS, Washington, D C. (1986). [2] Krishnan, A.V., Stathis, P., Permuth, S.F., Tokes, L. and Feldman, D.: Endocrinology, 132: (1993). [3] Lazear, N.R.: Adv. Mater. Processes. 147: (1995). [4] Brotons, J.A., Olea-Serrano, M.F., Villalobos, M., Pedraza, V. and Olea, N.: Environ. Health. Perspect. 13: (1995). [5] Goodson, A., Summerfield, W. and Cooper, I.: Food. Addit. Contam. 19: (22). [6] Olea, N., Pulgar, R., Perez, P., Olea-Serrano, M.F., Rivas, A., Novillo-Fertrell, A., Pedraza, V., Soto, A.M. and Sonnenschein, C.: Environ. Health. Perspect. 14: (1996). [7] Ackerman, L., Noonan, G., Heiserman, W., Roach, J., Limm, W. and Begley, T.: J. Agricul. Food. Chem., 58: (21). [8] Sun, Y., Irie, M., Kishikawa, N., Wada, M., Kuroda, N. and Nakashima, K.: Biomed. Chromatography, 18: (24). [9] Cheung, C.C.C., Zheng, G.J., Li, A.M.Y., Richardson, B.J. and Lam, P.K.S.: Aqua. Toxicol., 52: (21). [1] APHA. Standard methods for the examination of water and waste water, 2 th Edition, Washington, DC. (1998). [11] Finney, D.J.: Probit analysis, 3rd (Ed.), Cambridge University Press, London, pp 333 (1971). [12] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: J. Biol. Chem. 193: (1951). [13] Marklund, S. and Marklund, G.: Eur. J. Biochem. 47: (1974). [14] Claiborne, A.: In: CRC Handbook of methods for

8 J. Cell Tissue Research oxygen radical research. R Greenwald (ed.), CRC Press, Boca Raton, Florida, pp (1985). [15] Carlberg, I. and Mannervik, B.J.: J. Biol. Chem. 25: (1985). [16] Ohkawa, H., Ohishi, N. and Yagi, K.: Anal. Biochem. 95: (1979). [17] Slater, E.C. and Bonner, W.D.: Biochem. J. 52: (1952). [18] Mishra, V., Lal, H., Chawla, G. and Viswanatan, P.N.: Ecotoxicol. Environ. Saf. 1: (1985). [19] Mallatt, J.: Canadian. J. Fish. Aquat. Sci. 42: (1985). [2] Poleksic, V. and Mitrovic-Tutundzic, V.: Fish gills as a monitor of sublethal and chronic effects of pollution. In: Sublethal and Chronic effects of pollutants on freshwater fish (Müller, R. and Lloyd, R. eds.), Oxford, Fishing News Books, pp (1994). [21] Arellano, J.M., Ortiz, J.B., de Canales, M.L.G. and Sarasquete, C.: Histochem. J., 33: (21). [22] Pane, E.F., Haque, A. and Wood, C.M.: Aquat. Toxicol., 69: (24). [23] Arellano, J.M., Storch, V. and Sarasquete, C.: Ecotoxicol. Environ. Saf. 44: (1999). [24] Sikka, S.C.: Curr. Med. Chem. 8: (21). [25] Kono, Y. and Fridovich, I.: J. Biol. Chem. 257: (1982). [26] Bray, M.A., Gordon, D. and Morley, J.: British. J. Pharmacol. 52: 453 (1974). [27] Thiele, J.J., Freisleben, H.J., Fuchs, J. and Ochsendorf, F.R.: Human. Reprod. 1: (1995). [28] Halliwell, B. and Guteridge, J.M.C.: Free Radicals in Biology and Medicine. Clarendon Press Inc., Oxford (1985). [29] Rajeswari, K., Janardan Reddy, S., Rafi, G.M., Reddy, S.N. and Reddy, D.C.: Environ. Ecology. 74: (1989). 4226