Oxidative stress and histopathologic biomarkers of exposure to bisphenol-A in the freshwater fish , Ctenopharyngodon idella

Bisphenol-A (BPA) belongs to the family of endocrine disrupting chemicals (EDCs) and it is used in the production of polycarbonate plastic and epoxy resins. The reproductive toxicity of BPA is well documented but it also exerts its toxic effects through multiple pathways especially by inducing a state of oxidative stress and causing damage to the vital organs. In the present study, histopathologic and oxidative damage caused by BPA in liver and kidneys of fresh water cyprinid, Ctenopharyngodon idella was evaluated. LC50 of BPA for Ctenopharyngodon idella was determined by probit regression analysis. Fish were exposed to a sublethal concentration of BPA i.e. 3.2 ppm (1/2 LC50) for 14 days. Histologic studies revealed that BPA caused degenerative changes in liver and kidneys and exposure of sublethal concentration of BPA caused oxidative damage in both organs. Lipid peroxidation significantly increased in liver and kidneys of treated group. Catalase activity and reduced glutathione content significantly decreased in the group exposed to BPA compared to control and glutathione-S-transferase activity increased significantly in both organs exposed to the sublethal concentration of BPA. From this study it is concluded that BPA caused toxic effects in fish species by changing oxidative balance and damaging the vital organs.


INTRODUCTION
Waterbodies are a major sink for industrial, domestic and other anthropogenic compounds (Canli, Ay, Kalay, 1998).The aquatic pollution has far reaching impacts on organisms in the recipient environment.Fish as inhabitant of aquatic system cannot avoid the inimical effects of the pollutants.A set of biomarkers is generally used to evaluate the biological effects of pollutants.Such biomarkers act as an early warning of a specific detrimental biological endpoint.Oxidative stress and histopathologic biomarkers are used in ecotoxicology (Pandey et al., 2003).Histopathology of fish tissues is a reliable monitoring tool, which allows the assessment of the environmental stressor's effects.It is one of the most reliable indicator of the health impairment induced by the anthropogenic stressors in aquatic organism (Fernandes et al., 2008;Leonardi, Tarifeno, Vera, 2009).Enzymatic and non-enzymatic antioxidants are important defense mechanism of organisms which provide protection against environmental pro-oxidants by countering the impact of reactive oxygen species (Tabrez, Ahmad, 2009).Therefore, antioxidant parameters and oxidative stress indices are considered potential biomarkers and are frequently used as screening tools to assess the impacts of environmental stress.Important antioxidant enzymes are catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) and glutathione peroxidase (GPx).In addition, glutathione, vitamins and carotene also help the organism to mitigate the external pollutants and help the protective enzyme system of the organism.
Of many compounds released into the aquatic environment, endocrine disrupting chemicals (EDCs) have gained much attention.Bisphenol-A (BPA) is a well-known EDC.It is the highest volume chemical produced worldwide and mimics the naturally occurring estrogen, estradiol 17β.BPA is used in the production of epoxy resin lining of food and beverage containers, polycarbonate plastic (Vom Saal, Hughes, 2005), as a constituent of dental sealant (Olea et al., 1996;Vandenberg et al., 2007), as a flame retardant precursor and also used in the production of thermal papers and carbonless copy (Suzuki et al., 2000;Debenest et al., 2010;Liao, Kannan, 2011).Several studies suggest that BPA cause acute, short-term and sub-chronic toxicity (Tyl et al., 2002;Tyl, 2008).Bisphenol-A cause tissue injury by forming reactive oxygen species (Kabuto, Amakawa, Shishibori, 2004;Hassan et al., 2012;Aboul Ezz, Khadrawy, Mourad, 2015) Grass carp (Ctenopharyngodon idella) is a cosmopolitan species.It is an exotic species introduced in the Pakistani rivers and thrives well in river and pond system.It is now cultured with other major carps.The present study focused on the role of BPA in generating the reactive oxygen species (ROS) and inducing oxidative stress in fish (Ctenopharyngodon idella) liver and kidneys and inducing histopathologic alteration.The effect of bisphenol-A on a biogenic macromolecular peroxidation indicator (thio barbituric acid-reactive substance; TBARS), reactive oxygen species (ROS) regulating enzymes (catalase and GPx), an antioxidant (glutathione) and histology of vital organs was evaluated.

MATERIAL AND METHODS
Juvenile grass carp (Ctenopharyngodon idella hereafter C.idella) (10.5±2.1 cm; 50.3±3.57g) were purchased from a commercial fish farm.Fish were acclimatized for two weeks in 50 liter well aerated glass aquaria.Physico-chemical parameters of water e.g.dissolved oxygen, temperature and pH were recorded every day.Fish were kept under natural photoperiod and temperature, fed with commercial fish pallet (Oryza Organics, Lahore).

Acute study
For acute studies LC 50 (lethal concentration with 50% mortality) was calculated for 96 hours (APHA, 2005) by probit regression analysis (Finney, 1971), using IBM SPSS statistics (Version: 20).Fish were divided into 20 groups (6 fish per group) and exposed to graded concentrations of BPA (0.5 ppm to 10 ppm) with the increment of 0.5 ppm.Water was replaced every day and fresh toxicant was added after water renewal for 96 hours.Fish were monitored at regular intervals and dead fish were removed from aquaria.

Sublethal studies
For sublethal studies, 20 fish were divided into two groups (control and treated), having 10 fish per group.Bisphenol-A, 96 hour LC 50 for juvenile grass carp (Ctenopharyngodon idella) was 6.42385 ppm.A nonlethal concentration of 3.2 ppm was selected (1/2 LC 50 ) and fish were exposed to 1/2 LC 50 concentration for 14 days.After the stipulated time, five fish were randomly selected from the control and treatment group.Fish were anesthetized with clove oil (Kaiser et al., 2006), length and weight was recorded and fish were dissected humanely.Liver and kidneys were removed, cleaned of extraneous tissue and weighed to the nearest mg.A portion of tissues were preserved in 10% formalin for histologic studies and the remaining portions were washed with 0.9% ice-cold saline solution, blotted on filter paper, snap frozen in liquid nitrogen and stored at -80 o C until further biochemical analysis.All animal handling and experimental protocols were approved by research committee of Department of Zoology, GC University, Lahore.

Histologic studies
The preserved tissues were processed in various grades of ethanol, cleared in xylene and impregnated with wax (mp; 58 o C).Five microns thick sections were cut using rotary microtome (Leica RM 2165).Tissue sections were stained with hematoxylin and eosin (H&E).Stained slides were studied and photographed by high resolution microscope (Leica, Japan) fitted with a digital camera.

Biochemical analysis
Biochemical analysis was done using post mitochondrial supernatant (PMS).The tissues were ground in liquid nitrogen, homogenized in chilled 0.1 M potassium phosphate buffer (pH 7.4) containing 1.17% KCl and centrifuged for 30 mints at 10,500 rpm to obtain the PMS.

Measurement of lipid peroxidation
Lipid peroxidation was determined by using the method of Wright, Colby and Miles (1981) with slight modifications.Reaction mixture (3 mL) contained 10% tissue homogenate, 10% TCA and 0.67 % TBA.The reaction mixture was incubated in boiling water for 45 min, cooled and centrifuged at 2500×g for 10 min.Absorbance of the supernatant was taken at 532 nm at 37 o C using Hitachi U-2000 spectrophotometer.Rate of Lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances and expressed as nmol TBARS formed/g tissue at 37 o C by using a molar extinction coefficient of 1.56 x 10 5 /M/ cm.
After one hour the mixture was centrifuged for 15 min (1200 rpm, 4 o C).Supernatant was filtered.The final reaction mixture (3 mL) contained filtered aliquot, 0.1 M phosphate buffer and 10 mM DTNB.Absorbance was recorded at 412 nm.The results were expressed as nmol GSH/g of tissue using molar extinction coefficient of 1.36x10 4 /M/cm.

Catalase activity
The catalase (CAT) activity was measured using the method of Claiborne (1985) as described by Haque et al. (2003).The reaction mixture consisted of 0.09 M H 2 O 2, 0.1 M phosphate buffer and PMS (10%) in a total volume of 3 ml.Change in absorbance was recorded after every 30 seconds at 240 nm in a double beam spectrophotometer (Hitachi U-2000).Catalase activity was calculated in terms of nmol H 2 O 2 consumed/min/mg protein.

Protein estimation
Protein was estimated according to method by Lowry et al. (1951) using bovine serum albumin (BSA) as standard.

Statistical analysis
The statistical analysis was performed using IBM SPSS (version 20).Data for glutathione-S-transferase and lipid peroxidation were log transformed and all data are expressed as mean ± S.E.M. significant difference between control and treated group was computed using Student's t-test.p<0.05.

RESULTS
No mortality was observed during the sublethal studies.However, as the time passed, the fish showed altered swimming pattern, erratic swimming and loss of balance.The 96 hours LC 50 value of BPA according to the probit-regression analysis was 6.323 ppm with 95% lower and upper confidence limit as 6.801 and 5.683 ppm respectively.

Effect of BPA on liver and kidneys histology
Fish liver from the control group did not show any alteration in structure.Hepatocytes have central nuclei arranged around the central vein.Cords of hepatocytes are separated by sinusoids (Figure 1).The liver sections of fish exposed to sublethal concentration of BPA for 14 days showed various histopathologic changes including ruptured central vein, lipid like vacuolization, macrophage and lymphocytes infiltration, ruptured and degenerated hepatocytes (Figure 1).Photomicrograph of C. idella kidneys from the control group showed normal structure, the brush border of proximal tubules and the lumen of distal tubules were normal in appearance (Figure 2).Kidney sections from the group exposed to sublethal concentration of BPA showed damaged renal tubules, shrinkage of tubules and tubule lumen and degeneration of tubules and hematopoietic tissue (Figure 2).

Effect of BPA on enzyme activity
A significant decrease in liver and kidney catalase activity (p<0.05) was observed in the bisphenol-A treated groups compared to control (Figure 3).Significant enhancement in the level of lipid peroxidation was recorded in liver and kidney (p<0.05) of fish exposed to BPA in comparison with the control group (Figure 4).
The glutathione-S-transferase activity increased significantly in both tissues of fish exposed to sublethal BPA compared to the control (Figure 5).Bisphenol-A exposure for 14 days caused a significant decrease in reduced glutathione content (p<0.05) both in liver and kidney (Figure 6).

DISCUSSION
Various anthropogenic materials are released into bodies of water which affect aquatic life especially fish.Histopathology and enzyme status are useful to determine the effects of anthropogenic pollutants on the organisms (Khoshnood et al., 2010).The present study revealed that    Exposure to the sublethal concentration of bisphenol-A changed the normal architecture of liver, increase lipid like vacuolization and inflammation in sinusoids.Similar changes were recorded in the liver of Catla catla exposed to 1-4 ppm BPA (Faheem, Jahan, Lone, 2016).Dilated sinusoids and change in liver normal structure may be due to the loss of structural proteins after the toxicant exposure.One of the nonspecific responses of fish in toxic conditions is hepatocyte vacuolation (Roberts, 1978) this vacuolization is typical of lipid accumulation in liver.Similar changes in liver were reported by Peixoto et al. (2013) in Barbus bocagei caught from Vizela River, Portugal.Ameur et al. (2012) found similar histopathologic changes in liver of Mugil cephalus and Dicentrarchus labrax from Bizerte Lagoon, Tunisia.Kidney sections of fish exposed to BPA showed necrosis, degeneration of renal tubules, inflammation in hematopoietic tissue, shrinkage of tube lumen, and hemorrhage.Similar results were reported in Cyprinus carpio exposed to deltamethrin (Cengiz, 2006) and in Oreochromis niloticus exposed to heavy metals (Khidr, Mekkawy, 2008).
In the present study, the catalase activity was significantly decreased both in liver and kidney.Catalase is an active and first enzyme that shows alteration, following oxidative stress (Jin et al., 2010).The reason of decrease catalase activity may be the inactivation of enzyme by overproduction of ROS (Pigeolet et al., 1990).Sayeed et al. (2003) reported a significant decrease in catalase activity in all the organs of Channa punctatus exposed to deltamethrin.Chitra and Maiby (2014) studied decrease in catalase activity in the liver of fresh water fish Oreochromis mossambicus exposed to the sublethal concentration of BPA which is in accordance with the present study.Faheem et al. (2012) reported a decrease in the catalase activity in liver of Oreochromis niloticus exposed to the sublethal concentration of cadmium.Wu et al. (2011) reported a significantly reduced catalase activity in zebrafish exposed to graded concentration (0.1-1000μg/l) of BPA.A similar decrease was observed in gills of Oreochromis mossambicus exposed to 1 ppm BPA for 10 and 20 days (Chitra, Sajhita, 2014).A decrease in catalase activity was reported by Li et al. (2016) in all vital organs of Japanese medaka exposed to 1.5 mg/l BPA.Contrary to these findings, male medaka exposed to 10μg/l BPA showed 5-20 fold upregulation of catalase mRNA (Qiu et al., 2016).This may indicate that at lower concentrations fish respond quickly to remove BPA but as the exposure concentration increases the tissue systems of fish undergoes exhaustive necrosis thus causing a decrease in activity of enzymes.We have already reported above damage to the liver and kidney system of C. idella exposed to sublethal concentration of BPA (Figure 1 and 2).
The reactive oxygen species (ROS) produced  during oxidative stress reacts with unsaturated fatty acids that are present in membranes and cause lipid peroxidation.Therefore, increased lipid peroxidation is an indication of high level of ROS (Thiele et al., 1995) and used extensively to study oxidative stress (Huggett et al., 1992).A significant increase (p< 0.05) in lipid peroxidation was recorded in C. idella after 14 days exposure of bisphenol-A compared to control group.Chitra, Latchoumycandane and Mathur (2003)  Glutathione-S-transferase (GST) is an important phase II biotransformation enzyme and used as biomarker of exposure to pollutants in an aquatic system (Livingstone, 1998).In the present study, there was an increase in glutathione-S-transferase activity in liver and kidneys of C. idella.The increased GST activity may be to detoxify BPA because GST adds GSH-group to xenobiotics or their metabolites, making them more water-soluble and, thus, excreted more easily (Moorhouse, Casida, 1992).Increased liver GST activity has been demonstrated many times in various fish species as the result of exposure to PCBs (Gadagbui, Goksoyr, 2001) and other pollutants (Ahmad et al., 2000;Ansari, Ansari 2014).Increase in GST activity was observed in zebrafish embryo exposed to very low (0.1μg/l) BPA (Wu et al., 2011).Olsvik et al. (2009) found non-significant increase in gst mRNA in liver of Atlantic cod when exposed to 50µg/L of BPA for three weeks.Increase in GST activity was also observed in hepatocytes of pearl mullet exposed to BPA (Kaya, Kaptaner, 2016) similar increase in GST activity was reported by Li et al. (2016) in liver and gills of Japanese medaka exposed to 1.5 mg/L BPA.
Reduced glutathione (GSH) is the major nonprotein thiol and plays a pivotal role in cell viability protecting cells against lipid peroxidation either alone or in conjugation with other proteins (Anjum et al., 2011).In our study, we found a significant decrease in the amount of reduced GSH in liver and kidney of C.,idella exposed to sublethal concentration of BPA.This decrease may be due to GSH being used to scavenge the free radicals and ROS generated by bisphenol-A.Yazdani, Andresen and Gjoen (2016) reported a significant decrease in gsh mRNA level in Atlantic salmon kidney cell lines exposed to 100μM BPA.The reduced GSH content observed in the present study may be due to its lower transcription.Decreased GSH content was also observed in hepatocytes of pearl mullet exposed to 200 μM BPA for 24 hours (Kaya, Kaptaner, 2016).
The results of the present study revealed that the fish like other animals, undergo oxidative stress and respond by changes in activity of the antioxidant enzymes and morphological alterations in liver and kidney tissues.Therefore, the results obtained and reported here are the first for this EDC on C. idella, a cosmopolitan species, and will be useful for future work in elucidating the detailed effects of BPA in this species and other carp species.

FIGURE 2 -
FIGURE 2 -Kidney tissue of C. idella from control and treated group exposed to the sublethal concentration of BPA for 14 days.(H) hematopoietic tissue; (Dt) distal tubules; (L) tube lumen; (D) degeneration of tubules; (S) shrinkage of tube lumen.H & E stain, 40X.

FIGURE 3 -
FIGURE 3 -Catalase activity in the liver and kidneys of C.idella (n=5), exposed to the sublethal concentration of BPA for 14 days.The values given are mean ± S.E.M. *= p<0.05.

FIGURE 4 -
FIGURE 4 -Lipid peroxidation in the liver and kidneys of C. idella (n=5), exposed to the sublethal concentration of BPA for 14 days.Values given are mean ±S.E.M. * = p<0.05.

FIGURE 5 -
FIGURE 5 -Glutathione-S-transferase activity in the liver and kidneys of C.idella (n=5), exposed to the sublethal concentration of BPA for 14 days.The values given are mean ± S.E.M. *= p<0.05.

FIGURE 6 -
FIGURE 6 -Reduced glutathione content in the liver and kidneys of C. idella (n=5), exposed to the sublethal concentration of BPA for 14 days.The values give are mean ±S.E.M. * = p< 0.05.