Ecotoxicology and Environmental Safety 106 (2014) 33–39

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Sublethal toxicity of carbofuran on the African catfish Clarias gariepinus: Hormonal, enzymatic and antioxidant responses Ahmed Th.A. Ibrahim a,n, Ahmed S.A. Harabawy b,c a

Zoology Department, Faculty of Science, New Valley Branch, Assiut University, Assiut, Egypt Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt c Biology Department, Faculty of Science, North Jeddah, King Abdulaziz University, Jeddah, Saudi Arabia b

art ic l e i nf o

a b s t r a c t

Article history: Received 3 January 2014 Received in revised form 20 April 2014 Accepted 21 April 2014

The present study examined the impacts of carbofuran on endocrinology of the catfish, Clarias gariepinus, for the first time and evaluated cortisol (CRT), triiodothyronine (T3), thyroxin (T4), 17βestradiol (E2) and testosterone (TST) and the oxidative stress markers including SOD, CAT, GSTs, GSH. The toxic effects on the metabolic enzymes, G6PDH and LDH, in addition to lipid peroxidation (LPO) and DNA damage as biomarkers in Nile catfish, to sublethal exposures of carbofuran (0.16 and 0.49 mg/L, for 35 days) were studied. Statistically significant differences between selected parameters between control and carbofuran-treated fish were recorded. Carbofuran caused a significant (po0.05) increase in CRT and T3 levels; the mean levels of T4, TST, E2 exhibited significant decreases (po0.05) in carbofuran-treated fish. Toxicity of carbofuran on liver, kidney, gills, gonads and muscles after 35 days of exposure was found. Glycogen levels showed a highly significant decrease in liver and gills (po 0.001), a significant decrease (po 0.05) in kidney and muscles, and insignificant changes (p40.05) in gonads of treated fish. The two metabolic enzymes G6PDH and LDH in all tissues exhibited significant decreases (po0.05) in treated fish. SOD, CAT, GSH and GST levels showed significant decreases (po0.05) in all tissues of fish after exposure to carbofuran. LPO levels increased significantly (po0.05) in all tissues except gonads after 5 weeks of exposure to carbofuran. There was a significant (po0.05) increase in DNA fragmentation percentage in treated fish. Our results provide a clear evidence on the response of C. gariepinus to sublethal doses of carbofuran and allow us to consider catfish as a good bioindicator to reflect the endocrine disrupting impacts of carbofuran, and reflect the potential of this pesticide to cause disturbance in antioxidant defense system as well as metabolism and induction of lipid peroxidation (LPO) and DNA damage in contaminated ecosystems. & 2014 Elsevier Inc. All rights reserved.

Keywords: Carbofuran Clarias Hormones Antioxidants Oxidative stress Enzymes

1. Introduction The aquatic and terrestrial ecosystems are continuously contaminated with chemical pollutants from industrial, agricultural and domestic activities. Pesticides are a major category of toxicants, which have serious toxic impacts on aquatic life and still constitute a significant risk due to their toxicity on non-target organisms including fishes (Soloneski and Larramendy, 2012; Ghazala et al., 2014). The continuous contamination by pesticides is a problem of worldwide importance leading to great efforts, especially in the advanced countries, to assessment and hence prevent the harmful impacts of these pollutants to the extent of their banning (EUC, 2007). Carbofuran is a systemic and contact

n

Corresponding author. E-mail addresses: [email protected], [email protected], [email protected] (A.Th.A. Ibrahim). http://dx.doi.org/10.1016/j.ecoenv.2014.04.032 0147-6513/& 2014 Elsevier Inc. All rights reserved.

insecticide, acaricide and nematicide with activity against many agricultural pests; and has very toxic impacts on invertebrates and birds and relatively high mammalian toxicity as reported by Otieno et al. (2010). Carbofuran is very toxic to fish and the LC50 values appear to differ from one species to another within the same family due to species sensitivity to carbofuran (Dobšίková, 2003; Ghazala et al., 2014). Otieno et al. (2010) have determined the concentrations of carbofuran residues in water which are ranging from 0.005 to 0.495 mg/L. Carbofuran residues in uncultivated, unfertilized and unsterilized soil in Egypt and the bioaccumulation of carbofuran in tissues of C. gariepinus have been reported by Harabawy and Ibrahim (2014). Several pesticides including carbofuran behave as endocrine disruptors (EDCs) with a potential to change the expression of vital genes leading to disturbance in plasma hormones levels and reproductive dysfunction or immunosuppression (Lau et al., 2007; Jin et al., 2010). EDCs can interfere with synthesis, secretion, transport, metabolism or binding action of the natural blood-borne

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A.Th.A. Ibrahim, A.S.A. Harabawy / Ecotoxicology and Environmental Safety 106 (2014) 33–39

hormones that are naturally found in the body and responsible for homeostasis, reproduction, and developmental processes (DiamantiKandarakis et al., 2009). They enter the body through food, drinking water, air and skin contact and affect the health and reproduction (Lukáčová et al., 2013). EDCs may be natural hormones, from any animal and can exert hormonal actions on other animals; natural chemicals, such as toxins produced by algae, fungi; some synthetic pharmaceuticals; pesticides; plastics, phthalates; in addition to steroid hormones mimics, chemicals antagonize male or female hormones (Allinson, 2008). Hormones are regulatory biochemicals produced in all multicellular organisms and regulate a variety of physiological and behavioral activities. Cortisol is the main glucocorticoid hormone secreted by interrenal in fish; it plays a major regulatory role in normal metabolism and thus influences the behavior of an individual (Barton, 2002). Thyroid hormones in fish (thyroxine, T4 and triiodothyronine, T3) play a crucial role in growth regulation, metabolism and involved in reproduction; so, any disruption in the thyroid axis may lead to serious impacts on normal development, differentiation, growth or reproduction in several vertebrates including fishes (Brown et al., 2004). Sex steroid hormones such as testosterone and 17β-estradiol are known as key hormones that control and regulate the developmental stages of the organism including gametogenesis, fertilization, sexual development and development of primary and secondary sexual characteristics (Nelson, 2005). 17β-Estradiol (E2) plays a crucial role in ovarian development and induces the synthesis and release of vitellogenin by liver (Meunpol et al., 2007); and can exert a positive feedback on the hypothalamic-pituitary-gonadal axis stimulating the release of FSH that is important for gametogenesis and steroidogenesis (Young et al., 2005). Testosterone is the main androgen in males that is produced by testes. Testosterone is converted into E2 within granulosa cells via aromatization, where, aromatase is the enzyme that converts testosterone into estradiol (Huffman et al., 2013). Any significant change in serum hormones levels due to pollution could be used as valuable biomarkers and useful tools in monitoring the impacts of stressors on fish (Hedayati and Arsham, 2012; Ghazala et al., 2014). Oxidative stress is another mechanism for toxicity leading to cell death and disturb the physiological processes in fish (Banaee, 2013). Various environmental contaminants including metals and pesticides can induce oxidative stress by generating a variety of highly reactive oxygen species (ROS) such as H2O2, O2
and OH
and electrophilic free-radical metabolites that interact with nucleophilic sites in DNA and causing breaks and other DNA damages (Lukáčová et al., 2013; Harabawy and Ibrahim, 2014; Harabawy and Mosleh, 2014). ROS are naturally generated by aerobic organisms through oxidative metabolism such as mitochondrial respiration (Vinodhini and Narayanan, 2009); thus, may be produced during detoxification process of insecticides (Üner et al., 2006). When there is an imbalance between the cellular antioxidant enzyme activities and ROS production, the antioxidant system becomes unable to eliminate or neutralize the excess of ROS, and then leads to oxidative stress (Nishida, 2011). ROS are useful when used by the immune system as a means to attack and kill pathogens (Segal, 2005). Elevated ROS can cause protein oxidation, DNA damage, initiate lipid peroxidation in tissues, cause alterations in gene expression, and changes in cell redox status resulting in oxidative damage to organisms (Vinodhini and Narayanan, 2009). But, production of ROS can activate the expression of genes encoding antioxidant enzymes and elevation of ROS scavengers levels (Mahboob, 2013). Fishes have developed detoxification mechanisms to remove the toxic impacts of pollutants through releasing the xenobiotic metabolizing enzymes; this detoxification process involves two phases, in phase I, oxygen is added to the xenobiotic structure, and in phase II, most of

oxygenated metabolites formed in phase I, conjugate with glutathione; and finally the conjugated metabolites become very polar substances that cannot be resorbed, highly water-soluble and easier to eliminate (Banaee, 2013). Antioxidant defenses include antioxidant enzymes such as glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and nonenzymatic defense includes reduced glutathione (GSH), vitamins A, C and E, carotenes etc. (Filho, 1996). In addition to intracellular antioxidant enzymes, there are many extracellular antioxidant molecules (e.g. ascorbate, uric acid, etc.) that circulate in biological fluids scavenging free radicals and ROS (Valavanidis et al., 2006). In fish, SOD and CAT constitute the first line of defense against oxidative stress. SOD catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen while CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen (Banaee, 2013). GSTs are a group of cytosolic detoxifying enzymes found in different tissues, defend cells against the mutagenic, carcinogenic and toxic impacts of various pollutants; helping in prevention of lipid peroxidation and suppressing apoptosis (Jain et al., 2010; Banaee, 2013). GSTs contributes in phase II; they catalyze the conjugation of the thiol group of GSH with the reactive electrophilic compounds (phase I metabolites) leading to neutralization of their active electrophilic sites; and then the parent compound become inactive, nontoxic, more polar and water soluble compounds and become easy to excrete (Mofeed and Mosleh, 2013). Because xenobiotics can induce the antioxidant defenses, the enzymatic and non-enzymatic antioxidants are widely used as environmental biomarkers. LPO and DNA damage are good biomarkers on oxidative damage in cells and used to evaluate acute and chronic exposure to genotoxic agents (Bhuvaneshwari, et al., 2013). Also, pollutants can increase or inhibit the metabolic enzyme activities and change the enzyme levels to be enough to provide information of diagnostic values (Begum, 2009). Glucose-6Phosphate dehydrogenase (G6PDH) was recognized as an antioxidant enzyme; it is a key enzyme that catalyzes the pentose phosphate pathway producing the principal intracellular reductant NADPH for redox regulation that is vital in cell growth (Dube et al., 2013). Lactate dehydrogenase (LDH) is used as a well biomarker of organ or tissue lesions in toxicological studies reflecting water contamination (Osman et al., 2010). Fish are widely used to evaluate the environmental health and provide a good model to monitor the poisoning in aquatic ecosystems because they are very sensitive to toxins and able to metabolize and bioaccumulate xenobiotics (Fazio et al., 2013). The African catfish Clarias gariepinus was selected as an indicator species for the present study because it is easy to get from the natural environment and due to its sensitivity to toxicants (Harabawy and Ibrahim, 2014) and its ability to bioaccumulate high concentrations of residues in body tissues (Abu Zeid et al., 2005). Harabawy and Ibrahim (2014) have studied the toxic effects of sublethal doses of carbofuran (0.16 and 0.49 mg/L, for 35 days) on C. gariepinus. They have recorded that carbofuran caused significant alterations in the hematological and blood biochemical parameters including RBCs count, Hb, Hct, MCHC, MCV, MCH levels, WBCs, neutrophils, eosinophils, basophils, monocytes and lymphocytes counts; and significant alterations in activities of AST, ALT and ALP; also, plasma glucose, total lipids, urea, creatinin, total protein, albumin and globulin levels and consequently A/G ratio also were altered. In addition, they also recorded that carbofuran caused genotoxic and cytotoxic impacts such as formation of micronuclei (MN) and many morphological alterations (malformations) in the erythrocytes of C. gariepinus. Even now, there are no available literatures concerning the evaluation of toxic impacts of carbofuran on hormones of C. gariepinus. Accordingly, authors aimed to study the effects of carbofuran pesticide, as an endocrine disruptor, on endocrinology

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of the catfish, C. gariepinus for the first time including cortisol (CRT), triiodothyronine (T3), thyroxin (T4), 17β-estradiol (E2) and testosterone (TST), as well as, to evaluate the toxic potential of carbofuran on the antioxidant defense system including SOD, CAT, GSTs, GSH in addition to G6PDH, LDH, lipid peroxidation and DNA damage as biomarkers, which reflect the responses of C. gariepinus to the toxicity of carbofuran pesticide at two sublethal concentrations (0.16 and 0.49 mg/L, for 35 days).

at a wavelength of 340 nm and at 37 1C using kits, Stanbio LDH (UV-Rate) procedure no. 0940 USA for the quantitative determination of lactate dehydrogenase (Kachmar and Moss, 1976) and RANDOX Laboratories Ltd., PD410, UK BT294QY, for the quantitative determination of glucose-6-phosphate dehydrogenase (Kornberg, 1955).

2. Material and methods

2.2.2.4. Glutathione-S-transferase (GST) and reduced glutathione (GSH). GST activity was determined by the method of Habig et al. (1974) by following the increase in absorbance at 340 nm due to the formation of the conjugate 1-chloro-2, 4dinitrobenzene (CDNB) using as substrate at the presence of reduced glutathione (GSH). GSH was determined in PMS by the method of Jollow et al. (1974) as modified by Ahmad et al. (2000).

2.1. Materials 2.1.1. Specimens collection In the present work, 30 specimens of Nile catfish C. gariepinus were collected from the River Nile at Assiut (353712.9 g and 3773.15 cm). Fish specimens kept in 220 L glass tank (100 cm  40 cm  55 cm) containing tap water (temperature 22.370.2 1C; pH 7.570.18; dissolved oxygen (DO) 6.570.88 mg/1; photoperiod 12:12 Light:Dark) and were fed on a commercial pellet diet (3 percent of body weight per day). After two weeks acclimatization, fishes were classified into 3 groups (10 fish/tank, 5 males and 5 females): control and two carbofuran-treated groups. 2.1.2. Chemicals Carbofuran, 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl-N-methylcarbamate (98 percent purity), was obtained from Hebei Chinally International Trade Co., Hebei, China. 2.1.3. Experimental setup Two sublethal concentrations of carbofuran pesticide were applied (0.16 and 0.49 mg/L).These sublethal doses were selected based on study of Daryani (2003) that showed that the 96-h LC50 for carbofuran was 1.63 mg/L for C. gariepinus. Exposure duration was 35 days and the water and carbofuran were completely replenished each day. Stock solution (1000 mg/L) of carbofuran was prepared and stored in clean glass bottles and diluted to concentrations of 0.16 and 0.49 mg/L as 1/10 and 3/10 of LC50, respectively. 2.1.4. Ethical statement All experiments were carried out in accordance with the Egyptian laws and University guidelines for the care of experimental animals. All procedures of the current experiment have been approved by the Committee of the Faculty of Science, Assiut University, Egypt. 2.2. Methods After 35-day period, blood and tissue samples (liver, kidney, gills, gonads (testes and ovaries) and muscles) of the control and treated fish (10 fish/group) were collected. 2.2.1. Hormone assays Blood samples were collected from severance of the caudal peduncle and centrifuged (20 min at 1500 rpm), followed by collection of serum, freezing and storing at
70 1C before hormone analysis. Concentration of cortisol, T3, T4 in males and females, and Testosterone (Tes, only in males) and 17β-estradiol (E2, only in females) were determined using competitive chemiluminescent enzyme immunoassay (Immulite 1000, Siemens, Los Angeles, CA, USA). All samples were run in duplicate and assayed at the same time, in a single run with a single lot number of reagents and consumables employed by a single operator, with intra-assay coefficients of variation for all variables less than 5 percent. 2.2.2. Glycogen determination Tissues analyzed for glycogen levels were first wet weighed (1 g of each of selected tissues) and then placed into centrifuge tubes containing 3 ml of KOH solution (30 percent). The glycogen levels in the samples were determined by Anthron method (Plummer, 1971). 2.2.2.1. Tissues preparation. One gram of gills, kidney, liver, gonads (testes and ovaries) and muscles were carefully excised, surface dried with filter paper, thoroughly washed with 50 mM phosphate buffer pH 7.4 and homogenized with 50 mM phosphate buffer pH 7.4 containing, 1 mM EDTA, 1 mM DTT, 0.15 M KCl, 0.01 percent PMSF. Homogenization was carried out at 4 1C using 12–15 strokes of a motor driven Teflon Potter homogenizer and centrifuged at 10,000 rpm for 20 min at 4 1C. Supernatant was used for antioxidant activities and oxidative stress studies. 2.2.2.2. LDH and G6pDH. Enzymes activity were determined in the supernatant with a spectrophotometer (Micro Lab 200 Vital Scientific, Dieren, The Netherlands)

2.2.2.3. Catalase (CAT) and superoxide dismutase (SOD). Catalase activity was measured using Biodiagnostic Kit no. CA 25 17 that is based on the spectrophotometric method described by Aebi (1984). Determination of superoxide dismutase activity using Sigma-Aldrich Chemie Kit no. 19160 SOD determination. Results were expressed as U SOD/mg protein (Misra and Fridovich, 1972).

2.2.2.5. Lipid peroxidation measurements. Lipid peroxidation (LPO) in the selected tissues was determined by the procedure of Utley et al. (1967). The absorbance of each aliquot was measured at 535 nm. The rate of lipid peroxidation was expressed as nmol of thiobarbituric acid reactive substance (TBARS) formed per hour per milligram of protein using a molar extinction coefficient of 1.56 M
1 cm
1. (Buege and Aust, 1978). 2.2.2.6. DNA fragmentation measurement. DNA fragmentation was determined by the procedure of Kurita-Ochiai et al. (1999) using spectrophotometer (Micro lab 200 vital scientific Dieren, The Netherlands) at 575 nm or 600 nm against reagent blank. The percentage of fragmented DNA was estimated by the following formula: percent of fragmented DNA ¼ fragmented DNA/(fragmented þintact DNA)  100. 2.2.2.7. Statistical analysis. The basic statistics (means and standard errors) of the measured parameters were estimated. The patterns of variation due to carbofuran were tested by using one-way ANOVA, which determined the effects of carbofuran as the factor simultaneously tested. The differences between means were done by using the Tukey-HSD test. Range test was used as a post-hoc test to compare between means at Pr 0.05. The software SPSS, version 10 (SPSS, 1998) was used.

3. Results The mean hormonal indices, cortisol (CRT), triiodothyronine (T3), thyroxin (T4), 17β-estradiol (E2) and testosterone (TST) of the control and the treated African catfish C. gariepinus exposed to two sublethal concentrations of carbofuran, 0.16 and 0.49 mg/L, for 35 days are shown in Table 1. From the analyses, the differences between selected parameters of control and treated fish were statistically important. Table 1 shows that the cortisol levels (CRT) increased significantly (p o0.05) with the increase of carbofuran doses (0.16 mg/L and 0.49 mg/L) in males, females and both sexes together in comparison with the control group. The treatment with carbofuran caused a significant (po 0.05) increase in triiodothyronine concentration (T3) in males, females and both sexes together; but the increase of T3 was not related to carbofuran doses. The results showed that, exposure of the catfish, C. gariepinus, to sublethal concentration of carbofuran (0.16 mg/L for 35 days), had no effects (p4 0.05) on thyroxin levels (T4) in all cases of males, females and both sexes; while the higher sublethal dose (0.49 mg/L) of carbofuran induced significant (p o0.05) decreases in T4 levels in all cases of males, females and both sexes comparing to the control group. The value of mean testosterone level (TST) exhibited a significant decrease (p o0.05) in male fish exposed to 0.16 and 0.49 mg/L carbofuran (201.410 and 160.560 ng/dl respectively) comparing to the healthy control (263.000 ng/dl). In females, a significant decrease (p o0.05) in mean 17β-estradiol level (E2) was recorded in serum of fish exposed to 0.16 and 0.49 mg/L carbofuran (806.110 and 508.340 ng/dl, respectively) comparing to the control one (981.070 ng/dl). The effects of sublethal doses (0.16 and 0.49 mg/L) of carbofuran pesticide on liver, kidney, gills, gonads (testis and ovary) and muscles of C. gariepinus after 35 days of exposure are shown in Table 2. Glycogen levels showed a highly significant decrease in

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Table 1 The basic data of certain blood hormones of males, females and combined sexes of Clarias gariepinus exposed to carbofuran: T1 ¼ 0.16 mg/L and T2¼ 0.49 mg/L for 35 days of exposure. Parameters

Control

T1

T2

CRT (ng/ml) Males (N ¼ 5) Females (N ¼ 5) Combined sexes (N ¼10)

8.225 7 0.097A 8.5017 0.207A 8.4187 0.191A

11.432 70.365B 11.842 70.198B 11.734 70.265B

21.0657 1.413C 21.563 7 1.039C 21.383 7 1.223C

T3 (ng/ml) Males (N ¼ 5) Females (N ¼ 5) Combined sexes (N ¼10)

0.805 7 0.044A 0.828 7 0.022A 0.8217 0.019A

0.945 70.032B 0.912 70.014B 0.932 70.019B

0.9737 0.052B 1.004 7 0.043B 0.9977 0.019B

T4 (ng/ml) Males (N ¼ 5) Females (N ¼ 5) Combined sexes (N ¼10)

0.320 7 0.011A 0.3277 0.014A 0.323 7 0.007A

0.309 70.009A 0.302 70.008A 0.304 70.007A

0.249 7 0.013B 0.259 7 0.016B 0.255 7 0.007B

TST (ng/dl) Males (N ¼ 5)

263.0007 5.611A

201.410 74.379B

160.560 7 3.426C

E2 (ng/dl) Females (N ¼ 5)

981.0707 22.231A

806.110 718.229B

508.340 7 13.360C

The different letters (A, B and C) indicates there is a significant difference at p r 0.05.

liver and gills (p o 0.001); this decrease was related significantly with the increase of exposure dose. In kidney, significant (p o0.05) decreases in glycogen levels are recorded in both carbofurantreated fish groups (0.16 and 0.49 mg/L) comparing with those of control and are not related to carbofuran dose. In muscles, the lower sublethal dose of carbofuran (0.16 mg/L) had no effects (p 40.05) on glycogen content; while the higher sublethal dose (0.49 mg/L) of carbofuran induced significant (po 0.05) decreases in glycogen levels in comparison with the control. On the other hand, insignificant changes (p 4 0.05) are recorded in glycogen content in gonads (testes and ovaries) of treated fish when compared with the control. The results showed that the two metabolic enzymes G6PDH and LDH in the different tissues (liver, kidney, gills, testis, ovary and muscles) of C. gariepinus exhibited significant decreases (po 0.05) in carbofuran-treated groups when compared with the control group; and these decreases increased with the increase of carbofuran doses. The changes in SOD, CAT, GSH and GST levels in all tissues under investigation of C. gariepinus after 35-day exposure to sublethal doses (0.16 and 0.49 mg/L) are given in Table 2. The obtained results revealed that treatment of C. gariepinus with carbofuran induced significant decreases (p o0.05) in SOD, CAT, GSH and GST levels in liver, kidney, gills, testes, ovaries and muscles as compared to the control. All these decreases are significantly related to the increase of carbofuran dose through the exposure period (35 days) except GST in ovary and muscles which was not affected with the dose of carbofuran. On the other hand, the data (Table 2) presented that LPO level increased significantly (po 0.05) in all tissues except gonads after five weeks of exposure to carbofuran when compared to the control group. Also, there was a significant (po0.05) increase in DNA fragmentation percentage in all tissues under investigation of treated groups comparing with those of control group.

4. Discussion The endocrine system is a major homeostatic control system in the body, acting to maintain normal functions and development to face the constantly changing environment and working in tandem with the nervous system (Lukáčová et al., 2013). The impacts of endocrine disruptors on organisms may be attributed to their ability to mimicking endogenous hormones such as estrogens and

androgens; antagonizing normal endogenous hormones; changing the pattern of synthesis and metabolism of natural hormones; and modifying hormone receptor levels (Mueller, 2004; Lukáčová et al., 2013). Their effects can extend to the thyroid, nervous, immune system and metabolism in general (Hotchkiss et al., 2008). In the present work, serum estrogen 17β-estradiol (E2) decreased significantly after exposure to carbofuran sublethal doses. It is known that, in female, the ovary is the main site for estrogen production and aromatase, the enzyme catalyzing estrogen formation; and the mRNA transcripts of aromatase in follicles were dependent on the developmental stage of the ovarian follicle (Young et al., 1983; Nagahama, 1997). Chatterjee et al. (2001) have recorded that 17βEstradiol and vitellogenin levels in serum and ovary of catfish, Heteropneustes fossilis were reduced after exposure to sublethal concentration of carbofuran suggesting an antiestrogenic action of carbofuran; also, they have observed that carbofuran reduced gonadal maturation by arresting the development of ovarian follicles at stage I, which might have resulted in the depressed production of aromatase in the ovarian tissue. Inhibition of aromatase activity by fadrozole reduced secretion of estrogen by ovarian follicles in coho salmon in vitro (Afonso et al., 1997). Reduction of serum estrogen is also possible through the rapid degradation of the hormone to its metabolic product catecholestrogen (Chatterjee et al., 2001). Testosterone is the main androgen in males secreted by the testes. In the present work, serum testosteron decreased significantly after exposure to carbofuran sublethal doses; this may reflect the androgen mimicking properties of carbofuran. Change of sex steroids levels in serum may return to intervention with the steroid synthesis through the pituitary-gonadal axis, or to the impacts on steroid metabolism and excretion; therefore, the decrease of testosterone in contaminated organisms can be attributed to an inhibition of steroid biosynthesis (Hedayati and Arsham, 2012). Elevation of cortisol levels helps provide protection against the impacts of toxicants; however, prolonged cortisol elevation may lead to immunosuppression, reduced reproductive investment or production of sex steroids and reduced growth as reported by Marentette et al. (2012). In the present study, C. gariepinus, under carbofuran toxicity, exhibited a significant increase in serum cortisol level. Many metal and organic compounds can disrupt the hypothalamopituitary-interrenal axis that controls the response of cortisol to stress, and consequently alter normal fish behavior (Scott

2.206 7 0.047A 2.463 7 0.053B 2.886 7 0.062C 2.1777 0.046A 4.955 7 0.112B 10.229 7 0.232C 1.0747 0.023A 0.906 7 0.018B 0.8707 0.019B 1.2127 0.040A 1.034 7 0.034B 0.788 7 0.026C 165.1707 3.743A 149.250 7 3.382B 117.2707 2.657C The different letters (A, B and C) indicates there is a significant difference at p r 0.05.

3.383 7 0.077A 2.8277 0.094B 1.909 7 0.064C 7.701 70.175A 7.104 70.161B 5.699 70.129C 7.7617 0.176A 6.885 7 0.156B 4.889 7 0.111C CONT T1 T2 Muscles (N ¼ 10)

4.462 7 0.095A 4.2757 0.091A 3.950 7 0.084B

2.453 7 0.281A 2.682 7 0.096A 2.342 7 0.085A 4.1777 0.098A 7.094 7 0.453B 13.2437 0.945C 1.4247 0.087A 1.3007 0.008B 1.1937 0.041B 1.4427 0.122A 1.1587 0.009B 0.6447 0.198C 164.8127 1.134A 132.323 7 3.780B 117.1677 6.212C 1.834 70.032A 1.345 70.163B 1.311 70.124B CONT T1 T2 Ovaries (N¼ 5F)

12.2137 0.156A 11.8127 0.243A 11.2117 0.125A

2.583 7 0.165A 1.839 7 0.103B 1.532 7 0.112C

6.542 7 0.276A 5.2467 0.098B 3.564 7 0.134C

5.843 7 0.452A 5.468 7 0.222A 5.377 7 0.256A 9.6577 0.823A 17.2217 0.754B 25.1657 0.534C 1.894 7 0.049A 1.5417 0.112B 1.087 7 0.043C 1.8767 0.326A 1.2137 0.121B 0.928 7 0.329C 197.453 7 8.267A 151.654 7 4.734B 109.7777 5.369C 9.087 7 0.298A 6.456 7 0.271B 4.1237 0.241C 1.477 70.026A 1.008 70.019B 1.045 70.107B 2.1457 0.028A 1.7347 0.062B 1.1237 0.052C CONT T1 T2 Testes (N ¼ 5M)

18.8767 1.325A 17.2137 0.987A 17.1217 1.023A

2.305 7 0.049A 3.014 7 0.100B 4.630 7 0.154C 1.463 7 0.033A 5.802 7 0.193B 14.6177 0.487C 1.950 7 0.065A 1.645 7 0.055B 1.385 7 0.046C 2.1577 0.046A 1.832 7 0.039B 0.755 7 0.016C 242.3107 5.170A 204.9707 4.645B 176.230 7 3.993C 1.143 70.024A 0.986 70.021B 0.788 70.026C CONT T1 T2 Gills (N¼ 10)

4.6797 0.100A 4.0197 0.086B 3.1827 0.106C

2.039 7 0.044A 1.793 7 0.038B 1.223 7 0.026C

11.308 7 0.241A 8.796 7 0.188B 4.9757 0.106C

4.758 7 0.102A 8.205 7 0.175B 13.347 7 0.285C 3.5077 0.117A 13.662 7 0.455B 21.7477 0.493C 1.783 7 0.038A 1.409 7 0.030B 1.1057 0.024C 1.694 7 0.056A 1.1337 0.038B 0.542 7 0.018C 186.1607 3.972A 157.6007 3.363B 102.390 7 2.185C 8.6477 0.196A 6.388 7 0.145B 3.1997 0.072C 3.122 70.067A 2.699 70.058B 1.905 70.041C 4.285 7 0.091A 3.6357 0.078B 2.1427 0.046C CONT T1 T2 Kidney (N ¼ 10)

18.3317 0.610A 16.587 7 0.552AB 14.6477 0.488B

5.368 7 0.115A 12.368 7 0.280B 20.119 7 0.456C 3.9107 0.083A 19.040 7 0.406B 35.3327 1.176C 1.3007 0.028A 1.1827 0.039B 0.559 7 0.019C 1.881 7 0.040A 1.320 7 0.028B 0.4337 0.009C 150.2407 3.404A 118.4007 2.683B 73.620 7 1.668C 6.038 7 0.129A 4.383 7 0.094B 1.630 7 0.035C 2.669 70.089A 2.049 70.068B 1.415 70.047C 3.1427 0.067A 2.7287 0.058B 1.665 7 0.036C CONT T1 T2 Liver (N ¼ 10)

32.2787 0.689A 28.220 7 0.602B 20.665 7 0.688C

LPO (Nmol/mg) DNA fragmen. (%) GST (nmol/mg) GSH (nmol/mg) CAT (U/mg) SOD (U/mg) LDH (U/L) G6PDH (U/L) Glycogen (mg/g) Doses Organ

Table 2 The basic data of tissues constituent parameters (enzymatic, antioxidant and oxidative stress) of Clarias gariepinus exposed to carbofuran for 35 days: T1 ¼0.16 mg/L and T2 ¼0.49 mg/L; (N ¼ 10); M, male; F, female.

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and Sloman, 2004). Elevations in serum cortisol levels as stress response to pesticides and heavy metals were reported in different fishes, Oreochromis mossambicus (Fu et al., 1990) and Atlantic salmon, Salmo salar (Waring and Moore, 2004). Some xenobiotics share structural similarity to the thyroid hormones (T4 and T3), behave as endocrine disruptors and can interfere with the thyroid axis (Hedayati and Arsham, 2012). The action of these compounds is achieved by binding to transporter proteins such as transthyretin that transport TH in the blood (Morgado et al., 2007). In the present study, C. gariepinus, under carbofuran toxicity, exhibited a significant reduction in serum T4 levels and a significant increase in T3 levels. Similar results after exposed of Sarotherodon mossambicus to dimecron pesticide are recorded (Thangavel et al., 2005). Chronic exposure of Channa punctatus to 4.5 mg/L or 5 mg/L carbofuran for four or six months also caused thyroid cell hypertrophy (Saxena and Mani, 1988) and lower blood T4 levels (Bhattacharya, 1993). Also, carbaryl concentrations of 5 and 12 mg/L decreased plasma T4 levels and increased plasma T3 levels in Clarias batrachus (Sinha et al., 1991); this may be due to either increased conversion of T4 to T3 or decreased metabolism of T3. Reduction of T4 in the present study could be due to the competitive binding of carbofuran with T4, making it inactive, or with transthyretin (a plasma T4-binding protein) as reported in other fish (Ucan-Martin et al., 2009). Two important notes from previous studies are used for comparison with our results: the stress hormone cortisol stimulates conversion of T4 to T3 (Hontela et al., 1995); and 17β-estradiol can reduce plasma T3 (Leatherland, 1985). The results of the present study revealed that: cortisol, that stimulates the conversion of T4 to T3, increased significantly in serum of C. gariepinus with the increase of carbofuran concentration; (2) at the same time, T4 decreased and T3 increased significantly; and (3) 17β-estradiol was reduced significantly with the increase of carbofuran concentration. These findings agree with those of Hontela et al. (1995) and Leatherland (1985) in that, the high level of cortisol led to more conversion from T4 to T3, thereby T4 decreased and T3 increased; but the low levels of 17β-estradiol in our study may be not enough to decrease T3. In the present study, glycogen contents in liver, kidney, gills and to some degree in muscle of C. gariepinus decreased significantly after exposure to carbofuran doses, but insignificant changes in gonads was recorded. The decrease was greater in liver, gills and kidney than in muscle, as liver is the principal organ for glycogen storage. Leatherland (1985) has detected that 17βestradiol significantly reduced hepatic glycogen levels in rainbow trout, Salmo gairdneri. In our study, the decline of glycogen in different tissues of C. gariepinus may be a result of glycogen breakdown to meet the energy demand under toxic stress of carbofuran pesticide as proposed by Tendulkar and Kulkarni (2012). Glycogen can also be depleted in response to some physiological processes such as sexual maturation or nonchemical stresses such as temperature and hypoxia; and hence, glycogen is a non-specific parameter indicating stress of the organism (Khidr et al., 2008). Glycogen breakdown is rapidly activated by changes of both extracellular and intracellular environments (Dube et al., 2013). On the other hand, reduction of glycogen contents due to exposure to pesticides may be attributed to the interference with activity of key enzymes that are involved in glycogenesis and lead to inhibition of glycogen storage in liver (Rezg et al., 2006). Alteration of G6PDH and LDH activities could be well biomarkers reflecting water contamination (Osman et al., 2010). In the present study, the activities of G6pDH and LDH enzymes were decreased significantly in all tissues of carbofuran treated C. gariepinus in comparison with those of control fish. Similar results were observed in catfish exposed to concentrations of 21 ppb,

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there were significant depressions in gulucose-6-phosphatase in the gills, liver and kidneys (Verma et al., 1981). The changes in these metabolic key enzymes, G6PDH and LDH, in the tissues of C. gariepinus exposed to carbofuran indicate that great changes in carbohydrate and protein metabolism are occurred. The antioxidant defense system of treated C. gariepinus with carbofuran in this study was altered in terms of decreased activity of glutathione S-transferases (GST), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) and increased lipid peroxidation and DNA fragmentation. Kaur et al. (2012) recorded that lipid peroxidation, superoxide dismutase and catalase are decreased in kidney of rats after exposure to carbofuran. Carbofuran was found to inhibit GST of Gambusia yucatana tissues (Rendón-von Osten et al., 2005). The present study showed that GSH level decreased in the liver, kidney, gills, goands and muscles of C. gariepinus exposed to carbofuran dosages. These depletions in GSH activity in different tissues may reflect its utilization in countering the prevailing oxidative stress under the influence of ROS generated from carbofuran oxidative stress. Therefore, GSH provide the foremost defense against ROS-induced cellular damage (Regoli and Principato, 1995). Results of the present investigation revealed the decrease of SOD activity due to the increased production of ROS as evident by the increased LPO levels due to carbofuran treatment. Exposure to pesticides or various pollutants caused a significant reduction in SOD activities (Isik and Celik, 2008) and in CAT (Hai et al., 1997) in different fish. In addition, singlet oxygen and peroxyl radicals can also inhibit SOD and CAT activities (Ray, 1991).The activity of CAT may be related to the production of H2O2 during the detoxification process for xenobiotics (Monteiro et al., 2006). The superoxide radicals have been shown to inhibit the activities of CAT (Kale et al., 1999). CAT can protect DNA from damage by acting as ROS scavengers (Bhuvaneshwari et al., 2013). The higher levels of lipid peroxidation in liver, kidney, gills and muscles of C. gariepinus exposed to carbofuran in the present study suggests that ROS are not totally scavenged by the antioxidant enzymes and be considered as one of the main toxic effect of carbofuran. Also the increase of ROS reflects the alteration in cell membrane characteristics because lipid peroxidation leads to hydrolysis of phospholipids into hydroperoxy fatty acids (Mekkawy et al., 2013). Previous investigations have reported the induction of LPO by pesticides such as dichlorovos (Bebe and Panemangalore, 2003), butachlor (Farombi, et al., 2008) and atrazine (Mekkawy et al., 2013). ROS and oxidative stress have been demonstrated to trigger the apoptosis (Shen and Liu, 2006). Oxidative stress has the potential to attack the fluidity of cellular membrane systems in addition to the integrity of DNA in nucleus (Pašková, 2012). In the present study, there was a significant increase in DNA fragmentation percentage in all tissues of treated groups comparing with the control fish. This DNA damage reflects DNA strand breaks that occurred from oxidative stress on C. gariepinus exposed to carbofuran. Therefore, the current results provide a clear evidence on the response of C. gariepinus to sublethal doses of carbofuran and allow us to consider the catfish C. gariepinus as a good bioindicator to reflect the endocrine disrupting impacts of carbofuran pesticide, and reflect the potential of carbofuran to cause disturbance in the antioxidant defense system as well as the metabolism in terms of vital metabolic enzyme activities (G6pDH and LDH), induction of lipid peroxidation (LPO) and DNA damage in the fish in contaminated ecosystems. References Abu Zeid, I.E.M., Syed, M.A., Ramli, J., Arshad, J.H., Omar, I., Shamaan, N.A., 2005. Bioaccumulation of carbofuran and endosulfan in the mrican catfish Clarias gariepinus. Pertanika J. Sci. Technol. 13, 249–256.

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