Bull Environ Contam Toxicol (2014) 92:388–392 DOI 10.1007/s00128-014-1232-7
Hepatic Antioxidant Enzymes SOD and CAT of Nile Tilapia (Oreochromis niloticus) in Response to Pesticide Methomyl and Recovery Pattern Shun Long Meng • Jia Zhang Chen • Pao Xu • Jian Hong Qu • Li Min Fan Chao Song • Li Ping Qiu
•
Received: 1 July 2013 / Accepted: 7 February 2014 / Published online: 17 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Hepatic antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) of Nile tilapia in response to pesticide methomyl and recovery pattern were researched by exposing tilapia to sub-lethal methomyl concentrations of 0, 0.2, 2, 20 and 200 lg/L for 30 days, and then transferred to methomyl-free water for 18 days. Hepatic SOD and CAT were measured at 10 min (day 0), 6, 12, 18, 24 and 30 days after starting the experiment and at 18 days after transferring to methomyl-free water. The results showed hepatic SOD and CAT activities in 2, 20 and 200 lg/L groups were affected significantly, however, that in 0.2 lg/L group didn’t change significantly compared to control during 30-day exposure period. Thus it would appear the 0.2 lg/L methomyl might be considered the no observed adverse effect level. Recovery data showed that, for SOD, the effects produced by lower concentration of methomyl 2 lg/L were reversible but not at concentrations higher than 20 lg/L, however, for CAT, the effects produced by all the concentrations were reversible.
Shun Long Meng and Jia Zhang Chen have contributed equally to this work. S. L. Meng J. Z. Chen P. Xu J. H. Qu L. M. Fan C. Song L. P. Qiu Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China S. L. Meng J. Z. Chen (&) P. Xu (&) Freshwater Fisheries Research Center, Scientific Observing and Experimental Station of Fishery Resources and Environment in the Lower Reaches of the Changjiang River, Chinese Academy of Fishery Sciences, No. 9, East Shanshui Road, Binhu District, Wuxi 214081, Jiangsu, China e-mail: [email protected] P. Xu e-mail: [email protected]
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Keywords Methomyl Liver Tilapia Antioxidant enzymes Recovery Pesticides are used worldwide in agricultural activity, mostly to promote the harvest of products. However, these compounds are released into the environment and due to their physico-chemical properties, such as water solubility, vapor pressure or partition coefficients between organic matter (in soil or sediment) and water, they can disperse in various environmental media provoking serious health problems (Gramatica and Di Guardo 2002). Carbamates are systemic and contact pesticides used as substitutes for organochlorine insecticides because of their high efficiency and relative low persistence in the environment (Ribera and Narbonne 2001). Methomyl (C5H10N2O2S), S-methyl-1-N[(methylcarbamoyl)-oxy]-thioacetimidate, is an insecticide belonging to the family of carbamate pesticides, and it is one of the environmental estrogens having endocrine disrupting effects. Because of its broad biological activity, relatively rapid disappearance and high efficiency against insects, methomyl is widely used in many agricultural countries for crop protection and soil or plant treatment (WHO 1996). Methomyl has high water solubility (57.9 g/ L at 25°C) and a weak-to-moderate adsorption to soils, and therefore poses a contamination risk to surface and ground water, especially the methomyl applied in the agricultural area is expected to infiltrate into the groundwater and threatens the safety of the resource for drinking water (Strathmann and Stone 2001). In aerobic cells, reactive oxygen species (ROS), such as superoxide anion, hydroxyl radical etc., are generated during normal metabolism, particularly as a result of oxidative metabolism at mitochondrial membranes, and these intermediates might be detrimental to the cell, leading to a
Bull Environ Contam Toxicol (2014) 92:388–392
state called oxidative stress (Zhang et al. 2004). Most components of cellular structure and function are likely to be the potential targets of oxidative damage, and the most susceptible substrates for autoxidation are polyunsaturated fatty acids of the cell membrane, which undergo peroxidation rapidly. This may lead to muscle degradation, impairment of the nervous system, haemolysis, general deterioration of the cellular metabolism and eventual cell death (Zhang et al. 2004). However, aerobic organisms have evolved defense system to protect themselves from the toxic effects of the increased ROS production, activating the antioxidant system, such as antioxidant scavengers, e.g. glutathione, Phase II detoxification enzymes, e.g. GST, and specific antioxidant enzymes, e.g. SOD, CAT, GPx, GR (Freeman and Crapo 1982; Winston and Di Giulio 1991; Frei 1999; Hogg and Kalyanaraman 1999), and the first line of defense against oxidative stress consists of the antioxidant enzymes SOD and CAT (Stara et al. 2012). Defense systems protect against attack from either endogenous (physiological production) or exogenous (xenobiotic-related) sources of ROS. Thus, there is mediation between ROS production and antioxidant scavengers in aerobic cells. Under normal physiological situation, ROS is removed by antioxidant defense systems; however, a severe oxidative stress influences the activities of redox enzymes and lead to oxidative damage (Livingstone 2001). There were some reports about the acute toxicity of methomyl on aquatic organisms (Hashimoto and Nishiuchi 1981; Farre´ et al. 2002; Pereira and Gonc¸alves 2007; Li et al. 2008), however, the chronic toxic effects of methomyl on aquatic organisms, especially on fish, were scarcely investigated. Thus, the aim of the present study was to investigate the chronic toxic effects and recovery pattern of methomyl in tilapia Oreochromis niloticus, by analyzing the responses of the fish liver antioxidant defense enzymes SOD and CAT.
Materials and Methods Male Nile tilapia, O. niloticus, was chosen for this study because methomyl is one of the environmental estrogens having endocrine disrupting effects (The serum sex steroid hormones, such as 17b-estradiol, testosterone and 11-ketotestosterone, have been analyzed simultaneously, and the data are not presented in this paper) and tilapia is commonly available in most fish farms worldwide. Specimens of O. niloticus with an average weight of 150.7 ± 9.7 g and length of 19.0 ± 1.4 cm were supplied by the fish farm of freshwater fisheries research center, Chinese Academy of Fishery Science (Wuxi, China). Before the experiments fish were acclimated under laboratory conditions for 4 weeks at a population density of 30 specimens in 200-L
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glass aquaria supplied with dechlorinated tap water. The physicochemical characteristics of the water used in the aquaria were analyzed according to methods in ‘‘Standard method for the examination of water and wastewater’’ (State Environmental Protection Agency of China 2002). The water had a pH of 7.3 ± 0.3 and a temperature of 25 ± 0.5°C. Water hardness was 107 mg/L (as CaCO3), and dissolved oxygen concentration was 6.5–7.0 mg/L. Stock fish and experimental fish were fed 2 % body mass daily, with a commercial fish feed (Ningbo Tech-bank co., LTD, China) and submitted to a 12-h light and 12-h dark photoperiod. Fish were used when no mortality was observed in the acclimation population. Methomyl (97 % w/w) was produced by Shanghai Focus Biological Technology Co., Ltd, China. All other chemicals used were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA) and Sangon (Shanghai, China). Male tilapia were randomly distributed into 200-L glass aquaria containing different concentrations of methomyl (0, 0.2, 2, 20 and 200 lg/L), with 3 replicates per treatment, and the range of exposure concentrations was based on the information from the previous study on 96 h LC50 (430 lg/L) for tilapia (with an average weight of 3.9 ± 0.4 g and length of 6.3 ± 0.6 cm), the residue level (0–55.3 lg/L) of methomyl in environmental water (Van Scoy et al. 2013) and the U.S. EPA on drinking water quality established a maximum permissible concentration for methomyl of 200 lg/L (U.S.EPA 2012). The actual methomyl concentrations in the test water were measured by the method of Chen et al. (1996). The actual methomyl concentrations of 0, 0.2, 2, 20, 200 lg/L groups were 0, 0.24, 2.06, 21.55, 205.57 lg/L respectively at 0 h of exposure (the initial concentrations), and were 0, 0.20, 1.95, 19.55, 198.57 lg/L respectively after 24 h of exposure. And the results were discussed in relation to the nominal concentrations. Thirty fish were introduced into each concentration in a semi-static system and water was renewed daily. The experiment lasted for 48 days, after 30 days of exposure period the remaining fish were transferred to methomyl-free water for 18 days and then the same parameters were measured to study the recovery response. Sampling of exposed and control fish (n = 6/group, 2 tilapia per replicate) was done at 10 min (day 0), 6, 12, 18, 24, and 30 days during exposure and 18 days (R18) after transferring to methomyl-free water for recovery. Feed was withheld 24 h prior to sampling. Tilapia were euthanized in approximately 250 mg/L ethyl 3-aminobenzoate methanesulfonate salt (MS-222, TCI Inc., Japan), and thereafter measured and weighed. Then, fish were rapidly killed by decapitation and the liver immediately removed and processed, and then snap-frozen in liquid nitrogen and stored at -80°C for later assay.
123
123
Means significant difference between recovery group and 30-day exposure group #
* Means significant difference from the control
All data were expressed as mean ± standard deviation (n = 6). A two sample Student’s t test was used to test for significant differences between groups. Differences were statistically significant when p \ 0.05
105.42 ± 6.70# 133.43 ± 8.36* 135.78 ± 5.43* 165.67 ± 5.39* 355.67 ± 8.39* 100.86 ± 6.49 200
173.54 ± 5.39*
101.15 ± 7.43# 134.33 ± 7.21* 133.55 ± 7.81* 258.89 ± 8.11* 179.87 ± 7.83* 99.87 ± 7.07 20
138.45 ± 7.99*
101.23 ± 8.18#
99.78 ± 5.72 104.66 ± 7.04
195.78 ± 6.46* 179.86 ± 5.53*
105.65 ± 8.55 107.56 ± 7.86
163.24 ± 6.28* 127.76 ± 6.01*
106.23 ± 8.35 105.35 ± 8.30
109.89 ± 8.47
100.26 ± 8.47
100.49 ± 7.34
0.2
2
CAT
73.88 ± 4.13*# 56.71 ± 4.76* 51.33 ± 4.51* 53.56 ± 3.85* 197.56 ± 5.54* 99.65 ± 3.91 200
143.26 ± 4.47*
83.33 ± 4.97* 74.33 ± 4.88* 95.67 ± 4.22* 189.55 ± 3.34* 141.22 ± 5.06* 100.11 ± 4.94 20
118.52 ± 5.26*
98.78 ± 4.08#
101.22 ± 4.86 107.65 ± 5.54
176.22 ± 4.70* 163.22 ± 3.13*
105.94 ± 4.82 109.33 ± 4.9
145.62 ± 4.76* 118.23 ± 4.10*
105.31 ± 4.84 103.22 ± 4.52
106.39 ± 5.47
101.21 ± 4.39
100.33 ± 4.86
0.2 SOD
2
30 24 18 12 6 0
Exposure time (d) Methomyl (lg/L)
Neither mortality nor visible disease signals were observed in the tilapia exposed to sublethal concentrations of methomyl during the performance of the experiment. Changes in SOD and CAT activities in experimental fish are shown in Table 1, and the results of two-way ANOVA are shown in Table 2. The results of two-way ANOVA indicated that time, methomyl concentration and the interaction between the two factors had significant effects on SOD activities (Table 2). Compared to control, SOD activity presented a significant increase at 2, 20, and 200 lg/L of methomyl at days 12, 6 and 6 respectively. The highest induction (197.56 % of the control) of SOD activity was found following exposure to 200 lg/L methomyl at day 12. SOD is
Antioxidant (% of control)
Results and Discussion
Table 1 Liver SOD and CAT activities in O. niloticus exposed to sublethal concentrations of methomyl
Samples were stored at -80°C with an Ultra-low temperature freezer (Forma-86C, America). Samples were homogenized using an electrical homogenizer (Pro-200, America). Centrifugations were done with a refrigerated centrifuge (Sigma 2–16 K, Germany). Spectrophotometric readings were carried out with a UV–vis spectrophotometer (UV-759S, China). Tilapia livers were homogenized using an electrical homogenizer. About 0.30 g of liver tissue was homogenized after addition of 3.0 mL of 10.0 mM Tris buffer (pH 7.5) for detection of enzyme activities. The extracts were centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was used immediately as the enzyme analysis. All the above operations were carried out at 4°C. Catalase activity was assayed by ultraviolet spectrophotometry (Xu et al. 1997), and one unit of enzyme activity is defined as the amount of enzyme which decreased the concentration of H2O2 by 50 % in 100 s at 25°C. Superoxide dismutase (SOD) was assayed by the method of Marklund and Marklund (1974), and one unit of SOD activity is defined as the amount of the enzyme which gave 50 % inhibition of the oxidation rate of 0.1 mM pyrogallol in one ml of solution at 25°C. Protein levels were estimated by the method of Bradford (1976) using bovine serum albumin as a standard. The activities of CAT and SOD of treated tilapia were compared to control group in each sampling day, including recovery group (R18), and expressed as % of control. All data were expressed as mean ± SD (n = 6). Two-way analysis of variance (ANOVA) and two sample Student’s t test were used for statistical analysis with p \ 0.05 being considered significant. In all the tables, ‘‘*’’ means significant difference from the control, ‘‘#’’ means significant difference between recovery group and 30-day exposure group.
Bull Environ Contam Toxicol (2014) 92:388–392
R18
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Bull Environ Contam Toxicol (2014) 92:388–392
391
Table 2 Results of two-way ANOVA Source
SOD df
CAT SS
MS
F
p
df
SS
MS
F
p
Time
5
29,168.3
5,833.7
271.39
0.000
5
126,648
25,330
470.20
0.000
Concentration
3
26,282.0
8,760.7
407.56
0.000
3
101,077
33,692
625.44
0.000
15
157,457.5
10,497.2
488.34
0.000
15
261,504
17,434
323.62
0.000
21.5
120
6,464
54
143
495,694
Time 9 concentration Error
120
2,579.5
Total
143
215,487.3
the first enzyme to deal with oxyradicals (Zhang et al, 2004). SOD serves to protect cells against the oxidative damage of free radicals by catalysing the conversion of superoxide anion to molecular oxygen (O2) and hydrogen peroxide (H2O2), which is then catalyzed either by CAT or GPx. Induction of SOD could occur during high production ¨ ner 2000), the of superoxide anion radical (Oruc and U significant induction (p \ 0.05) of SOD in the present study might have occurred as a direct response to methomyl induced superoxide anion production as reported in the hepatocytes of rainbow trout exposed to selenite (Misra and Niyogi 2009). Velisek et al. (2011) observed increased SOD activity in common carp liver after long-term exposure to terbutryn. Jin et al. (2010) also observed increased SOD activity in zebrafish (Danio rerio) liver after 14 days atrazine exposure. However, after long-term exposure, SOD activities in the liver of fish exposed to 20 and 200 lg/L decreased at day 24 and 30 and at day 18 and 30 respectively, compared to control, a significant decrease (p \ 0.05) in SOD activity was observed after 30 and 18 days of methomyl exposure respectively (Table 1). ElKhawaga (2005) observed a reduction in hepatic SOD activity of mice injected with single dose of methomyl (7 mg/kg body weight) for 24 h. The decrease of SOD activity could be explained in two ways. Firstly, methomyl could not be metabolized completely by tilapia, especially at high concentrations and long-time exposures, so methomyl might accumulate in tilapia liver and consequently its conjugation with SOD, inactivating the enzyme. Secondly, if H2O2 is not enzymatically decomposed, it can be converted to the very short-lived and highly aggressive hydroxyl radical. The superoxide anion, H2O2 and hydroxyl radical could cause oxidation of SOD, reducing its activity (Dimitrova et al. 1994). The results of two-way ANOVA indicated that time, methomyl concentration and the interaction between the two factors had significant effects on CAT activities (Table 2). Compared to control, CAT presented a significant increase at 2, 20, and 200 lg/L of methomyl at days 12, 6 and 6 respectively. The highest CAT activity (355.67 % of control) was found following exposure to
200 lg/L methomyl at day12 (Table 1). Pesticide-induced inhibition and increment of CAT activity has been reported in studies of various fish species. Jin et al. (2010) reported increased CAT activity in liver of zebrafish after 14 days atrazine exposure. Ballesteros et al. (2009) stated that the activity of CAT was significantly decreased in liver of the onesided livebearer (Jenynsia multidentata) exposed to endosulfan. However, Moraes et al., (2009) reported a decrease in liver CAT activity in teleost fish (Leporinus obtusidens) and silver catfish (Schilbe intermedius) after exposure to herbicides. Keramati et al. (2010) reported that Diazinon caused fluctuated levels in fish Rutilus rutilus. It has also been reported that lower concentrations of endosulfan increase the CAT activity in Oncorhynchus mykiss while the higher concentrations reduce its activity (Sharbidre et al., 2011). The increase in CAT activities in the liver as observed in the present study might be in response to H2O2 produced by SOD activity since CAT is responsible for the detoxification of H2O2 to oxygen and water. Our results were accordant to the previous reports by Zhang et al. (2004) and Yi et al. (2007) where hepatic CAT activities were elevated in fish after chronic exposure to 2,4-dichlorophenol and alachlor. When tilapia was exposed to 0.2 lg/L methomyl, there were no significant changes in SOD and CAT activities compared to control (Table 1), which meant low methomyl concentration could not increase ROS. So we concluded 0.2 lg/L methomyl might be suggested as the threshold dose for no effect to tilapia (this threshold dose is only effective to this experiment tilapia size). When the tilapia exposed to 0.2, 2, 20 and 200 lg/L methomyl were transferred to methomyl-free water for 18 days, CAT activities in fish liver returned to control values (Table 1), however, SOD activities in 20 and 200 lg/L treatments could not return to the control values (Table 1). It could be concluded SOD was more sensitive to methomyl compared to CAT, and SOD may be better than CAT as a biomarker. In conclusion, exposure concentration and time affected SOD and CAT activities in fish exposed to methomyl, and 0.2 lg/L methomyl might be considered as the no observed adverse effect level to tilapia.
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392 Acknowledgments The authors acknowledge support from the Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (No. 2013A0303) and China Agriculture Research System (No. CARS-49) for the financial supports.
References Ballesteros ML, Wunderlin DA, Bistoni MA (2009) Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan. Ecotox Environ Safe 72(1):199–205 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 Chen YJ, Lu YH, Ren JL, Chen YL, Sun XH (1996) The liquid-solid extraction of methomyl in environmental water and gas chromatographic analysis. Environ Sci 17(2):68–70 Dimitrova MS, Tishinova V, Velcheva V (1994) Combined effect of zinc and lead on the hepatic superoxide dismutase-catalase system in carp (Cyprinus carpio). Comp Biochem Physiol C 108(1):43–46 El-Khawaga OAY (2005) Role of selenium on antioxidant capacity in methomyl-treated mice. J Physiol Biochem 61:501–506 Farre´ M, Fernandez J, Paez M, Granada L, Barba L, Gutierrez H, Barcelo D (2002) Analysis and toxicity of methomyl and ametryn after biodegradation. Anal Bioanal Chem 373(8):704–709 Freeman BA, Crapo JD (1982) Biology of disease: free radicals and tissue injury. Lab Invest 47:412–426 Frei B (1999) Molecular and biological mechanisms of antioxidant action. FASEB J 13:963–964 Gramatica P, Di Guardo A (2002) Screening of pesticides for environmental partitioning tendency. Chemosphere 47(9): 947–956 Hashimoto Y, Nishiuchi Y (1981) Establishment of bioassay methods for the evaluation of acute toxicity of pesticides to aquatic organisms. J Pestic Sci 6(2):257–264 Hogg N, Kalyanaraman B (1999) Nitric oxide and lipid peroxidation. Biochim Biophys Acta 1411:378–384 Jin Y, Zhang X, Shu L, Chen L, Sun L, Qian H, Fu Z (2010) Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio). Chemosphere 78(7):846–852 Keramati V, Jamili SH, Ramin M (2010) Effect of diazinon on catalase antioxidant enzyme activity in liver tissue of Rutilus rutilus. J Fish Aquat Sci 5(5):368–376 Li H, Jiang H, Gao X, Wang X, Qu W, Lin R, Chen J (2008) Acute toxicity of the pesticide methomyl on the topmouth gudgeon (Pseudorasbora parva): mortality and effects on four biomarkers. Fish Physiol Biochem 34:209–216 Livingstone DR (2001) Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar Pollut Bull 42(8):656–666 Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469–474 Misra S, Niyogi S (2009) Selenite causes cytotoxicity in rainbow trout (Oncorhynchus mykiss) hepatocytes by inducing oxidative stress. Toxicol In Vitro 23(7):1249–1258
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Bull Environ Contam Toxicol (2014) 92:388–392 Moraes BS, Loro VL, Pretto A, da Fonseca MB, Menezes C, Marchesan E, de Avila LA (2009) Toxicological and metabolic parameters of the teleost fish (Leporinus obtusidens) in response to commercial herbicides containing clomazone and propanil. Pestic Biochem Physiol 95(2):57–62 ¨, U ¨ ner N (2000) Combined effects of 2, 4-D and Oruc EO azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus. Comp Biochem Physiol C 127(3):291–296 Pereira JL, Gonc¸alves F (2007) Effects of food availability on the acute and chronic toxicity of the insecticide methomyl to Daphnia spp. Sci Total Environ 386:9–20 Ribera D, Narbonne JF (2001) Saint-Denis M, Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil, effects of carbaryl. Soil Biol Biochem 33: 1123–1130 Sharbidre AA, Metkari V, Patode P (2011) Effect of methyl parathion and chlorpyrifos on certain biomarkers in various tissues of guppy fish Poecilia reticulate. Pestic Biochem Physiol 101(2):132–141 Stara A, Machova J, Velisek J (2012) Effect of chronic exposure to simazine on oxidative stress and antioxidant response in common carp (Cyprinus carpio L.). Environ Toxicol Pharmacol 33(2):334–343 State Environmental Protection Agency of China (2002) Standard method for the examination of water and wastewater. Beijing: China Environmental Science Press 725–729 Strathmann TJ, Stone AT (2001) Reduction of the carbamate pesticides oxamyl and methomyl by dissolved FeII and CuI. Environ Sci Technol 35:2461–2469 U.S. EPA (2012) 2012 Edition of the Drinking Water Standards and Health Advisories. EPA 822-S-12-001. Office of WaterU.S. Environmental Protection Agency, Washington, DC Van Scoy AR, Yue M, Deng X, Tjeerdema RS (2013) Environmental fate and toxicology of methomyl. Rev Environ Contam T 222:93–109 Velisek J, Stara A, Kolarova J, Svobodova Z (2011) Biochemical, physiological and morphological responses in common carp (Cyprinus carpio L.) after long-term exposure to terbutryn in real environmental concentration. Pestic Biochem Physiol 100(3): 305–313 WHO (1996) Methomyl Environmental Health Criteria 178. WHO Geneva: 74 Winston GW, Di Giulio RT (1991) Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat Toxicol 19(2):137–161 Xu JB, Yuan XF, Lang PZ (1997) Determination of catalase activity and catalase inhibition by ultraviolet spectrophotometry. Chinese Environ Chem 16:73–76 Yi X, Ding H, Lu Y, Liu H, Zhang M, Jiang W (2007) Effects of longterm alachlor exposure on hepatic antioxidant defense and detoxifying enzyme activities in crucian carp (Carassius auratus). Chemosphere 68(8):1576–1581 Zhang J, Shen H, Wang X, Wu J, Xue Y (2004) Effects of chronic exposure of 2, 4-dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus. Chemosphere 55(2):167–174
Hepatic Antioxidant Enzymes SOD and CAT of Nile Tilapia (Oreochromis niloticus) in Response to Pesticide Methomyl and Recovery Pattern Shun Long Meng • Jia Zhang Chen • Pao Xu • Jian Hong Qu • Li Min Fan Chao Song • Li Ping Qiu
•
Received: 1 July 2013 / Accepted: 7 February 2014 / Published online: 17 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Hepatic antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) of Nile tilapia in response to pesticide methomyl and recovery pattern were researched by exposing tilapia to sub-lethal methomyl concentrations of 0, 0.2, 2, 20 and 200 lg/L for 30 days, and then transferred to methomyl-free water for 18 days. Hepatic SOD and CAT were measured at 10 min (day 0), 6, 12, 18, 24 and 30 days after starting the experiment and at 18 days after transferring to methomyl-free water. The results showed hepatic SOD and CAT activities in 2, 20 and 200 lg/L groups were affected significantly, however, that in 0.2 lg/L group didn’t change significantly compared to control during 30-day exposure period. Thus it would appear the 0.2 lg/L methomyl might be considered the no observed adverse effect level. Recovery data showed that, for SOD, the effects produced by lower concentration of methomyl 2 lg/L were reversible but not at concentrations higher than 20 lg/L, however, for CAT, the effects produced by all the concentrations were reversible.
Shun Long Meng and Jia Zhang Chen have contributed equally to this work. S. L. Meng J. Z. Chen P. Xu J. H. Qu L. M. Fan C. Song L. P. Qiu Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China S. L. Meng J. Z. Chen (&) P. Xu (&) Freshwater Fisheries Research Center, Scientific Observing and Experimental Station of Fishery Resources and Environment in the Lower Reaches of the Changjiang River, Chinese Academy of Fishery Sciences, No. 9, East Shanshui Road, Binhu District, Wuxi 214081, Jiangsu, China e-mail: [email protected] P. Xu e-mail: [email protected]
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Keywords Methomyl Liver Tilapia Antioxidant enzymes Recovery Pesticides are used worldwide in agricultural activity, mostly to promote the harvest of products. However, these compounds are released into the environment and due to their physico-chemical properties, such as water solubility, vapor pressure or partition coefficients between organic matter (in soil or sediment) and water, they can disperse in various environmental media provoking serious health problems (Gramatica and Di Guardo 2002). Carbamates are systemic and contact pesticides used as substitutes for organochlorine insecticides because of their high efficiency and relative low persistence in the environment (Ribera and Narbonne 2001). Methomyl (C5H10N2O2S), S-methyl-1-N[(methylcarbamoyl)-oxy]-thioacetimidate, is an insecticide belonging to the family of carbamate pesticides, and it is one of the environmental estrogens having endocrine disrupting effects. Because of its broad biological activity, relatively rapid disappearance and high efficiency against insects, methomyl is widely used in many agricultural countries for crop protection and soil or plant treatment (WHO 1996). Methomyl has high water solubility (57.9 g/ L at 25°C) and a weak-to-moderate adsorption to soils, and therefore poses a contamination risk to surface and ground water, especially the methomyl applied in the agricultural area is expected to infiltrate into the groundwater and threatens the safety of the resource for drinking water (Strathmann and Stone 2001). In aerobic cells, reactive oxygen species (ROS), such as superoxide anion, hydroxyl radical etc., are generated during normal metabolism, particularly as a result of oxidative metabolism at mitochondrial membranes, and these intermediates might be detrimental to the cell, leading to a
Bull Environ Contam Toxicol (2014) 92:388–392
state called oxidative stress (Zhang et al. 2004). Most components of cellular structure and function are likely to be the potential targets of oxidative damage, and the most susceptible substrates for autoxidation are polyunsaturated fatty acids of the cell membrane, which undergo peroxidation rapidly. This may lead to muscle degradation, impairment of the nervous system, haemolysis, general deterioration of the cellular metabolism and eventual cell death (Zhang et al. 2004). However, aerobic organisms have evolved defense system to protect themselves from the toxic effects of the increased ROS production, activating the antioxidant system, such as antioxidant scavengers, e.g. glutathione, Phase II detoxification enzymes, e.g. GST, and specific antioxidant enzymes, e.g. SOD, CAT, GPx, GR (Freeman and Crapo 1982; Winston and Di Giulio 1991; Frei 1999; Hogg and Kalyanaraman 1999), and the first line of defense against oxidative stress consists of the antioxidant enzymes SOD and CAT (Stara et al. 2012). Defense systems protect against attack from either endogenous (physiological production) or exogenous (xenobiotic-related) sources of ROS. Thus, there is mediation between ROS production and antioxidant scavengers in aerobic cells. Under normal physiological situation, ROS is removed by antioxidant defense systems; however, a severe oxidative stress influences the activities of redox enzymes and lead to oxidative damage (Livingstone 2001). There were some reports about the acute toxicity of methomyl on aquatic organisms (Hashimoto and Nishiuchi 1981; Farre´ et al. 2002; Pereira and Gonc¸alves 2007; Li et al. 2008), however, the chronic toxic effects of methomyl on aquatic organisms, especially on fish, were scarcely investigated. Thus, the aim of the present study was to investigate the chronic toxic effects and recovery pattern of methomyl in tilapia Oreochromis niloticus, by analyzing the responses of the fish liver antioxidant defense enzymes SOD and CAT.
Materials and Methods Male Nile tilapia, O. niloticus, was chosen for this study because methomyl is one of the environmental estrogens having endocrine disrupting effects (The serum sex steroid hormones, such as 17b-estradiol, testosterone and 11-ketotestosterone, have been analyzed simultaneously, and the data are not presented in this paper) and tilapia is commonly available in most fish farms worldwide. Specimens of O. niloticus with an average weight of 150.7 ± 9.7 g and length of 19.0 ± 1.4 cm were supplied by the fish farm of freshwater fisheries research center, Chinese Academy of Fishery Science (Wuxi, China). Before the experiments fish were acclimated under laboratory conditions for 4 weeks at a population density of 30 specimens in 200-L
389
glass aquaria supplied with dechlorinated tap water. The physicochemical characteristics of the water used in the aquaria were analyzed according to methods in ‘‘Standard method for the examination of water and wastewater’’ (State Environmental Protection Agency of China 2002). The water had a pH of 7.3 ± 0.3 and a temperature of 25 ± 0.5°C. Water hardness was 107 mg/L (as CaCO3), and dissolved oxygen concentration was 6.5–7.0 mg/L. Stock fish and experimental fish were fed 2 % body mass daily, with a commercial fish feed (Ningbo Tech-bank co., LTD, China) and submitted to a 12-h light and 12-h dark photoperiod. Fish were used when no mortality was observed in the acclimation population. Methomyl (97 % w/w) was produced by Shanghai Focus Biological Technology Co., Ltd, China. All other chemicals used were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA) and Sangon (Shanghai, China). Male tilapia were randomly distributed into 200-L glass aquaria containing different concentrations of methomyl (0, 0.2, 2, 20 and 200 lg/L), with 3 replicates per treatment, and the range of exposure concentrations was based on the information from the previous study on 96 h LC50 (430 lg/L) for tilapia (with an average weight of 3.9 ± 0.4 g and length of 6.3 ± 0.6 cm), the residue level (0–55.3 lg/L) of methomyl in environmental water (Van Scoy et al. 2013) and the U.S. EPA on drinking water quality established a maximum permissible concentration for methomyl of 200 lg/L (U.S.EPA 2012). The actual methomyl concentrations in the test water were measured by the method of Chen et al. (1996). The actual methomyl concentrations of 0, 0.2, 2, 20, 200 lg/L groups were 0, 0.24, 2.06, 21.55, 205.57 lg/L respectively at 0 h of exposure (the initial concentrations), and were 0, 0.20, 1.95, 19.55, 198.57 lg/L respectively after 24 h of exposure. And the results were discussed in relation to the nominal concentrations. Thirty fish were introduced into each concentration in a semi-static system and water was renewed daily. The experiment lasted for 48 days, after 30 days of exposure period the remaining fish were transferred to methomyl-free water for 18 days and then the same parameters were measured to study the recovery response. Sampling of exposed and control fish (n = 6/group, 2 tilapia per replicate) was done at 10 min (day 0), 6, 12, 18, 24, and 30 days during exposure and 18 days (R18) after transferring to methomyl-free water for recovery. Feed was withheld 24 h prior to sampling. Tilapia were euthanized in approximately 250 mg/L ethyl 3-aminobenzoate methanesulfonate salt (MS-222, TCI Inc., Japan), and thereafter measured and weighed. Then, fish were rapidly killed by decapitation and the liver immediately removed and processed, and then snap-frozen in liquid nitrogen and stored at -80°C for later assay.
123
123
Means significant difference between recovery group and 30-day exposure group #
* Means significant difference from the control
All data were expressed as mean ± standard deviation (n = 6). A two sample Student’s t test was used to test for significant differences between groups. Differences were statistically significant when p \ 0.05
105.42 ± 6.70# 133.43 ± 8.36* 135.78 ± 5.43* 165.67 ± 5.39* 355.67 ± 8.39* 100.86 ± 6.49 200
173.54 ± 5.39*
101.15 ± 7.43# 134.33 ± 7.21* 133.55 ± 7.81* 258.89 ± 8.11* 179.87 ± 7.83* 99.87 ± 7.07 20
138.45 ± 7.99*
101.23 ± 8.18#
99.78 ± 5.72 104.66 ± 7.04
195.78 ± 6.46* 179.86 ± 5.53*
105.65 ± 8.55 107.56 ± 7.86
163.24 ± 6.28* 127.76 ± 6.01*
106.23 ± 8.35 105.35 ± 8.30
109.89 ± 8.47
100.26 ± 8.47
100.49 ± 7.34
0.2
2
CAT
73.88 ± 4.13*# 56.71 ± 4.76* 51.33 ± 4.51* 53.56 ± 3.85* 197.56 ± 5.54* 99.65 ± 3.91 200
143.26 ± 4.47*
83.33 ± 4.97* 74.33 ± 4.88* 95.67 ± 4.22* 189.55 ± 3.34* 141.22 ± 5.06* 100.11 ± 4.94 20
118.52 ± 5.26*
98.78 ± 4.08#
101.22 ± 4.86 107.65 ± 5.54
176.22 ± 4.70* 163.22 ± 3.13*
105.94 ± 4.82 109.33 ± 4.9
145.62 ± 4.76* 118.23 ± 4.10*
105.31 ± 4.84 103.22 ± 4.52
106.39 ± 5.47
101.21 ± 4.39
100.33 ± 4.86
0.2 SOD
2
30 24 18 12 6 0
Exposure time (d) Methomyl (lg/L)
Neither mortality nor visible disease signals were observed in the tilapia exposed to sublethal concentrations of methomyl during the performance of the experiment. Changes in SOD and CAT activities in experimental fish are shown in Table 1, and the results of two-way ANOVA are shown in Table 2. The results of two-way ANOVA indicated that time, methomyl concentration and the interaction between the two factors had significant effects on SOD activities (Table 2). Compared to control, SOD activity presented a significant increase at 2, 20, and 200 lg/L of methomyl at days 12, 6 and 6 respectively. The highest induction (197.56 % of the control) of SOD activity was found following exposure to 200 lg/L methomyl at day 12. SOD is
Antioxidant (% of control)
Results and Discussion
Table 1 Liver SOD and CAT activities in O. niloticus exposed to sublethal concentrations of methomyl
Samples were stored at -80°C with an Ultra-low temperature freezer (Forma-86C, America). Samples were homogenized using an electrical homogenizer (Pro-200, America). Centrifugations were done with a refrigerated centrifuge (Sigma 2–16 K, Germany). Spectrophotometric readings were carried out with a UV–vis spectrophotometer (UV-759S, China). Tilapia livers were homogenized using an electrical homogenizer. About 0.30 g of liver tissue was homogenized after addition of 3.0 mL of 10.0 mM Tris buffer (pH 7.5) for detection of enzyme activities. The extracts were centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was used immediately as the enzyme analysis. All the above operations were carried out at 4°C. Catalase activity was assayed by ultraviolet spectrophotometry (Xu et al. 1997), and one unit of enzyme activity is defined as the amount of enzyme which decreased the concentration of H2O2 by 50 % in 100 s at 25°C. Superoxide dismutase (SOD) was assayed by the method of Marklund and Marklund (1974), and one unit of SOD activity is defined as the amount of the enzyme which gave 50 % inhibition of the oxidation rate of 0.1 mM pyrogallol in one ml of solution at 25°C. Protein levels were estimated by the method of Bradford (1976) using bovine serum albumin as a standard. The activities of CAT and SOD of treated tilapia were compared to control group in each sampling day, including recovery group (R18), and expressed as % of control. All data were expressed as mean ± SD (n = 6). Two-way analysis of variance (ANOVA) and two sample Student’s t test were used for statistical analysis with p \ 0.05 being considered significant. In all the tables, ‘‘*’’ means significant difference from the control, ‘‘#’’ means significant difference between recovery group and 30-day exposure group.
Bull Environ Contam Toxicol (2014) 92:388–392
R18
390
Bull Environ Contam Toxicol (2014) 92:388–392
391
Table 2 Results of two-way ANOVA Source
SOD df
CAT SS
MS
F
p
df
SS
MS
F
p
Time
5
29,168.3
5,833.7
271.39
0.000
5
126,648
25,330
470.20
0.000
Concentration
3
26,282.0
8,760.7
407.56
0.000
3
101,077
33,692
625.44
0.000
15
157,457.5
10,497.2
488.34
0.000
15
261,504
17,434
323.62
0.000
21.5
120
6,464
54
143
495,694
Time 9 concentration Error
120
2,579.5
Total
143
215,487.3
the first enzyme to deal with oxyradicals (Zhang et al, 2004). SOD serves to protect cells against the oxidative damage of free radicals by catalysing the conversion of superoxide anion to molecular oxygen (O2) and hydrogen peroxide (H2O2), which is then catalyzed either by CAT or GPx. Induction of SOD could occur during high production ¨ ner 2000), the of superoxide anion radical (Oruc and U significant induction (p \ 0.05) of SOD in the present study might have occurred as a direct response to methomyl induced superoxide anion production as reported in the hepatocytes of rainbow trout exposed to selenite (Misra and Niyogi 2009). Velisek et al. (2011) observed increased SOD activity in common carp liver after long-term exposure to terbutryn. Jin et al. (2010) also observed increased SOD activity in zebrafish (Danio rerio) liver after 14 days atrazine exposure. However, after long-term exposure, SOD activities in the liver of fish exposed to 20 and 200 lg/L decreased at day 24 and 30 and at day 18 and 30 respectively, compared to control, a significant decrease (p \ 0.05) in SOD activity was observed after 30 and 18 days of methomyl exposure respectively (Table 1). ElKhawaga (2005) observed a reduction in hepatic SOD activity of mice injected with single dose of methomyl (7 mg/kg body weight) for 24 h. The decrease of SOD activity could be explained in two ways. Firstly, methomyl could not be metabolized completely by tilapia, especially at high concentrations and long-time exposures, so methomyl might accumulate in tilapia liver and consequently its conjugation with SOD, inactivating the enzyme. Secondly, if H2O2 is not enzymatically decomposed, it can be converted to the very short-lived and highly aggressive hydroxyl radical. The superoxide anion, H2O2 and hydroxyl radical could cause oxidation of SOD, reducing its activity (Dimitrova et al. 1994). The results of two-way ANOVA indicated that time, methomyl concentration and the interaction between the two factors had significant effects on CAT activities (Table 2). Compared to control, CAT presented a significant increase at 2, 20, and 200 lg/L of methomyl at days 12, 6 and 6 respectively. The highest CAT activity (355.67 % of control) was found following exposure to
200 lg/L methomyl at day12 (Table 1). Pesticide-induced inhibition and increment of CAT activity has been reported in studies of various fish species. Jin et al. (2010) reported increased CAT activity in liver of zebrafish after 14 days atrazine exposure. Ballesteros et al. (2009) stated that the activity of CAT was significantly decreased in liver of the onesided livebearer (Jenynsia multidentata) exposed to endosulfan. However, Moraes et al., (2009) reported a decrease in liver CAT activity in teleost fish (Leporinus obtusidens) and silver catfish (Schilbe intermedius) after exposure to herbicides. Keramati et al. (2010) reported that Diazinon caused fluctuated levels in fish Rutilus rutilus. It has also been reported that lower concentrations of endosulfan increase the CAT activity in Oncorhynchus mykiss while the higher concentrations reduce its activity (Sharbidre et al., 2011). The increase in CAT activities in the liver as observed in the present study might be in response to H2O2 produced by SOD activity since CAT is responsible for the detoxification of H2O2 to oxygen and water. Our results were accordant to the previous reports by Zhang et al. (2004) and Yi et al. (2007) where hepatic CAT activities were elevated in fish after chronic exposure to 2,4-dichlorophenol and alachlor. When tilapia was exposed to 0.2 lg/L methomyl, there were no significant changes in SOD and CAT activities compared to control (Table 1), which meant low methomyl concentration could not increase ROS. So we concluded 0.2 lg/L methomyl might be suggested as the threshold dose for no effect to tilapia (this threshold dose is only effective to this experiment tilapia size). When the tilapia exposed to 0.2, 2, 20 and 200 lg/L methomyl were transferred to methomyl-free water for 18 days, CAT activities in fish liver returned to control values (Table 1), however, SOD activities in 20 and 200 lg/L treatments could not return to the control values (Table 1). It could be concluded SOD was more sensitive to methomyl compared to CAT, and SOD may be better than CAT as a biomarker. In conclusion, exposure concentration and time affected SOD and CAT activities in fish exposed to methomyl, and 0.2 lg/L methomyl might be considered as the no observed adverse effect level to tilapia.
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392 Acknowledgments The authors acknowledge support from the Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (No. 2013A0303) and China Agriculture Research System (No. CARS-49) for the financial supports.
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