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Subchronic intoxication with chlorfenvinphos, an organophosphate insecticide, affects rat brain antioxidative enzymes and glutathione level
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Food and Chemical Toxicology 46 (2008) 82–86 www.elsevier.com/locate/foodchemtox
Subchronic intoxication with chlorfenvinphos, an organophosphate insecticide, affects rat brain antioxidative enzymes and glutathione level Anna Lukaszewicz-Hussain Department of Toxicology, Medical University of Bialystok, 2c Mickiewicza Street, 15-222 Bialystok, Poland Received 20 April 2007; accepted 30 June 2007
Abstract Organophosphate pesticides (OP) belong to the class of xenobiotics that are intentionally released to the environment. Toxicity of these compounds is mainly due to inhibition of acetylcholinesterase (AChE), but many authors postulate that OP in acute as well as in chronic intoxication disturb the redox processes, changing the activities of antioxidative enzymes and causing enhancement of lipid peroxidation in many organs. Epidemiological studies have demonstrated a relationship of certain human diseases with pesticide exposure and with changes in antioxidative enzymes. There is also evidence that oxidative stress is an important pathomechanism of neurological disorders such as Alzheimer disease and Parkinson disease, cardiovascular disorders and many others. The study objective was to investigate the activities of brain antioxidative enzymes and reduced glutathione level in rats subchronically intoxicated with chlorfenvinphos. In the rat brain the activities of such enzymes as superoxide dismutase, catalase, glutathione peroxidase and reductase were found to increase, while reduced glutathione level decreased in chlorfenvinphos intoxication. Based on experimental findings of this study, it can be suggested that subchronic administration of chlorfenvinphos leads to a change in the brain oxidative status and that the change occurs at a dose of 0.3 mg/kg/day, i.e., twice smaller than LOAEL level for rats. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Brain; Oxidative stress; Organophosphate
1. Introduction Organophosphate pesticides (OP) are the class of xenobiotics that are intentionally released to the environment. Therefore, there is a growing public concern about the accumulation of these insecticides in food products and water supplies, as repeated or prolonged exposure is a likely cause of delayed toxicity (Costa, 2006; Toxicological profile for chlorfenvinphos, 1997; Zhang et al., 2004). Currently, the general population is primarily exposed to chlorfenvinphos as well as other organophosphates by ingesting food products containing these compounds, particularly fresh fruit and vegetables. In addition, the general population may be exposed to chlorfenvinphos dermally E-mail address: [email protected] 0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.06.038
because of its presence in lanolin and lanolin-containing pharmaceutical products (Toxicological profile for chlorfenvinphos, 1997). Chlorfenvinphos, as other organophosphates, inhibits acetylcholinesterase activity in the central and peripheral nervous system when ingested in acute exposure (Savolainen, 2001; Vidyasagar et al., 2004). Thus, the main target of OP action is the central and peripheral nervous system, although many authors postulate that these compounds in both acute and chronic intoxication disturb the redox processes, changing the activities of antioxidative enzymes and causing enhancement of lipid peroxidation in many organs (Costa, 2006; Poovala et al., 1999; Sharma et al., 2005; Zhang et al., 2004). The results of epidemiological studies demonstrated a relationship of certain human diseases with pesticide
A. Lukaszewicz-Hussain / Food and Chemical Toxicology 46 (2008) 82–86
exposure and with changes in antioxidative enzymes (Abdollahi et al., 2004; Chan et al., 1998; Checkoway and Nelson, 1999; Petrovitch et al., 2002). Also, there is evidence for oxidative stress as an important pathomechanism of neurological disorders such as Alzheimer disease and Parkinson disease, cardiovascular diseases and many others (Abdollahi et al., 2004; Mates et al., 1999; Petrovitch et al., 2002). The objective of the current study was to assess the influence of chlorfenvinphos subchronic administration on the activities of brain antioxidative enzymes and reduced glutathione level. 2. Material and methods
83
Portland, USA. The brain samples for this determination were prepared by homogenization in 8 volumes of cold buffer (50 mM Tris–HCl, pH 7.5, containing 5 mM EDTA and 1 mM 2-mercaptoethanol), and then centrifuged 8500g for 10 min., 4 °C. The brain activity of GR was determined with the use of BIOXYTECH GR-340 Assay kit produced by OXIS International, Inc., Portland, USA, in a supernatant of tissue homogenate obtained by mincing the tissue in cold buffer (50 mM potassium phosphate pH 7.5, containing 1 mM EDTA) and centrifuging 8500g for 10 min., 4 °C. For determination of brain GSH content with BIOXYTECH GSH400 Assay kit produced by OXIS International, Inc., Portland, USA, the tissues were minced in ice-cold metaphoshate acid solution and centrifuged at 3000g for 10 min. The assay was performed in a clear supernatant. The brain concentration of protein was measured according to the method of Lowry et al. (1951), using bovine serum as the standard. Activities of the enzymes and reduced glutathione level were expressed as units of the activity per gram of protein. TM
TM
2.1. Animals Male Wistar rats (from certificate Laboratory Animal House, Brwinow, Poland), 250–280 g body weight, were housed in metal cages with free access to drinking water and standard pellet diet.
2.2. Treatment and blood collection The animals received once daily, intragastrically with a stomach tube, 0.1 ml/100 g of olive oil (control groups) and oil solution of chlorfenvinphos i.e., 2-chloro-1-(2,4-dichlorophenyl) vinyldiethyl phosphate (CVP) at a dose of 0.02LD50; 0.3 mg/kg b.w. (experimental groups). The LD50 for chlorfenvinphos was 15 mg/kg b.w. Chlorfenvinphos (certified purity) was obtained from Institute of Organic Industrial Chemistry, Warsaw, Poland. The purity value – min. 98.2% (m/m), is based on determinations made on samples using the following methods: gas chromatography (GLC) and thin layer chromatography (TLC). The identity of product was established by infrared spectroscopy. The animals were sacrificed on day 14 and day 28 of the exposure. Brain samples were removed and washed with ice-cold 0.9% NaCl solution containing 0.16 mg/ml heparin. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reductase (GR) as well as reduced glutathione level (GSH) were determined in the brain. The study was approved by the Local Ethical Committee.
2.3. Biochemical estimation SOD activity was measured in 10% brain homogenates prepared in 0.25 M saccharose, centrifuged at 8500g, 4 °C for 10 min; next, the assay was performed in a supernatant using BIOXYTECH SOD-525 Assay kit produced by OXIS International, Inc., Portland, USA. CAT activity was measured in 10% brain homogenates prepared in phosphate buffer, centrifuged at 9000g, 4 °C for 15 min. The activity was determined in a supernatant as described by Aebi (1984). The brain activity of GPx was determined in a supernatant using BIOXYTECH GPx-340 Assay kit produced by OXIS International, Inc., TM
TM
2.4. Statistical analysis Data for all the groups of animals were compared using a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison tests. Pearson correlation between the study parameters was calculated. The probability of p < 0.05 was considered significant.
3. Results After chlorfenvinphos administration, no sign of toxicity was observed. Rats exhibited normal behaviour in comparison to the control groups. Chlorfenvinphos administration resulted in an increase in the activities of antioxidative enzymes in the rat brain after 14 days of insecticide administration and these activities remained elevated throughout the exposure. Brain SOD and CAT activities increased more than twice on day 14 as well as on day 28 of the exposure. These changes were statistically significant in comparison to the control groups. A positive correlation was found between CAT and SOD activities (r = 0.67, p = 0.000179) Table 1. The activities of glutathione related enzymes – GPx and GR were increased after 14 days as well as 28 days of chlorfenvinphos administration. The changes in GR activity were more pronounced then in GPx. At the same time GSH level was decreased compared to the control. All these changes were statistically significant in comparison to the control. Table 2.
Table 1 Activities of antioxidative enzymes in the brain of rats subchronically intoxicated with chlorfenvinphos Parameter
Days of exposure to CVP 14 days 0
Activity of SOD (U/mg protein) Activity of CAT (U/mg protein)
5.48 ± 0.70 n = 7 21.99 ± 1.25 n = 6
Data represent mean ± SD of 6–8 individual values. a Statistically significant compared to control.
28 days 0.02 LD50 a
12.04 ± 0.98 n = 6 46.34 ± 4.74a n = 7
0
0.02 LD50
6.16 ± 1.16 n = 6 19.51 ± 2.67 n = 6
12.77 ± 1.48a n = 8 45.20 ± 3.64a n = 8
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Table 2 Activities of glutathione related enzymes and glutathione level in the brain of rats subchronically intoxicated with chlorfenvinphos Parameter
Days of exposure to CVP 14 days 0
Activity of GPx (U/g protein) Activity of GR (U/g protein) Level of GSH (lmol/g protein)
56.16 ± 4.36 n = 7 8.75 ± 0.85 n = 7 30.50 ± 4.06 n = 7
28 days 0.02 LD50 a
62.20 ± 3.78 n = 7 16.60 ± 3.01a n = 8 24.87 ± 3.14a n = 8
0
0.02 LD50
52.93 ± 5.65 n = 5 8.01 ± 1.03 n = 8 31.85 ± 8.07 n = 8
66.64 ± 7.27a n = 7 14.94 ± 2.10a n = 7 23.58 ± 3.51a n = 7
Data represent mean ± SD of 5–8 individual values. a Statistically significant compared to control.
4. Discussion Chlorfenvinphos, an organophosphate insecticide, poses a risk to those professionally involved in its production and use in agriculture as well as to the general population exposed to this compound by the consumption of contaminated food products (Petrovitch et al., 2002; Toxicological profile for chlorfenvinphos, 1997). In the current study, the rats received chlorfenvinphos at a dose 0.3 mg/kg bw/day. The chronic oral minimum human risk level (MRL) for chlorfenvinphos was established at 0.0007 mg/kg/day and this level was developed from a LOAEL of 0.7 mg/kg/day, based on adverse neurological effects in rats (Toxicological profile for chlorfenvinphos, 1997). Thus, a dose of 0.3 mg/kg/day, used in this study, was more than twofold lower in comparison to LOAEL level for rats. In an earlier study, we observed a decrease in serum cholinesterase (ChE) activity on day 28 of rat exposition to insecticide at a dose of 0.02LD50 and an increase in the brain level of TBARS (tiobarbituric acid reactive substances), referred to as lipid peroxidation index, already on day 14 of the rat subchronic exposure to chlorfenvinphos. The concentration remained at this elevated level up to day 28 of exposition. Moreover, enhancement of lipid peroxidation was found to precede the reduction in serum cholinesterase activity, i.e., the index assessed in professional OP exposure (Lukaszewicz-Hussain et al., 2007). The increased level of TBARS suggests induction of oxidative stress in the brain of rats subchronically exposed to chlorfenvinphos (Lukaszewicz-Hussain et al., 2007). Oxidative stress occurs as a consequence of imbalance between the production of reactive oxygen species and the antioxidative process in favour of radical production (Dringen, 2000). In the present study, we observed increased activities of such brain antioxidative enzymes as SOD and CAT as well as glutathione related enzymes. There is no significant change between examined antioxidative parameters after 14 days and 28 days of chlorfenvinphos administration. This observation is not surprising and could be explained on the basis of our earlier results (Lukaszewicz-Hussain and Moniuszko-Jakoniuk, 2005). In the above mentioned paper we reported increased activities of liver SOD and CAT at the 1st hour after single dose exposition to chlor-
fenvinphos (dose 0.02LD50) and returned to the control value at the 24th hour for CAT and at the 48th hour for SOD. Because of this, according to other researchers, single oral dose of chlorfenvinphos reduces toxicity of the subsequent dose administered within 24 h period by causing increase in cytochrome P450 level and rising related enzymes (Ikeda et al., 1990,1991). This causes acceleration of chlorfenvinphos metabolism and may lead to the increased production of reactive oxygen species (Poovala et al., 1999). For these reasons, in the present study, the cumulative effect of chlorfenvinphos has not been observed. In the present study the brain activities of SOD and CAT were found to increase simultaneously and this increase was more pronounced than that of GPx. The increase in superoxide dismutase and catalase activities in the brain of chlorfenvinphos exposed rats appears to be due to the intensified generation of reactive oxygen species. Oxygen reactive species may attack all the major biomolecules, and among them, lipids are most susceptible to this attack (Vidyasagar et al., 2004). We observed the enhancement of brain lipid peroxidation in our previous study (Lukaszewicz-Hussain et al., 2007). The activities of SOD and CAT in the brain of chlorfenvinphos-treated rats were positively correlated. This suggests that the increased level of superoxide anion radical leads to the elevation of hydrogen peroxide level. SOD is an ‘‘incomplete antioxidant’’, which scavenges superoxide anion radical and contributes to overproduction of hydrogen peroxide – a more reactive particle then superoxide anion (Ho et al., 1998; Mao et al., 1993). Catalase is the major enzyme responsible for removal of hydrogen peroxide from normal tissues. By breaking down hydrogen peroxide, this enzyme does not cause generation of any other reactive oxygen species (Girotti, 1998; Mueller et al., 1997). The other hydrogen peroxide removing enzyme, i.e., GPx, converts not only hydrogen peroxide but also lipid peroxides to water and alcohol. Thus, GPx ‘‘monitors’’ the rate of lipid peroxidation and participates in cell membrane stabilization (Girotti, 1998). The enzyme converts reduced glutathione (GSH) to oxidized glutathione (GSSG). In return, to recycle GSSG to GSH, cells utilize glutathione reductase. Moreover, GSH alone exerts an antioxidative effect as it reacts with radicals such as superoxide anion radical, nitric oxide and hydroxyl radical
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(Dringen, 2000; Zasadowski et al., 2004). Thus, changes in the activities of GPx and GR are connected with changes in tissue GSH level. In the current study, the increase in brain GPx activity was accompanied by increased activity of GR and decreased level of GSH. There is evidence that reduced level of glutathione enhances the toxic effect of different insults, as glutathione plays an important role in detoxification of reactive oxygen species in the brain (Dringen, 2000). The observation of brain GSH decreased level in rats subchronically intoxicated with chlorfenvinphos seems to be a very important finding of the present study, since GSH depletion may enhance susceptibility of brain cells to other harmful events, including reduced production of mitochondrial energy, which has been described by Zeevalk et al., 1998. Results obtained by other authors suggest that a lowered glutathione level could contribute to neuronal loss (Dringen, 2000). However, it has been emphasized that in the brain of rats subchronically intoxicated with chlorfenvinphos the level of GSH is reduced despite increased activity of GR and only a small increase in GPx. In other words, a significant increase in GR activity is in contrast with the rate of change in GPx activity and GSH level. For this reason, we suggest glutathione involvement in non-enzymatic reaction with reactive oxygen species. The small rise in GPx activity would possibly result in the enhancement of brain oxidative stress (increased level of brain thiobarbituric acid reactive substances) as shown in our earlier study (Lukaszewicz-Hussain et al., 2007). Many authors have reported that both the increased production of reactive oxygen species and attenuation of the antioxidant barrier of the organism are likely to induce oxidative stress, thus leading to liver and brain damage, tubular necrosis and cardiotoxicity in acute as well as in subchronic OP intoxication (Abdollahi et al., 2004; Akhgari et al., 2003; Hai et al., 1997; Mates et al., 1999; Poovala et al., 1999). The main clinical effect of acute intoxication with organophosphate insecticides involves the inhibition of AChE activity in blood and nervous system, resulting in the accumulation of acetylcholine and cholinergic system overstimulation (Savolainen, 2001; Sharma et al., 2005; Vidyasagar et al., 2004). During AChE inhibition, excessive amounts of ROS may be generated, since high energy consumption coupled with inhibition of oxidative phosphorylation leads to decreased capacity of cells to maintain its energy levels. For this reason, the excessive amounts of reactive oxygen species may be generated in different organs, including the brain (Milatovic et al., 2006). The generation of reactive oxygen species and changes in antioxidative parameters as a mechanism involved in brain and muscle damage have been also described by other authors (Brocardo et al., 2005; Milatovic et al., 2006). Brain tissue is particularly susceptible to oxidative damage, as it is rich in polyunsaturated fatty acids which easily undergo peroxidation. Moreover, the brain uses a relatively
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large amount of oxygen at rather low activities of antioxidative enzymes (Akhgari et al., 2003; Dringen, 2000; Droge, 2002). Based on experimental findings of this study, it can be suggested that subchronic administration of chlorfenvinphos leads to induction of brain oxidative stress. Moreover, an effect on the brain oxidative status was observed at a dose 0.3 mg/kg/day, i.e., twice smaller than LOAEL level for rats (Toxicological profile for chlorfenvinphos, 1997). The current findings have shown increased activities of antioxidative enzymes and a reduced level of GSH in this organ. The lowered glutathione level appears to be the first indicator of oxidative stress in Parkinson disease progression (Dringen, 2000). There is also evidence that oxidative stress is an important pathomechanism in others neurodegenerative diseases (Abdollahi et al., 2004; Mates et al., 1999; Petrovitch et al., 2002). Therefore, evaluation of the effects of organophosphates on the brain antioxidative status seems to have important implications. References Abdollahi, M., Rainba, A., Shadnia, S., Nikfar, S., Rezaie, A., 2004. Pesticide and oxidative stress: a review. Med. Sci. Monitor 10 (6), RA141–RA147. Aebi, H.E., 1984. Catalase in vitro. Meth. Enzymol. 105, 121–126. Akhgari, M., Abdollahi, M., Kebryaeezadeh, A., Hosseini, R., Sabzevari, O., 2003. Biochemical evidence for free radical induced lipid peroxidation as a mechanism for subchronic toxicity of malathion in blood and liver of rats. Hum. Exp. Toxicol. 22, 205–208. Brocardo, P.S., Pandolfo, P., Takahashi, R.N., Rodrigues, A.L.S., Dafre, A.L., 2005. Antioxidant defenses and lipid peroxidation in the cerebral cortex and hippocampus following acute exposure to malathion and/or zinc chloride. Toxicology 207, 283–291. Chan, D.K.Y., Woo, J., Ho, S.C., Pang, C.P., Law, L.K., Ng, P.W., Hung, W.T., Kwok, T., Hui, E., Orr, K., Leung, M.F., Kay, R., 1998. Genetic and environmental risk factors for Parkinson’s disease in a Chinese population. Neurol. Neurosurg. Psyc. 65, 781–784. Checkoway, H., Nelson, L.M., 1999. Epidemiologic approaches to the Parkinson’ s disease etiology. Epidemiology. 10, 327–336. Costa, L.G., 2006. Current issues in organophosphate toxicology. Clin. Chim. Acta 306, 1–13. Dringen, R., 2000. Metabolism and functions glutathione in brain. Progr. Neurobiol. 62, 649–671. Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95. Girotti, A.W., 1998. Lipid hydroperoxide generation, turnover, and effector action in biological systems. J. Lipid Res. 39, 1529–1542. Hai, D.Q., Varga, Sz.I., Matkovits, B., 1997. Organophosphate effects on antioxidant system of Carp (Cyprinus carpio) and Catfish (Ictalurus nebulosus). Comp. Biochem. Pharmac. 117C, 83–88. Ho, Y.S., Gargano, M., Cao, J., Bronson, R.T., Wittman, T., Fazekas, T., 1998. Reduced fertility in female mice lacking copper–zinc dismutase. J. Biol. Chem. 203, 7765–7769. Ikeda, T., Kojima, T., Yoshida, M., Takahashi, H., Tsuda, S., Shirasu, Y., 1990. Pretreatment of rats with organophosophorus insecticide, chlorfenvinphos, protects against subsequent challenge with the same compound. Fundam. Appl. Toxicol. 14, 560–567. Ikeda, T., Tsuda, S., Shirasu, Y., 1991. Metabolic induction of the hepatic cytochrome P450 system by chlorfenvinphos in rats. Fund. Appl. Toxicol. 17, 361–367.
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Lowry, O.H., Rosenbrough, M.J., Farr, A.L., Randall, R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 263– 270. Lukaszewicz-Hussain, A., Moniuszko-Jakoniuk, J., 2005. A low dose of chlorfenvinphos affects hepatic enzymes in serum and antioxidant enzymes in erythrocytes and liver of the rat. Pol. J. Environ. Stud. 14, 199–202. Lukaszewicz-Hussain, A., Moniuszko-Jakoniuk, J., Rogalska, J., 2007. Assessment of lipid peroxidation in rat tissues in subacute chlorfenvinphos administration. Pol. J. Environ. Stud. 16, 233–236. Mao, G.D., Wittman, S., Thomas, P.D., Lopaschuk, G.D., Poznansky, M.J., 1993. Superoxide dismutase (SOD)–catalase conjugates. role of hydrogen peroxide and the Fenton reaction in SOD toxicity. J. Biol. Chem. 268, 416–420. Mates, J.M., Perez-Gomez, C., De Castro, I.N., 1999. Antioxidant enzymes and human diseases. Clin. Biochem. 32, 595–603. Milatovic, D., Gupta, R.C., Aschner, M., 2006. Anticholinesterase toxicity and oxidative stress. Sci. World J. 6, 295–310. Mueller, S., Riedel, H.D., Stremmel, W., 1997. Direct evidence for catalase as the predominant H2O2-removing enzyme in human erythrocytes. Blood 90, 4973–4978. Petrovitch, H., Ross, W., Abbott, R.D., Sanderson, W.T., Sharp, D.S., Tanner, C.M., Masaki, K.H., Blanchette, P.L., Popper, J.S., Foley, D., Launer, L., White, L.R., 2002. Plantation work and risk of Parkinson Disease in a population-based longitudinal study. Arch. Neurol. 59, 1787–1792.
Poovala, V.S., Huang, H., Salahudeen, A.K., 1999. Role of oxygen metabolites in organophosphate–bidrin-induced renal tubular cytotoxicity. J. Am. Soc. Nephr. 10, 1746–1752. Savolainen, K., 2001. Understanding the toxic action of organophosphates. In: Krieger, R.I. (Ed.), . In: Handbook of pesticide toxicology, vol. 2. Academic Press, USA, pp. 1013–1043. Sharma, Y., Bashir, S., Irshad, M., Gupta, S.D., Dogra, T.D., 2005. Effects of acute dimethoate administration on antioxidant status of liver and brain of experimental rats. Toxicology 206, 49–54. Toxicological profile for chlorfenvinphos, 1997. US Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, 163. Vidyasagar, J., Karunakar, N., Reddy, M.S., Rajnarayana, K., Surender, T., Krishna, D.R., 2004. Oxidative stress and antioxidant status in acute organophosphorous insecticide poisoning. Indian J. Pharmac. 36, 76–79. Zasadowski, A., Wysocki, A., Barski, D., Spodniewska, A., 2004. Some aspects of reactive oxygen species (ROS) and antioxidative system agent’s action. Short review. Acta Toxicol. 12, 5–21. Zeevalk, G.D., Bernard, L.P., Ehrhart, J., Niclas, W.J., 1998. Excitotoxicity and oxidative stress during inhibition of energy metabolism. Dev. Neurosci. 20, 444–453. Zhang, J.L., Qiao, C.L., Lan, W.S., 2004. Detoxification of organophosphorus compounds by recombinant carboxylesterase from an insecticide-resistant mosquito and oxime-induced amplification of enzyme activity. Inc. Environ. Toxicol. 19, 154–159.
Food and Chemical Toxicology 46 (2008) 82–86 www.elsevier.com/locate/foodchemtox
Subchronic intoxication with chlorfenvinphos, an organophosphate insecticide, affects rat brain antioxidative enzymes and glutathione level Anna Lukaszewicz-Hussain Department of Toxicology, Medical University of Bialystok, 2c Mickiewicza Street, 15-222 Bialystok, Poland Received 20 April 2007; accepted 30 June 2007
Abstract Organophosphate pesticides (OP) belong to the class of xenobiotics that are intentionally released to the environment. Toxicity of these compounds is mainly due to inhibition of acetylcholinesterase (AChE), but many authors postulate that OP in acute as well as in chronic intoxication disturb the redox processes, changing the activities of antioxidative enzymes and causing enhancement of lipid peroxidation in many organs. Epidemiological studies have demonstrated a relationship of certain human diseases with pesticide exposure and with changes in antioxidative enzymes. There is also evidence that oxidative stress is an important pathomechanism of neurological disorders such as Alzheimer disease and Parkinson disease, cardiovascular disorders and many others. The study objective was to investigate the activities of brain antioxidative enzymes and reduced glutathione level in rats subchronically intoxicated with chlorfenvinphos. In the rat brain the activities of such enzymes as superoxide dismutase, catalase, glutathione peroxidase and reductase were found to increase, while reduced glutathione level decreased in chlorfenvinphos intoxication. Based on experimental findings of this study, it can be suggested that subchronic administration of chlorfenvinphos leads to a change in the brain oxidative status and that the change occurs at a dose of 0.3 mg/kg/day, i.e., twice smaller than LOAEL level for rats. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Brain; Oxidative stress; Organophosphate
1. Introduction Organophosphate pesticides (OP) are the class of xenobiotics that are intentionally released to the environment. Therefore, there is a growing public concern about the accumulation of these insecticides in food products and water supplies, as repeated or prolonged exposure is a likely cause of delayed toxicity (Costa, 2006; Toxicological profile for chlorfenvinphos, 1997; Zhang et al., 2004). Currently, the general population is primarily exposed to chlorfenvinphos as well as other organophosphates by ingesting food products containing these compounds, particularly fresh fruit and vegetables. In addition, the general population may be exposed to chlorfenvinphos dermally E-mail address: [email protected] 0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.06.038
because of its presence in lanolin and lanolin-containing pharmaceutical products (Toxicological profile for chlorfenvinphos, 1997). Chlorfenvinphos, as other organophosphates, inhibits acetylcholinesterase activity in the central and peripheral nervous system when ingested in acute exposure (Savolainen, 2001; Vidyasagar et al., 2004). Thus, the main target of OP action is the central and peripheral nervous system, although many authors postulate that these compounds in both acute and chronic intoxication disturb the redox processes, changing the activities of antioxidative enzymes and causing enhancement of lipid peroxidation in many organs (Costa, 2006; Poovala et al., 1999; Sharma et al., 2005; Zhang et al., 2004). The results of epidemiological studies demonstrated a relationship of certain human diseases with pesticide
A. Lukaszewicz-Hussain / Food and Chemical Toxicology 46 (2008) 82–86
exposure and with changes in antioxidative enzymes (Abdollahi et al., 2004; Chan et al., 1998; Checkoway and Nelson, 1999; Petrovitch et al., 2002). Also, there is evidence for oxidative stress as an important pathomechanism of neurological disorders such as Alzheimer disease and Parkinson disease, cardiovascular diseases and many others (Abdollahi et al., 2004; Mates et al., 1999; Petrovitch et al., 2002). The objective of the current study was to assess the influence of chlorfenvinphos subchronic administration on the activities of brain antioxidative enzymes and reduced glutathione level. 2. Material and methods
83
Portland, USA. The brain samples for this determination were prepared by homogenization in 8 volumes of cold buffer (50 mM Tris–HCl, pH 7.5, containing 5 mM EDTA and 1 mM 2-mercaptoethanol), and then centrifuged 8500g for 10 min., 4 °C. The brain activity of GR was determined with the use of BIOXYTECH GR-340 Assay kit produced by OXIS International, Inc., Portland, USA, in a supernatant of tissue homogenate obtained by mincing the tissue in cold buffer (50 mM potassium phosphate pH 7.5, containing 1 mM EDTA) and centrifuging 8500g for 10 min., 4 °C. For determination of brain GSH content with BIOXYTECH GSH400 Assay kit produced by OXIS International, Inc., Portland, USA, the tissues were minced in ice-cold metaphoshate acid solution and centrifuged at 3000g for 10 min. The assay was performed in a clear supernatant. The brain concentration of protein was measured according to the method of Lowry et al. (1951), using bovine serum as the standard. Activities of the enzymes and reduced glutathione level were expressed as units of the activity per gram of protein. TM
TM
2.1. Animals Male Wistar rats (from certificate Laboratory Animal House, Brwinow, Poland), 250–280 g body weight, were housed in metal cages with free access to drinking water and standard pellet diet.
2.2. Treatment and blood collection The animals received once daily, intragastrically with a stomach tube, 0.1 ml/100 g of olive oil (control groups) and oil solution of chlorfenvinphos i.e., 2-chloro-1-(2,4-dichlorophenyl) vinyldiethyl phosphate (CVP) at a dose of 0.02LD50; 0.3 mg/kg b.w. (experimental groups). The LD50 for chlorfenvinphos was 15 mg/kg b.w. Chlorfenvinphos (certified purity) was obtained from Institute of Organic Industrial Chemistry, Warsaw, Poland. The purity value – min. 98.2% (m/m), is based on determinations made on samples using the following methods: gas chromatography (GLC) and thin layer chromatography (TLC). The identity of product was established by infrared spectroscopy. The animals were sacrificed on day 14 and day 28 of the exposure. Brain samples were removed and washed with ice-cold 0.9% NaCl solution containing 0.16 mg/ml heparin. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reductase (GR) as well as reduced glutathione level (GSH) were determined in the brain. The study was approved by the Local Ethical Committee.
2.3. Biochemical estimation SOD activity was measured in 10% brain homogenates prepared in 0.25 M saccharose, centrifuged at 8500g, 4 °C for 10 min; next, the assay was performed in a supernatant using BIOXYTECH SOD-525 Assay kit produced by OXIS International, Inc., Portland, USA. CAT activity was measured in 10% brain homogenates prepared in phosphate buffer, centrifuged at 9000g, 4 °C for 15 min. The activity was determined in a supernatant as described by Aebi (1984). The brain activity of GPx was determined in a supernatant using BIOXYTECH GPx-340 Assay kit produced by OXIS International, Inc., TM
TM
2.4. Statistical analysis Data for all the groups of animals were compared using a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison tests. Pearson correlation between the study parameters was calculated. The probability of p < 0.05 was considered significant.
3. Results After chlorfenvinphos administration, no sign of toxicity was observed. Rats exhibited normal behaviour in comparison to the control groups. Chlorfenvinphos administration resulted in an increase in the activities of antioxidative enzymes in the rat brain after 14 days of insecticide administration and these activities remained elevated throughout the exposure. Brain SOD and CAT activities increased more than twice on day 14 as well as on day 28 of the exposure. These changes were statistically significant in comparison to the control groups. A positive correlation was found between CAT and SOD activities (r = 0.67, p = 0.000179) Table 1. The activities of glutathione related enzymes – GPx and GR were increased after 14 days as well as 28 days of chlorfenvinphos administration. The changes in GR activity were more pronounced then in GPx. At the same time GSH level was decreased compared to the control. All these changes were statistically significant in comparison to the control. Table 2.
Table 1 Activities of antioxidative enzymes in the brain of rats subchronically intoxicated with chlorfenvinphos Parameter
Days of exposure to CVP 14 days 0
Activity of SOD (U/mg protein) Activity of CAT (U/mg protein)
5.48 ± 0.70 n = 7 21.99 ± 1.25 n = 6
Data represent mean ± SD of 6–8 individual values. a Statistically significant compared to control.
28 days 0.02 LD50 a
12.04 ± 0.98 n = 6 46.34 ± 4.74a n = 7
0
0.02 LD50
6.16 ± 1.16 n = 6 19.51 ± 2.67 n = 6
12.77 ± 1.48a n = 8 45.20 ± 3.64a n = 8
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Table 2 Activities of glutathione related enzymes and glutathione level in the brain of rats subchronically intoxicated with chlorfenvinphos Parameter
Days of exposure to CVP 14 days 0
Activity of GPx (U/g protein) Activity of GR (U/g protein) Level of GSH (lmol/g protein)
56.16 ± 4.36 n = 7 8.75 ± 0.85 n = 7 30.50 ± 4.06 n = 7
28 days 0.02 LD50 a
62.20 ± 3.78 n = 7 16.60 ± 3.01a n = 8 24.87 ± 3.14a n = 8
0
0.02 LD50
52.93 ± 5.65 n = 5 8.01 ± 1.03 n = 8 31.85 ± 8.07 n = 8
66.64 ± 7.27a n = 7 14.94 ± 2.10a n = 7 23.58 ± 3.51a n = 7
Data represent mean ± SD of 5–8 individual values. a Statistically significant compared to control.
4. Discussion Chlorfenvinphos, an organophosphate insecticide, poses a risk to those professionally involved in its production and use in agriculture as well as to the general population exposed to this compound by the consumption of contaminated food products (Petrovitch et al., 2002; Toxicological profile for chlorfenvinphos, 1997). In the current study, the rats received chlorfenvinphos at a dose 0.3 mg/kg bw/day. The chronic oral minimum human risk level (MRL) for chlorfenvinphos was established at 0.0007 mg/kg/day and this level was developed from a LOAEL of 0.7 mg/kg/day, based on adverse neurological effects in rats (Toxicological profile for chlorfenvinphos, 1997). Thus, a dose of 0.3 mg/kg/day, used in this study, was more than twofold lower in comparison to LOAEL level for rats. In an earlier study, we observed a decrease in serum cholinesterase (ChE) activity on day 28 of rat exposition to insecticide at a dose of 0.02LD50 and an increase in the brain level of TBARS (tiobarbituric acid reactive substances), referred to as lipid peroxidation index, already on day 14 of the rat subchronic exposure to chlorfenvinphos. The concentration remained at this elevated level up to day 28 of exposition. Moreover, enhancement of lipid peroxidation was found to precede the reduction in serum cholinesterase activity, i.e., the index assessed in professional OP exposure (Lukaszewicz-Hussain et al., 2007). The increased level of TBARS suggests induction of oxidative stress in the brain of rats subchronically exposed to chlorfenvinphos (Lukaszewicz-Hussain et al., 2007). Oxidative stress occurs as a consequence of imbalance between the production of reactive oxygen species and the antioxidative process in favour of radical production (Dringen, 2000). In the present study, we observed increased activities of such brain antioxidative enzymes as SOD and CAT as well as glutathione related enzymes. There is no significant change between examined antioxidative parameters after 14 days and 28 days of chlorfenvinphos administration. This observation is not surprising and could be explained on the basis of our earlier results (Lukaszewicz-Hussain and Moniuszko-Jakoniuk, 2005). In the above mentioned paper we reported increased activities of liver SOD and CAT at the 1st hour after single dose exposition to chlor-
fenvinphos (dose 0.02LD50) and returned to the control value at the 24th hour for CAT and at the 48th hour for SOD. Because of this, according to other researchers, single oral dose of chlorfenvinphos reduces toxicity of the subsequent dose administered within 24 h period by causing increase in cytochrome P450 level and rising related enzymes (Ikeda et al., 1990,1991). This causes acceleration of chlorfenvinphos metabolism and may lead to the increased production of reactive oxygen species (Poovala et al., 1999). For these reasons, in the present study, the cumulative effect of chlorfenvinphos has not been observed. In the present study the brain activities of SOD and CAT were found to increase simultaneously and this increase was more pronounced than that of GPx. The increase in superoxide dismutase and catalase activities in the brain of chlorfenvinphos exposed rats appears to be due to the intensified generation of reactive oxygen species. Oxygen reactive species may attack all the major biomolecules, and among them, lipids are most susceptible to this attack (Vidyasagar et al., 2004). We observed the enhancement of brain lipid peroxidation in our previous study (Lukaszewicz-Hussain et al., 2007). The activities of SOD and CAT in the brain of chlorfenvinphos-treated rats were positively correlated. This suggests that the increased level of superoxide anion radical leads to the elevation of hydrogen peroxide level. SOD is an ‘‘incomplete antioxidant’’, which scavenges superoxide anion radical and contributes to overproduction of hydrogen peroxide – a more reactive particle then superoxide anion (Ho et al., 1998; Mao et al., 1993). Catalase is the major enzyme responsible for removal of hydrogen peroxide from normal tissues. By breaking down hydrogen peroxide, this enzyme does not cause generation of any other reactive oxygen species (Girotti, 1998; Mueller et al., 1997). The other hydrogen peroxide removing enzyme, i.e., GPx, converts not only hydrogen peroxide but also lipid peroxides to water and alcohol. Thus, GPx ‘‘monitors’’ the rate of lipid peroxidation and participates in cell membrane stabilization (Girotti, 1998). The enzyme converts reduced glutathione (GSH) to oxidized glutathione (GSSG). In return, to recycle GSSG to GSH, cells utilize glutathione reductase. Moreover, GSH alone exerts an antioxidative effect as it reacts with radicals such as superoxide anion radical, nitric oxide and hydroxyl radical
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(Dringen, 2000; Zasadowski et al., 2004). Thus, changes in the activities of GPx and GR are connected with changes in tissue GSH level. In the current study, the increase in brain GPx activity was accompanied by increased activity of GR and decreased level of GSH. There is evidence that reduced level of glutathione enhances the toxic effect of different insults, as glutathione plays an important role in detoxification of reactive oxygen species in the brain (Dringen, 2000). The observation of brain GSH decreased level in rats subchronically intoxicated with chlorfenvinphos seems to be a very important finding of the present study, since GSH depletion may enhance susceptibility of brain cells to other harmful events, including reduced production of mitochondrial energy, which has been described by Zeevalk et al., 1998. Results obtained by other authors suggest that a lowered glutathione level could contribute to neuronal loss (Dringen, 2000). However, it has been emphasized that in the brain of rats subchronically intoxicated with chlorfenvinphos the level of GSH is reduced despite increased activity of GR and only a small increase in GPx. In other words, a significant increase in GR activity is in contrast with the rate of change in GPx activity and GSH level. For this reason, we suggest glutathione involvement in non-enzymatic reaction with reactive oxygen species. The small rise in GPx activity would possibly result in the enhancement of brain oxidative stress (increased level of brain thiobarbituric acid reactive substances) as shown in our earlier study (Lukaszewicz-Hussain et al., 2007). Many authors have reported that both the increased production of reactive oxygen species and attenuation of the antioxidant barrier of the organism are likely to induce oxidative stress, thus leading to liver and brain damage, tubular necrosis and cardiotoxicity in acute as well as in subchronic OP intoxication (Abdollahi et al., 2004; Akhgari et al., 2003; Hai et al., 1997; Mates et al., 1999; Poovala et al., 1999). The main clinical effect of acute intoxication with organophosphate insecticides involves the inhibition of AChE activity in blood and nervous system, resulting in the accumulation of acetylcholine and cholinergic system overstimulation (Savolainen, 2001; Sharma et al., 2005; Vidyasagar et al., 2004). During AChE inhibition, excessive amounts of ROS may be generated, since high energy consumption coupled with inhibition of oxidative phosphorylation leads to decreased capacity of cells to maintain its energy levels. For this reason, the excessive amounts of reactive oxygen species may be generated in different organs, including the brain (Milatovic et al., 2006). The generation of reactive oxygen species and changes in antioxidative parameters as a mechanism involved in brain and muscle damage have been also described by other authors (Brocardo et al., 2005; Milatovic et al., 2006). Brain tissue is particularly susceptible to oxidative damage, as it is rich in polyunsaturated fatty acids which easily undergo peroxidation. Moreover, the brain uses a relatively
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large amount of oxygen at rather low activities of antioxidative enzymes (Akhgari et al., 2003; Dringen, 2000; Droge, 2002). Based on experimental findings of this study, it can be suggested that subchronic administration of chlorfenvinphos leads to induction of brain oxidative stress. Moreover, an effect on the brain oxidative status was observed at a dose 0.3 mg/kg/day, i.e., twice smaller than LOAEL level for rats (Toxicological profile for chlorfenvinphos, 1997). The current findings have shown increased activities of antioxidative enzymes and a reduced level of GSH in this organ. The lowered glutathione level appears to be the first indicator of oxidative stress in Parkinson disease progression (Dringen, 2000). There is also evidence that oxidative stress is an important pathomechanism in others neurodegenerative diseases (Abdollahi et al., 2004; Mates et al., 1999; Petrovitch et al., 2002). Therefore, evaluation of the effects of organophosphates on the brain antioxidative status seems to have important implications. References Abdollahi, M., Rainba, A., Shadnia, S., Nikfar, S., Rezaie, A., 2004. Pesticide and oxidative stress: a review. Med. Sci. Monitor 10 (6), RA141–RA147. Aebi, H.E., 1984. Catalase in vitro. Meth. Enzymol. 105, 121–126. Akhgari, M., Abdollahi, M., Kebryaeezadeh, A., Hosseini, R., Sabzevari, O., 2003. Biochemical evidence for free radical induced lipid peroxidation as a mechanism for subchronic toxicity of malathion in blood and liver of rats. Hum. Exp. Toxicol. 22, 205–208. Brocardo, P.S., Pandolfo, P., Takahashi, R.N., Rodrigues, A.L.S., Dafre, A.L., 2005. Antioxidant defenses and lipid peroxidation in the cerebral cortex and hippocampus following acute exposure to malathion and/or zinc chloride. Toxicology 207, 283–291. Chan, D.K.Y., Woo, J., Ho, S.C., Pang, C.P., Law, L.K., Ng, P.W., Hung, W.T., Kwok, T., Hui, E., Orr, K., Leung, M.F., Kay, R., 1998. Genetic and environmental risk factors for Parkinson’s disease in a Chinese population. Neurol. Neurosurg. Psyc. 65, 781–784. Checkoway, H., Nelson, L.M., 1999. Epidemiologic approaches to the Parkinson’ s disease etiology. Epidemiology. 10, 327–336. Costa, L.G., 2006. Current issues in organophosphate toxicology. Clin. Chim. Acta 306, 1–13. Dringen, R., 2000. Metabolism and functions glutathione in brain. Progr. Neurobiol. 62, 649–671. Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95. Girotti, A.W., 1998. Lipid hydroperoxide generation, turnover, and effector action in biological systems. J. Lipid Res. 39, 1529–1542. Hai, D.Q., Varga, Sz.I., Matkovits, B., 1997. Organophosphate effects on antioxidant system of Carp (Cyprinus carpio) and Catfish (Ictalurus nebulosus). Comp. Biochem. Pharmac. 117C, 83–88. Ho, Y.S., Gargano, M., Cao, J., Bronson, R.T., Wittman, T., Fazekas, T., 1998. Reduced fertility in female mice lacking copper–zinc dismutase. J. Biol. Chem. 203, 7765–7769. Ikeda, T., Kojima, T., Yoshida, M., Takahashi, H., Tsuda, S., Shirasu, Y., 1990. Pretreatment of rats with organophosophorus insecticide, chlorfenvinphos, protects against subsequent challenge with the same compound. Fundam. Appl. Toxicol. 14, 560–567. Ikeda, T., Tsuda, S., Shirasu, Y., 1991. Metabolic induction of the hepatic cytochrome P450 system by chlorfenvinphos in rats. Fund. Appl. Toxicol. 17, 361–367.
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