- Email: [email protected]
or zinc chloride
Toxicology 207 (2005) 283–291
Antioxidant defenses and lipid peroxidation in the cerebral cortex and hippocampus following acute exposure to malathion and/or zinc chloride Patr´ıcia S. Brocardoa , Pablo Pandolfoa , Reinaldo N. Takahashib , Ana L´ucia S. Rodriguesa,∗ , Alcir L. Dafrec a
c
Departamento de Bioqu´ımica, Centro de Ciˆencias Biol´ogicas, Universidade Federal de Santa Catarina, SC 88040-900, Florian´opolis, Brazil b Departamento de Farmacologia, Centro de Ciˆ encias Biol´ogicas, Universidade Federal de Santa Catarina, SC 88040-900, Florian´opolis, Brazil Departamento de Ciˆencias Fisiol´ogicas, Centro de Ciˆencias Biol´ogicas, Universidade Federal de Santa Catarina, SC 88040-900, Florian´opolis, Brazil Received 18 August 2004; received in revised form 17 September 2004; accepted 25 September 2004 Available online 18 November 2004
Abstract This study investigates the effects of acute exposure to organophosphate insecticide malathion (250 mg/kg, i.p.) and/or ZnCl2 (5 mg/kg, i.p.), with the following parameters: lipid peroxidation and the activity of acetylcholinesterase (AChE), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH), and the levels of total glutathione (GSH-t) in the hippocampus and cerebral cortex of female rats. Malathion exposure elicited lipid peroxidation and reduced AChE activity in the cerebral cortex and hippocampus. It also reduced the activity of GR and GST, and increased G6PDH activity in the cerebral cortex, without changing the levels of GSH-t and GPx activity. ZnCl2 exposure reduced AChE activity and caused a mild pro-oxidative effect, since lipid peroxidation was increased in the hippocampus. ZnCl2 , individually or in combination with malathion, caused a reduction in GR and GST activity in the cerebral cortex. Malathion and/or ZnCl2 did not change the GSH-t levels. Moreover, ZnCl2 prevented the increase in G6PDH activity caused by malathion. It showed that ZnCl2 had little effect against the changes induced by malathion. In fact, zinc itself produced pro-oxidant action, such as the reduction in the activity of the antioxidant enzymes GR and GST. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Malathion; Zinc; Acetylcholinesterase; Lipid peroxidation; Antioxidant enzymes; Brain
∗
Corresponding author. Tel.: +55 48 331 9692; fax: +55 48 331 9672. E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues).
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.09.012
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1. Introduction Malathion [O,O-dimethyl-S-(1,2-dicarbethoxy-ethyl)phosphorodithioate] is an organophosphate (OP) insecticide of low mammalian toxicity which is used extensively throughout the world to control major arthropods in public health programs, animal ectoparasites, human head and body lice, household insects and to protect grain in storage (Maroni et al., 2000). Like other OP compounds, malathion is known to inhibit acetylcholinesterase (AChE) activity, an effect that is thought to underlie the neurotoxicity elicited by these compounds (Kwong, 2002). However, some studies indicate that other biochemical targets may be affected by OP insecticides (Ward and Mundy, 1996; Samimi and Last, 2001). Moreover, the lipophilic nature of OPs facilitates their interaction with the cell membrane and leads to perturbations of the phospholipid bilayer structure (Videira et al., 2001). The toxicity of OP agents may be due, at least in part, to the formation of reactive oxygen species (ROS), leading to lipid peroxidation, which is generally assessed by an increase in the levels of thiobarbituric acid reactive substances (TBARS) (Banerjee et al., 1999; Verma and Srivastava, 2001; Ranjbar et al., 2002). Several studies indicate that malathion exposure increases TBARS levels in the erythrocytes, liver, and brain of rats (Hazarika et al., 2003; Akhgari et al., 2003). Other studies indicate that antioxidant enzyme activity was either increased, reduced, or not changed in the liver, brain, and erythrocytes of animals treated with this OP compound (Srikanth and Seth, 1990; Pedrajas et al., 1995; Ahmed et al., 2000; John et al., 2001; Akhgari et al., 2003; Hazarika et al., 2003). The differences found may be due, e.g. to the malathion exposure regime employed, the tissue distribution, or the age of the animals. One area of increasing interest is the study of the capability of some essential mineral elements to modulate the effects of environmental toxicants. In that respect, several studies have shown that zinc, an essential nutrient, at least under certain conditions, may have antioxidant properties (Powell, 2000). Also, zinc deficiency causes a significant increase in glutathioneS-transferase (GST) activity and lipid peroxidation in the serum, liver, and brain of rats (Yousef et al., 2002), as well as a decrease of the activity of super-
oxide dismutase (SOD) in the liver of mice (Chen and Young, 1998). However, an excess of zinc may produce cytotoxic effects, which is easily demonstrated as zinc levels reach high concentrations in the extracellular fluid in the brain (Choi and Koh, 1998; Oteiza et al., 2004). In fact, some studies have demonstrated that zinc induces neuronal death in cultured cortical neurons, an effect that seems to be mediated by oxidative stress (Kim et al., 1999; Pong et al., 2002). In the present study, we have investigated the effect of acute treatment with malathion and/or ZnCl2 on antioxidant defense systems and lipid peroxidation in the cerebral cortex and hippocampus of rats.
2. Materials and methods 2.1. Chemicals The following chemicals were used: commercialgrade malathion (95% purity, CAS 121-75-5, Dipil Chemical Ind., Brazil); nicotinamide adenine dinucleotide phosphate (reduced form; NADPH); nicotinamide adenine dinucleotide phosphate (oxidized form; NADP+ ), glucose-6-phosphate (G6P), tertbutylhydroperoxide (tBOOH), magnesium chloride, oxidized (GSSG) and reduced (GSH) glutathione; 1-chloro-2,4-dinitrobenzene (CDNB); glutathione reductase; acetylthiocholine iodide; bovine serum albumin; 5,5-dithio bis(2-nitrobenzoic acid) (DTNB); malondialdehyde (MDA); and 2-thiobarbituric acid (TBA) from Sigma Chemical Co. (USA). Trichloroacetic acid (TCA) and zinc chloride (ZnCl2 ) were purchased from Merck (Germany). 2.2. Animals and treatments Adult female Wistar rats weighing 180 ± 20 g were kept with free access to food and water on a 12 h light/dark cycle. The rats were housed in plastic cages; they were randomly divided into four groups of eight animals each. The experiments were performed after approval of the protocol by the Ethics Committee of the Institution, and every effort was made to minimize animal suffering. Malathion (250 mg/kg, 1/5 LD50 ); ZnCl2 (5 mg/kg) and saline were administered intraperitoneally (i.p.) in
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a constant volume of 1 ml/kg of body weight. The dose of malathion employed caused no overt signs of cholinergic toxicity. All groups of rats received two contralateral i.p. injections. The second injection was given immediately after the first one. Rats were given: • • • •
Group I: saline and saline; Group II: ZnCl2 and saline; Group III: malathion and saline; Group IV: malathion and ZnCl2 .
Rats were killed by decapitation 24 h after treatment. Hippocampi and cerebral cortices were removed and homogenized 1:2 (w/v; 1 g tissue with 2 ml 20 mM Hepes buffer, pH 7.4). The homogenate was centrifuged at 15,000 × g, for 30 min at 4 ◦ C, and the resultant supernatant was used for different antioxidant enzyme assays. Acetylcholinesterase activity was measured in homogenates centrifuged at 2300 × g, for 15 min. Each sample was taken from one animal and assayed in triplicate.
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2.5. Antioxidant enzyme assays Glutathione peroxidase (GPx) activity was measured by the Wendel (1981) method, using tertbutylhydroperoxide as a substrate. NADPH disappearance was monitored by a spectrophotometer at 340 nm. Glutathione-S-transferase (GST) activity was assayed by the procedure of Habig and Jakoby (1981), using CDNB as a substrate. The assay was conducted by monitoring the appearance of the conjugated complex of CDNB and GSH at 340 nm. Glutathione reductase (GR) activity in brain homogenates was determined by the method described by Carlberg and Mannervik (1985). The reduction of GSSG in the presence of NADPH was measured spectrophotometrically at 340 nm. The activity of glucose-6-phosphate dehydrogenase (G6PDH) was determined by means of the absorbance increase induced by the reduction of NADP+ to NADPH, at 340 nm (Glock and McLean, 1953).
2.3. Glutathione (GSH) assay 2.6. Determination of acetylcholinesterase activity This method takes advantage of glutathione reductase (GR) activity to reduce glutathione disulfide or oxidized (GSSG) to the reduced form, using NADPH as an electron donor. The GSH formed in the previous reaction reacts with the Ellman reagent DTNB to form GSH–TNB, an asymmetric disulfide and the colorforming thionitrobenzoic acid (TNB), which is measured continuously at 412 nm. GR reduces GSH–TNB to GSH, and the second TNB is released, increasing color proportionally with GSH and/or GSSG in the assay. GSH reenters the cycle, reacting with another DTNB and continuing the recycling assay. The assay is performed using a standard curve with known GSSG concentration (Tietze, 1969, modified by Akerboom and Sies, 1981).
Acetylcholinesterase activity of hippocampus and cerebral cortex was estimated by the method of Ellman et al. (1961), using acetylthiocholine iodide as a substrate. The rate of hydrolysis of acetylthiocholine iodide is measured at 412 nm through the release of the thiol compound which, when reacted with DTNB, produces the color-forming compound TNB. 2.7. Determination of protein The protein content in hippocampal and cerebral cortical homogenates was quantified by the method of Bradford (1976), using bovine serum albumin as a standard.
2.4. Measurement of TBARS levels 2.8. Statistical analysis As an index of lipid peroxidation, the formation of TBARS was measured in hippocampal and cerebral cortical crude homogenates according to the method of Ohkawa et al. (1979). The results were expressed in terms of the extent of malondialdehyde (MDA) production.
All data are expressed as mean ± S.E.M. by twoway analysis of variance (ANOVA) (malathion × ZnCl2 ), followed by Newman–Keuls test when appropriate. Differences between groups were considered significant when P < 0.05.
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3. Results
3.3. Antioxidant enzyme activities
3.1. Acetylcholinesterase (AChE) activity
Table 3 shows that treatment with malathion and ZnCl2 , alone or in co-administration, caused a reduction in GST (about 25–35%) and GR (about 25–55%) activity in the cerebral cortex, but no synergistic reduction in enzyme activity was observed in rats treated with both compounds. The cerebral cortical activity of G6PDH increased slightly, yet significantly, in the malathion-treated group, while co-administration of ZnCl2 prevented that effect. These changes in G6PDH, GR and GST activity were not observed in the hippocampus of rats treated with malathion and/or ZnCl2 . No change in GPx activity was observed in the cerebral cortex or hippocampus of any group.
Table 1 shows the effect of acute administration of malathion and/or ZnCl2 on AChE activity. Malathion or ZnCl2 significantly reduced AChE activity in the cerebral cortex and hippocampus as compared with the saline-treated group (control). Moreover, when in combination, malathion and ZnCl2 significantly reduced AChE activity in the cerebral cortex, but not in the hippocampus. 3.2. Thiobarbituric acid reactive substance (TBARS) measurements A significant increase in the TBARS levels was found in the cerebral cortex of rats treated with malathion. In the hippocampus, an increase in TBARS levels was observed in all groups as compared with the control group (Table 2).
3.4. Measurement of total glutathione (GSH-t) levels Malathion and ZnCl2 , alone or in combination, did not significantly alter the levels of GSH in the cerebral cortex and hippocampus of rats (Table 4).
Table 1 Effect of treatment with malathion (250 mg/kg, i.p.) and/or zinc chloride (ZnCl2 ; 5 mg/kg i.p.) on AChE activity in the cerebral cortex and hippocampus of rats
4. Discussion
Treatment
Cerebral cortex
4.1. Malathion
Saline ZnCl2 Malathion Malathion + ZnCl2
47.4 33.9 40.5 35.4
± ± ± ±
1.7 1.5** 0.5** 2.0**
Hippocampus 28.3 21.6 21.2 24.5
± ± ± ±
2.0 1.8* 1.0* 2.2
Enzyme activity is expressed as nmol/min/mg protein. Values are given as mean ± S.E.M., n = 6–8. ∗ P < 0.05 as compared with saline-treated control. ∗∗ P < 0.01 as compared with saline-treated control. Table 2 TBARS levels measured in the cerebral cortex and hippocampus of animals treated with malathion (250 mg/kg, i.p.) and/or zinc chloride (5 mg/kg, i.p.) Treatment
Cerebral cortex
Saline ZnCl2 Malathion Malathion + ZnCl2
260 275 376 312
± ± ± ±
10 15 13** 32
Hippocampus 150 186 180 177
± ± ± ±
6.9 4.8* 8.8* 9.2*
TBARS levels are expressed as nmol/mg wet tissue. Values are expressed as mean ± S.E.M., n = 7–8. ∗ P < 0.05 as compared with saline-treated control. ∗∗ P < 0.01 as compared with saline-treated control.
The inhibition of AChE activity in target tissues is often taken as an indication of pesticide intoxication (Kwong, 2002). In our study, we used AChE activity as a marker of the effectiveness of exposure to malathion. We found that AChE activity was significantly reduced in the cerebral cortex (15%) and hippocampus (25%) of malathion-exposed rats. These results are somewhat similar to those found in the brain of rats exposed to malathion acutely (Hazarika et al., 2003) or chronically (Srikanth and Seth, 1990). Our results also demonstrate an increase on TBARS levels in the cerebral cortex and hippocampus of malathion-exposed rats, indicating that, in addition to AChE inhibition, oxidative stress may be a mechanism underlying the central effects of malathion exposure, as suggested in other studies with malathion (Hazarika et al., 2003) or other OP compounds (Bagchi et al., 1995; Yang et al., 1996; Yang and Dettbarn, 1996). To investigate the impact of malathion on antioxidant defenses in the CNS (cerebral cortex and hip-
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Table 3 Effect of malathion (250 mg/kg, i.p.) and/or zinc chloride (5 mg/kg, i.p.) exposure on antioxidant enzyme activity (GST, GR, G6PDH, GPx) in the cerebral cortex and hippocampus of rats Tissue/treatment
GST
Cerebral cortex Saline ZnCl2 Malathion Malathion + ZnCl2
GR
G6PDH
GPx
112.4 73.1 84.9 80.0
± ± ± ±
4.3 4.4** 7.0** 7.0**
52.0 39.5 22.8 33.7
± ± ± ±
1.7 3.6** 1.5** 4.2**
34.5 37.8 41.9 34.1
± ± ± ±
1.7 0.9 1.1** 1.8
32.9 38.2 34.6 37.3
± ± ± ±
2.4 2.9 1.9 1.6
Hippocampus Saline ZnCl2 Malathion Malathion + ZnCl2
63.3 56.2 59.8 53.2
± ± ± ±
4.9 4.8 6.4 3.3
29.8 33.9 29.3 34.9
± ± ± ±
1.1 2.0 1.9 1.3
17.7 21.9 21.6 20.4
± ± ± ±
2.4 2.6 2.3 1.7
31.4 28.6 30.7 31.5
± ± ± ±
1.8 2.3 1.6 2.7
Activities are expressed as nmol/min/mg protein. Values are expressed as mean ± S.E.M., n = 7–8. ∗∗ P < 0.01 as compared with saline-treated control.
pocampus), four antioxidant enzymes and GSH-t levels were measured in rats treated with a single dose of malathion. Overall, a significant decrease in the GST and GR levels in the cerebral cortex was observed, while the hippocampus was not affected. There were no changes in GPx or GSH-t in either cerebral cortex or hippocampus, while a small, but significant, increase in the G6PDH levels was observed in the cerebral cortex. Considering that glutathione S-transferases are detoxifying enzymes that catalyze the conjugation of a variety of electrophilic substrates to the thiol group of GSH, producing less toxic forms (Hayes and Pulford, 1995), the significant decrease of GST activity in the cerebral cortex after administration of malathion may indicate insufficient detoxification of malathion in this brain region. However, no effect of the treatment was observed in the hippocampus, indicating a regional vulnerability of the enzyme to the effects of malathion. Literature data relative to the effects of malathion exposure on GST activity are diTable 4 GSH-t levels (mol/g wet tissue) in the cerebral cortex and hippocampus of malathion (250 mg/kg, i.p.) and/or zinc chloride (5 mg/kg, i.p.)-exposed rats Treatment
Cerebral cortex
Saline ZnCl2 Malathion Malathion + ZnCl2
1.38 1.37 1.45 1.34
± ± ± ±
0.07 0.11 0.12 0.05
Values are expressed as mean ± S.E.M. (n = 7–8).
Hippocampus 1.03 0.94 1.14 1.16
± ± ± ±
0.07 0.08 0.11 0.08
verse. Some studies, similar to our study, also report a decrease of GST activity in the liver (Hazarika et al., 2003), and erythrocytes (John et al., 2001) of malathion-treated rats. Moreover, a reduction of the brain activity of GST was shown to occur in neonatal rats exposed to malathion, but not in adult female rats (Timur et al., 2003). Hazarika et al., 2003, too, did not report an effect on the brain activity of GST. An increase in the brain activity of GST was also reported in rats chronically exposed to malathion (Srikanth and Seth, 1990). The differences observed between our study and previous publications may be due to the fact that other studies did not determine enzyme activity in specific brain regions. The malathion treatment regime may also be responsible for the differences. Malathion, like other OPs, is detoxified, e.g. via conjugation reactions with GSH (Malik and Summer, 1982). GSH depletion is associated with oxidative stress and cytotoxicity. Results show that malathion exposure caused a reduction of the GSH-t content in blood (Banerjee et al., 1999; Ahmed et al., 2000; Seth et al., 2001). In the present study, acute treatment with the employed dose of malathion was not capable of inducing changes in cerebral GSH-t levels. A study supporting our findings has shown that acute oral administration of malathion to rats did not alter the CNS levels of non-protein thiols whose major component is GSH (Hazarika et al., 2003). However, we cannot rule out the possibility that GSSG had been increased by the malathion exposure in our study. Indeed, the activity of GR, the enzyme responsible for providing reduced
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GSH from its oxidized form (GSSG), was greatly inhibited in the cerebral cortex of malathion-treated rats. In line with this result, is the decrease of GR activity in the brain of rats chronically treated with quinalphos, an OP pesticide (Dwivedi et al., 1998). The activity of G6PDH, an enzyme of the pentose shunt pathway providing reductive equivalents to GR, was increased in the cerebral cortex of malathiontreated rats. This result might be due to a compensatory mechanism which is in line with the reduction of GR activity, since the antioxidant defense mechanisms ultimately rely on the adequate production of NADPH for reducing equivalents during oxidative stress (Minard and McAlister-Henn, 2001; Hashida et al., 2002; Jain et al., 2003). In fact, several studies have demonstrated an increase in G6PDH activity under oxidative stress conditions in the liver cells, tumor cells, and brain of patients with Alzheimer’s disease (Cramer et al., 1995; Ursini et al., 1997; Palmer, 1999; Jain et al., 2003). Related to our work, the treatment of pregnant rats with OP glyphosphate over 21 days produced an increase in G6PDH activity in the brains of the fetuses, while no changes were observed in the pregnant rats (Daruich et al., 2001). 4.2. Zinc Zinc is present in CNS at high concentrations, particularly in the hippocampus, and is known to regulate many physiological responses. It is an essential catalytic or structural element of many proteins, and a signaling messenger that is released by neural activity (Choi and Koh, 1998). Zinc has been shown to have antioxidant properties. Zinc deficiency results in an increase in the oxidative damage (Yousef et al., 2002; Oteiza et al., 2004; Ho et al., 2003). Moreover, zinc can protect against oxidative damage due to xenobiotics or in certain pathological conditions (Fukino et al., 1986; Farinati et al., 2003; Stehbens, 2003). Despite these antioxidant properties, excess zinc in the extracellular fluid may elicit neurotoxic effects (Choi and Koh, 1998; Kim et al., 1999; Pong et al., 2002; Oteiza et al., 2004). In our study, ZnCl2 treatment caused a significant reduction of AChE activity in the cerebral cortex and hippocampus, increased TBARS levels in the hippocampus only (not in the cerebral cortex), and led to a reduction of GST and GR activity only in the cerebral cor-
tex. Otherwise, the activities of GPx and G6PDH in the cerebral cortex, and of GR, GST, GPx, and G6PDH in the hippocampus, was unaffected by ZnCl2 treatment. The lower AChE activity observed in the cerebral cortex and hippocampus of ZnCl2 -exposed rats is in accordance with available data on the brain of zinctreated fish and rats (Kozik et al., 1980; Suresh et al., 1992). It is interesting to note that the region accumulating maximum zinc, i.e. the hippocampus (Choi and Koh, 1998), showed an elevation in lipid peroxidation, indicating that this region may be susceptible to damage by zinc. Although the hippocampal activity of the enzymes GR, GST, GPx and G6PDH was unaffected by zinc treatment, the increased TBARS levels observed in the hippocampus indicate that zinc may have pro-oxidative effects. One possibility to account for this result is the zinc-induced generation of free radicals whose mechanism is unknown (Kim et al., 1999; Pong et al., 2002; Oteiza et al., 2004). Some studies have found that zinc treatment increased TBARS levels in the brain (Padmaja and Ramamurthi, 1997; Lin et al., 2003) and in cortical neuron cultures (Kim et al., 1999; Pong et al., 2002). The absence of an effect of zinc on cortical TBARS levels shows the difference in the sensitivity of the brain regions to the pro-oxidative effects of zinc. The reduction of GST activity in the cerebral cortex of zinc-treated rats is an interesting finding. Very few studies are available in literature regarding the effects of zinc on GST activity. It is reported that a zinc-deficient diet leads to an increase in the activity of brain GST (Yousef et al., 2002). Corroborating our results regarding the reduction of GR activity by zinc treatment is the reported inhibitory effect of zinc on purified GR (Mize and Langdon, 1962) and GR from cultured lung cells (Walther et al., 2003; Wilhelm et al., 2001). The inhibition of GR activity is taken to be one of the major causes of zinc toxicity, which leads to a consequent consumption of GSH and to GSSG accumulation (Walther et al., 2003; Wilhelm et al., 2001). In our study, however, the acute treatment with ZnCl2 did not change GSH-t levels in the hippocampus and cerebral cortex. It is clear from literature that zinc may elicit prooxidative effects (Choi and Koh, 1998; Kim et al., 1999; Pong et al., 2002; Oteiza et al., 2004). That corroborates our results, i.e. reduction of the antioxidant protection
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afforded by GR and GST and increase in lipid peroxidation in the hippocampus. However, more studies will be necessary for a better understanding of the mechanism of zinc-induced oxidative damage. 4.3. Co-administration of malathion and ZnCl2 Co-administration of malathion and ZnCl2 shows a similar reduction of AChE activity in the cerebral cortex and an increase in TBARS levels in the hippocampus. The lipid peroxidation induced by malathion exposure was not prevented by the co-administration of ZnCl2 in the hippocampus, but only in the cerebral cortex. Apart from this small effect on TBARS levels, the co-administration of malathion/ZnCl2 did not produce a synergistic effect on the analyzed parameters. It should be noted that ZnCl2 administration was able to prevent an increase in cortical G6PDH activity. Contrary to initial hypothesis, there was little evidence for interaction between malathion and ZnCl2 except for block of increased cortical TBARS levels and G6PDH activity following malathion in combined group. In fact, it is important to note that zinc itself produced pro-oxidant action, such as the reduction of the level of antioxidant enzymes GR and GST in the cerebral cortex and the increase in TBARS levels in the hippocampus. In that context, the use of zinc as an antioxidant agent deserves more investigation.
Acknowledgements This study was supported by grants from Plano Sul Pesquisa e P´os-Graduac¸a˜ o (PSPPG; 520684/990); Coordenadoria de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES), Conselho de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq), Brazil. The authors thank Barbara Uecker for the English revision of the manuscript.
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Antioxidant defenses and lipid peroxidation in the cerebral cortex and hippocampus following acute exposure to malathion and/or zinc chloride Patr´ıcia S. Brocardoa , Pablo Pandolfoa , Reinaldo N. Takahashib , Ana L´ucia S. Rodriguesa,∗ , Alcir L. Dafrec a
c
Departamento de Bioqu´ımica, Centro de Ciˆencias Biol´ogicas, Universidade Federal de Santa Catarina, SC 88040-900, Florian´opolis, Brazil b Departamento de Farmacologia, Centro de Ciˆ encias Biol´ogicas, Universidade Federal de Santa Catarina, SC 88040-900, Florian´opolis, Brazil Departamento de Ciˆencias Fisiol´ogicas, Centro de Ciˆencias Biol´ogicas, Universidade Federal de Santa Catarina, SC 88040-900, Florian´opolis, Brazil Received 18 August 2004; received in revised form 17 September 2004; accepted 25 September 2004 Available online 18 November 2004
Abstract This study investigates the effects of acute exposure to organophosphate insecticide malathion (250 mg/kg, i.p.) and/or ZnCl2 (5 mg/kg, i.p.), with the following parameters: lipid peroxidation and the activity of acetylcholinesterase (AChE), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH), and the levels of total glutathione (GSH-t) in the hippocampus and cerebral cortex of female rats. Malathion exposure elicited lipid peroxidation and reduced AChE activity in the cerebral cortex and hippocampus. It also reduced the activity of GR and GST, and increased G6PDH activity in the cerebral cortex, without changing the levels of GSH-t and GPx activity. ZnCl2 exposure reduced AChE activity and caused a mild pro-oxidative effect, since lipid peroxidation was increased in the hippocampus. ZnCl2 , individually or in combination with malathion, caused a reduction in GR and GST activity in the cerebral cortex. Malathion and/or ZnCl2 did not change the GSH-t levels. Moreover, ZnCl2 prevented the increase in G6PDH activity caused by malathion. It showed that ZnCl2 had little effect against the changes induced by malathion. In fact, zinc itself produced pro-oxidant action, such as the reduction in the activity of the antioxidant enzymes GR and GST. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Malathion; Zinc; Acetylcholinesterase; Lipid peroxidation; Antioxidant enzymes; Brain
∗
Corresponding author. Tel.: +55 48 331 9692; fax: +55 48 331 9672. E-mail addresses: [email protected], [email protected] (A.L.S. Rodrigues).
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.09.012
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1. Introduction Malathion [O,O-dimethyl-S-(1,2-dicarbethoxy-ethyl)phosphorodithioate] is an organophosphate (OP) insecticide of low mammalian toxicity which is used extensively throughout the world to control major arthropods in public health programs, animal ectoparasites, human head and body lice, household insects and to protect grain in storage (Maroni et al., 2000). Like other OP compounds, malathion is known to inhibit acetylcholinesterase (AChE) activity, an effect that is thought to underlie the neurotoxicity elicited by these compounds (Kwong, 2002). However, some studies indicate that other biochemical targets may be affected by OP insecticides (Ward and Mundy, 1996; Samimi and Last, 2001). Moreover, the lipophilic nature of OPs facilitates their interaction with the cell membrane and leads to perturbations of the phospholipid bilayer structure (Videira et al., 2001). The toxicity of OP agents may be due, at least in part, to the formation of reactive oxygen species (ROS), leading to lipid peroxidation, which is generally assessed by an increase in the levels of thiobarbituric acid reactive substances (TBARS) (Banerjee et al., 1999; Verma and Srivastava, 2001; Ranjbar et al., 2002). Several studies indicate that malathion exposure increases TBARS levels in the erythrocytes, liver, and brain of rats (Hazarika et al., 2003; Akhgari et al., 2003). Other studies indicate that antioxidant enzyme activity was either increased, reduced, or not changed in the liver, brain, and erythrocytes of animals treated with this OP compound (Srikanth and Seth, 1990; Pedrajas et al., 1995; Ahmed et al., 2000; John et al., 2001; Akhgari et al., 2003; Hazarika et al., 2003). The differences found may be due, e.g. to the malathion exposure regime employed, the tissue distribution, or the age of the animals. One area of increasing interest is the study of the capability of some essential mineral elements to modulate the effects of environmental toxicants. In that respect, several studies have shown that zinc, an essential nutrient, at least under certain conditions, may have antioxidant properties (Powell, 2000). Also, zinc deficiency causes a significant increase in glutathioneS-transferase (GST) activity and lipid peroxidation in the serum, liver, and brain of rats (Yousef et al., 2002), as well as a decrease of the activity of super-
oxide dismutase (SOD) in the liver of mice (Chen and Young, 1998). However, an excess of zinc may produce cytotoxic effects, which is easily demonstrated as zinc levels reach high concentrations in the extracellular fluid in the brain (Choi and Koh, 1998; Oteiza et al., 2004). In fact, some studies have demonstrated that zinc induces neuronal death in cultured cortical neurons, an effect that seems to be mediated by oxidative stress (Kim et al., 1999; Pong et al., 2002). In the present study, we have investigated the effect of acute treatment with malathion and/or ZnCl2 on antioxidant defense systems and lipid peroxidation in the cerebral cortex and hippocampus of rats.
2. Materials and methods 2.1. Chemicals The following chemicals were used: commercialgrade malathion (95% purity, CAS 121-75-5, Dipil Chemical Ind., Brazil); nicotinamide adenine dinucleotide phosphate (reduced form; NADPH); nicotinamide adenine dinucleotide phosphate (oxidized form; NADP+ ), glucose-6-phosphate (G6P), tertbutylhydroperoxide (tBOOH), magnesium chloride, oxidized (GSSG) and reduced (GSH) glutathione; 1-chloro-2,4-dinitrobenzene (CDNB); glutathione reductase; acetylthiocholine iodide; bovine serum albumin; 5,5-dithio bis(2-nitrobenzoic acid) (DTNB); malondialdehyde (MDA); and 2-thiobarbituric acid (TBA) from Sigma Chemical Co. (USA). Trichloroacetic acid (TCA) and zinc chloride (ZnCl2 ) were purchased from Merck (Germany). 2.2. Animals and treatments Adult female Wistar rats weighing 180 ± 20 g were kept with free access to food and water on a 12 h light/dark cycle. The rats were housed in plastic cages; they were randomly divided into four groups of eight animals each. The experiments were performed after approval of the protocol by the Ethics Committee of the Institution, and every effort was made to minimize animal suffering. Malathion (250 mg/kg, 1/5 LD50 ); ZnCl2 (5 mg/kg) and saline were administered intraperitoneally (i.p.) in
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a constant volume of 1 ml/kg of body weight. The dose of malathion employed caused no overt signs of cholinergic toxicity. All groups of rats received two contralateral i.p. injections. The second injection was given immediately after the first one. Rats were given: • • • •
Group I: saline and saline; Group II: ZnCl2 and saline; Group III: malathion and saline; Group IV: malathion and ZnCl2 .
Rats were killed by decapitation 24 h after treatment. Hippocampi and cerebral cortices were removed and homogenized 1:2 (w/v; 1 g tissue with 2 ml 20 mM Hepes buffer, pH 7.4). The homogenate was centrifuged at 15,000 × g, for 30 min at 4 ◦ C, and the resultant supernatant was used for different antioxidant enzyme assays. Acetylcholinesterase activity was measured in homogenates centrifuged at 2300 × g, for 15 min. Each sample was taken from one animal and assayed in triplicate.
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2.5. Antioxidant enzyme assays Glutathione peroxidase (GPx) activity was measured by the Wendel (1981) method, using tertbutylhydroperoxide as a substrate. NADPH disappearance was monitored by a spectrophotometer at 340 nm. Glutathione-S-transferase (GST) activity was assayed by the procedure of Habig and Jakoby (1981), using CDNB as a substrate. The assay was conducted by monitoring the appearance of the conjugated complex of CDNB and GSH at 340 nm. Glutathione reductase (GR) activity in brain homogenates was determined by the method described by Carlberg and Mannervik (1985). The reduction of GSSG in the presence of NADPH was measured spectrophotometrically at 340 nm. The activity of glucose-6-phosphate dehydrogenase (G6PDH) was determined by means of the absorbance increase induced by the reduction of NADP+ to NADPH, at 340 nm (Glock and McLean, 1953).
2.3. Glutathione (GSH) assay 2.6. Determination of acetylcholinesterase activity This method takes advantage of glutathione reductase (GR) activity to reduce glutathione disulfide or oxidized (GSSG) to the reduced form, using NADPH as an electron donor. The GSH formed in the previous reaction reacts with the Ellman reagent DTNB to form GSH–TNB, an asymmetric disulfide and the colorforming thionitrobenzoic acid (TNB), which is measured continuously at 412 nm. GR reduces GSH–TNB to GSH, and the second TNB is released, increasing color proportionally with GSH and/or GSSG in the assay. GSH reenters the cycle, reacting with another DTNB and continuing the recycling assay. The assay is performed using a standard curve with known GSSG concentration (Tietze, 1969, modified by Akerboom and Sies, 1981).
Acetylcholinesterase activity of hippocampus and cerebral cortex was estimated by the method of Ellman et al. (1961), using acetylthiocholine iodide as a substrate. The rate of hydrolysis of acetylthiocholine iodide is measured at 412 nm through the release of the thiol compound which, when reacted with DTNB, produces the color-forming compound TNB. 2.7. Determination of protein The protein content in hippocampal and cerebral cortical homogenates was quantified by the method of Bradford (1976), using bovine serum albumin as a standard.
2.4. Measurement of TBARS levels 2.8. Statistical analysis As an index of lipid peroxidation, the formation of TBARS was measured in hippocampal and cerebral cortical crude homogenates according to the method of Ohkawa et al. (1979). The results were expressed in terms of the extent of malondialdehyde (MDA) production.
All data are expressed as mean ± S.E.M. by twoway analysis of variance (ANOVA) (malathion × ZnCl2 ), followed by Newman–Keuls test when appropriate. Differences between groups were considered significant when P < 0.05.
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3. Results
3.3. Antioxidant enzyme activities
3.1. Acetylcholinesterase (AChE) activity
Table 3 shows that treatment with malathion and ZnCl2 , alone or in co-administration, caused a reduction in GST (about 25–35%) and GR (about 25–55%) activity in the cerebral cortex, but no synergistic reduction in enzyme activity was observed in rats treated with both compounds. The cerebral cortical activity of G6PDH increased slightly, yet significantly, in the malathion-treated group, while co-administration of ZnCl2 prevented that effect. These changes in G6PDH, GR and GST activity were not observed in the hippocampus of rats treated with malathion and/or ZnCl2 . No change in GPx activity was observed in the cerebral cortex or hippocampus of any group.
Table 1 shows the effect of acute administration of malathion and/or ZnCl2 on AChE activity. Malathion or ZnCl2 significantly reduced AChE activity in the cerebral cortex and hippocampus as compared with the saline-treated group (control). Moreover, when in combination, malathion and ZnCl2 significantly reduced AChE activity in the cerebral cortex, but not in the hippocampus. 3.2. Thiobarbituric acid reactive substance (TBARS) measurements A significant increase in the TBARS levels was found in the cerebral cortex of rats treated with malathion. In the hippocampus, an increase in TBARS levels was observed in all groups as compared with the control group (Table 2).
3.4. Measurement of total glutathione (GSH-t) levels Malathion and ZnCl2 , alone or in combination, did not significantly alter the levels of GSH in the cerebral cortex and hippocampus of rats (Table 4).
Table 1 Effect of treatment with malathion (250 mg/kg, i.p.) and/or zinc chloride (ZnCl2 ; 5 mg/kg i.p.) on AChE activity in the cerebral cortex and hippocampus of rats
4. Discussion
Treatment
Cerebral cortex
4.1. Malathion
Saline ZnCl2 Malathion Malathion + ZnCl2
47.4 33.9 40.5 35.4
± ± ± ±
1.7 1.5** 0.5** 2.0**
Hippocampus 28.3 21.6 21.2 24.5
± ± ± ±
2.0 1.8* 1.0* 2.2
Enzyme activity is expressed as nmol/min/mg protein. Values are given as mean ± S.E.M., n = 6–8. ∗ P < 0.05 as compared with saline-treated control. ∗∗ P < 0.01 as compared with saline-treated control. Table 2 TBARS levels measured in the cerebral cortex and hippocampus of animals treated with malathion (250 mg/kg, i.p.) and/or zinc chloride (5 mg/kg, i.p.) Treatment
Cerebral cortex
Saline ZnCl2 Malathion Malathion + ZnCl2
260 275 376 312
± ± ± ±
10 15 13** 32
Hippocampus 150 186 180 177
± ± ± ±
6.9 4.8* 8.8* 9.2*
TBARS levels are expressed as nmol/mg wet tissue. Values are expressed as mean ± S.E.M., n = 7–8. ∗ P < 0.05 as compared with saline-treated control. ∗∗ P < 0.01 as compared with saline-treated control.
The inhibition of AChE activity in target tissues is often taken as an indication of pesticide intoxication (Kwong, 2002). In our study, we used AChE activity as a marker of the effectiveness of exposure to malathion. We found that AChE activity was significantly reduced in the cerebral cortex (15%) and hippocampus (25%) of malathion-exposed rats. These results are somewhat similar to those found in the brain of rats exposed to malathion acutely (Hazarika et al., 2003) or chronically (Srikanth and Seth, 1990). Our results also demonstrate an increase on TBARS levels in the cerebral cortex and hippocampus of malathion-exposed rats, indicating that, in addition to AChE inhibition, oxidative stress may be a mechanism underlying the central effects of malathion exposure, as suggested in other studies with malathion (Hazarika et al., 2003) or other OP compounds (Bagchi et al., 1995; Yang et al., 1996; Yang and Dettbarn, 1996). To investigate the impact of malathion on antioxidant defenses in the CNS (cerebral cortex and hip-
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Table 3 Effect of malathion (250 mg/kg, i.p.) and/or zinc chloride (5 mg/kg, i.p.) exposure on antioxidant enzyme activity (GST, GR, G6PDH, GPx) in the cerebral cortex and hippocampus of rats Tissue/treatment
GST
Cerebral cortex Saline ZnCl2 Malathion Malathion + ZnCl2
GR
G6PDH
GPx
112.4 73.1 84.9 80.0
± ± ± ±
4.3 4.4** 7.0** 7.0**
52.0 39.5 22.8 33.7
± ± ± ±
1.7 3.6** 1.5** 4.2**
34.5 37.8 41.9 34.1
± ± ± ±
1.7 0.9 1.1** 1.8
32.9 38.2 34.6 37.3
± ± ± ±
2.4 2.9 1.9 1.6
Hippocampus Saline ZnCl2 Malathion Malathion + ZnCl2
63.3 56.2 59.8 53.2
± ± ± ±
4.9 4.8 6.4 3.3
29.8 33.9 29.3 34.9
± ± ± ±
1.1 2.0 1.9 1.3
17.7 21.9 21.6 20.4
± ± ± ±
2.4 2.6 2.3 1.7
31.4 28.6 30.7 31.5
± ± ± ±
1.8 2.3 1.6 2.7
Activities are expressed as nmol/min/mg protein. Values are expressed as mean ± S.E.M., n = 7–8. ∗∗ P < 0.01 as compared with saline-treated control.
pocampus), four antioxidant enzymes and GSH-t levels were measured in rats treated with a single dose of malathion. Overall, a significant decrease in the GST and GR levels in the cerebral cortex was observed, while the hippocampus was not affected. There were no changes in GPx or GSH-t in either cerebral cortex or hippocampus, while a small, but significant, increase in the G6PDH levels was observed in the cerebral cortex. Considering that glutathione S-transferases are detoxifying enzymes that catalyze the conjugation of a variety of electrophilic substrates to the thiol group of GSH, producing less toxic forms (Hayes and Pulford, 1995), the significant decrease of GST activity in the cerebral cortex after administration of malathion may indicate insufficient detoxification of malathion in this brain region. However, no effect of the treatment was observed in the hippocampus, indicating a regional vulnerability of the enzyme to the effects of malathion. Literature data relative to the effects of malathion exposure on GST activity are diTable 4 GSH-t levels (mol/g wet tissue) in the cerebral cortex and hippocampus of malathion (250 mg/kg, i.p.) and/or zinc chloride (5 mg/kg, i.p.)-exposed rats Treatment
Cerebral cortex
Saline ZnCl2 Malathion Malathion + ZnCl2
1.38 1.37 1.45 1.34
± ± ± ±
0.07 0.11 0.12 0.05
Values are expressed as mean ± S.E.M. (n = 7–8).
Hippocampus 1.03 0.94 1.14 1.16
± ± ± ±
0.07 0.08 0.11 0.08
verse. Some studies, similar to our study, also report a decrease of GST activity in the liver (Hazarika et al., 2003), and erythrocytes (John et al., 2001) of malathion-treated rats. Moreover, a reduction of the brain activity of GST was shown to occur in neonatal rats exposed to malathion, but not in adult female rats (Timur et al., 2003). Hazarika et al., 2003, too, did not report an effect on the brain activity of GST. An increase in the brain activity of GST was also reported in rats chronically exposed to malathion (Srikanth and Seth, 1990). The differences observed between our study and previous publications may be due to the fact that other studies did not determine enzyme activity in specific brain regions. The malathion treatment regime may also be responsible for the differences. Malathion, like other OPs, is detoxified, e.g. via conjugation reactions with GSH (Malik and Summer, 1982). GSH depletion is associated with oxidative stress and cytotoxicity. Results show that malathion exposure caused a reduction of the GSH-t content in blood (Banerjee et al., 1999; Ahmed et al., 2000; Seth et al., 2001). In the present study, acute treatment with the employed dose of malathion was not capable of inducing changes in cerebral GSH-t levels. A study supporting our findings has shown that acute oral administration of malathion to rats did not alter the CNS levels of non-protein thiols whose major component is GSH (Hazarika et al., 2003). However, we cannot rule out the possibility that GSSG had been increased by the malathion exposure in our study. Indeed, the activity of GR, the enzyme responsible for providing reduced
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GSH from its oxidized form (GSSG), was greatly inhibited in the cerebral cortex of malathion-treated rats. In line with this result, is the decrease of GR activity in the brain of rats chronically treated with quinalphos, an OP pesticide (Dwivedi et al., 1998). The activity of G6PDH, an enzyme of the pentose shunt pathway providing reductive equivalents to GR, was increased in the cerebral cortex of malathiontreated rats. This result might be due to a compensatory mechanism which is in line with the reduction of GR activity, since the antioxidant defense mechanisms ultimately rely on the adequate production of NADPH for reducing equivalents during oxidative stress (Minard and McAlister-Henn, 2001; Hashida et al., 2002; Jain et al., 2003). In fact, several studies have demonstrated an increase in G6PDH activity under oxidative stress conditions in the liver cells, tumor cells, and brain of patients with Alzheimer’s disease (Cramer et al., 1995; Ursini et al., 1997; Palmer, 1999; Jain et al., 2003). Related to our work, the treatment of pregnant rats with OP glyphosphate over 21 days produced an increase in G6PDH activity in the brains of the fetuses, while no changes were observed in the pregnant rats (Daruich et al., 2001). 4.2. Zinc Zinc is present in CNS at high concentrations, particularly in the hippocampus, and is known to regulate many physiological responses. It is an essential catalytic or structural element of many proteins, and a signaling messenger that is released by neural activity (Choi and Koh, 1998). Zinc has been shown to have antioxidant properties. Zinc deficiency results in an increase in the oxidative damage (Yousef et al., 2002; Oteiza et al., 2004; Ho et al., 2003). Moreover, zinc can protect against oxidative damage due to xenobiotics or in certain pathological conditions (Fukino et al., 1986; Farinati et al., 2003; Stehbens, 2003). Despite these antioxidant properties, excess zinc in the extracellular fluid may elicit neurotoxic effects (Choi and Koh, 1998; Kim et al., 1999; Pong et al., 2002; Oteiza et al., 2004). In our study, ZnCl2 treatment caused a significant reduction of AChE activity in the cerebral cortex and hippocampus, increased TBARS levels in the hippocampus only (not in the cerebral cortex), and led to a reduction of GST and GR activity only in the cerebral cor-
tex. Otherwise, the activities of GPx and G6PDH in the cerebral cortex, and of GR, GST, GPx, and G6PDH in the hippocampus, was unaffected by ZnCl2 treatment. The lower AChE activity observed in the cerebral cortex and hippocampus of ZnCl2 -exposed rats is in accordance with available data on the brain of zinctreated fish and rats (Kozik et al., 1980; Suresh et al., 1992). It is interesting to note that the region accumulating maximum zinc, i.e. the hippocampus (Choi and Koh, 1998), showed an elevation in lipid peroxidation, indicating that this region may be susceptible to damage by zinc. Although the hippocampal activity of the enzymes GR, GST, GPx and G6PDH was unaffected by zinc treatment, the increased TBARS levels observed in the hippocampus indicate that zinc may have pro-oxidative effects. One possibility to account for this result is the zinc-induced generation of free radicals whose mechanism is unknown (Kim et al., 1999; Pong et al., 2002; Oteiza et al., 2004). Some studies have found that zinc treatment increased TBARS levels in the brain (Padmaja and Ramamurthi, 1997; Lin et al., 2003) and in cortical neuron cultures (Kim et al., 1999; Pong et al., 2002). The absence of an effect of zinc on cortical TBARS levels shows the difference in the sensitivity of the brain regions to the pro-oxidative effects of zinc. The reduction of GST activity in the cerebral cortex of zinc-treated rats is an interesting finding. Very few studies are available in literature regarding the effects of zinc on GST activity. It is reported that a zinc-deficient diet leads to an increase in the activity of brain GST (Yousef et al., 2002). Corroborating our results regarding the reduction of GR activity by zinc treatment is the reported inhibitory effect of zinc on purified GR (Mize and Langdon, 1962) and GR from cultured lung cells (Walther et al., 2003; Wilhelm et al., 2001). The inhibition of GR activity is taken to be one of the major causes of zinc toxicity, which leads to a consequent consumption of GSH and to GSSG accumulation (Walther et al., 2003; Wilhelm et al., 2001). In our study, however, the acute treatment with ZnCl2 did not change GSH-t levels in the hippocampus and cerebral cortex. It is clear from literature that zinc may elicit prooxidative effects (Choi and Koh, 1998; Kim et al., 1999; Pong et al., 2002; Oteiza et al., 2004). That corroborates our results, i.e. reduction of the antioxidant protection
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afforded by GR and GST and increase in lipid peroxidation in the hippocampus. However, more studies will be necessary for a better understanding of the mechanism of zinc-induced oxidative damage. 4.3. Co-administration of malathion and ZnCl2 Co-administration of malathion and ZnCl2 shows a similar reduction of AChE activity in the cerebral cortex and an increase in TBARS levels in the hippocampus. The lipid peroxidation induced by malathion exposure was not prevented by the co-administration of ZnCl2 in the hippocampus, but only in the cerebral cortex. Apart from this small effect on TBARS levels, the co-administration of malathion/ZnCl2 did not produce a synergistic effect on the analyzed parameters. It should be noted that ZnCl2 administration was able to prevent an increase in cortical G6PDH activity. Contrary to initial hypothesis, there was little evidence for interaction between malathion and ZnCl2 except for block of increased cortical TBARS levels and G6PDH activity following malathion in combined group. In fact, it is important to note that zinc itself produced pro-oxidant action, such as the reduction of the level of antioxidant enzymes GR and GST in the cerebral cortex and the increase in TBARS levels in the hippocampus. In that context, the use of zinc as an antioxidant agent deserves more investigation.
Acknowledgements This study was supported by grants from Plano Sul Pesquisa e P´os-Graduac¸a˜ o (PSPPG; 520684/990); Coordenadoria de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES), Conselho de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq), Brazil. The authors thank Barbara Uecker for the English revision of the manuscript.
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