Protective effect of melatonin on propoxur-induced impairment of memory and oxidative stress in rats
Abstract
Propoxur (a carbamate pesticide) has been shown to adversely affect memory and induce oxidative stress on both acute and chronic exposure. This study was designed to explore the modulation of the effects of propoxur over cognitive function by melatonin (MEL). Cognitive function was assessed using step-down latency (SDL) on a passive avoidance apparatus, and transfer latency (TL) on an elevated plus maze. Oxidative stress was assessed by examining brain malondialdehyde (MDA) and reduced glutathione (GSH) levels and catalase (CAT) activity. A significant reduction in SDL and prolongation of TL was observed for the propoxur (10 mg/kg/d; p.o.) treated group at weeks 6 and 7 when compared with control. One week treatment with MEL (50 mg/kg/d; i.p.) antagonized the effect of propoxur on SDL, as well as TL. Propoxur produced a statistically significant increase in the brain MDA levels and decrease in the brain GSH levels and CAT activity. Treatment with MEL attenuated the effect of propoxur on oxidative stress. The results of the present study thus show that MEL has the potential to attenuate cognitive dysfunction and oxidative stress induced by toxicants like propoxur in the brain. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 705–713, 2014.
INTRODUCTION
Propoxur, a carbamate insecticide has been shown to produce neurotoxicity apart from a number of other adverse effects. It has been associated with decrease in memory and hand eye co-ordination. Earlier studies performed in our laboratory have shown that the toxicity of several pesticides like lindane, phosphamidon, and propoxur result from increased lipid peroxidation in the brain regions accompanied by decreased levels of glutathione, and an accompanied decrease in the activity of glutathione peroxidase, glutathione reductase, catalase (CAT), and superoxide dismutase (SOD) (Sahaya et al., 2007; Suke et al., 2008; Gupta et al., 2009; Joshi et al., 2010; Kosta et al., in press; Mehta et al., 2010; Sharma et al., 2011, in press). Carbamates primarily interact with sulfhydryl groups, most likely by interfering with glycolysis and the construction of fibrillary proteins like neurofilament. The damaged caused to the nervous system has been linked to inhibition of activities of acetylcholinesterase and gamma amino butyric acid (GABA) (Tilson et al., 1987; Smuldeas et al., 2003). Propoxur also causes oxidative stress and suppression of humoral immune response in rats. It causes lipid peroxidation by generating oxygen free radicals and superoxide ions. SOD, CAT, and glutathione are also altered following propoxur exposure (Suke et al., 2006; Eraslan et al., 2009; Maran et al., 2009).
Melatonin (MEL), also known as N-acetyl-5-methoxytryptamine, is synthesized mainly by pineal gland, and it participates in many important physiological processes. It has been reported in earlier studies that this neurohormone has potent antioxidant properties (Tan et al., 1993; Gulcin et al., 2002; Gulcin et al., 2003; Peyrot and Ducrocq, 2008; Hardeland et al., 2009; Reiter et al., 2009; Jou et al., 2010; Kaur et al., 2010; Nopparat et al., 2010), besides exerting immune system modulation and anti-inflammatory activity (Pappolla et al., 1998; Rosales-Corral et al., 2003; Reiter et al., 2008). It is also a potent scavenger of hydroxyl radical (Tan et al., 1993), and readily crosses the blood–brain barrier (Menedez-palaez et al., 1993) where it provides oxidative protection (Reiter, 1998; Reiter et al., 2000). It also quenches other reactive species, such as hydrogen peroxide (Tan et al., 2000), singlet oxygen (Cagnoli et al., 1995), peroxynitrite (Gilad et al., 1997; Blanchard et al., 2000), and nitric oxide (Noda et al., 1999; Blanchard et al., 2000; Trujanski et al., 2000). MEL may reduce oxidative stress by stimulating certain important detoxifying enzymes, such as SOD (Antolin et al., 1996) and glutathione peroxidase (Barlow-Walden et al., 1995), a key protective enzyme in brain. MEL alleviates lipid peroxidation in brain from many free radical–generating toxicants (Melchiorri et al., 1995; Yamamoto and Tang, 1996; Reiter et al., 1997). More recently, it has been shown that the MEL metabolites, N1-acetyl-N2-5-formyl-methoxykynuramine and N1-acetyl-5-methoxykynuramine, in the CNS have the ability to downregulate pro-oxidative and proinflammatory enzymes (Hardeland et al., 2009). The present study was therefore designed to investigate the effect of MEL on propoxur-induced modulation of cognitive function and oxidative stress in rats.
MATERIALS AND METHODS
Animals
Male Wistar rats, weighing between 120 and 220 g, were used in the study. The animals were procured from the Central Animal House, University College of Medical Sciences. The animals were housed in standard laboratory conditions with pellet diet and water available ad libitum. Appropriate permissions were obtained from Institutional Animal Ethics Committee, and care of the animals was as per guidelines of ‘Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India,’ for laboratory animal facilities.
Chemicals and Treatment Schedule
Propoxur (98% purity) and MEL (98% purity) were obtained from Sigma chemicals (USA). All other chemicals used were of laboratory grade. Propoxur was given per orally (p.o.) with groundnut oil as a vehicle. To limit the weight gain of animals due to oil consumption, the concentration of propoxur was maintained such that no animal received more than 0.5 mL of oil per day, anytime during the study period. MEL was administered intraperitoneally (i.p.) in a dose of 50 mg/kg/day, after dissolving in distilled water with two drops of Tween-80 added per 10 mL of suspension. Each animal received 0.5 mL of suspension per 100 g of body weight per day.
Animals were randomly divided into six groups having 10 rats per group. All animals received propoxur or vehicle for propoxur (p.o.) for 6 weeks. MEL or its vehicle was administered i.p. for one week after 6-week treatment with propoxur (Table 1). Different groups were evaluated for cognitive function 1 day before the start of treatment, at 6 weeks and on the day of completion of treatment schedule. Animals were trained on each day prior to assessment of cognition. Animals were tested after 30 min for acquisition (training) and after 24 h for retention (testing for retention of the learned task) of memory. Finally, at the end of study period the animals were sacrificed using deep ether anesthesia and brain taken out to assess oxidative stress. All the experiments were conducted in the Neuropharmacology Laboratory, Department of Pharmacology, University College of Medical Sciences, Delhi between 0900 and 1600 hrs.
| S. No. | Group | Propoxur/Vehicle for 6 weeks | MEL/ Vehicle for 1 week |
|---|---|---|---|
| 1 | Control | Vehicle | Vehicle |
| 2 | Propoxur | Propoxur (10 mg/kg/d, p.o.) | Vehicle |
| 3 | Only MEL | Vehicle | MEL (50 mg/kg/d, i.p.) |
| 4 | Propoxur + MEL | Propoxur (10 mg/kg/d, p.o.) | MEL (50 mg/kg/d, i.p.) |
- MEL, Melatonin.
Assessment of Cognition
Step Down Latency in Passive Avoidance Apparatus
This apparatus consists of a grid floor on the center of which a wooden block is placed. The block served as a shock free zone (SFZ). The rat was placed on the SFZ and on stepping down was given electric shock (20 V, 50 Hz, 1 mA, 1 s) through the grid floor. The experiment was repeated after half hour and the time taken by the rat to step down was observed (step-down latency, SDL; acquisition of memory). The procedure was repeated after 24 h without shock (retention of memory). A cut-off time of 180 s was taken and for the animal which did not step down during this period, SDL was taken as 180 s (Mondadori et al., 1994; Izquierdo et al., 1995; Joshi and Parle, 2006).
Transfer Latency on Elevated Plus Maze
The elevated plus maze consisting of two open arms (50 cm × 10 cm) and two closed arms (50 cm × 10 cm × 40 cm) with an open roof was used. The maze was elevated to a height of 50 cm from the floor. The animals were placed individually at either ends of the open arms and allowed to enter the closed arms. During first time screening, if the animal did not enter the closed arm within 180 s, it was not included in the experiment. To become acquainted with the maze, the animals were allowed to explore the maze for 20 s after reaching the closed arm and then returned to their home cage. The learning was tested 30 min later on the same day and the animals were retested 24 h after the first day training to test the retention of memory. The time which the animal takes to move from the open arm to the closed arm is taken as transfer latency (TL). A time of 180 s was taken as cut-off and animals not entering the closed arm during this period were assigned the TL of 180 s (Itoh et al., 1990; Dhingra et al., 2003; Naidu et al., 2004).
Determination of Oxidative Stress
At the end of study period, animals were sacrificed by deep ether anesthesia; brain was quickly dissected out in toto, washed with ice-cold sodium phosphate buffer, weighed and stored over ice. The brains were further processed within half hour of dissection, and the estimation of oxidative stress done on the same working day. Brain tissue was homogenized with 10 times (w/v) sodium phosphate buffer (pH 7.4, ice cold, mixture of KH2PO4 and Na2HPO4). The homogenate was centrifuged at 3000 rpm for 15 min and the supernatant was used for estimation of malondialdehyde (MDA), reduced glutathione and CAT.
MDA Estimation
MDA, an indicator of lipid peroxidation was estimated as described by Okhawa et al. (1979). A sample containing 0.5 mL of supernatant was mixed with 1 mL trichloroacetic acid (20%, pH 3.5), 1.5 mL thiobarbituric acid (0.8%) and 0.2 mL sodium lauryl sulfate (8.1%) and heated at 100°C for 1 h. After cooling with tap water, 5 mL of butanol: pyridine (15:1, v/v) and 1 mL of distilled water were added. The mixture was vortexed vigorously and was centrifuged at 4000 rpm for 10 min. Thereafter, the organic layer was withdrawn and absorbance measured at 532 nm using a spectrophotometer.
Reduced Glutathione Estimation
GSH was estimated by the method as described by Ellman ( 1959). To 0.5 mL of the supernatant obtained above 1 mL trichloroacetic acid (5%) was added and the mixture centrifuged to remove the proteins. To 0.1 mL of this homogenate, 4 mL of phosphate buffer (pH 8.4), 0.5 mL of DTNB (5,5-dithiobis 2-nitrobenzoic acid) and 0.4 mL double distilled water were added. The mixture was vortexed and absorbance read at 412 nm within 15 min.
CAT Estimation
Brain CAT activity was estimated by the colorimetric method described by Clairborne ( 1985). A total of 189 μL of 30% H2O2 was taken and mixed with 100 mL of phosphate buffer. A total of 2.95 mL of this solution was taken and 50 μL of supernatant was added and the mixture was immediately put into spectrophotometer and kinetics was read for 90 s (with three readings of 30 s each) at 240 nm.
Statistical Analysis
All the values are expressed as mean ± standard error of the mean (SEM). Data were analyzed using one-way ANOVA with post hoc Tukey's test (α = 0.05). All statistical analyses were carried out using the SPSS v13.0 software, in consultation with the Department of Biostatistics and Medical Informatics, University College of Medical Sciences, Delhi.
RESULTS
Cognitive Assessment Parameters
Step-Down Latency
At the start of the experiment (i.e., on day 0), no significant differences were found among the SDL values of all the experimental groups. A significant reduction in both acquisition as well as retention in SDL paradigm was observed for the propoxur treated group at weeks 6 and 7 when compared with SDL values of both control (p < 0.001) and day 0 of propoxur-treated groups (p < 0.001) [Fig. 1(A,B)]. A significant reduction in SDL values was also noted for propoxur + MEL group at week 6 (p < 0.001). After 1 week treatment (i.e., at the end of 7th week), MEL was observed to antagonize the effect of propoxur on SDL in both acquisition as well as retention paradigms [Fig. 1(A,B)].
Effect of propoxur, and melatonin (MEL) on (A) acquisition on SDL; (B) retention on SDL. All animals received propoxur or vehicle for propoxur (10 mg/kg/d, p.o.) for 6 weeks. MEL or its vehicle was administered i.p. for 1 week after treatment with propoxur in a dose of 50 mg/kg/d. The different groups were evaluated for cognitive function 1 day before the start of treatment, at 6 weeks and on the day of completion of treatment schedule. Animals were trained on each day prior to assessment of cognition. Animals were tested after 30 min for acquisition and after 24 h for retention of memory. Values are expressed as mean ± SEM, for 10 animals in each group. ap < 0.001 when compared with normal control group; bp < 0.001 as compared to propoxur only group.
Transfer Latency
At day 0, no significant difference was found among the TLs of all the groups. In the propoxur treated group, a significant (p < 0.001) prolongation in both acquisition and retention was observed when compared with control [Fig. 2(A,B)]. Same effect was also noted in the propoxur + MEL group at 6th week when compared with control, and MEL only groups. There was marked reduction in TL of propoxur+MEL group (p < 0.001) when compared with propoxur only group at 7th week [Fig. 2(A,B)].
Effect of propoxur, and melatonin (MEL) on (A) acquisition on TL; (B) retention on TL. All animals received propoxur or vehicle for propoxur (10 mg/kg/d, p.o.) for 6 weeks. MEL or its vehicle was administered i.p. for 1 week after treatment with propoxur in a dose of 50 mg/kg/d. The different groups were evaluated for cognitive function 1 day before the start of treatment, at 6 weeks and on the day of completion of treatment schedule. Animals were trained on each day prior to assessment of cognition. Animals were tested after 30 min for acquisition and after 24 h for retention of memory. Values are expressed as mean ± SEM, for 10 animals in each group. ap < 0.001 as compared to normal control group; bp < 0.001 as compared to propoxur only group.
Oxidative Stress Parameters
Lipid Peroxidation
There was a marked and statistically significant (p < 0.001) increase in the brain MDA levels of group treated with only propoxur (Table 2). Treatment with MEL attenuated the effect of propoxur on MDA level, the difference between propoxur alone and propoxur+MEL was found to be significant (p < 0.001). No difference was observed in the brain MDA levels of MEL only, and control group (Table 2).
| Groups/Weeks | Treatment (6 weeks + 1 week) | MDA (nmol/g wet brain tissue) | GSH (μg/g wet brain tissue) | CAT (nmol of H2O2 consumed/min/mg) |
|---|---|---|---|---|
| Control | Vehicle (groundnut oil) | 166.70 ± 5.32 | 393.00 ± 11.66 | 26.14 ± 1.30 |
| Propoxur | Propoxur (10 mg/kg/d, p.o.) | 508.22 ± 4.35a | 208.62 ± 10.72a | 16.12 ± 0.82a |
| Only MEL | MEL (50 mg/kg/d, i.p.) | 173.50 ± 17.35 | 567.37 ± 16.35a | 17.06 ± 0.92 |
| Propoxur + MEL | Propoxur (10 mg/kg/d, p.o.) + MEL (50 mg/kg/d, i.p.) | 181.50 ± 7.43b | 533.50 ± 13.16a, b | 18.96 ± 0.57c |
- Values are expressed as mean ± SEM.
- a Values differ significantly from normal control (p < 0.001).
- b Values differ significantly from propoxur only group (p < 0.001).
- c Values differ significantly from propoxur only group (p < 0.05).
Reduced Glutathione
A significant (p < 0.001) decrease was found in the brain GSH levels of propoxur treated group when compared with control (Table 2). A significant increase was noted for MEL (p < 0.001), and propoxur + MEL (p < 0.001) treated groups when compared with the control group for propoxur (Table 2). MEL antagonized the effect of propoxur on GSH. The difference between propoxur alone and propoxur + MEL group was found to be statistically significant (p < 0.001).
Catalase
There was a marked and statistically significant decrease in the brain CAT activity of group treated with only propoxur (p < 0.001). Treatment with MEL attenuated the effect of propoxur on CAT, the difference between propoxur alone and propoxur+MEL was found to be significant (p < 0.01). No difference was observed in the brain CAT activity of MEL only, and control groups (Table 2).
DISCUSSION
The present study was designed to explore the effect of MEL on propoxur-induced cognitive dysfunction by measuring the SDL in continuous avoidance paradigm, and TL in the plus-maze apparatus. Since, oxidative stress can affect memory and both MEL and propoxur have influence on the oxidative stress, parameters of oxidative stress viz. MDA, GSH and CAT were also estimated in the brain at the end of the study period.
The results of the present study demonstrate that SDL was reduced and TL was prolonged on long-term (6 weeks) administration of propoxur. MEL was able to reverse the impairment in memory caused by propoxur when administered for 1 week following pretreatment with propoxur for 6 weeks.
In the present study, it was observed that MEL had attenuating effect on propoxur-induced cognitive dysfunction. MEL and its metabolites have strong antioxidant properties that act as endogenous buffering agents against oxidative stress (Manda et al., 2008; Peyrot and Ducrocq, 2008; Hardeland et al., 2009). This suggests there may be physiological alterations when MEL secretion is reduced in old age. In earlier studies performed in our laboratory, we observed that the administration of MEL, piracetam, ascorbic acid, 4′-chlorodiazepam, pregnenolone sulfate and vitamin E attenuated cognitive dysfunction and oxidative stress induced by phosphamidon, lindane or propoxur (Sahaya et al., 2007; Gupta et al., 2009; Mehta et al., 2010; Kosta et al., in press; Sharma et al., in press). Moreover, progesterone was observed to be effective in modulating cognitive dysfunction induced by phosphamidon, whereas it failed to modulate the same effect when induced by lindane or propoxur (Sahaya et al., 2007; Mehta et al., 2010; Sharma et al., 2011). In an earlier report, MEL decreased the oxidative stress produced by 2,4-dichlorophenoxyacetic acid in rat cerebellar granule cells (Bongiovanni et al., 2007). MEL has also been observed to reduce protein, lipid, and DNA oxidation induced by phosphine (Hsu et al., 2000, 2002), X-rays (Manda et al., 2007), or hypochlorous acid (Zavodnik et al., 2004) because of its free radical–scavenging property. This may also in part relate to the free radical theory of aging (Harman, 1956). Besides being a direct scavenger of radicals, MEL has indirect antioxidative actions as well (Rodriguez et al., 2004). Oxidative damage is considered a likely cause of brain dysfunction because the brain is believed to be particularly vulnerable to oxidative stress owing to a relatively high rate of oxygen free radical generation without suitable levels of antioxidant defenses compared with other somatic tissues (Coyle and Puttfarken, 1993; Reiter, 1998). Exogenous MEL may prevent the increased production of lipid peroxidation products and might have a potential role for retardation of oxidative events (Akbulut et al., 2008).
Propoxur resulted in induction of oxidative stress in brain as evidenced by reduction in brain GSH and CAT activity and increase in brain MDA levels. Lipid peroxidation has been postulated as the primary event mediating the toxicity of a broad spectrum of pesticides. Dowla and coworkers ( 1996) observed that in vitro activities of delta-amino levulinic acid dehydratase and Cu-Zn SOD in human red blood cells were inhibited following methamidophos exposure. In pesticide poisoning cases, tissue glutathione reductase (GR), glutathione peroxidase, SOD and CAT activities, as well as MDA production are increased but GSH levels are decreased suggesting adaptive measure to tackle any insecticide accumulation (Banerjee et al., 1999). These enzymes efficiently scavenge toxic free radicals and partly protect against lipid peroxidation from pesticide exposure. The generation of oxidative stress by propoxur in the present study is in strong agreement with the findings of Suke et al. ( 2006).
In the present study, propoxur adversely affected the parameters of memory and oxidative stress. MEL demonstrated favorable effects on these parameters. The results of the present study provided evidence that SDL was reduced and TL prolonged on long-term (6 weeks) administration of propoxur. MEL was able to reverse the impairment in memory caused by propoxur when administered for 1 week following pretreatment with propoxur for 6 weeks. Furthermore, propoxur alone increased the oxidative stress, whereas MEL was able to reverse this increase by decreasing the MDA levels and increasing the NP-SH levels. These results are in line with earlier studies, which have reported attenuating effects of MEL on oxidative damage following pesticide exposure (Melchiorri et al., 1995; Hsu et al., 2000; Hsu et al., 2002; Suke et al., 2006; Bongiovanni et al., 2007; Omurtag et al., 2008). Thus, the study reveals a possible correlation between memory impairment and oxidative stress in brain signifying yet another potential role of MEL in the functioning of nervous system and possible use of this group of drugs in reversing the damage induced by pesticides like propoxur in the brain.
Oxidative stress has been intimately related to cognitive dysfunction. Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease in humans and several animal models of Alzheimer's disease (Veinbergs et al., 2000; Sharma and Gupta, 2002; Parle and Dhingra, 2003; Aslan and Ozben, 2004). At the same time, the mechanism of influence of propoxur over cognition and behavior is not solely limited to oxidative stress. It has been postulated that propoxur achieves its behavioral effects and neurotoxicity through several mechanisms like inhibiting acetylcholinesterase, interfering with GABA, affecting energy supply, glucose utilization and SH-groups in neurons (Tilson et al., 1987; Smuldeas et al., 2003; Schmuck and Mihail, 2004). In the present study, propoxur adversely affected the parameters of memory and oxidative stress. MEL demonstrated favorable effects on these parameters. Hence, a co-relation between oxidative stress and memory impairment could be a possibility. However, the effect of MEL on other classes of pesticides such as organochlorines, organophosphates, etc. needs to be studied.
CONCLUSION
MEL was able to reverse the propoxur induced cognitive impairment in the SDL and TL paradigms. It also reversed the derangement in oxidative stress parameters of MDA, GSH and CAT caused by propoxur. This study reveals a possible correlation between memory impairment and oxidative stress in brain signifying a potential role of MEL in the functioning of nervous system and possible use of this chemical in reversing the damage induced by pesticides like propoxur in the brain.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
