Introduction

Organophosphate (OP) compounds are the most common chemical agents to kill pest and insects. Recently, more than 100 different OP compounds have been synthesized and are extensively used worldwide in agriculture, homes, gardens, veterinary practices, medicine and industry (Kwong, 2002; Yurumez et al., 2007). The most well-known OP compounds group are malathion, parathion, fenthion, diazinon, dimethoate, dichlorvos, dimefox, chlorpyrifos, paraoxon, soman, sarin, tabun, echothiophate, isoflurophate, tribufos, merphos and trichlorfon (Kwong, 2002; Pena-Llopis, 2005). Evidences suggest that the formation of oxygen free radicals can be a major factor in the toxicity of organophosphorous insecticides thus producing oxidative stress (Ciccheti et al., 2003 and Abdollahi et al., 2004). Chlorpyrifos, a sulphur containing organophosphate has been used widely in agriculture and animal husbandry. The main mechanism of chlorpyrifos (CPF) toxicity involves inhibition of acetylcholinesterase (AChE) enzyme and it is bio-transformed to its oxygen analogue-chlorpyrifos oxon, before it functions as potent cholinesterase inhibitor (O’ Brien, 1967). Chlorpyrifos produced oxidative stress results in the accumulation of lipid peroxidation products in different organs of rats (Verma and Srivastava, 2003) and has also been shown to damage DNA (Shadnia et al., 2005). Oxidative stress has been implicated as a contributing mechanism in development of different neurotoxic effects of chlorpyrifos such as seizures (Gupta, 2001), developmental neurotoxicity (Qiao, 2005), retinal toxicity (Yu 2008), hepatotoxicity and nephrotoxicity. The diversity of mechanisms underlying the toxicity of chlorpyrifos is one of the reasons that urge investigators for identification of target organs that may contribute to the understanding of the mechanism of action. Since there is limited research investigating the oxidative stress changes induced by chlorpyrifos at tissue level, so the objective of this study was to evaluate the toxicity of the administered dose of chlorpyrifos in adult rats based on the results of the histological oxidative stress changes.

Organophosphorus insecticides (OPI’s) including chlorpyrifos form chelating complexes with metals. Lead is one of such metallic environmental pollutants, particularly widespread in industrial areas. Animals are exposed to lead from numerous sources as well as from the general environment. The main sources of contamination of feed by lead are soil, industrial pollution, agricultural technology and feed processing. Ingested lead has resulted in poisoning, poor performance and death in animals (Swarup et al., 2007). Studies have reported that lead has a potential for inducing oxidative stress and acts as a catalyst in the oxidative reactions of biological macromolecules. Therefore, the toxicities associated with this metal might be due to oxidative tissue damage (Donaldson, 1991). Although lead toxicity has been extensively studied in animals, there are only a limited number of studies on the effects of lead on tissue oxidative stress changes. Therefore, the purpose of the present study was to evaluate the effects of dietary lead exposure on, lipid peroxidation status in the serum, brain, lungs, liver, heart and kidneys of wistar rats. To prevent peroxidative tissue damage, there are protective mechanisms in vivo, such as an enzymatic defense system (antioxidant enzymes) and free-radical scavengers (antioxidants). Ascorbic acid is a well-known antioxidant vitamin involved in several biochemical processes in biological systems. This vitamin breaks the chain of lipid peroxidation in cell membranes and scavenges free radicals such as reactive oxygen species (Carr and Frei, 1999; Kucuk, 2003). Therefore during our current study we aimed to determine whether the negative effects of lead and chlorpyrifos, attributed to tissue peroxidation, could be reversed by adding ascorbic acid.

Material and Methods

Experimental Animals

The wister rats weighing between 150-200 gm used in the present study were procured from IIIM (CSIR, Lab) Jammu, India and were maintained under standard environmental conditions with ad lib feed and water. The animals were treated humanely during the whole period of experimental study and the work was considered by the institutional Animal Ethics Committee vide No.AU/FVSc/C-11/2456-68 on ethical standards in animal experimentation.

Chemicals

Chlorpyrifos (20%w/v), used was commercially obtained from Tata Rallis India Limited, Mumbai as Tafaban in 1litre pack. Lead acetate (99.9%) and ascorbic acid (99.9%) were purchased from Hi-Media Labs Mumbai. All other chemicals used in the study were of extra pure quality and purchased from Hi-media, S.D. Fine Chem. Pvt. Ltd., Qualigens Chem. (Mumbai) and E. Merck (Mumbai).

Experimental Design

These rats were randomly allocated to eight groups of six rats each and subjected to various daily treatment regimes for 98 days. Group I served as control receiving only corn oil, group II received chlorpyrifos @ 5.5 mg/ kg.(1/25^th LD₅₀) in corn oil, group III received lead acetate @100 ppm in water, whereas animals in group IV received a combination of chlorpyrifos @ 5.5mg/kg in corn oil and lead acetate @ 100 ppm in water. Group V received vitamin C @ 100mg/kg in water, group VI received a combination of chlorpyrifos @ 5.5mg/kg and vitamin C @ 100mg/kg, group VII received lead acetate @ 100 ppm in water and vitamin C @ 100mg/kg and group VIII received chlorpyrifos @ 5.5mg/kg, lead acetate @100ppm in water and vitamin C @ 100mg/kg. The administration of the toxicants was carried out between 9:30-10:30 AM daily upto 98 days. All the rats were weighed at weekly intervals during exposure with toxicants and necessary corrections in chlorpyrifos dosage were made according to the changes in the body weight. After collection of blood on 98^th day, animals of all groups were killed by cervical dislocation. The organs were examined for any gross abnormality. Organs like liver, kidneys, heart, lungs and brain were removed, cleaned free from extraneous material and 1g of tissue was taken in 10 ml ice-cold 0.1M potassium phosphate buffer (pH 7.4) and its homogenate was prepared under cold conditions using tissue homogenizer. These homogenates were centrifuged at 4000 rpm for 10-15 min and the supernatant was collected and used for determination of membrane peroxidation and activities of enzymes indicative of oxidative stress.

Statistical Analysis

Standard statistical procedures were followed and the data collected during the experiment was subjected to analysis of variance which was carried in completely randomized design (CRD) and the significance was tested using Duncan Multiple Range Test (Duncan 1955). The significance was assayed at 5% (p<0.05) levels.

Results and Discussion

Most of OP compounds are highly lipid–soluble agents and are well absorbed from the skin, oral mucous membranes, conjunctiva and gastrointestinal and respiratory tracts. The onset, severity and duration of poisoning is determined by the dose, route of exposure, physicochemical properties of the OP (e.g. lipid solubility), rate of metabolism (whether transformation in the liver is required before the compound becomes toxic) and whether the organophosphorylated cholinesterase ages rapidly (Karalliedd et al., 2003). Furthermore, recent studies have revealed that oxidative stress could be an important component of the mechanism of OP compound poisoning [Altuntas et al., 2004; Dandapani et al., 2003]. Oxidative stress is induced in both acute and chronic intoxication with organophosphate compounds in humans and experimental animals, which is manifested by changes in the activity of antioxidative enzymes and/or altered levels of non enzymatic antioxidants in various organs of the organism. Several antioxidant enzymes and molecules have been used to evaluate oxidative damage in animal and human studies. Reduced glutathione (GSH) and glutathione disulfide (GSSG) concentrations, as well as modifications in superoxide dismutase (SOD) activity are the most frequently used markers in tissues or in blood. Moreover, many authors have shown the effects of oxidative stress in the form of increased concentration of e.g. malonyldialdehyde (MDA), being a lipid peroxidation marker as well as increased level of (ROS) (Abdollahi et al., 2004). Lipid peroxidation has been measured by quantifying the thiobarbituric acid reactive substances. The cell has several ways to alleviate the effects of oxidative stress, either by repairing the damage (damaged nucleotides and LPO by-products) or by directly diminishing the occurrence of oxidative damage by means of enzymatic and non-enzymatic antioxidants. Enzymatic and non-enzymatic antioxidants have also been shown to scavenge free radicals and ROS (Gultekin et al. 2001). Non-enzymatic antioxidants such as vitamin E and vitamin C can also act to overcome oxidative stress, being a part of the total antioxidant system. Vitamin C is hydrophilic and is a very important free-radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and protecting biomembranes from peroxidative damage (Harapanhalli 1996). In addition to its antioxidant effects, vitamin C is involved in the regeneration of tocopherol from tocopheroxyl radicals in the membrane. During the current study a significant increase of MDA levels (Table 1) was observed in liver, lung, kidney and brain tissues of groups II and III as compared to group I. Also a significant increase was observed in liver MDA levels in groups IV and VII. MDA levels in kidneys showed a significant increase in group VI while as a significant increase was observed in MDA levels in brain tissue of groups IV, VII and VIII. Similar types of results were reported from the studies of Gao et al., 2003 and Verma and Srivastava (2001) using chlorpyrifos and Patra and Swarup (2001), Gurer et al., 1999 using lead in rats. In the present study vitamin C ameliorated toxic lipid peroxidative effects as has also been reported by Bashandy (2006) and Altuntas and Delübas (2002) in lead and fenthion and treated rats.

Table 1: Effect of Repeated Oral Administration of Chlorpyrifos, Lead Acetate, Vitamin C Alone and in Combinations on Tissue Lipid Peroxidation (nmol MDA formed/ g tissue) in Rats

Values given are mean ± SE of the results obtained from 6 animals; Means with at least one common superscript do not differ significantly at 5% (P<0.05)

SOD is the first line of defense against the action of O₂^– and other reactive oxygen species (ROS). Superoxide radicals are produced in mitochondria and endoplasmic reticulum as a consequence of auto-oxidation of electron transport chain components. These superoxide free radicals are generated during monovalent reduction of oxygen and are toxic to biological systems. SOD catalyses the conversion of superoxide radical (O^-2) to hydrogen peroxide (H₂O₂), while catalase converts H₂O₂ to H₂O. So, these enzymatic antioxidants may counteract oxidative stress and can alleviate the toxic effects of reactive oxygen species, ROS (Bagchi et al., 1995). During the current study a significant decrease in SOD levels (Table 2) of liver, lungs and kidneys was observed in groups II and IV. Also a significant decrease in SOD levels in liver, heart and brain was observed in group VI along with a significant decrease in kidney SOD levels in group VII while as group III showed significant decrease of SOD levels in lung and kidney tissues. This decrease in SOD activity could be due to the increased production of ROS as evident from the increased LPO levels. However, a few studies have indicated that superoxide radicals can also inhibit catalase (CAT) activity and the increased H₂O₂ levels resulting from CAT inhibition could finally inhibit SOD activity. Thus, increased formation of MDA could be due to both increase in pesticide induced ROS formation and SOD inhibition (Gultekin et al., 2001).These findings are in consonance with the studies of Verma and Srivastava (2003) in CPF and El-Nekeety et al., 2009 and Gurer et al., 2000 in lead treated rats. Ameliorative effect of vitamin C in the present study is in accordance with studies of Verma et al., 2007 and El-Tohamy and El-Nattat (2010) on chlorpyrifos and lead acetate respectively.

Table 2: Effect of Oral Administration of Chlorpyrifos, Lead Acetate, Vitamin C Alone and in Combinations on Tissue Superoxide Dismutase Activity (U/g Tissue) in Rats

Values given are mean ± SE of the results obtained from 6 animals; Means with at least one common superscript do not differ significantly at 5% (P<0.05)

Catalase a haeme-containing enzyme in present in all aerobic eukaryotes, important for the removal of hydrogen peroxide generated in peroxisomes (microbodies) by oxidases, involved in ß-oxidation of fatty acids, the glyoxylate cycle (photo-respiration) and purine catabolism. There was no significant change in lung and heart tissue levels of catalase (Table 3) in any of the treatment groups as compared to control group on 98^th day of experimentation.

Table 3: Effect of Oral Administration of Chlorpyrifos, Lead Acetate, Vitamin C Alone and in Combinations on Tissue Catalase Activity (mmol H₂o₂ decomposed/min/g Tissue) in Rats

Values given are mean±SE of the results obtained from 6 animals. Means with at least one common superscript do not differ significantly at 5% (P<0.0)

Catalase levels in liver and kidneys decreased significantly in groups I, II, III. A significant decrease was also observed in brain tissue of groups II and VI. These findings are in agreement with previous studies of Gultekin et al. (2001) and Verma et al. (2007) on CPF treated rats. Decreased catalase levels during lead acetate treatment in current study and ameliorative effect of vitamin C over lead induced alterations in catalase activity are in agreement with studies of El-Tohamy and El-Nattat (2010) in male rabbits. Glutathione peroxidase is a selenium containing enzyme which reduces hydrogen peroxide forming GSSG and thereby serves as an alternative means of detoxifying activated oxygen. The activity of GPx is dependent upon glutathione level. Decreased glutathione activity in co-administered group might be the reason for decreased activity of GPx.

During the current study liver, heart and brain tissues of group IV showed a significant decrease in GPx levels (Table 4) on 98^th day of experimentation. Lung and kidney tissues in group III and liver tissue of group II also showed a significant decrease in GPx levels as compared to that of control group on 98^th day of experimentation. Present findings of decreased GPx levels are in agreement with studies of Verma and Srivastava (2003) and Jackie et al., 2011 on chlorpyrifos and lead treated rats, respectively. The ameliorative effects of vitamin C observed in the present study are in agreement with studies of Nagat et al., 2011 on CPF in rats and El-Tohamy and El-Nattat, 2010 on lead acetate in rabbits.

Table 4: Effect of Oral Administration of Chlorpyrifos, Lead Acetate, Vitamin C Alone in Combinations on Tissue Glutathione–S-Transferase Activity (mmol of Conjugate of gsh-cdnb /min/g Tissue) in Rats

Values given are mean±SE of the results obtained from 6 animals; Means with at least one common superscript do not differ significantly at 5% (P<0.05)

Glutathione-S-Transferses (GSTs) enzymes constitute about 10 % cytosolic protein in some mammalian organs. GST catalyze the conjugation of reduced glutathione via the sulfhydryl group to electrophilic centers on a wide variety of substances. A significant decrease in GST levels (Table 5) was observed in lung, liver and heart tissues of groups II and IV and kidney and brain tissues of group II. These findings are in agreement with studies of Jackie et al., 2011 and Verma and Srivastava (2003) on lead and CPF, respectively. Ameliorative effect of vitamin C on decreasing GST levels in the present study are in agreement with studies of El-Tohamy and El-Nattat, 2010 in lead treated rabbits and Nagat et al., 2011 in CPF treated rats.

Table 5: Effect of Oral Administration of Chlorpyrifos, Lead Acetate, Vitamin C Alone and in Combinations on Tissue Glutathione Peroxidase Activity (U/g Tissue) in Rats

Values given are mean±SE of the results obtained from 6 animals; Means with at least one common superscript do not differ significantly at 5% (P<0.05)

Conflict of Interest

The authors declare they have no competing interest and no financial relationship with the organization that sponsored the research.

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