Experimental study on the effect of vitamin C administration on lipid peroxidation and antioxidant enzyme activity in rats exposed to chlorpyriphos and lead acetate

Aim : To evaluate the effects of chlorpyriphos, lead acetate, vitamin C alone, and in combination on the activity of oxidative stress parameters in wistar rats. Marerial and Methods: Rats of 150-200g body weight were divided into eight groups of six animals each and were subjected to various daily oral treatment regimes for 98 days. Group I served as control receiving only corn oil, group II received th chlorpyriphos @ 5.5 mg/ kg in corn oil, group III received lead acetate @100 ppm in water, whereas animals in group IV th received a combination of chlorpyriphos @ 5.5mg/kg in corn oil and lead acetate @ 100 ppm in water. Group V received th vitamin C @ 100mg/kg in water, group VI received a combination of chlorpyriphos @ 5.5mg/kg and vitamin C @ th th 100mg/kg , group VII received lead acetate @ 100 ppm in water and vitamin C @ 100mg/kg and group VIII received chlorpyriphos @ 5.5mg/kg , lead acetate @100ppm in water and vitamin C @ 100mg/kg. Results: Administration of both chlorpyriphos and lead acetate caused a significant decrease in oxidative stress parameters viz. blood glutathione, catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-s-transferase (GST) along with a significant increase in lipid peroxidation level when given alone or in combination. Conclusions: The study demonstrated that treatment of chlorpyriphos and lead treated rats with vitamin C significantly improved some of altered oxidative stress parameters revealing the protective effect of this vitamin C against oxidative stress induced by chlorpyriphos and lead.


Introduction
Chlorpyriphos, a phosphorothioate organophosphorus insecticide is metabolically activated through Organophosphates (OP) were first synthesized in oxidative desulfuration to chlorpy-riphos oxon by Germany before the Second World War and now there cytochrome P .Chlorpyriphos oxon binds to acetyl-450 have been an estimated 300,000 severe pesticide cholinesterase, inhibiting its ability to hydrolyze the poisoning events reported worldwide, mostly due to neurotransmitter acetylcholine.Both chlorpyriphos them.Organophosphates, among other pesticides are and its oxon are metabolized to 3, 5, 6-trichloro-2the most toxic to the vertebrates [1,2].Poisoning pyridinol by mixed functions oxidase system and induce occurs as a result of agricultural use, suicide or oxidative stress which may constitute significantly to accidental exposure [3].Apart from inhibition of overall toxicity [4].Chlorpyriphos produced oxidative cholinesterase and presence of cholinergic effects, stress results in the accumulation of lipid peroxidation oxidative stress has been reported by many authors as products in different organs of rats [5] and has also been one of the adverse effects in poisoning by OP in both shown to damage DNA [1].humans and animals.On the basis of relevant literature It is now well recognized that humans and animals it is concluded, that determination of oxidative stress are exposed to more than one chemical concurrently parameters can be useful for monitoring people exposed from various sources such as food, air, water and to OP professionally.consumer products (including some heavy metals and pesticides).Organophosphorus insecticides (OPI's) like other insecticides form chelating complexes with some heavy metals like lead, mercury and copper.
Oxidative stress changes are the sequelae of toxicities of both organophosphates and some heavy metals.In purchased from Hi-media, S.d.Fine Chem.Pvt. Ltd., recent years, lead has become a regulatory concern and Qualigens Chem.(Mumbai, India) and E. Merck subject of much interest among pharmacologists, (Mumbai, India).environmental scientists and clinicians due to its Experimental Design: These rats were randomly continuous emission from industrial sources and allocated to eight groups of six rats each and subjected automobile exhausts and its pharmacological behavior to various daily treatment regimes for 98days.Group I to remain bound to mammalian tissues, particularly in served as control receiving only corn oil, group II bones for a long duration.Organic lead is more evenly th received chlorpyriphos @ 5.5 mg/ kg.(1/25 LD ) in 50 distributed between erythrocytes and soft tissue and corn oil, group III received lead acetate @100 ppm in less likely to accumulate in bone than inorganic lead th water, whereas animals in group IV received a due to its higher lipid solubility and thus cause combination of chlorpyriphos @ 5.5mg/kg in corn oil oxidative stress.th and lead acetate @ 100 ppm in water.Group V There are several mechanisms to counteract the th damage caused by reactive oxygen species (ROS) in received vitamin C @ 100mg/kg in water, group VI the human and animal organism.One of them is the received a combination of chlorpyriphos @ 5.5mg/kg th enzymatic system which consists of such enzymes as and vitamin C @ 100mg/kg, group VII received lead superoxide dismutase (SOD) (manganese in the active acetate @ 100 ppm in water and vitamin C @ th enzymatic centre or copper and zinc), catalase (CAT), 100mg/kg and group VIII received chlorpyriphos @ glutathione peroxidase (GPx) and glutathione 5.5mg/kg, lead acetate @100ppm in water and vitamin reductase (GR).Another antioxidative system is non-C @ 100mg/kg.The administration of the toxicants enzymatic and consists of a reduced form of glutathione was carried out between 9:30-10:30 AM daily upto 98 (GSH) and vitamins such as vitamin C, vitamin E and days.beta-carotene.Each of these antioxidative systems has All the rats were weighed at weekly intervals a specific activity/concentration, but they work during exposure with toxicants and necessary synergistically generated.Vitamin C and Vitamin E are corrections in dosages were made according to the reported to act as effective antioxidants for protection changes in the body weight.Blood samples were th th th against diseases and degenerative process caused by collected at zero, 30 , 60 and 98 day of experimental oxidative stress [6].Vitamin C has been studied study, for which the animals were anaesthetized with extensively in modulating lead intoxication.It acts diethyl ether.Blood samples were collected from retromainly as an antioxidant molecule and its beneficial orbital fossa using capillary tubes in aliquots effects could be attributed to its ability to complex with containing heparin @10 IU/ml of blood.The red blood lead [7].Thus, the administration of vitamin C may cells were washed with normal saline solution thrice, augment the function of endogenous free radical before preparing the RBC lysate.RBC sediment scavengers and consequently decrease the deleterious obtained after harvesting of plasma was diluted with effects of free radicals on body cells.
normal saline solution in the ratio of 1:1 on v/v basis The present study was thus conducted to access and mixed gently and thoroughly.The diluted the ameliorative effect of vitamin C on the oxidative erythrocytes were centrifuged for 10 min.After stress changes induced by administration of chlorpyricentrifugation the supernatant was discarded along phos and lead acetate.
with buffy coat and again NSS was added to the RBC

Materials and Methods
on v/v basis, mixed gently and then centrifuged.This process was repeated thrice.After final washing 1 per Experimental animals: The Wister rats weighing cent haemolysate (100µl washed RBC + 9.9 ml PBS) between 150-200 gm used in the present study were and 33 per cent hemolysate (330µl washed RBC+ procured from Indian institute of integrative medicine 670µl PBS) in phosphate buffer solution (PBS), pH 7.4 (IIIM), Council of scientific and industrial research, were prepared.The 1 percent haemolysate was used for (CSIR) Lab, Jammu, India.
the estimation of catalase, superoxide-dismutase, Ethical approval: All rats were maintained under glutathione-peroxidase and glutathione-s-transferase standard environmental conditions with ad libitum and 33 per cent haemolysate was used for estimation of feed and water.The animals were treated humanely lipid peroxidation.during the whole period of experimental study and the of diseases such as cardiovascular, respiratory, results were obtained from studies of Verma and neurological as well as for the general ageing process.
Srivastava [5]; Patra and Swarup [8] in rats using Several drugs, xenobiotics and environmental chlorpyriphos and lead respectively.pollutants are known to cause this imbalance between Blood glutathione (GSH) is an important formation and removal of reactive oxygen species naturally occurring antioxidant, which prevents free (ROS).Xenobiotics comprise an important source of radical damage and helps in detoxification by conjugating ROS, which are produced in cells during normal with chemicals.In addition, GSH is pivotal to the metabolic processes involving oxygen.However cellular antioxidant defenses by acting as an essential presence of ROS may be significantly increased by co-factor for antioxidant enzymes including glutathione exposure to different environmental toxicants peroxidase (GPx) and glutathione-s-transferase (GST) produced from the industry, agriculture, tobacco [9,10].Under oxidative stress, GSH is depleted by smoke or pollution accidents.Biological antioxidants GSH related enzymes to detoxify the peroxides produced including vitamins can prevent the uncontrolled due to increased lipid peroxidation [11].Decrease in formation of free radicals and activated oxygen species glutathione results in the impairment of mechanism of or inhibit their reaction with biological structures.The metabolic detoxification [12].A significant decrease in destruction of most free radicals and activated oxygen blood glutathione level (  [14] in chlorpyriphos and lead treated rats respectively.mutations in tumor suppressor gene or the genes of Vitamin C treatment was found to increase the blood antioxidant enzymes.Malondialdehyde (MDA) is the glutathione levels towards normal both in lead acetate end point of lipid peroxidation process which may be and chlorpyriphos treated animals, which is in defined as an oxidative deterioration of polyunsaagreement with interactive studies of Verma et al. [13] turated lipids.Lipid peroxidation has been measured [chlorpyriphos and vitamin C] and Bashandy [15] [lead by quantifying the thiobarbituric acid reactive and vitamin C].The primary role of vitamin C is to substances.During the current study it was observed neutralize free radicals, both inside and outside the that lipid peroxidation level (Table-1) in group II th th th cells.A free radical will seek out an electron to regain showed significant increase on 30 , 60 and 98 day as their stability.Vitamin C being an excellent source of compared to group I on said dates.Also a significant th electrons can donate electrons to free radicals such as increase in lipid peroxidation was observed on 60 and th th th th th hydroxyl and superoxide radicals and quench their 98 day in groups III, IV , VI , VII and VIII as reactivity [16].compared to group I on these days respectively.Similar  Superoxide radicals are produced in mitochondria and 60 day post exposure.These findings are in and endoplasmic reticulum as a consequence of autoagreement with previous study of and Verma et al. [13] oxidation of electron transport chain components.The using chlorpyriphos in rats.Ameliorative effect of major enzyme that protects against superoxide vitamin C in the current study against decreased production in the body is superoxide dismutase which catalase activity is in agreement with studies of Eldisproportionates the superoxide to hydrogen peroxide Tohamy and El-Nattat [19] in male rabbits and Verma and oxygen [17].Decrease in SOD activity is et al. [21] in rats using lead and chlorpyriphos respectively.suggestive of excess free radical generation which Glutathione peroxidase (GPx) is a selenium th impairs natural defense mechanism of the body.On 30 containing enzyme which reduces hydrogen peroxide day of experimentation there was a significant decrease forming GSH and thereby serves as an alternative th means of detoxifying activated oxygen.The activity of in SOD level in groups II and VII as compared to GPx is dependent upon glutathione level.A significant group I.A significant decrease was observed in SOD th th th th decrease in GPx level (Table -5) was observed on 30 level (Table -3) of groups II, III, IV and VI on 60 and th th th day in group III, on 60 day in groups II, III and IV and 98 day of study.These findings are in consonance with th th the studies of Verma and Srivastava [5] in chlorpyriphos on 98 day of experimentation in group VI on as treated rats and El-Nekeety et al. [18] in lead treated compared to control group.Present findings of rats.The results of ameliorative effect of vitamin C in decreased GPx and GST levels are in agreement with the present study are in accordance with studies of studies of Verma and Srivastava [5] and Jackie et al.Verma et al. [13] and El-Tohamy and El-Nattat [19] [22] in chlorpyriphos and lead treated rats, respectively.using chlorpyriphos and lead acetate respectively in The ameliorative effects of vitamin C observed in the rats.Vitamin C ameliorates the inhibitory action of lead present study are in agreement with studies of Nagat et acetate by removing ROS once formed, thus preventing al. [23] on chlorpyriphos in rats and El-Tohamy and free radical chain reactions.
El-Nattat [19] on lead acetate in rabbits.Catalase is a haeme-containing enzyme that catalyzes GSTs are a major group of enzymes that constitute the dismutation of hydrogen peroxide into water and about 10 per cent cytosolic protein in some mammalian oxygen.The enzyme is found in all aerobic eukaryotic organs.GST catalyze the conjugation of reduced cells and is important for the removal of hydrogen glutathione via the sulfhydryl group to electrophilic peroxide generated in peroxisomes (microbodies) by centers on a wide variety of substances.This catalytic action of oxidases which are involved in ß-oxidation of activity of combined glutathione with electrophiles fatty acids and purine catabolism.Stress conditions in helps in excretion of toxicant from the cells and which there is a large free radical generation also result protects the tissues from oxidative stress [10].In the in the depletion in catalase activity [20].A significant current study a significant decrease in GST level th decrease in catalase activity (

Table -
[13] as compared to control group.Similar results increased production may cause cellular and molecular were observed by Verma et al.[13]and Tandon et al. damage leading to lipid peroxidation as well as th

Table - 1
. Effect of repeated oral administration of chlorpyriphos, lead acetate, vitamin C alone, and in combinations on erythrocyte lipid peroxidation (n mole MDA formed/ml erythrocytes) in rats.

Table - 2
. Effect of oral administration of chlorpyriphos, lead acetate, vitamin C alone, and in combinations on blood glutathione levels (nmol/ml) 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)doi:10.5455/vetworld.2013.461-466

Table - 3
. Effect of oral administration of chlorpyriphos, lead acetate, vitamin C alone, and in combinations on blood superoxide dismutase activity (U/mg protein) in rats.

Table - 4
. Effect of oral administration of chlorpyriphos, lead acetate, vitamin C alone, and in combinations on blood catalase activity (ìmole H O decomposed/min/mg protein) 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)

Table - 5
. Effect of oral administration of chlorpyriphos, lead acetate, vitamin C alone, and in combinations on blood glutglutathione peroxidase activity (U/mg protein) 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)

Table - 6
. Effect of oral administration of chlorpyriphos, lead acetate, vitamin C alone, and in combinations on blood glutathione-S-transferase activity (ìmole of conjugate of GSH-CDNB /min/ mg plasma protein) 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)