Vitamin C Alleviates Chronic Chlorpyrifos Induced Alterations in Serum Lipids and Oxidative Parameters in Male Wistar Rats

Problem statement: Although the increase incidence of cardiovascular diseases, obesity and diabetes mellitus has been attributed to changi ng life style, the role of environmental contaminan ts is currently being given considerable attention. Pe sticides, including Organophosphate (OP) insecticides are one of the most pervasive environm ental contaminants that are deliberately released into the environment. Chlorpyrifos (CPF), a widely used OP insecticide has been shown to alter lipid profiles, thereby increasing the risk of atheroscle rosis. Apart from inhibition of acetyl cholinestera se, oxidative stress is one of the molecular mechanisms nvolved in CPF-evoked toxicity. Approach: The present study was aimed at evaluating the mitigatin effect of vitamin C on chronic CPF-evoked alteration in lipid profiles in Wistar rats. Twenty young adult male Wistar rats were assigned at rand om to four groups of five rats each. Groups I, II and III were administered S/oil (2 mL kg ), vitamin C (100 mg kg) and CPF (10.6 mg kg ~1/8th LD50), respectively while group IV was given vitamin C (100 mg kg) followed by CPF (10.6 mg kg ), 30 min later. The regimens were administered by gavage once daily for 17 weeks. The sera obtained f rom blood samples were evaluated for the levels of Cholesterol (TC), Triglycerides (TG), High Density Lipoproteins-Cholesterol (HDL-c), Malonaldehyde (MDA) and the activities of superoxid e ismutase and catalase. The levels of low Density Lipoprotein-Cholesterol (LDL-c) and Very Lo w Density Lipoprotein-Cholesterol (VLDL-c) and atherogenic index, were subsequently calculated . R sults: The increased levels of TC, LDL-c, MDA and atherogenic index and the decrease in the l ev ls of TG, HDL-c, VLDL-c, SOD and CAT evoked by CPF were mitigated by vitamin C. Conclusion: It is concluded that vitamin C alleviated the CPF induced deleterious alterations of lipid profil es, partly due to its antioxidant property.


INTRODUCTION
There has been increasing incidence of cardiovascular diseases, obesity and diabetes mellitus in the last few decades. Although changing life style is one of the risk factors implicated in some of these diseases, the contribution of environmental contaminants cannot be overemphasized. Pesticides are one of the most common contaminants that are deliberately released to the environment to combat the menace of pests that affect the quality and quantity of food resources and serve as vector of insect borne diseases. Organophosphate (OP) insecticides are one of the most widely used as 15 million kilogram were used in agriculture in the United States of America in 2007, representing 36% of all insecticides used (Grube et al., 2004). Chlorpyrifos, a chlorinated OP insecticide is one of the most widely used (Meeker et al., 2004; California Department of Pesticide Regulation (CDPR), 2010, despite the ban placed on some of its domestic uses by Unites States Environmental Protection Agency in 2000. Although their primary mechanism of systemic toxicity is due to Acetylcholinesterase (AChE) inhibition resulting in cholinergism, it is increasingly becoming clear that OP toxicity may involve multiple mechanisms, since toxicity occurs at doses that do not inhibit AChE or long after its restoration (Pope, 1999;Slotkin 2004a;2004b;Costa, 2006). Oxidative stress is one of the molecular mechanisms implicated in OP toxicity (Gultekin et al., 2001;Goel et al., 2005;Ambali et al., 2007;2010a;2010b;Ambali and Ayo, 2011). Lipids are essential for energy homeostasis, reproductive and organ physiology and numerous aspects of cellular biology. They are also linked with many pathological processes, such as obesity, diabetes, heart disease and inflammation. To meet the different demands from a variety of tissues, the body has evolved a sophisticated lipoprotein transport system to deliver cholesterol and fatty acids to the periphery (Lee et al., 2003). Alterations in the concentrations of these lipoproteins affect lipid metabolism with resultant dyslipidemia, a risk factor in atherosclerosis.
The liver, which is responsible for lipid metabolism, is one of the organs that are mostly affected by OP insecticides (Goel et al., 2005;Ambali et al., 2007;Aly et al., 2010;Tripathi and Srivastav, 2010) due to their role in the detoxification of xenobiotics. Furthermore, the liver is also the site for oxidative desulfuration of CPF to its more active metabolites, CPF-oxon, a more potent AChE inhibitor. Therefore, the liver, due to these functional roles is vulnerable to injury, which may alter its ability to metabolize lipids. Studies have partly implicated oxidative stress in the molecular mechanism of CPFinduced hepatotoxicity (Goel et al., 2005;Ambali et al., 2007;. A number of studies have shown alterations in lipid profiles following OP exposure (McGill et al., 1981;Quistad et al., 2001;Ibrahim and El-Gamal, 2003;El-Banna et al., 2009;El-Mazoudy et al., 2011). However, studies involving the mitigation of OP induced alterations of lipid profiles by readily available and cheap antioxidant molecules such as vitamin C become essential in the light of the current environmental and health challenges posed by OP insecticides. The purpose of the present study is to evaluate the mitigating effect of antioxidant vitamin C on chronic CPF-evoked alterations of lipid profiles in male Wistar rats.

MATERIALS AND METHODS
Experimental animals: Twenty young adult male Wistar rats (102±3. 2 g) used for this study were obtained from the laboratory animal house of the Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria. The animals were housed in metallic cages and were allowed to acclimatize for two weeks in the laboratory prior to the commencement of the experiment. They were fed on standard rat chow and water was provided ad libitum.

Chemical
acquisition and preparations: Commercial grade CPF (20% EC, Termicot ® , Sabero organics, Gujarat Limited, India), was prepared by reconstituting in soya oil (Grand Cereals and Oil Mills Ltd., Jos, Nigeria) to make 10% stock solution. Ascorbic acid (100 mg −1 ; Dol-Med Laboratories Limited, Lagos, Nigeria) was reconstituted in distilled water just prior to its daily administration. All other reagents used in this study were of analytical grade obtained from Sigma Inc., (USA).

Experimental protocol:
The rats were weighed and then assigned at random into 4 groups of 5 rats in each group. Group I (S/oil) served as the control group and was given only soya oil (2 mL kg −1 b.w.) while group II (VC) was given vitamin C (100 mg kg -1 b.w.) Group III (CPF) was administered with reconstituted CPF only (10.6 mg kg −1 b.w.~1/8th LD 50 , Ambali (2009) while group IV (VC+CPF) was pretreated with vitamin C (100 mg kg −1 b.w.) and then dosed with reconstituted CPF (10.6 mg kg −1 b.w.), 30 min later. The regimens were administered once daily by gavage for a period of 17 weeks. The study was carried out in accordance with the national research council NRC (2011). At the end of the treatment period, the rats were killed by jugular venesection after light ether anesthesia and blood samples were collected into centrifuge tubes, incubated on the shelf for 30 min and thereafter centrifuged at 800 xg for 10 min to obtain the sera samples used to evaluate the lipid profiles and oxidative stress markers.
Evaluation of the effect of treatments on lipid profiles: Serum lipid profiles such as Triglycerides (TG), Total Cholesterol (TC) and High Density Lipoprotein Cholesterol (HDL-c) analyzed. Serum TG concentration was determined by the enzymatic colorimetric method of Bergmeyer (1983). TC was evaluated using the enzymatic colorimetry method of Kayamori et al. (1999), while HDL-c was assessed using the colorimetry method of Finley et al. (1978). Low-Density Cholesterol (LDL-c) and Very Low Density Cholesterol (VLDL-c) were calculated using the formula of Friedewald et al. (1972) thus: LDL-c = Total cholesterol-HDL Cholesterol-(0.20 × triglycerides) VLDL-c = TG/5 Atherogenic Index (AI) was calculated according to Lee and Nieman (1996)

Evaluation of the effect of treatments on serum lipoperoxidation:
The level of thiobarbituric acid reactive substance, Malonaldehyde (MDA) as an index of lipid peroxidation was evaluated in the sera samples using the method of Draper and Hadley (1990). The principle of the method was based on spectrophotometric measurement of the color developed during the reaction of Thiobarbituric Acid (TBA) with Malonaldehyde (MDA). The MDA concentration in each sample was calculated by the absorbance coefficient of MDA-TBA complex 1.56× 10 5 cm −1 M −1 and expressed as nmol mL −1 .
Evaluation of the effect of treatments on serum superoxide dismutase activity: Superoxide Dismutase (SOD) activity was evaluated using NWLSSTM superoxide dismutase activity assay kit (Northwest Life Science Specialities, Vancouver, WA 98662) as stated by the manufacturer.
Evaluation of the effect of treatments on serum catalase activity: Catalase (CAT) activity was evaluated using NWLSS TM catalase activity assay kit (Northwest Life Science Specialities, LLC, Vancouver, WA 98662) as stated by the manufacturer.
Statistical analysis: All data were expressed as mean±SEM and then subjected to one-way analysis of variance followed by Tukey's multiple comparism tests. Values of p<0.05 were considered significant.

Serum lipid profiles:
The effect of treatments on TC is shown in Fig. 1. There was a significant (p<0.05) increase serum TC concentrations in the VC group relative to the CPF group. Although not significant (p>0.05), the TC concentration in the CPF group was relatively higher than that of the C/oil (9%) and VC (15%) groups, respectively.
The serum TG concentration in the CPF group was significantly lower compared to that of S/oil (p<0.05), VC (p<0.01) and VC+CPF (p<0.05) groups, respectively. There was no significant (p>0.05) change in the TG concentration in the VC+CPF group relative to that of S/oil or VC group (Fig. 2).
There was a significant reduction in HDL-c concentration in the CPF group relative to that of S/oil (p<0.05) and VC (p<0.01) groups, respectively. Although not significant (p>0.05), the HDL-c concentration in the VC+CPF group was 23% higher than that of the CPF group. The HDL-c concentration in the VC+CPF group was significantly (p<0.05) lower than that recorded in the VC group but no significant p>0.05) change when compared to that of S/oil group (Fig. 3).  The LDL-c concentration in the CPF group was significantly higher relative to that of S/oil (p<0.05), VC (p<0.01) and VC+CPF (p<0.05) groups, respectively. The LDL-c concentration in the VC group was significantly (p<0.05) lower compared to that of S/oil group but no significant (p>0.05) change when compared to that of VC+CPF group (Fig. 4).
There were no significant (p>0.05) changes in the VLDL-c concentrations in between the groups. However, the VLDL-c concentration in the CPF group was relatively lower in the CPF group relative to that of S/oil (12%), VC (24%) and VC+CPF (22%) groups, respectively (Fig. 5).
The AI in CPF group was significantly (p<0.05) higher compared to that of VC group. Although not significant (p>0.05), the AI in the CPF group was relatively higher than that of the S/oil (8%) or VC+CPF (14%) group. There was no significant (p>0.05) change in the AI in the VC+CPF group relative to that recorded in the S/oil or VC group (Fig. 6).

Malonaldehyde concentration:
The serum MDA concentration was significantly higher in the CPF group relative to that of S/oil (p<0.05), VC (p<0.01) and VC+CPF (p<0.05) groups, respectively. There was no significant (p>0.05) change in the MDA concentration in the VC+CPF group relative to that of S/oil or VC group (Fig. 7).

Superoxide dismutase activity:
The serum SOD activity in the CPF group was significantly lower relative to that of C/oil (p<0.05), VC (p<0.01) and VC+CPF (p<0.05) groups, respectively. There was no significant (p>0.05) change in the SOD activity in the VC+CPF group relative to that of S/oil or VC group (Fig. 8). Catalase activity: There was a significant decrease in serum CAT activity in the CPF group compared to that of S/oil (p<0.05), VC (p<0.01) and VC+CPF (p<0.05) groups, respectively. The CAT activity in the VC+CPF group was not significantly (p>0.05) different from that of S/oil or VC group (Fig. 9).

DISCUSSION
The increase serum TC observed in the CPF group in the present study was in accord with the results obtained from earlier studies (Barna-Lloyd et al., 1990;El-Mazoudy et al., 2011) that recorded an increase in TC concentration following repeated CPF exposure. The elevation in serum TC level observed in the CPF group could be due to blockage of liver bile ducts causing reduction or cessation of its secretion to the duodenum (Aldana et al., 2001). Similarly, the ability of the pesticide to interfere with the permeability of the liver cell membrane (Kallender et al., 2005) may have been partly responsible for the high TC. CPF-evoked hepatotoxicity has been previously reported by many workers (Goel et al., 2005;Ambali et al., 2007;. Hypercholestrolemia is a sign of liver damage (Abdou and El-Mazoudy, 2007). Pretreatment with vitamin C was shown in the present study to have apparently reversed the CPF-induced hypercholestrolemia. This may be due the ability of the vitamin to mitigate the hepatic damage evoked by the insecticide, which has been partly attributed to its antioxidant effect (Ambali et al., 2007;2011a;2011b). The improved liver function promotes efficient hepatic metabolism of cholesterol.
The present study has also demonstrated a relatively lower TG level in the CPF group and this observation agreed with those obtained in rats by Barna-Lloyd et al. (1990). The relative hypotriglyceridemia in the CPF group may have resulted from liver damage or hypolipoproteinemia (Kaneko et al., 2008;Stojevic et al., 2008) causing reduced interaction of TG with the lipoproteins, especially VLDL. The CPF evoked hypotriglyceridemia may have been due to impaired fatty acids synthesis (Bopanna et al., 1997), enhanced catabolism of VLDL, activation of Lecithin: Cholesterol Acyltransferase (LCAT) and tissue lipases (Khanna et al., 2002), inhibition of acetyl-CoA carboxylase (McCarty, 2001) and production of triglycerides precursors such acetyl-CoA and glycerol phosphate. Pretreatment with the antioxidant vitamin caused apparent normalization in the TG level. This may be due to the ability of the antioxidant to protect against oxidative damage to the liver by CPF (Ambali et al., 2007), which brings about an improvement in the entire processes that are associated with lipid metabolism.
The study also revealed a lower HDL-c concentration in CPF group, which was in agreement with the findings of El-Mazoudy et al. (2011). Similarly, low-HDL-c has also been reported in rats dosed repeatedly with diazinon (Ibrahim and El-Gamal, 2003). HDL, which is mainly synthesized in the liver and intestinal cells plays an important role in cholesterol efflux from tissues and carries it back to the liver for removal as bile acids (Shakoori et al., 1988). It has been established that the elevated serum or plasma HDLs levels have antiatherogenic effect (McGill et al., 1981), whereas the reduced levels are associated with an increased risk for coronary artery disease (Tietz, 1987). HDLs have been proposed to act in concert with LCAT to remove excess cholesterol from arterial tissue and thus prevent atherosclerosis (Glomset, 1979;Fielding and Fielding, 1995). Several lines of evidence suggest that oxidative modification of plasma LDL plays a major role in the pathogenesis of atherosclerosis (Fogelman et al., 1980;Steinberg et al., 1989) and that HDL protects LDL from oxidation (Banka, 1996;Parthasarthy et al., 1990). In addition, the low HDL-c may also be due to CPF induced hepatic damage (Goel et al., 2005;Ambali et al., 2007; causing impairment of lipoprotein synthesis. The implication of low HDL-c observed in rats chronically exposed to CPF only indicates that prolonged exposure to this insecticide may increase the risk of individuals developing coronary heart disease (Tietz, 1987).
The improvement in HDL-c concentration following vitamin C pretreatment is a demonstration of the antiatherogenic ability of the antioxidant vitamin. HDL is one of the most important protective factors against arteriosclerosis. This has been attributed to its active participation in the reverse transport of cholesterol (Tomas et al., 2004). In addition, HDL competes with LDL receptor sites on smooth muscle, resulting in partial inhibition of the uptake and degradation of LDL (Attia and Nasr, 2009). The improvement in the HDL-c in the VC+CPF group may be partly due to the hepatoprotective effect of the antioxidant vitamin (Ambali et al., 2007;. The present study has also demonstrated an increase in LDL-c in rats repeatedly exposed to CPF. Similar results were observed by El-Banna et al. (2009) and El-Mazoudy et al. (2011) following CPF exposure. The increased LDL-c may be due to hypercholestrolemia, which suppresses the formation of new LDL receptors thereby decreasing the cellular intake of cholesterol in the form of LDL. The increase in LDL-c recorded in the CPF group, which was accompanied by a low VLDL-c, suggests that CPF exposure promotes the deleterious conversion of some of the VLDL-c to LDL-c. Although hypercholesterolemia is universally accepted as a major risk factor for atherosclerosis, but at any given serum cholesterol concentration, there is variability in the occurrence of cardiovascular events, as it has been shown that the oxidative modification of LDL may be a crucially important step in the development of atherosclerotic plaque (Heinecke, 1997). LDLs transport cholesterol to the arteries where it is retained by arterial proteoglycans, which initiate the formation of plaques. LDL oxidation is thought to be the first step of atherogenesis, followed by foam cell, fatty streakand plaque formation (Nakamura et al., 2008). It has been hypothesized that LDLs can be transported through endothelial tight junctions and/or endothelial transcytosis from the lumen into the intima (Cancel et al., 2007), in which blood antioxidants are unlikely to be available and therefore prone to undergo atherogenic oxidative changes (Nakamura et al., 2008). The lack of access to antioxidant by LDL leads to their oxidation. Furthermore the increased MDA concentration and decreased SOD and CAT activities, indicative of oxidative stress in the CPF group further increases the likelyhood of LDL oxidation. Therefore, the increased levels of oxidised LDLs as observed in the present study increased the susceptibility of the rats to atherosclerosis and consequently, heart attack, stroke and peripheral vascular diseases Pretreatment with vitamin C was shown in the present study to reduce the serum LDL-c concentration. Therefore, vitamin C may be able to reduce the susceptibility of individual to CPF evoked increased susceptibility to atherosclerosis. Although, LDL-c is harmless until oxidized by FR (Teissedre et al., 1996), it is postulated that ingesting antioxidants and minimizing free radical exposure may reduce LDL's contribution to atherosclerosis (Esterbauer et al., 1991;Reaven et al., 1993). Furthermore, the ability of vitamin C to induce the activity of paraoxonase (Jarvik et al., 2002), a multitask protein that not only hydrolyzes OPs, but also prevents the oxidation of LDLs and HDLs (Chambers, 2008) may enhance protection from atherosclerosis, although oxidation of LDL-c can also occur via nonradical mechanisms such as direct oxidation by lipoxygenase (Carroll, 1989), hypochloric acid and aldehydes (Niki, 2004).
The low VLDL-c in the CPF group may be related to the hypotriglyceridemia recorded, since VLDL is assembled from TG. This finding was in agreement with those of El-Mazoudy et al. (2011). The high conversion of VLDL-c to LDL-c through increased activity of LDL-synthetase complex may have been responsible for its low concentration. Similarly, the increased activity of lipoprotein lipase not only hydrolyzes TGs but also has the effect of reducing VLDL and HDL to smaller fragments (Campbell and Smith, 2000) may have contributed to low VLDL. CPF evoked hepatotoxicity (Goel et al., 2005;Ambali et al., 2007; may have also contributed to the low VLDL since its synthesis occurs in the liver. VLDL, which is released by the liver, delivers fatty acids to adipocytes for storage and to cardiac and skeletal muscle for energy consumption (Lee et al., 2003). The implication of low VLDL in the CPF group is impairment of fatty acid storage. Pretreatment with vitamin C was able to increase the VLDL, therefore increasing the propensity of fatty acid storage and metabolism. This may be partly due to mitigation of CPF-induced hepatotoxicity by vitamin C (Ambali et al., 2007;Ambali, 2009), which may have improve the lipoprotein synthesis and metabolism. Furthermore, the relatively lowered LDL-c concentration in the vitamin C pretreated group is a demonstration of a reduction in the rate of conversion of VLDL to LDL.
Atherogenic index, which is used to predict the risk of coronary heart disease, was shown to be significantly higher in the CPF group. This shows that individuals chronically exposed to CPF are predisposed to developing coronary heart disease (Lee and Niemann, 2010). The reduction in atherogenic index by vitamin C is an indication of the antiatherogenic effect of the vitamin. This may be due to its ability to improve lipid metabolism through its hepatoprotective effect. The antioxidant effect, which decreases the propensity of LDL-coxidation, has contributed to the low atherogenic index. Furthermore, vitamin C has been shown to increase the activity of Paraoxonase I (PON I) (Jarvik et al., 2002), an arylesterase, synthesized primarily n the liver and is secreted into the plasma, where it is exclusively bound to High-Density Lipoprotein (HDL) particles containing apoA-I and apoA-j (Kujiraoka et al., 2006), which may have contributed to its antiatherogenic effect. Besides detoxificating OPs, PON1 displays at least two other very important functions including the prevention of unsaturated lipid oxidation and decomposition of lipid peroxidation products of oxidized LDL and HDL in the plasma (Mackness et al., 1998;Shih et al., 1998;Sozmen et al., 2002;Rice et al., 2007). PON1 may also confer protection against damages of vessel wall by antioxidation and destroying oxidative productions.
The increased MDA concentration and increased activities of SOD and CAT in CPF intoxicated rats is a demonstration of increased lipoperoxidation and oxidative stress. MDA is important and most commonly indicator of lipid peroxidation. Therefore, the increased MDA concentration in the CPF group may be due to peroxidation of polyunsaturated fatty acids, leading to degradation of phospholipids and ultimately cellular deterioration (Tappel, 1973). Earlier studies have shown that CPF caused increased lipoperoxidation (Gultekin et al., 2001;Goel et al., 2005;Ambali et al., 2010a and2010b;El-Mazoudy et al., 2011;Shittu et al., 2012a and2012b). Cholesterol, cholesterol esters and triglycerides compartments of the lipoprotein fractions can be oxidized by toxic radicals, resulting in the loss of chemical structures and cellular functions (El-Banna et al., 2009). Lipid peroxidation is accepted as a free radical process implicated in the formation of atherosclerosis (Wen et al., 1996). The decreased MDA concentration in the group pretreated with vitamin C is a demonstration of the antilipoperoxidative property of the vitamin, which was in agreement with previous findings (Aly et al., 2010;Ambali and Ayo, 2011).
The decreased SOD and CAT activities in the CPF group is an indication of oxidative stress, which was in agreement with previous studies (Gultekin et al., 2001;Tuzmen et al., 2007;Ambali and Ayo, 2011;Shittu et al., 2012a;2012b). The SOD is involved in the dismutation of O 2 oto H 2 O 2 , while CAT hydrolyzes the H 2 O 2 to H 2 O and molecular oxygen. The decreased activity of the antioxidant enzymes may have resulted from imbalance in the rate of synthesis to that of utilization. Oxidative stress compromises cellular integrity and function. Furthermore, the increased oxidative stress in the CPF group may result in the oxidation of LDL-c and HDL-c, thereby increasing the risk of cardiovascular diseases. Vitamin C has been shown in the present study to increase the activities of SOD and CAT, due to its antioxidant property and thereby decreasing the risk of lipoprotein oxidation, hence atherosclerosis.

CONCLUSION
The present study has shown that chronic CPF exposure causes oxidative tress and deleteriously alters lipid profiles, thereby increasing the risk of atherosclerosis. Vitamin C has also been demonstrated in the present study to mitigate the CPF-evoked deleterious alteration of lipid profiles, atherogenic index and oxidative stress, thereby reducing the risk of atherosclerosis.

Conflict of interest:
There is no conflict of interest in this study.