Protective effect of vitamin C on biochemical alterations induced by subchronic co-administration of chlorpyrifos and lead in Wistar rats

The advancement in technology and the need to satisfy the basic needs of man has led to an increasing number of chemicals being deliberately released into the environment. These chemicals interact with each other and also with the environment resulting in adverse health and environmental consequences [1]. Current understanding of the toxicity of many environmental toxicants/pollutants is based primarily on toxicity studies performed on laboratory animals exposed to a single agent [2,3]. However, with the environment being perverted with multiple chemical contaminants interacting with each other, it is increasingly becoming clearer that animal models focusing on the effect of single contaminant will be most misleading because it does not reflect environmental reality.


Introduction
The advancement in technology and the need to satisfy the basic needs of man has led to an increasing number of chemicals being deliberately released into the environment. These chemicals interact with each other and also with the environment resulting in adverse health and environmental consequences [1]. Current understanding of the toxicity of many environmental toxicants/pollutants is based primarily on toxicity studies performed on laboratory animals exposed to a single agent [2,3]. However, with the environment being perverted with multiple chemical contaminants interacting with each other, it is increasingly becoming clearer that animal models focusing on the effect of single contaminant will be most misleading because it does not reflect environmental reality.
Pesticides are one of the most pervasive groups of environmental chemicals that are being increasingly used due to the compelling need to feed the ever-growing human and animal population and to control public health. Organophosphate (OP) insecticides are one of the most widely used, accounting for about 50% global insecticide use [4]. Its widespread use has however been associated with adverse consequences on human and animal health. Following its introduction into the United States of America's market in 1965, chlorpyrifos (CPF) has been one of the most widely used OP insecticides in agriculture, horticulture and public health. Although some of its domestic uses have been banned by United States Environmental Protection Agency in 2000 [5], there is ample evidence that CPF remains one of the most common environmental contaminants [6,7].
Lead is the most ubiquitous heavy metal contaminant [8] because of its widespread industrial and domestic uses. Exposure to lead adversely affects human and animal health [9]. Lead is used in industries and in the homes, and it is present in the soil where it poses a lot of risk to humans and animals [10]. Lead is a major source of environmental pollution and residues have been detected in children toys and household kitchen materials, watering cans and pipes [11].
From the foregoing, therefore, CPF and lead are among the most pervasive environmental contaminants, especially considering their persistent nature. Although both contaminants are known to cause multiple organ toxicity, the mechanisms by which each of these agents causes toxicity are quite different, some of them are interrelated. The induction of oxidative stress is a common biochemical mechanism implicated in CPF [1,[12][13][14][15][16][17][18][19], and lead [8,[20][21][22] poisonings. CPF and lead have been shown to increase the production of free radical and decrease the availability of antioxidant molecules that mitigate the resultant damage. Vitamin C is a low-molecular weight antioxidant molecule that protects the cellular compartment from hydrophilic oxygen-nitrogen radicals; a property that makes it an efficacious antioxidant of the hydrophilic phase [23,24]. Earlier studies have shown the ability of vitamin C to mitigate toxicity induced by CPF [1,14,16,19,25] and lead [26][27][28][29]. Although very few studies have shown that co-exposure to CPF and lead induces toxicity [30,31], there has been paucity of information on the role of vitamin C in mitigating biochemical alterations induced by subchronic co-administration of CPF and lead. Therefore, the present study investigated the role of vitamin C in mitigating the biochemical alterations induced by

Animal treatments
Forty weaned male Wistar rats were divided at random into four groups of 10 animals per group. Group I was administered corn oil (2 ml/kg), while group II was given vitamin C (100 mg/kg). Group III was co-administered CPF (4.25 mg/kg~1/20 th LD 50 [25]) and lead acetate (225 mg/kg~1/20 th LD 50 [29]), respectively. Group IV were pretreated vitamin C (100 mg/kg) and then co-administered CPF (4.25 mg/kg) and Pb (225 mg/kg), 30 min later. These regimens were administered orally by gavage once daily for a period of 9 weeks. The rats were examined for toxic signs. At the termination of the dosing, the animals were killed via jugular venesection after light chloroform anesthesia. Blood samples were collected into labeled test tubes and then incubated on the shelf for 30 min. These were then centrifuged at 800 x g for 10 minutes, to obtain sera samples used for the biochemical assays.

Evaluation of serum biochemical parameters
The sera samples were evaluated for the concentration of electrolytes (Na + , K + and Cl -), total proteins (TP), albumin, urea, and the activities of aspartate aminotranferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and lactic dehydrogenase (LDH). These parameters were analyzed using an auto analyzer (Bayer Express Plus, Germany). Globulin concentration was obtained by subtracting albumin concentration from that of the total serum proteins, and consequently the albumin:globulin ratio was calculated.

Assessment of hepatic lipoperoxidation
The assessment of the level of hepatic lipoperoxidation was done by evaluating the malonaldehyde (MDA) concentration using the double heating method of Draper and Hadley [33] as modified by Yavuz et al. [34]. The MDA concentration was calculated by the absorbance coefficient, MDA-TBA complex 1.56x10 5 /cm and expressed in nmol/ mg of protein. The protein concentration was determined using the method described by Lowry et al. [35].

Statistical analysis
Data were expressed as mean ±SEM and subjected to one-way analysis of variance followed by Tukey's multiple comparison test using Graph Pad Prism version 4.0. Values of P<0.05 were considered significant.

Clinical signs
There was no clinical manifestation observed in C/oil and VC groups. However, rats in the CPF+Pb group showed lacrimation, congested ocular membranes, rough hair coat, piloerection, reduced feed intake and death in one animal. Lacrimation was the only apparent sign recorded in the VC+CPF+Pb group.

Serum electrolytes
There was no significant difference (P>0.05) in the Na + , K + and Clconcentrations in between the groups. However, the serum concentration of each of these electrolytes was marginally higher in the CPF+Pb group compared to those recorded in the other groups ( Figure  1).

Effect of treatments on serum proteins
The effect of treatments on serum protein concentrations is shown in Figure 2. There was a significant (P<0.01) decrease in the serum TP concentration in the CPF+Pb group compared to either C/oil or VC group. Although not significant (P>0.05), the TP concentration in the VC+CPF+Pb group increased by 9.4% relative to the CPF+Pb group. There was a significant (P<0.05) decrease in the TP concentration in the VC+CPF+Pb group compared to the VC group but no significant (P>0.05) change relative to that obtained in the C/oil group. There was no significant (P>0.05) change in the TP concentration in the C/oil group relative to those recorded in the VC group.
The albumin concentration in the CPF+Pb group decreased significantly (P<0.01) compared to that recorded in the C/oil or VC group. Although, not significant (P>0.05), the albumin concentration in the VC+CPF+Pb group increased by 9.4% compared to that recorded in the CPF+Pb group. There was no significant (P>0.05) change in the albumin concentration in the VC+CPF+Pb group compared to either C/oil or VC group. The albumin concentration in the C/oil group was not significantly different (P>0.05) from those obtained in the VC group ( Figure 2).
The globulin concentration in the CPF+Pb group decreased significantly (P<0.05) relative to the VC group but no significant change (P>0.05) compared to either the C/oil or VC+CPF+Pb group. However, the globulin concentration in the CPF+Pb group was comparatively lower when compared to that of C/oil (16%) or VC+CPF+Pb (9%) group. There was no significant (P>0.05) change in the globulin concentration in the VC+CPF+Pb group relative to C/oil or VC group ( Figure 2).
The effect of treatments on albumin/globulin ratio is shown in Figure 3. Although there was no significant (P>0.05) change in between the groups, the albumin/globulin ratio in the CPF+Pb group decreased marginally when compared to either C/oil (5%) and VC+CPF+Pb (1%) group but increased by 1% when compared to the VC group.

Effect of treatments on urea concentration
The effect of treatments on urea concentration is shown in Figure  4. There was a significant increase in urea concentration in the CPF+Pb group relative to either the C/oil (P<0.01) or VC (P<0.05) group but no significant change (P>0.05) was recorded when compared to that of VC+CPF+Pb group. However, the urea concentration in the CPF+Pb group increased by 16% relative to VC+CPF+Pb group. There was a significant (P<0.05) increase in urea concentration in the VC+CPF+Pb group relative to C/oil group but no significant (P>0.05) change was recorded when compared to the VC group. There was no significant (P>0.05) change in the urea concentration in the C/oil group relative to VC group.

Effect of treatments on hepatic enzymes activities
The effect of treatments on serum hepatic enzymes activities is shown in Figure 5. There was a significant (P<0.05) increase in AST activity in the CPF group relative to either C/oil or VC group. Although there was no significant change (P>0.05), the AST activity in the CPF+Pb group increased by 18% relative to VC+CPF+Pb group. There was no significant (P>0.05) change in the AST activity in the VC+CPF+Pb group relative to those recorded in either the C/oil or VC group. There was no significant (P>0.05) change in the AST activity in the C/oil group relative to that of VC group.
There was a significant increase in the ALT activity in the CPF+Pb group relative to C/oil (P<0.01), VC (P<0.01) or CPF (P<0.05) group. There was no significant (P>0.05) change in the ALT activity in the VC+CPF+Pb group compared to those recorded in either the C/oil or VC group. There was no significant (P>0.05) change in the ALT activity in the C/oil group relative to VC group.
There was a significant (P<0.05) increase in ALP activity in the CPF+Pb group compared to that in the C/oil, VC or VC+CPF+Pb group. There was no significant (P>0.05) change in the ALP activity in the VC+CPF+Pb group relative to that of C/oil or VC group. The ALP activity in the C/oil group was not significantly (P>0.05) different from that recorded in the VC group.
The GGT activity in the CPF+Pb group was significantly (P<0.01) higher compared to C/oil, VC or VC+CPF+Pb group. The GGT activity in the VC+CPF+Pb group was not significantly (P>0.05) different from those recorded in either the C/oil or the VC group. The AST activity in the C/oil group was not significantly different (P>0.05) compared to those in the VC group.
The LDH activity in the CPF+Pb group increased significantly (P<0.05) compared to that obtained in the C/oil, VC or VC+CPF+Pb group. There was no significant change (P>0.05) in the LDH activity in the VC+CPF+Pb group compared to that recorded either C/oil or VC group. There was no significant (P>0.05) change in the LDH activity in the C/oil group compared to that recorded in the VC group.

Effect of treatments on hepatic lipoperoxidation
There was a significant increase in the hepatic MDA concentration in the CPF+Pb group relative to those obtained in the C/oil (P<0.05), VC (P<0.01) or VC+CPF+Pb (P<0.05) group. There was no significant (P>0.05) change in the MDA concentration in the VC+CPF+Pb group relative to that in the C/oil or VC group. The MDA concentration in the C/oil group was not significantly (P>0.05) different compared to that recorded in the VC group ( Figure 6).

Discussion
The clinical signs recorded in the group co-administered CPF and lead reflect cholinergism apparently due to the ability of both CPF   [12,14,15,17] and lead [36][37][38] to inhibit AChE activity. The inhibition of AChE activity results in accumulation of acetylcholine in the cholinergic receptors in the peripheral and central nervous systems [39]. This results in initial stimulation and then subsequent paralysis of the cholinergic receptors. Anorexia recorded in the CPF+Pb group may be due to lead component of the combination [40,41].The ability of vitamin C to reduce toxic signs associated with co-exposure to CPF and lead may be due to its ability to restore AChE activity [16,34] and complex to lead [27] thereby reducing its free form in the body. These effects and those related to its radical scavenging properties may have contributed to the reduction in toxic signs in group pretreated with vitamin C.
The marginal increase in the serum concentration of Na + , K + and Clin the group co-administered CPF and Pb shows that these environmental contaminants slightly alters metabolic status. Alterations in the serum electrolytes concentration had earlier been reported following co-administration of CPF and lead [30]. Pretreatment with vitamin C was able to mitigate the apparent increase in the serum concentrations of these electrolytes, indicating apparent normalization of the metabolic alterations induced by co-exposure to CPF and lead.
The study revealed that subchronic co-exposure to CPF and lead caused a decrease in serum total proteins concentration apparently due to hypoalbuminemia. The hypoalbuminemia may be due to a decrease in serum albumin synthesis as a result of hepatic impairment and/ or increase urinary excretion due to impaired renal function arising from renal injury. Earlier studies have shown that CPF causes hepatic [14,18,42,43] and renal [14,25] impairments. Similarly, hepatic [29,44] and renal [45] impairments have been recorded in lead poisoning. Besides, albumin as an antioxidant [46] may have been used up in the process of combating oxidative stress evoked by both CPF and lead, thereby contributing to the low albumin concentration in the CPF+Pb group. Furthermore, the ability of albumin to directly scavenge OP and bind to it, therefore reducing the measurable free albumin available in the system may have contributed to the decline in its concentration. Earlier studies [25,[47][48][49] have demonstrated hypoproteinemia following CPF exposure. Similarly, an apparent hypoglobulinemia was recorded in the group exposed to CPF and lead. Hypoglobulinemia recorded following CPF exposure in earlier studies [14,18] has been partly attributed to lymphocytic leukopenia [14,18,50]. Apoptotic damage to the immune cells has also been described following pesticide exposure [51]. Similarly, leucopenia has been reported in lead poisoning [52].
Pretreatment with vitamin C has been shown by the present study to mitigate hypoproteinemia, hypoalbuminemia and the relative hypoglobulinemia associated with co-exposure to CPF and lead. The mitigation of the hypoalbuminemia by vitamin C may be due to protection of the liver from oxidative damage provoked by co-exposure to CPF and lead, apparently due to its antioxidant effect. The improved globulin concentration by pretreatment with vitamin C may also be due to reduction in apoptotic damage to the WBC due to its antioxidant properties. Vitamin C has been shown to mitigate leukopenia induced by CPF poisoning [25].
The increase in serum urea concentration in the group coadministered CPF and lead is suggestive of damage to the kidneys. Ambali [25] reported that a significant increase in serum urea concentration is observed when there is damage to the kidneys. The liver is responsible for the production of urea in the body, while the kidneys excrete it. A compromised kidney will not be able to perform the urea excretion function leading to uremia. This observation agreed with those obtained by Ambali [25] following chronic CPF-intoxication in Wistar rats. Pretreatment with vitamin C resulted in a decrease in urea concentration apparently due to protection of the kidneys from oxidative damage induced by subchronic co-administration of CPF and Pb.
The result also showed that subchronic co-administration of CPF and lead caused a significant increase in the activity of serum AST, an enzyme found not only in the liver but also in the skeletal muscle and myocardial cells. The significant increase in AST activity observed in this study may indicate hepatic or muscular damage induced by coadministration of CPF and lead thereby provoking increased AST liberation into the peripheral circulation. Pre-treatment with vitamin C caused a decrease in the activity of AST suggesting amelioration of hepatic or muscular damage. This shows that vitamin C protect the organs from the damage caused by co-administration of CPF and lead. Similarly, the significant increase in serum ALT activity recorded in the group co-administered CPF and lead is an indication of hepatic damage since the enzyme is more liver specific than AST [53]. Liver damage has been associated with CPF [14,18,25,42] and lead [44,54] poisonings. AST and ALT play essential roles in the intermediary metabolism of amino acids and in gluconeogenesis, and their concentration in liver is high relative to other tissues. ALT is found primarily in the cytosol of the hepatocytes, while AST is present in both cytosol and mitochondria [55]. In hepatic injury, there is leakage of the enzymes into the plasma thereby increasing its serum concentration [43].
Elevated ALP activity was also recorded in the group co-exposed to CPF and lead. ALP is a hydrolase enzyme for removing many types of molecules, including nucleotides, proteins and alkaloids, and therefore its activity is high in tissue involved in high level of metabolism. Increased ALP activity is not limited to liver damage only, it is also associated with pathological changes in the bone, kidneys, biles and placenta. Therefore, the high level of ALT activity may be due to damage or perturbations in any of these organs. Increased ALP activity has been associated with CPF [14,25,42,43] and lead [44] poisonings.
The study also showed a significant increase in the level of GGT in group co-administered CPF and lead. GGT is a liver-specific enzyme involved in transportation of amino acids and peptides into the cells, and it also takes part in glutathione metabolism. GGT is a good indicator of hepatobiliary disease [56] and its main use is to confirm if the elevations in ALP levels is of hepatic etiology, since the two enzymes have been termed cholestatic liver enzyme because of their increased activity in inflamed or damaged bile duct. Its activity in serum is higher when the epithelia of the bilious channels in the peripheral regions are damaged, in necrotic changes of bile channels, bile obstructions, chollangiohepatitis, cirrhosis and fatty liver [57]. The higher GGT activity recorded in the group co-administered with CPF and lead is an indication that the higher ALP activity in this group resulted significantly from lesions in the liver and bile duct.
The study also revealed a high LDH activity in the group coadministered CPF and lead. LDH catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD + [58]. LDH converts pyruvate, the final product of glycolysis to lactate when oxygen is absent or in short supply and it performs the reverse reaction during the Cori cycle in the liver. At high concentrations of lactate, the enzyme exhibits feedback inhibition and the rate of conversion of pyruvate to lactate is decreased. This is an important step in energy production [59]. Organs that are rich in this enzyme include the liver, heart, kidney and muscle, and death or damage to any of these organs results in its leakage into the blood stream [58]. Therefore, the high serum activity of this enzyme in the group co-administered CPF and lead may be due to damage to any of these organs. Oxidative stress, which is a mechanism involved in both CPF and lead toxicity causes multiple organ injury. Therefore, the high LDH activity may not be attributed to damage to only one organ but has been particularly linked to hepatocellular necrosis leading to leakage of the enzyme into the blood stream [8].
In summary, the increase in the activities of AST, ALT, ALP, GGT and LDH is a strong pointer to hepatotoxicity evoked by both CPF and lead. Exposure to either CPF [14,18,25,42,43] or lead [44,54] has been shown to evoke hepatic damage. The hepatoxicity in the group co-administered CPF and lead may have arisen from increased lipoperoxidative changes as shown by high hepatic lipoperoxidation.
Pre-treatment with vitamin C has been shown in the present study to cause a decrease in the activities of AST, ALT, ALP, GGT and LDH. This shows the protective effect of vitamin C on tissue damage caused by co-exposure to CPF and lead. This may be partly due to the antioxidant role of the vitamin resulted in scavenging the reactive oxygen and nitrogen species and neutralizing their effect before they could cause damage to the organs. Studies have shown the ability of vitamin C to mitigate organ toxicity provoked by CPF [1,14,19,25,60] and lead [29,44]. The organ shown to have been mostly protected from oxidative damage induced by co-administration of CPF and lead in the present study is the liver. The ability of vitamin C to protect the liver from peroxidative changes may have contributed to the mitigation of the hepatotoxicity recorded in the group pretreated with the vitamin.
The study showed that subchronic co-administration of CPF and lead caused an increase in liver MDA concentration. MDA is a biomarker of lipid oxidative damage and is a major oxidative product of peroxidized polyunsaturated fatty acids [61,62]. An increase in MDA concentration indicates lipoperoxidative changes in the hepatocytes, hence oxidative stress. Indeed, lipoperoxidative changes have been implicated in pesticide toxicity [63]. The ability of pesticides to cause multiple organ damage may be due to its pro-oxidative effect [64]. CPF has been shown by earlier studies to induce membrane lipoperoxidation hence oxidative stress [12,[14][15][16][17][18]42]. Similarly, lead has been shown to cause oxidative damage to tissues [8,20,21,22]. Apart from damaging lipids, oxidative stress alters the fluidity of the cell membranes and the DNA [65,66]. The consequence of oxidative damage to the cell is the alteration of the cellular integrity and functions. These may have been partly responsible for the altered biochemical parameters recorded in the CPF+Pb group in the present study.
Pre-treatment with vitamin C has been shown to significantly decrease the MDA concentration in the liver indicating its amelioration of the oxidative damage induced by co-administration of CPF and lead. Vitamin C, a potent hydrophilic antioxidant has been shown in many studies to protect the biological system from the ravaging effect of free radical induced by CPF [14,19,60]. In addition, vitamin C increases the activity of paraoxonase (PON), especially PON I [67], an esterase enzyme involved in the metabolism of OP compounds. Apart from protecting tissue against lead-evoked oxidative stress [29,44,68], vitamin C is also known to complex with lead [27], thereby mitigating the toxicity induced by the heavy metal [26,27,69,70].
In conclusion, the study has shown that subchronic co- administration of CPF and lead causes an alteration in biochemical parameters indicating damages to some organs such as the liver and kidneys. The study equally revealed that oxidative damage to the liver may have been partly involved in the molecular mechanism associated with these biochemical alterations. Pretreatment with vitamin C for the first time has equally been shown to apparently restore the biochemical alterations caused by co-administration of CPF and lead partly due to its antioxidant effect.