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The Effect Of Vitamin E On Phostoxin-induced Changes In Hemato-biochemical Parameters In Adult Wistar Rats

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Ibegbu Augustine Oseloka, Julicit Samnan Dassah, Alatise Ahmad Tajudeen,
Babatunde Abudullateef Olaniyi, Umana Uduak Emmanuel,
Hamman Wilson Oliver, Musa Sunday Abraham, Akpulu Peter Stephen.
Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria, Kaduna State-Nigeria. 81006.

All Correspondence to: Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria,
Kaduna State-Nigeria. 81006. E-mail: aoibegbu@yahoo.com

ABSTRACT
The effects of antioxidant Vitamin E on phostoxin-induced changes in the hemato-biochemical parameters in adult Wistar rats were studied. Thirty adult Wistar rats of both sexes were grouped into six groups of five rats each. Group 1 was the Control and was given normal saline, Group 2 was exposed to phostoxin for 3 hours with vitamin E and Group 3 was exposed to phostoxin for 1½ hours with vitamin E. Groups 4 and 5 were exposed to phostoxin for 3 hours and 1½ hours respectively and Group 6 was treated with vitamin E only. The rats were exposed to phostoxin through inhalational method for one week. At the end of the exposure period, the rats were sacrificed, the blood were collected for analysis. The results showed increase in the body weight of the rats which could be as a result of the effect of phostoxin leading to increase in appetite of the rats. The results of biochemical parameters showed there was significant increase in AST and ALP (P<0.05), but the change in ALT in the serum of treated animals was not significant. The result of other biochemical parameters, showed some significant differences (P<0.05) between the Control and the Experimental groups dependent on the exposure time to phostoxin. The result from this study showed that Vitamin E may play a role as an antioxidant resulting in ameliorative effects in phostoxin-induced toxicity in adult Wistar rats.

KeyWords: Vitamin E; Phostoxin; Biochemical Parameters; Oxidative stress markers; Liver enzymes; Wistar rats.

INTRODUCTION
Phostoxin is mainly used as a fumigant in pest control, for stored grains and provides an effective alternative to traditional control methods of rabbits, moles and rats (Sudakin, 2005). Inhalation of phostoxin may cause severe pulmonary irritation leading to acute pulmonary edema, cardiovascular dysfunction, CNS excitation, coma and possibly death. Gastrointestinal tract disorders, renal damage and leucopenia and hepatic damage may also develop (Sudakin and Power, 2007; Turkez and Togar, 2013).
Phostoxin emits a colorless gas which is odorless when pure, but the technical product has a foul odor, like that of a fish or garlic, because of the presence of substituted phosphine and diphosphine (Proudfoot , 2009; O’Malley et al., 2013). Phostoxin is flammable and explosive in air and can auto ignite at ambient temperatures. It is slightly soluble in water and soluble in most organic solvents and supplied in cylinders either as pure phosphide or diluted with nitrogen (Singh et al., 1996; Shadnia et al., 2008; Bumbrah et al., 2012). Aluminum phosphide which is really available as a fumigant for stored grains is highly toxic when consumed from freshly opened containers. It is
available in pellet and tablet form and also available in porous blister packs, sachets or as dusts (Shaheen, 1996; Shadnia et al., 2009; Easterwood et al., 2010).
Aluminum phosphide is an inorganic phosphide used to control insects and rodents in a variety of settings. It is mainly used as an indoor fumigant during crop transport, storage or in processing facilities for both food and non-food crops. It may also be used as outdoor fumigant for burrowing rodent and in mole control or in bait for rodent control in crops (Meister, 1992; O’Malley et al., 2013).
Phostoxin is one of the market names for aluminum phosphide products which emit a colorless gas and is odorless when pure, but the technical product has a foul smell (Degesch, 2011). It is soluble in water and in most organic solvents and supplied in cylinders either as pure phosphide or diluted with Nitrogen (Shaheen, 1996; Sudakin and Power, 2007). Phostoxin may be formulated as 55% active ingredient along with aluminum carbamate and inert ingredients (Gehring et al., 1991; Easterwood et al., 2010).
The pathology of phostoxin intoxication is characteristic of hypoxia or oxygen deficiency in body tissues. Frequent exposure to concentrations above permissible levels over a period of days or weeks may cause poisoning and treatment is symptomatic depending on the signs presented (Lall et al., 2000; Saleki et al., 2007).
Antioxidants may protect cells from the damage caused by unstable molecules, the free radicals which may cause to cancer. Antioxidants interact with, stabilize free radicals, and may prevent some of the damage free radicals might otherwise cause. Examples of antioxidants include beta–carotene, lycopene, Vitamins C, E, and A (Herrera and Barbas, 2001).
Vitamin E is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties (Packer et al 2001). Of these, a-tocopherol has been the most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolizing this form (Regina, 1999). The aim of the present study was to evaluate the effects of Vitamin E on phostoxin-induced changes in the hemato-biochemical parameters in adult Wistar rats.

Materials and Method
Chemicals
Phostoxin tablet used was manufactured by D & D Holdings Inc. USA. The tablets weigh 3g and releases 1g of phosphine gas, was purchased from Agro Allied Store, Zaria, Kaduna State-Nigeria. It takes an average of 3 days to completely decompose leaving a gray-white powder of aluminum hydroxide and inert ingredients of the ammonium carbarnate (Degesch, 2011). Vitamin E manufactured by Medizen USA was purchased from Beautiful Gate Pharmaceutical Store, Zaria Kaduna State-Nigeria. Vitamin E used was soft gelatin capsules containing 100mg of Vitamin acetate.
Experimental Animals
Thirty Wistar rats were obtained and acclimatized for three weeks in the animal house of Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria. The animals of average weight of 140gms and were randomly separated into six groups of five animals each and were fed with standard pellets and water was provided ad-bilitum.
Phostoxin was given through inhalation by using lightly suspended cotton wool in an enclosed box for one and a half hours and three hours respectively for a period of seven days. Vitamin E was administered at 800mg/kg body weight, orally by means of insulin syringe for animals in Groups 2, 3 and 6.
Experimental Protocol
The animals in Group 1, were used as the Control and given distill water, Group 2 was exposed to Phostoxin for one and half hours and administered Vitamin E at a concentration of 112mg/kg body weight, Group 3 was exposed to phostoxin for three hours and administered with Vitamin E at concentration o­f 112mg/kg body weight, Group 4 was exposed to phostoxin only for three hours, Group 5 was exposed to phostoxin only for one and half hours and Group 6 was administered with Vitamin E only at the concentration of 112mg/kg body weight. The administration lasted for seven days and on the ninth day, the rats were humanely sacrificed after anesthetizing them with chloroform and blood was collected in properly labeled EDTA bottles for hematological and biochemical analyses.

Biochemical Assay
The levels of biochemical parameters were determined by the use of an auto analyzer made by Roche-Hittachi, Japan, using commercial assay kits manufactured by Roche, Basel, Switzerland.
Estimation of Plasma Proteins
The levels of plasma proteins in the Blood was estimated using Okutucu et al. (2007) method of which the level of plasma protein could be altered in kidney diseases. The estimation of Uric acid, Urea and Creatinine values in the Blood was done according to the methods of Sembulingam and Sembulingam (2010). The blood level of these substances has been shown to increase in renal diseases and renal failure (Sembulingam and Sembulingam, 2010).
Estimation of Liver Enzymes
Liver secretes different serum enzymes, acting as biomarkers of liver cell damage (Vasudevan and Sreekumari, 2007). These enzymes include Alanine Amino Transferase (ALT) of which moderate increase from the normal may be seen in chronic liver disease such as cirrhosis, hepatitis and non-alcoholic steato-hepatitis (Vasudevan and Sreekumari, 2007). Aspartate Amino Transferases (AST) which is moderately elevated in liver diseases and a marked increase in AST may be seen in primary hepatoma, (Vasudevan and Sreekumari, 2007). Alkaline Phosphatase (ALP) is a nonspecific enzyme which hydrolyses aliphatic, aromatic or heterocyclic compounds of which the upper level of Normal serum value may be more in children because of the increased osteoblastic activity in children (Vasudevan and Sreekumari, 2007).

Estimation of Oxidative Parameters
Oxidative Stress markers such as Catalase activity, Superoxide Dismutase Activity, Assessment of Lipid Peroxidation and Glutathione Concentration were studied using the respective assay methods according to the instructions of the manufacturers. Catalase activity was determined using the method described by Sinha (1992). The absorbance was read at 570 nm and Standard cure was made using the absorbance obtained at various levels. Superoxide Dismutase (SOD) activity was determined by the method described by Fridovich (1989) and the absorbance was measured every 30s up to 150 s at 48Onm. Lipid peroxidation as evidenced by the formation of TBARS was measured by the modified method of Niehaus & Samuelson (1968) as described by Adhikari et al. (2009). The absorbance of the pink supernatant was measured against a reference blank using spectrophotometer at 535nm. Reduced glutathione (GSH) concentration measured according to the methods Ellman (1959) as modified by Rajagopalan et al. (2004) and Seiler et al. (2008). The absorbance was read at 412 nm.

Statistical Analysis
The data were expressed as means ± standard deviation (SD). Differences between group means were estimated using Students’ T-test and one-way analysis of variance (ANOVA) followed by Post-hock Turkey’s test using SPSS 12.0 for windows. A P value less than or equal to 0.05 was considered to be significant.

Results
Physical Observation….

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