AHMED K. SALAMA AND OMRAN A. OMRAN Medical Laboratories Dept., Faculty of Science, Majmaah University, Kingdom of Saudi Arabia, 1434 H This report is based upon work supported by the Essential and Health Sciences Research Center, Scientific Research Deanship, Majmaah University 1433.

 Pesticides are commonly used for achieving better quality products, increased production rate and controlling pest population.  On the other hand, pesticides are known to increase the production of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), superoxide(O 2 -), and hydroxyl (OH) radicals, which in turn prompt oxidative stress in different tissues.  A major form of cellular oxidation damage is lipid peroxidation, which is initiated by -OH free radical through the extraction of hydrogen atom from unsaturated fatty acids of membrane phospholipids causing disturbance of the biochemical and physiological functions of RBCs, damaging membranes and other tissues. Free radical generation is expected to induce hepatotoxicity.

The aim of the present study was planned to establish the antioxidant role of selenium and a combination of vitamin E and vitamin C on oxidative stress induced in rat RBCs and hepatocytes by some pesticides such as atrazine, dimethoate, or endosulfan. Vitamin E is a lipid soluble, chain-breaking antioxidant playing a major protective role against oxidative stress and prevents the production of lipid peroxides by scavenging free radicals in biological membranes. Vitamin C has antiradical activity indicating that it could provide an important dietary source of antioxidants. Vitamin C is a well-known low molecular weight antioxidant that protects the cellular compartment from water-soluble oxygen nitrogen radicals. Selenium is an essential element for biological systems, It is present in the active center of glutathione peroxidase (GPx), an antioxidant enzyme, which protects lipid membranes and macromolecules from oxidative damage produced by peroxides. Selenium also has the ability to counteract free radicals and protect the structure and function of proteins, DNA and chromosomes against oxidation injury. Selenium also controlling the deficiency of vitamin E and facilitate its absorption.

Animals and treatments:  Blood was obtained from rat by heart puncture using EDTA-Na salt and centrifuged at 3000 rpm for 5 min at 4°C.  RBC’s were taken and washed with phosphate buffered saline (PBS), pH 7.2. The final red cell suspension was taken in test tubes for treatment.  Liver was also dissected out and homogenized in saline solution (1:10 w/v). The homogenates were taken in a test tube for treatment. Pesticides, vitamins and selenium (AT, DM, ES, VE, VC, Se, AT + VE, DM + VE, ES + VE, AT + VC, DM + VC, ES + VC, AT + VE + Se, DM + VE + Se, ES + VE + Se, AT + VC + Se, DM + VC + Se, ES + VC + Se) were dissolved in 5% DMSO.  Also, 5% DMSO was dissolved/mixed in control group. RBC's or liver homogenate were incubated for 3 hours at 37 °C in a shaking water bath. At the end of incubation, the tubes will removed and subjected to biochemical analysis.

Lipid peroxidation and GSH contents in erythrocytes and hepatocytes: The levels of lipid peroxides (LPOs) and GSH in erythrocyte hemolysate and liver homogenate were determined. Lipid peroxidation was calculated as nanomoles of malondialdehyde (MDA)/mg protein. GSH was determined and calculated as µmole/mg protein. Antioxidant enzymes: The erythrocyte and hepatocyte homogenate was used for analysis of antioxidant enzymes. Activity of superoxide dismutase (SOD) was measured as units/mg protein. Catalase (CAT) activity was estimated as units/mg protein. Glutathione-S-transferase (GST-Px) activity was estimated and expressed as units/mg protein.

Treatment GSH content µmole/mg protein MDA nmoles/ mg protein SOD units/mg protein CAT units/mg protein GSH-Px units/mg protein Control 2.96 ± ± ± ± ± 2.54 AT 1.92 ± ± ± ± ± 4.34 AT + VE 2.50 ± ± ± ± ± 2.56 AT + VC 2.61 ± ± ± ± ± 3.22 AT + VE + Se 2.70 ± ± ± ± ± 1.19 AT + VC + Se 2.82 ± ± ± ± ± 3.44 Table (1): Levels of GSH, MDA and antioxidant enzymes of control and treated erythrocytes with AT alone or its combination with VE or VC and Se.

Treatment GSH content µmole/mg protein MDA nmoles/ mg protein SOD units/mg protein CAT units/mg protein GSH-Px units/mg protein Control 2.96 ± ± ± ± ± 2.54 DM 1.89 ± ± ± ± ± 3.28 DM + VE 2.13 ± ± ± ± ± 1.50 DM + VC 2.10 ± ± ± ± ± 1.45 DM + VE + Se 2.14 ± ± ± ± ± 3.98 DM + VC + Se2.72 ± ± ± ± ± 4.33 Table (2): Levels of GSH, MDA and antioxidant enzymes of control and treated erythrocytes with DM alone or its combination with VE or VC and Se.

Treatment GSH content µmole/mg protein MDA nmoles/ mg protein SOD units/mg protein CAT units/mg protein GSH-Px units/mg protein Control 2.96 ± ± ± ± ± 2.54 ES 1.82 ± ± ± ± ± 1.30 ES + VE 2.70 ± ± ± ± ± 3.34 ES + VC 2.97 ± ± ± ± ± 3.13 ES + VE + Se 2.82 ± ± ± ± ± 1.91 ES + VC + Se 2.96 ± ± ± ± ± 2.12 Table (3): Levels of GSH, MDA and antioxidant enzymes of control and treated erythrocytes with ES alone or its combination with VE or VC and Se.

Treatment GSH content µmole/mg protein MDA nmoles/ mg protein SOD units/mg protein CAT units/mg protein GSH-Px units/mg protein Control ± ± ± ± ± 0.54 AT ± ± ± ± ± 0.14 AT + VE ± ± ± ± ± 0.59 AT + VC ± ± ± ± ± 0.44 AT + VE + Se ± ± ± ± ± 0.24 AT + VC + Se ± ± ± ± ± 0.56 Table (4): Levels of GSH, MDA and antioxidant enzymes of control and treated hepatocytes with AT alone or its combination with VE or VC and Se.

Treatment GSH content µmole/mg protein MDA nmoles/ mg protein SOD units/mg protein CAT units/mg protein GSH-Px units/mg protein Control22.33 ± ± ± ± ± 0.54 DM19.12 ± ± ± ± ± 0.90 DM + VE21.10 ± ± ± ± ± 0.81 DM + VC22.12 ± ± ± ± ± 0.11 DM + VE + Se22.90 ± ± ± ± ± 0.22 DM + VC + Se22.89 ± ± ± ± ± 0.13 Table (5): Levels of GSH, MDA and antioxidant enzymes of control and treated hepatocytes with DM alone or its combination with VE or VC and Se.

Treatment GSH content µmole/mg protein MDA nmoles/ mg protein SOD units/mg protein CAT units/mg protein GSH-Px units/mg protein Control22.33 ± ± ± ± ± 0.54 ES15.22 ± ± ± ± ± 0.09 ES + VE21.11 ± ± ± ± ± 0.09 ES + VC21.15 ± ± ± ± ± 0.32 ES + VE + Se21.15 ± ± ± ± ± 0.31 ES + VC + Se21.99 ± ± ± ± ± 0.08 Table (6): Levels of GSH, MDA and antioxidant enzymes of control and treated hepatocytes with ES alone or its combination with VE or VC and Se.

The results of the study indicated that the in vitro lipo-peroxidative effect induced by pesticides such as atrazine, dimethoate, or endosulfan in male albino rat could be reduced using selenium and a combination of vitamin E and vitamin C. The treatment with selenium and a combination of vitamin E and vitamin C is potentially reduced the free radicals in erythrocytes or hepatocytes and ameliorated the oxidative stress as evidenced from lower concentrations of LPOs, higher contents of GSH and higher activities of SOD and CAT or GSH-Px in erythrocytes or hepatocytes. The efficacy of Vitamin E + Se in ameliorating pesticide-induced oxidative stress was higher for dimethoate or endosulfan than for Atrazin either in case of erythrocytes or hepatocytes.