1. PESTICIDES-INDUCED OXIDATIVE DAMAGE: POSSIBLE PROTECTION BY Ahmed k. Salama and Omran A. Omran Medical Laboratories Dept., Faculty of Science, Majmaah University, Kingdom of Saudi Arabia, 2013 This work supported by the Essential and Health Sciences Research Center, Scientific Research Deanship, Majmaah University 1433-1434

2. THE PROBLEM: Agrochemicals such as pesticides are used for achieving better quality and quantity products but they have many adverse effects on human. The toxic action of pesticides may include the induction of oxidative stress and accumulation of free radicals in the cell via increasing the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide, and hydroxyl radicals. A major form of oxidative damage is lipid peroxidation, which is initiated by hydroxyl free radical through the extraction of hydrogen atom from unsaturated fatty acids of membrane phospholipids causing disturbance of the biochemical and physiological functions of cells.

3. OBJECTIVES: The aim of the study was planned to investigate: 1.The capability of the pesticides atrazine, dimethoate and endosulfan to induce oxidative stress in male rat following in vitro treatment at different levels of each pesticide. 2.The possible protection of the oxidative damage induced by these pesticides in rat erythrocytes and hepatocytes using selenium and a combination of vitamin E or vitamin C.

4. EXPERIMENTAL METHODS Blood was obtained from rat by heart puncture and then centrifuged at 3000 rpm for 5 min at 4°C. RBC’s were taken and washed with phosphate buffered saline, pH 7.2. Liver was also dissected out and homogenized in saline solution (1:10 w/v). The homogenates were taken for treatment. RBC's or liver homogenate treated with pesticides, vitamins, selenium alone or with different combinations and then incubated for 3 hours at 37°C in a shaking water bath. At the end of incubation, all samples were subjected to biochemical analysis.

5. BIOCHEMICAL ANALYSIS: Lipid peroxidation level was determined and expressed as nanomoles of malondialdehyde (MDA)/mg protein GSH content was determined and expressed as µmole/mg protein. Glutathione-S-transferase (GST-Px) activity was estimated and expressed as units/mg protein.

6. Table (1): LPO, GSH and GSH-Px Levels of control (5% DMSO) or treated erythrocytes and hepatocytes with VE, VC or Se. GSH-Px Level (µmol NADPH/min/mg protein) GSH content (µmole/mg protein) LPO level (nmol MDA/mg protein) Treatment HepatocytesRBC’sHepatocytesRBC’sHepatocytesRBC’s 2.14±0.880.69±0.10233.14±0.8852.55±6.1012.54±0.968.96±0.19 5% DMSO 2.80±0.110.85±0.11244.70±10.1560.22±9.1011.84±0.118.55±0.11 VE (5mg) 2.22±0.770.77±0.23255.00±10.7055.17±8.8813.22±0.779.16±0.23 VC (5mg) 2.30±0.100.90±0.40247.30±10.2060.10±7.5613.16±0.908.92±0.44 Se (1mg) 2.370.80245.0457.0112.698.90 Average

7. Table (2): LPO Level (nmoles MDA/mg protein) of treated erythrocytes and hepatocytes with atrazine alone or combined with VE, VC and/or Se. LPO level in Hepatocytes LPO level in Erythrocytes Treatment % of Control 10mM % of Control 10mM 126.95%16.11±0.20135.96%12.10±0.76 AT 106.93%13.57±0.16107.53%9.57±0.16 AT + VE 88.26%11.20±0.4696.40%8.58±0.99 AT + VE + Se 98.66%12.52±0.22101.01%8.99±0.02 AT + VC 90.15%11.44±0.6079.89%7.11±0.88 AT + VC + Se

8. Table (3): LPO Level (nmoles MDA/mg protein) of treated erythrocytes and hepatocytes with dimethoate alone or combined with VE, VC and/or Se. LPO Level in Hepatocytes LPO Level in Erythrocytes Treatment % of Control 10mM % of Control 10mM 147.05% 18.66±2.24 114.61% 10.20±0.10 DM 105.91% 13.44±0.70 102.25% 9.10±0.40 DM + VE 79.67% 10.11±0.60 91.12% 8.11±0.80 DM + VE + Se 78.88% 10.01±0.40 91.21% 8.13±0.40 DM + VC 71.79% 9.11±1.40 79.89% 7.11±0.40 DM + VC + Se

9. Table (4): LPO Level (nmoles MDA/mg protein) of treated erythrocytes and hepatocytes with endosulfan alone or combined with VE, VC and/or Se. LP Level in Hepatocytes LPO Level in Erythrocytes Treatment % of Control 10mM % of Control 10mM 142.63% 18.10±0.76 158.43% 14.10±0.60 ES 118.99% 15.10±0.44 113.48% 10.10±0.11 ES + VE 87.47% 11.10±0.22 79.78% 7.10±0.46 ES + VE + Se 103.23% 13.10±0.88 124.72% 11.10±0.00 ES + VC 87.47% 11.10±0.16 102.25% 9.10±0.10 ES + VC + Se

10. Table (5): GSH content (µmole/mg protein) of treated erythrocytes and hepatocytes with atrazine alone or combined with VE, VC and/or Se.. Glutathione content in Hepatocytes Glutathione content in Erythrocytes Treatment % of Control 10mM % of Control 10mM 226.25% 554.41±15.60 176.81% 100.80±12.80 AT 184.52% 452.17±55.16 154.53% 88.10±5.10 AT + VE 122.76% 300.80±20.14 70.47% 40.18±6.19 AT + VE + Se 125.25% 306.92±33.12 105.58% 60.19±5.02 AT + VC 68.41% 167.64±18.90 57.88% 33.00±4.68 AT + VC + Se

11. Table (6): GSH content (µmole/mg protein) of treated erythrocytes and hepatocytes with dimethoate alone or combined with VE, VC and/or S.. Glutathione content in Hepatocytes Glutathione content in Erythrocytes Treatment % of Control 10mM % of Control 10mM 210.89% 516.77±18.24 313.45% 178.70±21.20 DM 153.91% 377.14±16.90 246.97% 140.80±12.10 DM + VE 122.06% 299.10±18.50 196.91% 111.99±9.60 DM + VE + Se 103.66% 254.01±20.10 217.35% 123.91±0.10 DM + VC 31.79% 77.91±0.10 97.01% 55.31±7.19 DM + VC + Se

12. Table (7): GSH content (µmole/mg protein) of treated erythrocytes and hepatocytes with endosulfan alone or combined with VE, VC and/or Se... Glutathione content in Hepatocytes Glutathione content in Erythrocytes Treatment % of Control 10mM % of Control 10mM 186.13% 456.10 ± 44.16 271.01% 154.50 ± 11.80 ES 135.13% 331.10 ± 18.14 37.89% 21.60 ± 9.25 ES + VE 58.85% 144.20 ± 14.29 18.24% 10.40 ± 0.46 ES + VE + Se 113.94% 279.19 ± 12.89 54.71% 31.19 ± 3.30 ES + VC 42.48% 104.10 ± 9.10 17.71% 10.10 ± 1.10 ES + VC + Se

13. Table (8): Glutathione Peroxidase Level (µmoles NADPH/min/ mg protein) of treated erythrocytes and hepatocytes with atrazine alone or combined with VE, VC and/or Se... GSH-Px level in Hepatocytes GSH-Px level in Erythrocytes Treatment % of Control 10mM % of Control 10mM 228.27% 5.41 ± 0.20 225.00% 1.80 ± 0.16 AT 133.76% 3.17 ± 0.16 150.00% 1.20 ± 0.10 AT + VE 75.95% 1.80 ± 0.66 72.50% 0.58 ± 0.19 AT + VE + Se 38.82% 0.92 ± 0.12 123.75% 0.99 ± 0.02 AT + VC 27.00% 0.64 ± 0.60 82.50% 0.66 ± 0.18 AT + VC + Se

14. Table (9): Glutathione Peroxidase Level (µmoles NADPH/min/ mg protein) of treated erythrocytes and hepatocytes with dimethoate alone or combined with VE, VC and/or Se... GSH-Px level in Hepatocytes GSH-Px level in Erythrocytes Treatment % of Control 10mM % of Control 10mM 115.19% 2.73 ± 0.29 225.00% 1.80 ± 0.20 DM 71.31% 1.69 ± 0.08 137.50% 1.10 ± 0.10 DM + VE 71.30% 1.60 ± 0.10 38.75% 0.31 ± 0.11 DM + VE + Se 46.41% 1.10 ± 0.10 101.25% 0.81 ± 0.10 DM + VC 29.54% 0.70 ± 0.16 13.75% 0.11 ± 0.10 DM + VC + Se

15. Table (10): Glutathione Peroxidase Level (µmoles NADPH/min/ mg protein) of treated erythrocytes and hepatocytes with endosulfan alone or combined with VE, VC and/or Se.... GSH-Px level in Hepatocytes GSH-Px level in Erythrocytes Treatment % of Control 10mM % of Control 10mM 257.38% 6.10 ± 0.16 262.50% 2.10 ± 0.40 ES 130.81% 3.10 ± 0.14 137.50% 1.10 ± 0.21 ES + VE 50.63% 1.20 ± 0.22 50.00% 0.40 ± 0.46 ES + VE + Se 130.80% 3.10 ± 0.88 148.75% 1.19 ± 0.01 ES + VC 46.41% 1.10 ± 0.10 112.50% 0.90 ± 0.10 ES + VC + Se

19. CONCLUSION: The results indicated that all treatments with pesticides, enhanced the LPO level, GSH content and GSH-Px activity via increasing oxidative stress in erythrocytes and hepatocytes of male rats. The treatment with selenium and a combination of VE or VC was potentially reduced the free radicals and declined the lipid peroxidation level, GSH content and GSH-Px activity in erythrocytes or hepatocytes and ameliorated the oxidative stress induced by such pesticides and thus reduced the lipo-peroxidative effect.

20. THANK YOU