M.Sc., PG & Research Department of Biochemistry, Muthayammal College of Arts & Science,

1. DELTAMETHRIN INDUCED OXIDATIVE STRESS IN ERYTHROCYTE AND AMELIORATION BY ADHATHODA VASICAbyF.BADRIYA M.Sc., PG & Research Department of Biochemistry, Muthayammal College of Arts & Science, Rasipuram, Nammakkal District, Salem.

2. INTRODUCTION • The erythrocytes in normal physiological conditions are resistance to oxidative damage because of their efficient protective mechanisms. • However under oxidative stress, the erythrocytes and their membranes are very sensitive to oxidative damage due to their content of unsaturated fatty acids, which are continuously exposed to high concentration of oxygen. • The work was designed to evaluate the protective effect of Adathoda vasica (Adathoda) against Deltamethrin induced oxidative damage, injury in human erythrocyte.

3. Lipid peroxidation was induced in human erythrocyte in vitro by Deltamethrin. Deltamethrin induced peroxidation, there is an increase in TBARS and conjugated dienes levels in erythrocytes with a decrease in glutathione level. • Adathoda vasica (Adathoda) supplementation reduced TBARS and conjucated diene levels in deltamethrininduced human RBC. • The glutathione level increased significantly on Adathoda vasica (Adathoda) treatment. Erythrocyte membrane-bound enzymes Na+-K+-ATPase and Mg2+-ATPase activities were decreased in Deltamethrin incubated erythroyctes. But Adathoda vasica (Adathoda) supplementation ameliorates these enzymes activities.

4. PESTICIDES • These results suggest that Adathoda vasica (Adathoda) has influence in controlling oxidative stress in human erythrocytes. Due to its antioxidant property they may have a good future in preventing or mitigating various oxidative stress associated diseases and ageing. •  The FAO has defined the term of pesticide as ``any substance or mixture of substances intended for preventing, destroying or controlling any pest, including vectors of human or animal disease, unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of food, agricultural commodities, wood and wood products or animal feedstuffs, or substances which may be administered to animals for the control of insects, arachnids or other pests in or on their bodies.

5. The World Health Organization (WHO) has reported that roughly three million pesticide poisonings occur annually, resulting in 220,000 deaths worldwide, some cases, it has been suggested that diseases such as cancer, allergies, neurological disorders and reproductive disorders may be connected to pesticide exposure. • Exposure to pesticides can occur via numerous pathways, including household use of pesticide products, dietary exposure to pesticide residues, and exposure to agricultural drift. Biological monitoring studies indicate that pesticide exposures are widespread in the human population.

6. PYRETHROID PESTICIDES • Pyrethroids are structural derivatives of naturally occurring pyrethrins, Pyrethroids are a class of neurotoxic pesticides registered for agricultural and residential use in the United States. Use of pyrethroids has continuously increased during the last two decades (Freedonia, 2006). • Exposure of deltamethrin has been shown to induce OS and cause perturbations in various biochemical parameters including LPO, antioxidant and neutrotransmission enzymes; the toxicity however, has been shown to be reduced by treatment with vitamin E.

7. FREE RADICALS • The term “reactive oxygen species” (ROS) collectively denotes oxygen-centered radicals such as superoxide (O2. ) and hydroxyl (.OH) as well as nonradical species derived from oxygen, such as hydrogen peroxide (H2O2), singlet oxygen (O2.), and hypochlorous acid (HOCl). ROS play a pivotal role in the action of numerous foreign compounds (xenobiotics). • A free radical is a molecule that contains an unpaired electron in its outer orbit and that can exist independently. Molecular oxygen is a diradical, containing 2 unpaired electrons with parallel spin configurations. Because electrons must have opposite spin to occupy the same orbit, electrons added to molecular oxygen must be transferred one at a time during its reduction. Sen CK 1995; Alessio HM 1993; Packer L 1997; Yu BP 1994).

8. ANTIOXIDANTS • Detoxification of ROS in the cell is provided by both enzymatic and nonenzymatic systems which constitute the antioxidant defense systems. Enzymatic systems include extensively studied enzymes such as SOD, catalase, glutathione peroxidases and glutathione-regenerating enzyme systems (Sies, 1985, 1991; Krinsky, 1992).

9. O2 + e- → O2→ super oxide radical • O2 - +H2O → HO2 + OH – hydroperoxyl radical • HO2 + e - + H → H2O2 hydrogen peroxide • H2O2 + e - → OH + OH – hydroxyl radical

10. Adhatoda Vasica Plant

11. Adhatoda vasica, also known as malabar nut tree is part of the Acanthaceae plant family. It is a small evergreen, sub-herbacious bush which grows commonly in open plains. • The medicinal properties of Adathoda Vasica Nees (Natural Order: Acanthaceae) have been known in India and several other countries for thousands of years. The plant has been recommended by Ayurvedic physicians for the management of various types of respiratory disorders. • It is used as expectorant, antispasmodic, bronchodilator, anti-histaminic, uterine stimulant, used in the treatment of menstrual disorders, eye infections, skin diseases, sore throat,bleeding diarrhoea and has sedative properties

12. BLOOD COLLECTION • Blood samples were collected into tubes containing EDTA-2Na from healthy adult individuals after informed consent. They were free of any medication, drugs or nutrient supplementation.

13. EXPERIMENTAL DESIGN • Erythrocyte suspensions were divided into four groups. • Group I: Served as Normal. • Group II: Erythrocyte suspension was incubated for 15 min at a concentration of 100μg/ml of Deltamethrin at room temperature. • Group III: Adosa extract alone incubated, at a concentration of 25μM for 15 min at room temperature . • Group IV: Adosa extract, incubated, followed by Deltamethrin incubation at indicated concentration.

14. Enzyme assay’s • PROTEIN • GLUTATHIONE (GSH) • MALONDIALDEHYDE (MDA) • LIPID PEROXIDATION (LPO) • SUPEROXIDE DISMUTASE (SOD) • CATALASE (CAT)

15. MEMBRANE BOUND ENZYMES • Na+K+ATPase • Mg+ ATPase STATISTICAL ANALYSIS • All data were analyzed with SPSS/10 student software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by LSD. The values are expressed as the mean ± SD for 5 different sets of experiments and results were considered significantly different if p < 0.05.

16. RESULTS Table:1levels (means±sd) of hemolysis, tbars and gsh of control and experimental erythrocytes.

17. RESULTS Table:2levels (mean ± SD) of Antioxidant and Membrane bound enzymes of control and experimental erythrocytes.

18. CONCLUSION • Most investigations in animals have examined the levels of enzymatic antioxidants and the lipid hydrocarbons, thiobarbituric acid reactive substances (TBARS), such as malondialdehyde (MDA), or lipid peroxides to infer oxidative stress. • In the present investigation, following in vitro exposure, Deltamethrin caused a significant induction of oxidative damage in erythrocytes as evidenced by increased percentage of Hemolysis and increased levels of thiobarbituric acid reactive substances (TBARS).

19. The activities of superoxide dismutase (SOD), catalase (CAT) and Glutathione peroxidase (GPx) were found to be significantly reduced in Deltamethrin treated erythrocytes compared with the control erythrocytes. • However, Adosa extract pretreatment significantly restored the activities of antioxidant enzymes and the levels of TBARS. • The activity of Na+-K+ & Mg2+ATPase was reduced in the erythrocytes treated with Deltamethrin alone while pre incubation with Adosa extract before the incubation of Deltamethrin prevented this fall in the activity of Na+ -K+ & Mg2+ATPase.

20. It can be concluded that, Adhatoda vasica extract was able to prevent the oxidative stress generated by deltamethrin in erythrocytes probably due to its antioxidant potential. • This study is a preliminary effort and requires further investigation at different levels.

21. Thank you