DELTAMETHRIN INDUCED OXIDATIVE STRESS IN ERYTHROCYTE AND AMELIORATION BY ADHATHODA VASICA

BY

F.BADRIYA

M.Sc.,

PG & Research Department of Biochemistry,

Muthayammal College of Arts & Science,

Rasipuram, Nammakkal District, Salem.

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.

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 deltamethrin induced 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.

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.

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.

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.

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).

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).

O2 + e- → O2 → super oxide radical

O2 - +H2O → HO2 + OH – hydroperoxyl radical

HO2 + e - + H → H2O2 hydrogen peroxide

  H2O2 + e - → OH + OH – hydroxyl radical

ADHATODA VASICA PLANT 

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

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.

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.

ENZYME ASSAY’S

PROTEIN

GLUTATHIONE (GSH)

MALONDIALDEHYDE (MDA)

LIPID PEROXIDATION (LPO)

SUPEROXIDE DISMUTASE (SOD)

CATALASE (CAT)

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.

RESULTS TABLE:1

LEVELS (MEANS±SD) OF HEMOLYSIS, TBARS AND GSH OF CONTROL AND EXPERIMENTAL ERYTHROCYTES.

Control Erythrocyte Erythrocytes + Adosa

Erythrycytes + Deltamethrin

Erythrocytes + Adosa + Deltamethrin

% Hemolysis 10.12+0.63 a10.08+1.1b54.02+1.5 c28.32+1.20

Lipid peroxides (µmoles of TBARS formed/l)

3.32+0.14 a3.14+0.29a8.44+1.45

c5.45+0.30

GSH (µmoles/mg protein)3.45+0.12

a3.6+1.21b2.35+0.65

c3.0+0.95

aNon significant

 

RESULTS TABLE:2

LEVELS (MEAN ± SD) OF ANTIOXIDANT AND MEMBRANE BOUND ENZYMES OF CONTROL AND EXPERIMENTAL ERYTHROCYTES.  

Control ErythrocytesErythrocytes + Adosa

Erythrocytes + Deltamethrin

Erythrocytes + Adosa + Deltamethrin

SOD (units/mg protein)

2.31+0.12 a2.13+0.41 b1.25+0.20 c1.7±0.20

CAT (mole/min/mg/protein)

31.2+1.78 a33.2+2.11b24.2+3.3 c27.22±3.20

GPX(mole/min/mg/ protein)

134.2+8.5a139.2+10.4

b84.8+10.5 c119±9.5

Na+K+ATPase (units/mg/protein)

 6.25±0.14

a6.30±0.71

b4.5±0.80

c5.5±0.60

Mg+ATPase (units/mg/protein)

8.5±1.44

a9.1±1.82

b5.36±1.30 c8.36±1.19

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).

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.

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.