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 →
O2 - +H2O → HO2 + OH –
radical

HO2 + e - + H → H2O2

H2O2 + e - → OH + OH –
super oxide radical
hydroperoxyl
hydrogen peroxide
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
% Hemolysis
10.12+0.63
a10.08+1.1
b54.02+1.5
Lipid peroxides (µmoles
of TBARS formed/l)
3.32+0.14
a3.14+0.29
a8.44+1.45
3.45+0.12
a3.6+1.21
b2.35+0.65
GSH (µmoles/mg protein)
aNon
significant
Erythrocytes + Adosa +
Deltamethrin
c28.32+1.20
c5.45+0.30
c3.0+0.95
RESULTS TABLE:2
LEVELS (MEAN ± SD) OF ANTIOXIDANT AND
EXPERIMENTAL ERYTHROCYTES.
MEMBRANE BOUND ENZYMES OF CONTROL AND
Erythrocytes + Adosa
Control Erythrocytes
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+2.11
b24.2+3.3
c27.22±3.20
b84.8+10.5
c119±9.5
GPX(mole/min/mg/
protein)
134.2+8.5
Na+K+ATPase
(units/mg/protein)
6.25±0.14
Mg+ATPase
(units/mg/protein)
8.5±1.44
a139.2+10.4
a6.30±0.71
a9.1±1.82
b4.5±0.80
b5.36±1.30
c5.5±0.60
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.