Pesticide Biochemistry and Physiology 102 (2012) 9194

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Pesticide Biochemistry and Physiology

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Effect of antioxidant supplementation on free radical scavenging system

and immune response in lindane treated scabies patients
Mythily Subramaneyaan a, Sachin Rustagi a, Sambit N. Bhattacharya b, Asok K. Tripathi a,
Basu D. Banerjee a, Rafat S. Ahmed a,
a
Department of Biochemistry, University College of Medical Sciences and Guru Teg Bahadur Hospital (University of Delhi), Delhi 110095, India
b
Department of Dermatology, University College of Medical Sciences and Guru Teg Bahadur Hospital (University of Delhi), Delhi 110095, India

a r t i c l e i n f o a b s t r a c t

Article history: The scabicide, lindane induces oxidative stress and immunological alterations. The present study was
Received 12 July 2011 undertaken to assess the ameliorative effects of antioxidant supplementation in lindane treated scabies
Accepted 5 November 2011 patients. Scabies patients were treated with either 1% lindane or 1% lindane along with antioxidant
Available online 19 November 2011
(Lycored or Vitamin-E). Oxidative stress and immunological parameters were evaluated in blood samples
and compared with healthy controls. Lindane caused a signicant increase in malonedialdehyde (MDA)
Keywords: levels and decrease in reduced glutathione (GSH) and ferric reducing ability of plasma (FRAP), which was
Scabies
attenuated by anti-oxidant therapy. The IL-1a levels were signicantly enhanced in scabies patients per
Lindane
Oxidative stress
se and remained unaffected after lindane/anti-oxidant treatment. The TNF-a and nitroblue tetrazolium
Immune response (NBT) reduction levels were not signicantly different in all the groups. Topical application of lindane
Lycored induces signicant free radical generation and may cause immunological alterations which can be
Vitamin-E reversed by antioxidant therapy.
2011 Elsevier Inc. All rights reserved.

1. Introduction
Scabies is a vesico-pustular inammation of the skin caused by
active penetration and intracutaneous burrowing of the impreg-
nated female and egg layings of the ectoparasite, Sarcoptes scabiei
var hominis, an itch mite [1] resulting in papular, intensely pruritic
eruption involving the interdigital spaces and exure creases [2]. It
is a common public health problem in poor communities and is
widespread in many underdeveloped countries [3]. There are about
300 million cases of scabies in the world each year. Common pre-
disposing factors are overcrowding, immigration, poor hygiene,
poor nutritional status, homelessness, dementia, and sexual contact
[4]. Many drugs, particularly insecticides, are used to treat scabies.
However, most of them have an innate toxicity. Lindane 1% cream
or lotion is still the treatment of choice in because of its low cost,
non-irritating nature and ease of application. Eradication of scabies
with a single application of 1% lindane has been reported [5]. Its
Abbreviations: TBA, 2-thiobarbituric acid; TPTZ, 2,4,6-tris pyridyl triazine;
DTNB, 5,50 -dithiobis-2-nitrobenzoic acid; CNS, central nervous system; FRAP, ferric
reducing ability of plasma; GPx, glutathione peroxidase; IL-1a, interleukin1a;
MDA, malonedialdehyde; NBT, nitroblue tetrazolium; ROS, reactive oxygen species;
GSH, reduced glutathione; TCA, trichloroacetic acid; TNF a, tumor necrosis factor a.
Corresponding author. Address: Department of Biochemistry, UCMS & GTB
Hospital, Dilshad Garden, Delhi 110095, India. Fax: +91 11 22590495.
E-mail address: rafatnizam@rediffmail.com (R.S. Ahmed).
disadvantage is that it can cause central nervous system (CNS) tox-
icity leading to increased excitability, convulsions, and death in rare
cases [6] and also may be associated with idiosyncratic aplastic
anemia [7]. Lindane is absorbed through mucous membranes and
is distributed to all body compartments with the highest concentra-
tion in lipid rich tissues and the skin [6]. Involvement of reactive
oxygen species (ROS) is currently postulated as one of the mecha-
nisms through which xenobiotics like lindane, exert their deleteri-
ous effects on animal tissues [8]. Most of the studies have been
performed on experimental animals, with few dealing with oxida-
tive stress in human cases following exposure to these pesticides
[9]. However, very little, clinically relevant information is available
on the presence of oxidative stress due to percutaneous absorption,
prime route of systemic exposure in therapeutic use of lindane. We
have earlier reported that topical application of lindane in scabies
patients induces signicant oxidative stress as compared to per-
methrin [10]. Once the S. scabiei penetrates the skin, it releases sub-
stances in response to contact with keratinocytes and langerhans
cells initiating an inammatory and immune reaction resulting in
the development of Type I and Type IV hypersensitivity [4]. Neutro-
phils constitute a greater proportion of the inammatory response
in secondary infections of scabies mites [11]. The oxidative burst of
neutrophils play a vital role in eliminating the scabies mite [12].
The present study was thus designed to explore the induction of
oxidative stress and immunological alterations if any, in scabies

0048-3575/$ - see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.pestbp.2011.11.002
92 M. Subramaneyaan et al. / Pesticide Biochemistry and Physiology 102 (2012) 9194
patients following lindane treatment, and whether systemic
administration of Lycored, a commercially available antioxidant
containing lycopene and Vitamin-E can ameliorate these effects.
2. Materials and methods
2.1. Chemicals
Commercially available antioxidant, brand name Lycored (Jag-
sonpal Pharmaceuticals Ltd., India) containing Lycopene (2000 lg),
Vitamin-A (2500 IU), Tocopherol (10 IU), Vitamin C (25 mg), zinc
sulphate (27.4 mg) and Selenium dioxide (70 lg) and a-Tocopherol
was used for antioxidant therapy. 5,50 -dithiobis-2-nitrobenzoic acid
(DTNB), 2,4,6-tris pyridyl triazine (TPTZ) and nitroblue tetrazolium
(NBT) were obtained from SigmaAldrich (St. Louis, MO, USA). 2-
Thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were ob-
tained from E. Merck (Mumbai, India). All other reagents used were
of analytical grade and obtained either from BDH or SISCO chemi-
cals, Mumbai, India.
2.2. Study subjects
The study group consisted of ninety self reporting adult patients
(1850 years) attending outpatient clinic of the department of Der-
matology, Guru Teg Bahadur Hospital, Dilshad garden, Delhi. Pa-
tients having nocturnal pruritis, burrows, scabietic lesions at
classical sites, family history of similar illness and clinically diag-
nosed by microscopy to have scabies were included in the study.
Clinical diagnosis was conrmed independently by two consul-
tants. They were alternatively assigned to treatment either with
1% lindane lotion (30 patients, Group B) or 1% lindane lotion along
with Lycored (30 patients, Group C) or 1% lindane lotion along with
Vitamin-E (30 patients, Group D). Pregnant and lactating mothers
and patients with active infection, eczematous or excoriated skin
lesions or receiving any other medication leading to oxidative stress
or history of occupational exposure to pesticides were excluded
from the study. Thirty age and sex matched (Table 1) adult healthy
volunteers, not suffering from any clinically detectable illness con-
stituted the control group (Group A). An informed consent was ob-
tained from every participant and the study was approved by the
Institutional Ethics Committee for Human Research (UCMS/IHEC/
18/2009). After hospitalization, Group B received 1% lindane lotion
(Gamascab) as a single overnight application. Group C received two
capsules of antioxidant (Lycored), one capsule 12 h before the lin-
dane application and the other along with application of 1% lindane
lotion. Group D received two capsules of Vitamin-E (400 mg a-
tocopherol acetate), one capsule 12 h before the lindane application
and the other along with lindane application. The lindane lotion
was applied topically all over the body excluding head and neck.
Each subject was instructed to have a thorough bath 8 h after appli-
cation. No other medication, topical or systemic, was allowed
Table 1
Age and sex distribution in study and control groups.
Group A (healthy control) Group B (lindane)
Age in years
2130 11 10
3140 10 11
4150 9 9
Mean SD 33.05 8.95 34.25 8.51
Sex
Male 15 15
Female 15 15
Total 30 30
The four groups were matched in terms of age and sex and no statistically signicant difference was found between them.
during this period. Group A healthy volunteers were not subjected
to topical/systemic administration of lindane or any other drug.
2.3. Blood sampling
Blood was drawn before and 1214 h after the application of
the drug in the study groups B, C and D. Two blood samples 12 h
apart were also analyzed from the control group for adequate sta-
tistical comparison. Three milliliter of blood was collected in a
plain vial and sera separated for estimation of MDA, IL-1a and
TNF-a levels. Five milliliter of heparinised blood samples were col-
lected and processed for neutrophil isolation and NBT assay, and
also for estimation of GSH content and FRAP levels.
2.4. Estimation of oxidative stress parameters
MDA level in serum (as an index of in vivo lipid peroxidation)
was determined as per method described by Satoh [13] using thio-
barbituric acid reagent (TBA). The MDATBA adduct formation was
measured spectrophotometrically at 532 nm and concentration of
MDA was expressed as nmol/ml of serum. Glutathione content in
blood was quantitated by the method of Tietze [14]. The chromo-
phoric product resulting from reaction of the reagent DTNB with
GSH was measured spectrophotometrically at 412 nm. The concen-
tration of GSH in blood was expressed as lmol/ml. Anti-oxidant
capacity of blood was determined by measuring the ferric reducing
ability of plasma (FRAP). The complex between Fe2+ and TPTZ gives
a blue color with absorbance at 593 nm [15].
2.5. Estimation of immune response
Neutrophils were isolated from heparinised venous blood after
erythrocytes gravity sedimentation in 6% dextran followed by
hypotonic lysis. Respiratory burst was assayed by the reduction
of nitroblue tetrazolium according to the method described by Pick
[16]. Quantitative determination of IL-1a and TNF-a was carried
out by commercially available ELISA kits as per manufacturers
protocol (Diaclone Research, France).
2.6. Statistical analysis
All assays were performed in duplicate. The values are ex-
pressed as means SD. The data were analyzed by one way ANOVA
followed by Tukeys test for inter-group comparison and paired
t-test for intra-group comparison. p < 0.05 was considered as level
of signicance in all statistical tests.
3. Results
The oxidative stress and inammatory markers were analyzed
in the study group both before (Group B1, C1 and D1) and 12 h
Group C (lindane + lycored) Group D (lindane + vitamin-E)
11 11
9 11
10 8
34.00 8.95 33.75 8.72
14 16
16 14
30 30
 M. Subramaneyaan et al. / Pesticide Biochemistry and Physiology 102 (2012) 9194
after application of lindane (Group B2, C2 and D2). The control
group was analysed for the same parameters for two samples ta-
ken 12 h apart (Group A1 and A2) to enable statistical analysis.
There was no signicant difference between the mean baseline
MDA, GSH levels and FRAP in control as well as all pretreatment
scabies groups. Lindane treatment alone signicantly increased
the MDA levels and decreased the GSH levels as well as FRAP as
compared to control (Table 2). Antioxidant supplementation ame-
liorated the effect of lindane treatment as evidenced by a signi-
cant decrease in MDA levels and increase in GSH and FRAP levels
to near control values.
The NBT reduction values as well as TNF-a levels remained un-
changed in all the pretreated as well as treated groups (Table 3).
However scabies per se resulted in a signicant increase in the
IL-1 a levels, which was not attenuated even after antioxidant
supplementation.
4. Discussion
Certain pesticides are known to disturb the biochemical and
physiological function of cells by affecting the membrane integrity
and thereby resulting in the pathophysiological changes in the organ
system [17]. Lindane, an environmentally persistent organochlorine
pesticide and a potent scabicide, induces lipid peroxidation and free
radical scavengers in human poisoning cases [18]. Due to its lipo-
philic nature, it tends to accumulate and interact with membrane
lipids. Lindane exposure also induces a derangement of many pro-
and anti-oxidant parameters in liver [19]. Detoxication of xenobi-
otics requires GSH, thereby further depleting reducing equivalents
making erythrocytes susceptible to lower amounts of oxidative
stress in terms of exposure to oxygen free radicals [20].
Free radical induced lipid peroxidation can be initiated by a
number of environmental triggers. MDA is one of several aldehydes
and ketones that result from peroxidation of mono and poly unsat-
urated fatty acids [21]. The percutaneous absorption of lindane has
been extensively investigated. Franz et al. [22] demonstrated that
human skin is inherently more permeable to lindane than guinea
pig skin. Plasma levels of lindane were found to average 10.3 ng/
ml (range 424 ng/ml) 72 h after whole body application in normal
volunteers with subsequent wash-off at 12 h. Hosler et al. [23]
have recorded similar levels. Their ndings indicate signicant
transcutaneous absorption of lindane occurs following a single
application and is compatible with the toxicity reported following
the topical application of lindane cream. Therefore, the increased
MDA levels in the present study can be explained on the basis of
high percutaneous absorption leading to free radical production
large enough to cause lipid peroxidation. Lindane application alone
resulted in a signicant reduction in GSH content (Group B2). This
Table 2
Oxidative stress parameters in control subjects and scabies cases.
Groups MDA (nM/ml serum)
Pretreatment
Control (A1) 2.56 0.31
Lindane (B1) 2.47 0.34
Lindane + Lycored (C1) 2.49 0.33
Lindane + Vitamin-E (D1) 2.45 0.37
Posttreatment
Control (A2) 2.57 0.27
Lindane (B2) 3.61 0.45a
Lindane + Lycored (C2) 2.52 0.28b
Lindane + Vitamin-E (D2) 2.61 0.33b
Values are mean SD (n = 30).
a
Signicantly different from control and antioxidant treated groups.
b
Signicantly different from lindane treated groups, p < 0.05.
93
decrease in GSH content may have resulted from the activity of
Glutathione peroxidase (GPx) in reducing lipid hydroperoxides to
stable non-radical lipid alcohols, utilizing GSH as a source of reduc-
ing equivalents.
Free radicals are usually inactivated, destroyed, scavenged
in vivo by a group of antioxidants. The FRAP assay measures the
combined antioxidant effect in biological uids and may be useful
in providing an index of ability to resist oxidative damage [15]. Fol-
lowing the application of study drugs, only post-treatment lindane
group (B2) showed a statistically signicant reduction in the FRAP
in comparison to both control and anti-oxidant treated groups.
This decrease in FRAP may be due to increased oxidative stress
and reduced GSH, induced by lindane. Lycored and Vitamin-E
administration signicantly replenished the GSH levels, thereby
indicating that oxidative stress due to lindane application in sca-
bies patients was signicantly attenuated by the antioxidants as
evidenced by the increased FRAP levels.
Scabies is characterized by an immune allergic response to infes-
tation of the skin by the mite S. scabiei [24]. Understanding the im-
mune response mechanisms to scabies mites in nave hosts and in
hosts with induced immunity is important to understand the
pathogenecity in the host [25]. Immune cells particularly neutro-
phils and monocytes have key functions in killing microbial invad-
ers and the regulation of inammatory response [26]. Respiratory
burst function resulting in the release of ROS such as superoxide an-
ion (O2) from neutrophils is one of the key mechanisms of the in-
nate immune system and maladaptive control of this mechanism is
thought to play a pivotal role in the development of pathogenesis
such as acute lung injury and sepsis. The ability of neutrophils to
destroy bacteria or other pathogens is dependant upon the ability
to mount an effective respiratory burst [27]. Invasion of scabies
mites triggers the inammatory response resulting in increased
production of pro-inammatory cytokines such as IL-1 [12].
IL-1a and TNF-a induces responses by neutrophils, macro-
phages in scabietic lesions in response to scabies antigen. The im-
mune system is regulated by an intricate mechanism; free radicals
play an important role in immune regulation. Pesticides are capa-
ble of modulating immune responses and affecting lymphocytes in
both experimental animals and cases of human poisoning [9]. Lym-
phocyte dysfunction may be an integral part of pesticide-induced
oxidative stress, presenting an approach that may serve to delin-
eate the possible mode of action [20]. Increased oxygen free radical
generation by pesticide exposure exerts deleterious effects on dif-
ferent components of immune system.
The mean NBT reduction values in both pre- and post-treatment
groups did not differ signicantly in scabies patients showing that
lindane and antioxidants (Lycored and Vitamin-E) have no effect
on respiratory burst in neutrophils indicating that inammation
by S. scabiei mite is not statistically signicant in terms of
GSH (lM/ml blood) FRAP (lM/ml serum)
6.37 0.23 1350.25 126.6
6.43 0.27 1337.65 107.5
6.45 0.29 1374.25 126.6
6.24 0.31 1349.75 119.3
6.39 0.24 1354.1 82.5
4.21 0.46a 1264.5 107.5a
6.41 0.27b 1385.0 139.8b
6.27 0.29b 1340.0 123.5b
94 M. Subramaneyaan et al. / Pesticide Biochemistry and Physiology 102 (2012) 9194

Table 3
Immune response in control subjects and scabies cases.

Groups NBT (OD540/106 cells/30 min) IL-1a (pg/ml serum) TNF-a (pg/ml serum)
Pretreatment
Control (A1) 0.070 0.01 22.26 1.71 13.33 1.07
Lindane (B1) 0.078 0.01 37.64 6.08a 13.25 0.99
Lindane + Lycored (C1) 0.072 0.01 34.29 3.62a 13.77 2.35
Lindane + Vitamin-E (D1) 0.075 0.01 32.57 3.25a 13.64 2.12
Posttreatment
Control (A2) 0.071 0.01 22.08 1.49 13.46 1.16
Lindane (B2) 0.079 0.01 35.84 6.15a 13.09 0.56
Lindane + Lycored (C2) 0.072 0.01 33.88 2.97a 13.61 1.97
Lindane + Vitamin-E (D2) 0.073 0.01 31.29 3.01a 13.59 1.85

Values are mean SD (n = 30).

a
Signicantly different from control group, p < 0.05.

neutrophil function. The mean baseline IL-1a levels differ signi-
cantly in control and scabies groups indicating that scabies per
se induces the production of immuno-modulatory cytokine, IL-
1a, a potent inducer of inammation. TNF-a levels were not statis-
tically signicant from the control group indicating that infection
by S. scabiei mite does not induce the production of TNF-a. Further,
anti
-oxidant therapy failed to have any effect on both IL-1a and TNF-a
levels.
On consolidating the results, our observations indicate that per-
cutaneous application of lindane does not cause immunotoxicity in
scabies patients. Lindane application does cause oxidative stress
which can be effectively attenuated by Lycored and/or Vitamin-E,
and suggests the incorporation of antioxidant drugs in the treat-
ment protocol of the disease. However, the effects are marginally
signicant which may be due to the small sample size. Further val-
idation of the results in large number of samples needs to be car-
ried out.
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