Vol.1, No.3, 167-172 (2009)
doi:10.4236/health.2009.13027
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Protective role of buffalo pineal proteins on
arsenic-induced oxidative stress in blood and
kidney of rats
Vijay K. Bharti*, R. S. Srivastava
Neurophysiology Laboratory, Division of Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, U.P., India;
*DIHAR (FRL), DRDO, C/o-56 APO, Leh-194101, India
Received 26 September 2009; revised 12 October 2009; accepted 13 October 2009.
ABSTRACT
Objective: Exposure to various toxic metals has
become an increasingly recognized source of ill-
ness in human and animals, worldwide. Arsenic
(As) and its compounds cause adverse health
effects in animals and humans. Recently, it has
been suggested that the pineal gland may also
have antioxidants role due to secretary product
other than melatonin. With keeping this view, pre-
sent investigation tested effect of buffalo (Bubalus
bubalis) pineal proteins (PP) on arsenic-induced
oxidative stress in RBCs (Red blood cells) and
kidney of rats. Methods: Eighteen adult female
Wistar rats were grouped into group-I (Control),
group-II (Arsenic control), and group-III (Arsenic
+ Pineal proteins). Experimental rats were given
100 ppm arsenic (p.o.) for 4 weeks alone or along
with pineal proteins at a dose of 100 µg/kg body
weight (i.p.). Results: Interestingly, arsenic ex-
posure led to the stimulation of kidney catalase
(CAT) activity, but inhibition of RBCs CAT activ-
ity and significantly (P<0.05) increased the RBCs
and kidney lipid peroxidation level (LPO). How-
ever, arsenic treatment caused depletion of glu-
tathione (GSH), superoxide dismutase (SOD),
glutathione peroxidase (GPx), and glutathione
reductase (GR) in kidney tissues. In RBCs, only
GR and CAT activity were significantly (P<0.05)
declined. These changes were significantly (P<0.05)
reversed by PP treatment in arsenic exposed
animals. Conclusion: Therefore, present study
indicated the significant protecting effect of buf-
falo (Bubalus bubalis) PP against arsenic in-
duced-oxidative stress through antioxidant de-
fense systems in rats.
Keywords: Arsenic Toxicity; Blood; Buffalo Pineal
Proteins; Kidney; Oxidative Stress; Free-Radicals; Rat
1. INTRODUCTION
Recent studies by Flora, 1999 [1], Bhadauria and Flora,
2007 [2] and Modi et al., 2007 [3] have suggested that
arsenic toxicity is associated with the induction of oxida-
tive stress in vital organs through overproduction of re-
active free radicals (ROS) and inhibition of antioxidant
enzymes activity. Research performed by Halliwell and
Gutteridge, 1992 [4], shows the importance of certain
enzymes in oxidative defense systems as they metabolize
either free radicals or reactive oxygen intermediates to
no-radical products. These enzymes play important role
in free radical scavenging and amelioration of oxidative
stress (OS). Study done by Halliwell and Gutteridge,
1992 [4] and Halliwell, 2006 [5], further revealed that
antioxidant defense mechanisms in our vital organs are
not sufficient to prevent toxicity-related increase in oxi-
dative damage and therefore exogenous intake of anti-
oxidants might have protecting role for preserving their
physiological functions. A growing number of studies
have been designed to test the antioxidant effect of some
agents to prevent toxicity-related degenerative changes in
vital organs. Studies done by Varner et al., 1998 [6] have
shown the vital role of kidney in xenobiotics metabolism.
Since, kidney and blood are among the most vulnerable
biological system during oxidative stress and any xeno-
biotics metabolic disturbances. Therefore, kidney damage
due to OS further aggravates the kidney dysfunction. This
may result in additional toxic metabolites load to renal
system and may cause more insult to kidney due to high
OS. Hence, in present study these organs are sampled for
study. As per studies of Reiter, 1991 [7] and Halliwell,
2006 [5], supplementations of antioxidants are needed to
enhance these antioxidative enzymes level for preventing
OS. Studies of Reiter et al., 1995 [8] illustrated the neu-
roendocrine functions of the pineal affect on wide variety
of physiological role in our body. Few studies of Si-
monneaux and Ribelayga, 2003 [9] and Tandon et al.,
2006 [10] revealed that beside melatonin (MEL), the

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168
Table 1. Distribution of experimental rats to different treatments.
Groups Body weight (g)Treatments Dose Route of administration
I Control (28 D) 141.67 a
±3.57
Drinking water
+ Normal saline Adlibitum Oral Intra peritoneal
II As
218.33 b
±11.53
Arsenic +
Normal saline 100 ppm As Oral with drinking water Intra peritoneal
III As+PP
145.00 a
±6.58
Arsenic +
Pineal Proteins
100 ppm As
100 µg/kg BW
Oral with drinking water
Intra peritoneal
Values (n=6); Means ±S.E) in the same column bearing no superscript (a, b, c) common vary significantly (P<0.05).
As: arsenic; PP: Buffalo pineal proteins
pineal gland secretes and expresses certain proteins es-
sential for various physiological functions. The study of
Bharti and Srivastava, 2009a [11] has suggested that the
pineal gland may also have antioxidants role due to sec-
retory product other than MEL. Literature regarding the
physiological role of PP and peptides in arsenic-induced
OS is completely lacking. However, earlier laboratory
findings revealed that, these buffalo PP and peptides have
certain physiological functions viz. immunopotentiation
by Ramasamy, 2006 [12], heat stress by Sejian, 2006 [13],
amelioration of fluoride-induced OS and apoptosis by
Bharti, 2008 [14] and Bharti and Srivastava, 2009b [15].
These findings suggest the test for protecting role of PP
against As-induced OS in kidney and blood. Therefore,
the present study will investigate the physiological role of
buffalo pineal proteins in amelioration of arsenic-induced
oxidative stress through modulation of certain antioxidant
defense systems. The present study will further substan-
tiate the physiological role of PP on antioxidant defense
systems, and findings may have significant implications
in elucidating the therapeutic use of pineal proteins as
antioxidants drugs and management of toxicity.
2. MATERIALS AND METHODS
All the procedures conducted on the experimental ani-
mals were duly approved by the Institutional Animal
Ethics Committee (IAEC) and Committee for the Purpose
of Control and Supervision of Experiments on Animals
(CPCSEA).
2.1. Chemicals
The chemical, glass and plastic were procured as per
requirement from different suppliers. All these metal salts
are soluble in water. All other chemicals used in the study
were of analytical grade from HiMedia, Loba Chemie
(Mumbai, India); Sigma Chemical Co., St. Louis; USA;
Genei, Bangalore; SRL Chemicals, India.
2.2. Experimental Animals
Experimental adult female Wistar rats (123-142 g BW)
were procured from the Laboratory Animal Resource
(LAR) Section, Indian Veterinary Research Institute,
Izatnagar-243122, U.P., India. Rats on arrival were ex-
amined for any abnormality or overt ill health. After an
acclimatization period of one week, they were weighed
and randomly assigned to various groups so as to give
approximately equal initial group mean body weights.
Rats were housed in polypropylene cages and rice husk
was used as the nesting material. Animal room tempera-
ture and relative humidity were set at 21±2 oC and
50±10%, respectively and lighting was controlled to give
12 h light and 12 h darkness. All the animals had free
access to standard laboratory animal diet and clean water.
The animals were checked daily for the health and hus-
bandry conditions.
2.3. Experimental Design
Eighteen adult female Wistar rats were grouped into
control (Group I), arsenic control (Group II), and arsenic
and buffalo pineal proteins (PP) (Group III) and given
different treatment (see Table 1). Before the start of ex-
periment, appropriate dose of arsenic and pineal proteins
were optimized in pilot trial. Arsenic (As) level in
drinking water was calculated and thereafter-required
concentration of As (100 ppm) was made by additional
sodium arsenite daily for 28 days. We used buffalo PP as
our agent since we have previously published this reagent.
Dose of pineal proteins was calculated based on animal
body weight and thereafter, they were dissolved in vehicle
(normal saline) and administered (daily for 28 day) in-
traperitoneally (i.p.). Solutions for administration in ex-
perimental animals were prepared daily to minimize
possible instability of the chemicals in the mixture.
2.4. Sample Collection
Daily observations were taken for the behavioral changes,
clinical signs of toxicity and mortality, if any, throughout
the experimental period. The rats were euthanized by
using ether at the end of experiments (28 day), then blood
and kidney were collected.
The heparinised blood samples were centrifuged at
2000 rpm for 15 min, and RBCs (Red blood cells) were
separated. The resulting erythrocyte (RBCs) pellet was
washed thrice with phosphate buffer saline (PBS, pH 7.4)
and dilution (33%) was made in PBS (pH 7.4). This 33%
packed RBCs was used for the estimation of lipid per-
oxidation (LPO), reduced glutathione (GSH), glutathione
reductase (GR), glutathione peroxidase (GPx) and hae-

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169
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moglobin (Hb). Another 1:10 dilution of packed RBCs in
PBS was used for the estimation of catalase (CAT) and
superoxide dismutase (SOD).
Immediately, kidney was cleaned, rinsed in chilled sa-
line, blotted and weighed. Thereafter, 200 mg of sample
was weighed and taken in 2 ml of ice-cold saline. Another
200 mg of sample was weighed separately and taken in 2
ml of 0.02 M EDTA for GSH estimation. Kidney ho-
mogenates were prepared using IKA homogenizer (Ger-
many), under ice-cold condition. Homogenate were col-
lected and centrifuged for 10 min at 3000 rpm at 4˚C.
Thereafter, cell free supernatant were collected and trans-
ferred to pre-cooled microfuge tube in duplicate and stored
at below -20 oC. These supernatants were used for estima-
tion of total proteins, LPO, CAT, SOD, GPx, GR activity,
and GSH concentration.
2.5. Analytical Procedures
Enzymatic (CAT, SOD, GR, GPx) and non-enzymatic
(GSH) antioxidant defense systems are essential to
combat OS in the body. These parameters are up regulated
by antioxidants supplementation. Therefore, to test anti-
oxidant properties of PP, we examined these parameters.
Absorbance of all the tissue biochemical estimations was
read, using Double Beam UV-VIS Spectrophotometer
(UV 5704 SS, ECIL, India).
2.5.1. Lipid Peroxidation (LPO)
Membrane peroxidative damage in RBCs and kidney
tissues due to free radicals was determined in terms of
malondialdehyde (MDA) production by the method of
Rehman, 1984 [16]. Lipid peroxidation in blood was
expressed as n M of MDA per ml of RBCs homogenates
and in kidney tissues as nM of MDA formed per g of wet
tissue.
2.5.2. Reduced Glutathione (GSH)
The concentration of GSH in kidney homogenates was
estimated by evaluating free-SH groups, using DTNB
method described by Sedlak and Lindsay, 1968 [17] and
the results have been expressed as µM/g of GSH per g of
wet tissue. However, in blood, GSH was estimated by the
5, 5’dithiobis (2-nitrobenzoic acid; DTNB) method of
Prins and Loos, 1969 [18] and expressed as mM GSH per
ml of packed RBCs.
2.5.3. Catalase (CAT)
Activities of catalase enzymes in blood and kidney tissues
were estimated by spectrophotometric method as describ-
ed by Bergmeyer, 1983 [19] and were expressed as nM
H2O2 utilized /min /mg protein and in blood, mM H2O2
utilized /min /mg Hb
2.5.4. Superoxide Dismutase (SOD)
Superoxide dismutase activities in blood and kidney
homogenates were estimated as per the method described
by Madesh and Balasubramanian, 1998 [20]. It involves
generation of superoxide by Pyrogallol autoxidation and
the inhibition of superoxide-dependent reduction of the
tetrazolium dye MTT [3-(4-5 dimethyl thiazol 2-xl) 2, 5
diphenyl tetrazolium bromide] to its formazan, measured
at 570 nm. Superoxide dismutase activity in blood was
expressed as U/g of Hb and as U/g of protein in kidney
tissues.
2.5.5. Glutathione Peroxidase (GPx)
Glutathione peroxidase (GPx) activities in blood and
kidney were determined by the method of Paglia and
Valantine, 1967 [21]. The enzyme activity in kidney has
been expressed as U/mg of protein, and one unit of en-
zyme activity is defined as 1 nM of substrate (NADPH)
utilized/min/mg protein and in blood as µmole NADPH
oxidized to NADP/ g of Hb/min.
2.5.6. Glutathione Reductase (GR)
The GR activities in kidney and blood were assayed by
the method of Goldberg and Spooner, 1983 [22]. The GR
activity in kidney tissues has been expressed as nM
NADPH oxidized to NADP/ g of protein/min and in
blood as nM NADPH oxidized to NADP/ g of Hb/min.
2.5.7. Protein Assay
Protein contents in kidney homogenates were determined
and calculated by the method of Lowry et al., 1951 [23]
and result has been expressed as mg/g of tissue.
2.5.8. Hb Estimation
Haemoglobin concentrations in packed RBCs were esti-
mated by spectrophotometric method using Drabkin’s
solution (Span India Kit).
2.5.9. Statistical Analysis
Differences between groups were statistically analyzed
by one-way ANOVA, and the differences between the
means of groups were separated by least significant dif-
ference (LSD) test. All data were presented as
mean±standard error. Values of p< 0.05 were regarded as
significant. A computer program (SPSS 10.01, SPSS Inc.
Chicago, IL, USA) was used for statistical analysis.
3. RESULTS
We evaluated different enzymatic and non-enzymatic
antioxidant defense system to assess the As-induced OS and
its amelioration by PP. All the comparisons were made
between vehicle and PP with control animals (28 days).
We did not find any abnormal behavior in animals during
the entire period of investigation and none of the animals
died in any group. We did not observe any gross abnor-
malities in the organs of female rats in different treat-
ments. Experimental data obtained in present study were
presented in tabular form (Means ±S.E) (see Tables 2, 3).
A significant (P<0.05) increase in lipid peroxides level
along with a concomitant decrease in the activities of CAT
and GR antioxidants enzymes were observed in RBC (Red
blood cells) of arsenic administered (100 ppm As/day in

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Table 2. Effect of different treatments on lipid peroxidation (LPO), catalase (CAT), superoxide dismutase (SOD), glutathione reductase
(GR), glutathione peroxidase (GPx), and reduced glutathione (GSH) level in RBCs of female rats.
Group Parameters
LPO
(nM MDA/ml)
CAT
(mM/min
/mg Hb)
SOD
(U)
GR
(µM/min
/g Hb)
GPx
(µM/min
/g Hb)
GSH
(µM
/ml)
I 10.23 a
±0.86
105.70 b
±7.85
8.12 a
±0.41
0.66 b
±0.58
8.35
±1.04
0.37
±0.22
II 16.23 b
±2.88
73.12 a
±6.95
9.29 a
±1.31
0.45 a
±0.39
9.08
±1.11
0.44
±0.18
III 6.51 a
±0.99
96.03 a b
±10.85
16.84 b
±1.32
0.68 b
±0.82
11.83
±1.29
0.43
±0.30
Values (n=6); Means ±S.E) in the same column bearing no superscript (a, b, c) common vary significantly (P<0.05).
Table 3. Effect of different treatments on lipid peroxidation (LPO), catalase (CAT), superoxide dismutase (SOD), glutathione reductase
(GR), glutathione peroxidase (GPx), and reduced glutathione (GSH) level in kidney of female rats.
Group Parameters
LPO
(nM MDA/g)
CAT
(nM/min
/mg protein)
SOD
(U)
GR
(nM/min
/mg protein)
GPx
(nM/min
/mg protein)
GSH
(µM
/g tissue)
I 5.00 a
±0.13
251.70 a
±4.58
5.98 c
±0.17
126.64 c
±8.47
20.13 b
±1.11
3.50 b
±0.63
II 16.17 c
±0.18
428.76 c
±13.17
3.33 a
±0.12
46.57 a
±8.43
13.23 a
±1.66
1.92 a
±0.46
III 7.24 b
±0.83
286.86 b
±7.59
5.05 b
±0.26
95.10 a
±9.33
18.58 b
±0.82
3.54 b
±0.11
Values (n=6); Means ±S.E) in the same column bearing no superscript (a, b, c) common vary significantly (P<0.05).
drinking water for 28 days) rats (see Table 2). In RBCs,
only GR and CAT activity were significantly (P<0.05)
declined in arsenic alone treated group. However, GPx,
GSH, SOD levels were unchanged in arsenic treated ani-
mals compared to control animals (see Table 2).
Arsenic treatment caused depletion of glutathione
(GSH), superoxide dismutase (SOD), glutathione per-
oxidase (GPx), and glutathione reductase (GR) in kidney
tissues. On the other hand, the increased LPO and CAT
level were recorded in kidney of arsenic exposed rats
(see Table 3).
All the adverse changes in LPO, CAT, SOD, GPx, GR,
and GSH of RBCs, and kidney brought by arsenic-
induced oxidative stress were significantly (P<0.05) re-
duced by PP treatment in As+PP administered rats (see
Tables 2, 3).
It was interesting to see the highest activity of SOD in
RBCs of As+PP treated rats compared to control (see
Table 2). These effects seem to be beneficial and indicate
antioxidant potential of pineal proteins. The results sug-
gested that the pineal proteins inhibit depletion of anti-
oxidant enzymes and concomitant decrease in the levels
of lipid peroxidation differentially in rats’ RBCs and
kidney of As treated animals.
4. DISCUSSIONS
Although there is inconclusive proof for an altered oxi-
dative stress and antioxidant balance in blood and renal
arsenic toxicity. Modi et al., 2007 [3] reported beneficial
effect of antioxidants in combating the toxic effects of
arsenic. Josephy et al., 1997 [24] also said that uncon-
trolled lipid peroxidation is a toxic process resulting in the
deterioration of biological membranes. Flora, 1999 [1]
and Bharti, 2008 [14] stated that the increased LPO in the
blood and kidney can be due to increased oxidative stress
in the cell as a result of depletion of antioxidant scavenger
system. This was reflected in the present study, as As-
induced oxidative stress caused more LPO in blood and
kidney.
As per study of Josephy et al., 1997 [24], alternatively,
the radical chain reaction may be broken by the action of
antioxidants and thereby controls of oxidative stress. Pineal
proteins reversed the adverse effect of oxidative stress
and reduced the LPO level and enhanced the antioxidant
defense system as well. Super oxide dismutase, GR, GPx,
GSH, and CAT activities increased to significant levels
than control in As+PP treated rats. These results were
equally comparable with control groups. These findings
suggest the antioxidant properties of pineal proteins, and
therefore present study elucidated the antioxidant prop-
erties of pineal proteins. This might be due to scavenging
of free radicals generated by As-exposure and breakage of
radical chain reaction, thereby reduction of lipid peroxi-
dation as stated by Bharti and Srivastava, 2009a [11].
Glutathione peroxidase (GPx) is the most important
enzyme for extraperoxisomal inactivation of H2O2, espe-
cially in the kidney and liver. Reduced glutathione (GSH)
plays a very important role as an intracellular antioxidant.
Renal concentration of reduced glutathione (GSH) and
SOD decreased in As treated rats compared to control and
As+PP reversed this decreased. We speculate the mecha-

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nism of protective effect of PP against As-induced oxi-
dative stress through scavenging of free radicals gener-
ated by As-exposure and breakage of radical chain reac-
tion, thereby reduction of lipid peroxidation. As per ex-
perimental study of Bharti and Srivastava, 2009a [11], this
physiological role of PP may be due to presence of certain
enzymes influencing it or via melatonin secretion on body
antioxidant defense systems. Therefore, administration of
pineal proteins is proved to be beneficial on vital organs
antioxidant defense system during As-induced oxidative
stress in rats.
Therefore, these findings support the hypothesis of
protecting role of buffalo pineal proteins against arse-
nic-induced oxidative stress through its antioxidant
properties and also find agreement with earlier findings of
Bharti and Srivastava, 2009b [15] about free radical scav-
enging ability of pineal proteins.
5. CONCLUSIONS
Pineal proteins reduced the arsenic-induced OS level in
kidney and blood as reflected by low LPO and higher
activities of catalase, GPx, GR, SOD, and GSH level.
Hence, these findings indicate the protecting role of PP
against As-induced OS in kidney and blood through
modulation of certain antioxidant defense systems. The
present study further substantiate the physiological role of
PP on antioxidant defense systems and findings have
significant implications in elucidating the therapeutic use
of pineal proteins as antioxidants drugs and management
of toxicity.
6. ACKNOWLEDGEMENTS
Project grant and facilities provided by Indian Veterinary
Research Institute for conducting this study is duly ac-
knowledged. I also acknowledge the tireless efforts of our
lab and animal shed assistants.
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