Food and Chemical Toxicology 45 (2007) 879–887

www.elsevier.com/locate/foodchemtox

Evaluation of protective efficacy of Spirulina fusiformis against

mercury induced nephrotoxicity in Swiss albino mice
a,b
Mukesh Kumar Sharma , Ambika Sharma a, Ashok Kumar a, Madhu Kumar a,*

a
Department of Zoology, University of Rajasthan, Jaipur 302004, India
b
Department of Zoology, R.L.S. Govt. (P.G.) College, Kaladera, Jaipur 303801, India

Received 7 December 2005; accepted 10 November 2006

Abstract

The toxicity of mercury to animals and man is well established and this depends greatly on the form of the mercury compounds. In
most animals’ species, including man, the kidney is the main site of deposition of inorganic mercury and target organ for its toxicity. In
the present study Spirulina fusiformis (a cyanobacterium, belongs to family – Oscillatoriaceae) has been investigated as a possible
modifier of mercury induced renal damages in Swiss albino mice. Animals were divided into four groups. (i) Control group – only vehicle
(0.9% NaCl) was administered as i.p. (ii) HgCl2 treated group – 5.0 mg/kg b.wt. HgCl2 was administered as i.p. (iii) Spirulina treated
group – 800 mg/kg b.wt. Spirulina extract was administered orally. (iv) Combination group – S. fusiformis was administered 10 days
before mercuric chloride administration and continued upto 30 days after mercuric chloride administration (5.0 mg/kg b.wt.). The ani-
mals were autopsied on 1, 3, 7, 15 and 30 days after treatment and the activity of alkaline phosphatase (ALP), acid phosphatase (ACP),
lactate dehydrogenase (LDH) and MDA (malondialdehyde) level were measured in kidney homogenates. The results indicated that there
was a time-dependent significant enhancement in MDA content and ACP activity and decrease in LDH and ALP activity observed after
HgCl2 treatment. Mercury intoxication also induces pathological alterations in the kidney such as degeneration of glomerulus, proximal
and distal tubules. A dose-dependent mortality was also observed following administration of different doses of HgCl2. In combined
treatment of Spirulina with HgCl2, a significant decrease in MDA content and ACP activity and elevation in LDH and ALP activity
was observed as compared to HgCl2 treated group. Spirulina pre- and post-treatment with mercury also significantly reduces pathological
alterations in kidney. Thus, the results from the present study suggest that S. fusiformis can significantly modify the renal damages
against mercuric chloride induced toxicity.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Mercuric chloride; Swiss albino mice; Spirulina fusiformis; Kidney; MDA content; Phosphatase; Lactate dehydrogenase

1. Introduction inorganic mercury causes severe kidney damage after acute

Mercury has been recognized as a highly toxic metal to
man for many years. It is widely used in industry, agricul-
ture and medical services. The toxicity of mercury depends
greatly on the forms of the mercury compounds (elemental,
inorganic and organic). Inorganic mercury present in the
environment is a well-established toxicant to human health
(Clarkson et al., 1988; WHO, 1991). It is well known that
*
Corresponding author.
E-mail address: mkshrma@hotmail.com (M.K. Sharma).
and chronic exposure (Zalups and Diamond, 1987a,b;
Zalups, 1997; Sharma et al., 2005a).
It is important to develop an effective drug for mercury
to prevent the mercury-induced renal damages. In recent
years, an extensive resecarch work has been carried out
on chemical protection. Some chemical substances, i.e.
sodium 2,3-dimercapto propane-1-sulfonate, 2,3-dimerca-
pto succinic acid (Tripathi and Flora, 1998; FDA, 1999;
Marcus, 2001), and dimercaprol (also known as BAL or
British Anti-Lewisite) (Micromedex, 1999; Wentz, 2000)
have been tested for their protective activity against heavy

0278-6915/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2006.11.009
880 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887
metal toxicity and found to be promising in the field of
chemical protection, but all these chemicals could not have
practical utility in human beings due to their inherent
toxicity at effective doses.
Historically, plants have been used as folk medicine
against various type of disease. Remedies from plant
sources (Indian system of medicine the ‘Ayurveda’) have
proved to be very popular in primary health care in India
for a long time.
In recent years, Spirulina is gaining more attention from
medical scientists as a nutraceutical and source of potential
pharmaceuticals. It is blue green algae (mycobacterium)
belonging to the family Oscillatoriaceae. It is known to
have high protein content (Kapoor and Mehta, 1994), nat-
ural biochelated vitamins, especially b-carotene (Prescott,
1978; Seshadri et al., 1991) and an antioxidant enzyme
superoxide dismutase (SOD). Spirulina fusiformis possess
potent antiviral activity (Hayashi et al., 1996.), anticancer
effects (Mittal et al., 1999), strengthens immune system
(Qureshi et al., 1995, 1996), radioprotective (Verma,
2000) and metalloprotective effects (Shastri, 1999).
In our laboratory, we have already reported that the S.
fusiformis extract was effective in reducing the mercury
toxicity in terms of mortality rate, liver damages and blood
biochemical alterations (Kumar et al., 2001, 2005; Sharma
et al., 2001, 2005b; Sharma, 2002). The present investiga-
tion has been carried out to evaluate the role of S. fusifor-
mis in modifying the mercury-induced nephrotoxicity in
Swiss albino mice.
2. Materials and methods
2.1. Test systems
Random-bred, male Swiss albino mice (6–8 weeks old) were obtained
from animal facility, IVRI, Izatnagar. They were kept under controlled
environment conditions with provision of a 12 h light: 12 h dark regimens.
The animals were provided with pelleted standard mice feed (Hindustan
Lever Ltd., India) and tap water ad libitum.
2.2. Chemicals
Pyridine, TMP, sodium dodecyl sulfate, thio barbituric acid, sodium
b-glycerophosphate, diethyl barbituric acid and ANSA are procured from
Sigma Chemical Company, Hyderabad, India and ammonium molybdate,
dinitrophenyl hydrazine (DNPH) and sodium pyruvate procured from
SRL Pvt. Ltd., Mumbai, India and tri chloro acetic acid procured from
Qualigens, Mumbai, India.
2.3. Spirulina fusiformis
Spirulina fusiformis in the form of powder was obtained from RECON
Ltd., Banglore, India. It was suspended in vehicle (olive oil) at different
dose level and 0.05 ml of Spirulina suspension was given to each mouse by
oral gavage daily.
2.4. Dose selection
Dose selection of S. fusiformis was done on the basis of drug tolerance
study. Various doses of S. fusiformis (200, 400, 600, 800, 1000 and
1200 mg/kg body weight in olive oil) were administered orally for 30 days.
The Spirulina did not show any sign of toxicity upto maximum dose and
from these doses 800 mg/kg body weight dose was most suitable in terms
of maximum protective effects.
2.5. Toxicant
Mercury in the form of inorganic mercury (mercuric chloride) was used
for the present study. It was dissolved in 0.9% NaCl at various dose levels
and administered intraperitoneally at once.
2.6. Experimental protocol
To evaluate the survival rate in Hg intoxicated mice and modulatory
influence of S. fusiformis against mercury induced renal damages in mice,
the animals Swiss albino mice were divided into the following groups.
2.6.1. Group I – mortality rate
To evaluate the mortality rate and LD50/30 of HgCl2 the animals Swiss
albino mice were administered different doses of HgCl2 viz. 0.1, 1.0, 2.5,
5.0, 6.0, 7.5, 10.0 mg/kg body weight in 0.9% NaCl. The survival rate of
these animals was recorded and LD50/30 was calculated by regression
analysis.
2.6.2. Group II – modulation of mercury toxicity
To evaluate the toxic effect of mercury and modulatory effect of S.
fusiformis against toxic effect of mercury in kidney, the animals Swiss
albino mice were divided into four subgroups:
Subgroup I Only vehicle (0.9% NaCl) in equal volume to mercury
(n = 30) treated animals, was given to these animals to serve as
control
Subgroup II The animals were administered HgCl2 5.0 mg/kg body
(n = 30) weight in 0.9% NaCl intraperitoneally at once
Subgroup III The animals were administered orally S. fusiformis
(n = 30) extract (800 mg/kg body weight) for 30 consecutive
days
Subgroup IV The animals were administered Spirulina extract
(n = 30) 800 mg/kg body weight orally for 10 days before
mercuric chloride (5.0 mg/kg body weight) and until 30
days after mercuric chloride administration
The animals from all above subgroups were autopsied on 1, 3, 7, 15 and
30th days and kidney were removed and processed for histological and
various biochemical parameters.
2.7. Histological studies
Kidneys from autopsied animals were excised out and fixed in Bouins
fixatives for 24–48 h. The fixed tissue was further embedded in paraffin
and 5-lm thick sections were prepared by using microtome. These sections
were stained with haematoxyline and eosin (H&E) and observed under
light microscope for histological alterations. The stained sections were
read only for the presence and absence of nephrosis. For this study,
nephrosis was defined as the presence of tubular epithelial degeneration
and/or necrosis. Individual cell necrosis was the criterion for identification
of the earliest histopathologic change. The severity of nephrosis was
recorded using a grading scale of 0–4 which was related to a subjective
impression of the extent of cortical tubular involvement (NTP, 1993), as
follows:
0 = indistinguishable from controls
1 = minimal, 625% tubules affected
2 = mild, >25% 6 50% tubules affected
3 = moderate, >50% 6 75% tubules affected
4 = marked, >75% tubules affected
 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887 881

2.8. Biochemical studies 3. Results

2.8.1. Alkaline phosphatase activity
The alkaline phosphatase activity in the kidney was estimated by Fiske
and Subbarow (1925) method. The alkaline phosphatase activity is the dif-
ference between inorganic phosphatase content of the incubated and control
samples expressed as Bodansky unit. One Bodansky unit corresponds to the
liberation of 1 mg of inorganic phosphorous from the tissue in 1 mg/gm/h.
In this method, the proteins content of the tissue homogenates was first
precipitated with trichloroacetic acid. The filtrate was then treated with
molybdate solution. This resulted in the formation of phosphomolybdic
acid from the phosphate present in the tissue. The phosphomolybdic acid
was then reduced to produce a blue colour whose intensity was propor-
tional to the amount of phosphate liberated.
2.8.2. Acid phosphatase activity
Acid phosphatase activity in the kidney was also estimated by the
method of Fiske and Subbarow (1925) for the determination of phosphate
liberation.
2.8.3. Lactate dehydrogenase activity
Lactate dehydrogenase activity in the kidney was estimated by
‘‘Wroblewski procedure’’ given in the sigma technical bulletin no. 500
(Sep. 1967). The substrate used was sodium pyruvate; pH of the buffer was
maintained at 7.5 at 37 °C. In the presence of enzyme pyruvic acid is
converted into lactic acid. Further pyruvic acid reacts with 2,4-dinitro-
phenyl hydrazine and forms an intensely colored brown hydrazone, which
has a high OD at 400–500 nm.
2.8.4. MDA (malondialdehyde) assay
MDA or lipid peroxides level in the kidney was measured by the
method of Ohkawa et al. (1979) as thiobarbituric acid reactive substances
(TBARS). The concentration of TBARS was expressed as n moles of
malondialdehyde per mg of tissue using 1,1,3,3-tetramethoxypropane
(TMP) as standard.
2.9. Statistical analysis
Data are expressed as mean ± SE. Statistical significance of difference
between groups was determined by Student’s t-test.
3.1. Mortality rate
A dose-dependent mortality was observed in animals
treated with different doses of mercuric chloride. No mor-
tality was observed upto 30 days from 0.10 to 2.5 mg/kg
body weight dose of HgCl2. The animals received 5.0 mg/
kg body weight of HgCl2 showed only 10% mortality till
30 days. At the dose of 6.0 mg/kg body weight 60% morta-
lity was observed within a period of 30 days. At the dose of
7.5 mg/kg body weight 100% mortality was observed and
at 10.0 and 12.5 mg/kg body weight 100% mortality
occurred within 48 and 12–16 h after the exposure of
mercury.
On the basis of above data, LD50/30 of the mercuric
chloride was calculated by extrapolating with the regres-
sion line. At Y-axis mortality % was taken and on X-axis
doses of HgCl2 were taken. The LD50/30 of HgCl2 was
5.6214 mg/kg body weight (Fig. 1).
3.2. Histological studies
The present investigation showed widespread morpho-
logical alterations in kidney of HgCl2 intoxicated mice
1-day post-administration. Widespread proximal tubular
necrosis with absence of brush border and desquamated
necrotic epithelial cells in the lumen was observed follow-
ing Hg exposure. Remnants of cellular debris were
observed in the lumen of both proximal and distal tubules.
Epithelial cell nuclei showed pyknosis, karyolysis and kar-
yorhexsis, indicating that they were in the process of dye-
ing. The time-dependent nephrotic changes were observed
in Hg intoxicated mice (Table 1). At day 7th severity of

140
130
120
Slope = 20.1886; Intercept = -63.4905
110
LD50/30 = 5.6214 mg/kg body weight
100
90
80
70
60
Mortality %

50
40
30
20
10
0
-10 1 2.5 5 6 7.5 10
-20
-30
-40
-50
-60
Dose of mercuric chloride (mg/kg b.wt)

Fig. 1. Determination of LD 50/30 of mercuric chloride.

882 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887

Table 1 3.3. Biochemical studies

Showing nephrosis severity at various time intervals following HgCl2
administration in Swiss albino mice
3.3.1. Alkaline phosphatase
Days and number of mice Nephrosis severity grade The alkaline phosphatase activity in HgCl2 treated
0 1 2 3 4 group was found to be highly significant (P < 0.001)
1d (n = 6) 0 4 2 0 0 decrease at all autopsy intervals as compared to the control
3d (n = 6) 0 0 4 1 1 group. When normal healthy mice were fed with Spirulina
7d (n = 6) 0 0 1 2 3 alone the alkaline phosphatase activity apparently similar
15d (n = 6) 0 0 1 3 2
30d (n = 6) 0 0 1 4 1
to the control group. However, the combined treatment
of Spirulina with mercury results in highly significant rise
Note. Mice were administered a single, intraperitoneal injection of 5 mg/
kg body weight HgCl2 and histopathology was evaluated at various times
in alkaline phosphatase activity at all autopsy intervals
after exposure. Nephropathy severity grade scale: 0 = none, indistin- with compared to HgCl2 intoxicated mice (Fig. 2).
guishable from controls; 1 = minimal, 625% tubules affected; 2 = mild,
>25% 6 50% tubules affected; 3 = moderate, >50% 6 75% tubules affec- 3.3.2. Acid phosphatase
ted; and 4 = marked, >75% tubules affected. The values in each cell rep- A highly significant (P < 0.001) increase in acid phos-
resent the number of animals within each treatment group with the
indicated grade of nephrosis. Scale adapted from NTP Technical Report
phatase activity was noticed in HgCl2 treated mice
(1993). throughout the experiment. Spirulina alone treatment did
not show any remarkable alteration in acid phosphatase
activity as compared to control. On the other hand, in com-
bined treatment of Spirulina with mercury there was a
nephrosis was prominent (>75% tubules were affected). On highly significant decline in acid phosphatase activity
day 30th it was difficult to distinguish between different observed at all autopsy intervals, indicating gradual recov-
tubules and the whole kidney appeared necrotic. The glo- ery as compared to HgCl2 intoxicated mice (Fig. 3).
merulus showed degenerative changes at all autopsy
intervals. 3.3.3. Lactate dehydrogenase (LDH)
In Spirulina alone treated animals kidney showed nearly Lactate dehydrogenase activity was significant
normal histoarchitecture. Kidney showed reparative ten- (P < 0.001) decrease in HgCl2 intoxicated mice with respect
dencies in Spirulina pre- and post-treated group. On day to control animals. However, Spirulina alone treatment did
1 proximal convoluted tubules and distal convoluted not shows any significant alteration in LDH activity at all
tubules showed slight degeneration at a few places. At autopsy intervals. Whereas, a significant rise in LDH activ-
day 1 nearly 50% animals showed no nephrosis (0% tubules ity was noticed in combined treatment of Spirulina with
affected). Upto day 30th the sign of reparation was mercury at all autopsy interval with respect to Hg intoxi-
observed with normal epithelial lining and normal nuclei cated mice (Fig. 4).
as compared to HgCl2 treated group. On this day 5 animals
showed less than 25% affected tubules. From day 1 upto 3.3.4. MDA assay (lipid peroxidation)
day 30th, glomerulus appeared normal without any epithe- The MDA level in kidney of HgCl2 treated mice showed
lium damage (Table 2). a highly significant (P < 0.001) increase at all autopsy inter-
val as compared to the control group. Mice treated with
Spirulina alone exhibited a significant lowering in MDA
Table 2 formation. Whereas, Spirulina pre- and post-treatment
Showing nephrosis severity at various time intervals following Spirulina
resulted in a highly significant decline in HgCl2 intoxicated
pre- and post-treatment with HgCl2 administration in Swiss albino mice
mice (Fig. 5).
Days and number of mice Nephrosis severity grade
0 1 2 3 4 4. Discussion
1d (n = 6) 3 2 1 0 0
3d (n = 6) 1 3 2 0 0 The kidneys are the primary target organ for accumula-
7d (n = 6) 0 2 3 1 0 tion and toxicity of inorganic mercury. The present investi-
15d (n = 6) 0 4 2 0 0
30d (n = 6) 1 5 0 0 0
gation suggests widespread proximal tubular necrosis with
absence of brush border and desquamated necrotic epithe-
Note. Mice were administered Spirulina 800 mg/kg body weight continu-
ously for 10 days before mercuric chloride (5.0 mg/kg body weight) and lial cells in the lumen following Hg exposure. Proximal
continued for 30 days following mercuric chloride administration and tubule is the most common site of toxicant-induced cell
histopathology was evaluated at various times after exposure. Nephrop- injury. Zalups et al. (1991) and Zalups and Barfuss
athy severity grade scale: 0 = none, indistinguishable from controls; (1996) also investigated that pars recta (straight segment)
1 = minimal, 625% tubules affected; 2 = mild, >25% 6 50% tubules
of the proximal tubule (particularly the portion at the junc-
affected; 3 = moderate, >50% 6 75% tubules affected; and 4 = marked,
>75% tubules affected. The values in each cell represent the number of tion of the cortex and outer medulla) is the segment of the
animals within each treatment group with the indicated grade of nephron that is the most vulnerable to the toxic effects of
nephrosis. Scale adapted from NTP Technical Report (1993). both inorganic and organic forms of mercury. This might
 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887 883

35

Enzyme activity (mg Pi/gm/hr) 30

c
25 c
c c
20 c
c c
c
15
c
c
10

0
1 3 7 15 30
aP<0.05
bP<0.01 Autopsy intervals (Days)
cP<0.001
Normal Spirulina Mercuric chloride Spirulina+Mercury+Spirulina

Fig. 2. Variation in activity of alkaline phosphatase in kidney of various experimental groups.

7
c
c
6
Enzyme activity (mg Pi/gm/hr)

c c
5
c
4 c
c
c
3 c

0
1 3 7 15 30
aP<0.05
Autopsy intervals (Days)
bP<0.01
cP<0.001
Normal Spirulina Mercuric chloride Spirulina+Mercury+Spirulina

Fig. 3. Variation in activity of acid phosphatase in kidney of various experimental groups.

be due to selective accumulation of xenobiotics into this
segment of the nephron. In contrast to the distal tubule,
which is characterized by a relatively tight epithelium with
high electrical resistance, the proximal tubule is a leaky epi-
thelium, favoring the flux of mercury compounds into
proximal tubular cells.
It is likely that the luminal and/or basolateral transport
of mercury into the proximal tubular epithelial cells is
through co-transport of mercury with an endogenous
ligand such as glutathione, cysteine or albumin, or through
some plasma membrane mercury-ligand complex. Thus
transport of GSH conjugates heavy metals and organic
anions and cations. It is localized mainly to the proximal
tubule than other segments, resulting in proximal tubular
accumulation and cellular toxicity of these xenobiotics
(Zalups, 1995). Depending on the severity of the nephrop-
athy induced by mercury, cellular injury and necrosis can
occur along the entire length of the pars recta and distal
tubules.
In the present study glomerulus degeneration was
observed. Carmignani et al. (1992) also observed that
administration of large doses of mercuric chloride (28 mg
884 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887

250
Enzyme activity (Units/mg of tissue/hr)

200 c
c c
c
c c
150 c
c
c
c
100

50

0
aP<0.05 1 3 7 15 30
bP<0.01
Autopsy intervals (days)
cP<0.001

Normal Spirulina Mercuric chloride Spirulina+Mercury+Spirulina

Fig. 4. Variation in lactate dehydrogenase activity of kidney in different experimental groups.

25

20 c
TBARS (nmol of MDA/mg tissue)

c
c c
15
c
c
c

10 c

c
a b
c
c
5 c

0
1 3 7 15 30
aP<0.05 Autopsy intervals (Days)
bP<0.01
cP<0.001 Normal Spirulina Mercuric chloride Spirulina+Mercury+Spirulina

Fig. 5. Variation in kidney lipid peroxidation (MDA assay) content in various experimental groups.

Hg/kg/day) in the drinking water for 6 months also to the glomerular basement membrane, followed by the
resulted in degenerative changes in glomerulus. Exposure deposition of immune complexes in the glomerulus (Biga-
of inorganic mercury can lead to the production of anti- zzi, 1988, 1992). The deposited immune complexes induce
bodies against the glomerular basement membrane and binding of complement, attraction of neutrophils and
results in an immunologically mediated membranous phagocytosis may results. Neutrophils and macrophages
glomerular nephritis (Bigazzi, 1992). This glomerular are commonly observed within glomeruli in glomerulone-
nephropathy is characterized by the binding of antibodies phritis and the local release of cytokines and reactive
 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887
oxygen species may contribute to glomerular injury
(Goldstein and Schnellmann, 1995).
The results of present investigation establish that
mercury intoxication causes dose-dependent mortality,
significant decline in alkaline phosphatase and lactate
dehydrogenase activity and significant elevate MDA level
and acid phosphatase activity.
There was a decrease in LDH and alkaline phosphatase
activity observed following the exposure of mercuric chlo-
ride. The enzyme lactate dehydrogenase is an important
enzyme, catalyses the interconversion of lactate and pyru-
vate in the glycolytic pathway and occurs as a tetrameric
molecule. Alkaline phosphatase enzyme is associated
with transfer of phosphates and it is linked with transpor-
tation of intermediate compounds in glycogenesis or
glycogenolysis.
The decrease in LDH and alkaline phosphatase activity
in the present investigation may be due to loss of brush
border of the proximal tubular cells, at an early stage of
renal epithelial cellular necrosis. Further, inorganic mer-
cury also directly inhibits LDH activity (Smith et al.,
1986). The sensitivity of LDH to inhibition by Hg suggests
measurement of inhibition of LDH activity does correlate
well with HgCl2-induced cellular toxicity (Smith et al.,
1986). These findings are in agreement with the findings
of Mehra and Kanwar (1986), Chung and Lee (1987), Lash
and Zalups (1992) and Sharma et al. (2005a).
Further, acid phosphatase activity showed highly signif-
icant elevation after HgCl2 exposure. Similar reports have
been also reported by Mehra and Kanwar (1986) and
Sharma et al. (2005a) following HgCl2 administration.
Acid phosphatase activity is localized in cellular lysosomes.
An enhanced peroxidation of lysosomal membranes due to
HgCl2 intoxication causes lysis of membrane and oozing
out of the enzyme hence results in an increased acid phos-
phatase activity.
One of the most important mechanisms for Hg-induced
oxidative damage is its known sulfhydryl reactivity. Once
absorbed in the cell, both Hg2+ and MeHg form covalent
bonds with GSH and the cysteine residues of proteins. A
single Hg ion can bind to and cause irreversible excretion
of up to two GSH molecules (Zalups and Lash, 1996; Quig,
1998). GSH, the primary intracellular antioxidant and the
conjugating agent, was shown to be depleted and to have
impaired function in Hg toxicity. Releasing the Hg ions
form complexes with GSH and cysteine results in greater
activity of the free Hg ions disturbing GSH metabolism
and damaging cells (Hultberg et al., 2001). We have also
reported that mercury causes significant depletion of
hepatic GSH (Sharma et al., 2002). It also promotes the
formation of reactive oxygen species by Fenton transition
equation, such as hydrogen peroxides and enhances the
subsequent iron and copper-induced production of lipid
peroxides and the highly reactive hydroxyl radical (Miller
et al., 1991; Halliwell and Gutteridge, 1989; Hussain
et al., 1999; Kim and Sharma, 2003; Sener et al., 2003;
Perottoni et al., 2004). Lipid peroxides alter membrane
885
structure and are highly disruptive of mitochondrial struc-
ture. Mercury also inhibits the activities of the free radical
quenching enzymes catalase, superoxide dismutase, and
perhaps the GSH peroxidase (Benov et al., 1990). From
the present investigation, it can be suggested that HgCl2
treatment significantly reduces the GSH content and the
antioxidant potential and thus accelerates the MDA pro-
duction, resulting in renal tubular damage.
It was observed that an extract of Spirulina when given
in combination significantly decline MDA content and
reduces the mercury induced nephrotoxicity which in turn
is reflected by significantly decrease in acid phosphatase
activity and increase in alkaline phosphatase and LDH
activity.
Several active components in S. fusiformis may provokes
the activity of free radical scavenging enzyme systems and
renders protection against mercury induced renal damages.
The metallo-protective role of Spirulina may be attributed
to the presence of b-carotene (Prescott, 1978; Seshadri
et al., 1991), vitamins C and E (Mathew et al., 1995),
enzyme superoxide dismutase (Ben-Amotz, 1997; Henrik-
son, 1989), selenium (Henrikson, 1989) and brilliant blue
polypeptide pigment phycocyanin (Shimamatsu, 1989).
b-Carotene of Spirulina may scavenge free radicals gen-
erated by mercury and reduces the lipid peroxidation. The
antioxidant mechanism of b-carotene has been suggested to
be singlet oxygen quenching, free radical scavenging and
chain breaking during lipid peroxidation (Foote and
Denny, 1968; Foote et al., 1970; Krinsky and Deneke,
1982; Gerster, 1993). Luxia et al. (1996) reported that b-
carotene of Spirulina may reduce cell damage, especially
the damage to DNA molecules, thus playing the role in
the repair of regeneration process of damaged cells.
Vitamin E functions as a trap for lipid peroxyl (LOO)
and other radicals, effectively inhibiting the peroxidation
of cellular membranes. Vitamin E prevents lipid peroxida-
tion and maintains GSH and ascorbic acid levels in dam-
aged tissue by inhibiting free radical formation (Duval
and Poelman, 1994; Kulkarni and Byczkowski, 1994).
According to Rao and Sharma (2001), vitamin E showed
protective effect against HgCl2 may be due to impaired
absorption of mercury in the gastrointestinal tract. Rana
et al. (1996) postulated that vitamin E has a protective
effect against mercury toxicity. Vitamin E inhibits oxidative
damage caused by mercury and cadmium intoxication
(Rana et al., 1996; Patil and Rao, 1999).
Vitamin C is an antioxidant vitamin, which protects the
cells from free radical attack. Upasani et al. (2001) reported
that administration of vitamins C and E and Spirulina
significantly reduces the lead induced MDA conjugates in
liver, lung and kidney of rats.
Selenium present in Spirulina-induced selenium contain-
ing enzyme GSH peroxidase, proteins or compounds such
as selenodiglutathione, selenocysteine and dimethylsele-
nide, which are known to modulate the toxic effects of
heavy metals. El-Demerdash (2001) reported that when
selenium in the form of sodium selenite is given in
886 M.K. Sharma et al. / Food and Chemical Toxicology 45 (2007) 879–887
combined treatment with Hg, it significantly modulates the
toxic effects of mercury on the various enzymatic activities
in rats.
Superoxide dismutase is a mitochondrial enzyme, which
is found to quench free radicals and prevents tissue
damage.
Phycocyanin significantly inhibited peroxyl radical-
induced lipid peroxidation in rat liver microsomes (Bhat
and Madyastha, 2000). Fukino et al. (1990) reported that
phycocyanin content of Spirulina significantly reduced the
renal toxicity in rats caused by para-amino phenol (pain
reliever) and cisplatin (anti-cancer). Thus, phycocyanin
compound of Spirulina may reduce the lipid peroxidation
and reduces the nephrotoxicity in mice.
Spirulina also induces the activity of immune system. It
builds up both the cellular and humoral arms of the
immune systems and thus improving their ability to func-
tion in spite of stresses from environmental toxins and
infectious agents (Hayashi et al., 1994; Qureshi et al., 1995).
5. Conclusion
From the present study it can be concluded that pre- and
post-treatment of Spirulina significantly protect the neph-
rotoxicity induced by mercury intoxication.
Acknowledgements
Financial assistance provided by CSIR, New Delhi to
one of us (M.K.S.) is highly acknowledged. Authors are
also thankful to Recon Limited, Bangalore for providing
Spirulina fusiformis.
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