J Appl Phycol (2010) 22:495–501

DOI 10.1007/s10811-009-9484-z

Phycobiliproteins from Pseudanabaena tenuis rich

in c-phycoerythrin protect against HgCl2-caused
oxidative stress and cellular damage in the kidney
Edgar Cano-Europa & Rocio Ortiz-Butrón & Carlos Angel Gallardo-Casas &
Vanessa Blas-Valdivia & Marisol Pineda-Reynoso & Roxana Olvera-Ramírez &
Margarita Franco-Colin

Received: 3 August 2009 / Revised and accepted: 30 October 2009 / Published online: 21 November 2009
# Springer Science + Business Media B.V. 2009

Abstract Our objective was to study if the phycobilipro-
teins of the cyanobacterium Pseudanabanea tenuis rich in
phycoerythrin protect renal cells against mercury-caused
oxidative stress and cellular damage in the kidney. We used
40 male mice that were assigned into five groups: a control
group that received phosphate buffer (PB) and saline and four
treatment groups which received either PB+HgCl 2 ,
PB+phycobiliproteins, or HgCl2+phycobiliproteins. The
kidneys of the mice were used to determine lipid peroxidation
and quantification of reactive oxygen species, oxidized
glutathione, and peroxidase activities (catalase and glutathi-
one peroxidase) and were also examined histologically. Our
results demonstrated that HgCl2 causes oxidative stress and
cellular damage and that all doses of phycobiliproteins
prevented the increase of oxidative markers and partially
protected against HgCl2-caused cell damage. This is the first
report which applied phycobiliproteins of P. tenuis rich in c-
phycoerythrin, like antioxidants against mercury chloride-
caused oxidative stress and renal damage.
Keywords Environmental health . Pollutants . Mechanism
of action . Mercury . Renal damage . Phycobiliproteins

Introduction
E. Cano-Europa (*) : R. Ortiz-Butrón : V. Blas-Valdivia :
M. Pineda-Reynoso
Mercury (Hg) is one of the heavy metals known to be toxic
Lab. de Neurobiología, Departamento de Fisiología, “Mauricio
Russek Berman” Escuela Nacional de Ciencias Biológicas, I.P.N., for mammals. Mercury has been recognized as a hazardous
Carpio y Plan de Ayala, environmental pollutant, and it is widely used in certain
México, D.F. 11340, México types of batteries and continues to be an essential
e-mail: edgarcanoeuropa@yahoo.com.mx
component of fluorescent light bulbs. The toxicity of
C. A. Gallardo-Casas mercury depends on the chemical form of the mercury
Departamento de Biología, Centro de Estudios Científicos y compounds (World Health Organization 2008). Specifically,
Tecnológicos No. 6 “Miguel Othon de Mendizabal”, inorganic mercury as Hg2+ has a great affinity for the
Av. Jardín s/n, Col del Gas. Del. Atzcapotzalco,
sulfhydryl groups of proteins and low-molecular-weight
México, D. F. 02050, México
thiols such as reduced glutathione (GSH). Mercuric
R. Olvera-Ramírez chloride (HgCl2) is a potent nephrotoxicant that has been
Departamento de Botánica, widely used in animal models for studying acute renal
Escuela Nacional de Ciencias Biológicas, I.P.N.,
failure because it causes oxidative stress and renal damage
Carpio y Plan de Ayala,
México, D.F. 11340, México (Sharma et al. 2007; Sener et al. 2007; Mahboob et al.
2001). It is important to develop an effective therapy to
M. Franco-Colin prevent mercury-caused renal damage.
Lab. de Metabolismo I, Departamento de Fisiología, “Mauricio
Cyanobacteria have been used as antioxidant protectors
Russek Berman” Escuela Nacional de Ciencias Biológicas, I.P.N.,
Carpio y Plan de Ayala, in different toxicological models (Bermenjo et al. 2008;
México, D.F. 11340, México Bermejo-Bescos et al. 2008; Sharma et al. 2007). Because
496
cyanobacteria contain β-carotene, vitamins, ω-3- and ω-6-
polyunsaturated fatty acids, and other phytochemicals and
phycobiliproteins, they are a potent antioxidant (Chamorro
et al. 2002).
Phycobiliproteins are antenna protein pigments involved
in the harvesting of light in cyanobacteria. Two main uses
of phycobiliproteins are as fluorescent markers of cells and
macromolecules in biomedical research and as a natural
colorant in food and cosmetics (Glazer 1985). Pseudana-
baena tenuis possesses three phycobiliproteins: allophyco-
cyanin, phycocyanin, and phycoerythrin. Our work
described here has shown that the phycobiliproteins of P.
tenuis, which are rich in phycoerythrin, are potent anti-
oxidants in a murine model with acute renal failure caused
by intoxication with HgCl2 because they prevent alteration
of the REDOX environment.
The REDOX environment is produced by the balance
between the rate of production of oxidants and their
removal by the antioxidant system. It is well established
that the REDOX environment is principally maintained by
GSH and oxidized glutathione (GSSG). Some workers
estimate the redox state of the system by the ratio GSH2/
GSSG (Schafer and Buettner 2001; Cano-Europa et al.
2008). Oxidative stress occurs when the REDOX environ-
ment changes to a more oxidized state and the oxidants
cannot be removed by the antioxidant system. The
generated oxidants interact chemically with biomolecules,
modifying their function or activating intracellular signals
that cause cellular death (Valko et al. 2007; Halliwell and
Gutteridge 2007).
Methods
Animals
Forty male NIH mice were used. Animals weighed between
25 and 30 g. They were housed in groups of eight in a
Plexiglas cage, with food and water ad libitum, in a room
with constant temperature (21±2°C) and a 12-h light/
12-h dark cycle (lights on at 0800). All experimental
procedures described in this study are in accordance with
the guidelines of the Laws and Codes of Mexico in The
Seventh Title of the Regulations of the General Law of
Health Regarding Health Research and the Mexican
Official Standard NOM-082-ZOO-1999 of the technical
specifications for production, care, and use of laboratory
animals. The animals were randomly assigned into five
groups: (1) a control group that received 100 mM phos-
phate buffer (PB) i.g. and 0.9% saline i.p. (n=8), (2)
PB +5 mg kg−1 HgCl2 i.p. (n=8), (3) PB +100 mg kg−1
phycobiliproteins i.g. (n=8), (4) 50 mg kg−1 phycobilipro-
teins +5 mg kg−1 HgCl2 i.p. (n=8), and (5) 100 mg kg−1
J Appl Phycol (2010) 22:495–501
phycobiliproteins +5 mg kg−1 HgCl2 (n=8). The adminis-
tration of phycobiliproteins or PB was made 30 min before
the injection of saline or mercury chloride, and the process
was repeated for the next 5 days at the same hour (1500).
Every day, the animals were weighed. Five days after
mercury administration, the mice were killed by cervical
dislocation and their kidneys were quickly removed and
weighed. The left kidney was frozen at −70°C until it was
used to determine lipid peroxidation, and the quantification
of reactive oxygen species (ROS), GSSG, catalase, and
glutathione peroxidase activities, as previously described
(Cano-Europa et al. 2008). The right kidneys were put into
Buoin’s fixer to later determine cellular damage by
histology.
Cyanobacteria and culture condition
Pseudanabanea tenuis is a high phycoerythrin producer
and was kindly provided by Dra. Roxana Olvera Ramírez.
It was grown in repeated batches in BG-11 medium at 25±
2°C with constant aeration. The culture was supplied with
constant illumination of 180μmol photons m−2 s−1 pro-
vided by a halogen lamp for 14 h per day.
Obtaining phycobiliproteins
Ten grams of the washed cell mass was solubilized in 10 mL
of 100 mM phosphate buffer, pH 7.4. It was frozen five times
at 0°C during 30 min and then thawed at room temperature.
The resultant slurry was centrifuged twice at 2,500×g for
30 min to remove the cell debris. The phycobiliproteins in
the supernatant were spectrophotometrically characterized as
described (Bermejo Roman et al. 2002). Pseudoanabaena
tenuis water extract contained 2% phycocyanin, 9% allo-
phycocyanin, and 89% phycoerythrin.
Histological study
The kidneys were fixed for 12 h and then were embedded in
paraffin. Coronal cuts of 7 μm were obtained with a standard
microtome. Each section was stained with hematoxylin–eosin,
dehydrated, and mounted with resin. The microscopic
description of the section was made by a person blind
to the experiment who checked almost 20 sections for
each animal. The presence or absence of renal damage
was determined. The severity of renal damage was
recorded using a graded scale (0–4) as previously
described (Sharma et al. 2007) as follows: 0 indistinguishable
from controls, 1 = minimal, ≤25% tubules and glomeruli
affected, 2 = mild, >25% and ≤50% tubules and glomeruli
affected, 3 = moderate, >50% and ≤75% tubules and
glomeruli affected, and 4 = marked, >75% tubules and
glomeruli affected.
J Appl Phycol (2010) 22:495–501
Biochemical techniques
All kidneys were homogenized in 3 mL of 10 mM phosphate
buffer, pH 7.4, to be used for all biochemical tests.
Lipid peroxidation assay
Lipid peroxidation (LP) was evaluated by the formation of
a lipid-soluble fluorescence, as described (Cano-Europa et
al. 2008) with some modifications. Briefly, 1-mL aliquots
were added to 7 mL of chloroform/methanol (2:1, v/v).
After stirring for 15 s, the mixture was cooled in ice for
30 min to allow phase separation. The chloroform phase was
measured in a Perkin-Elmer LS55 luminescence spectropho-
tometer at 370 nm (excitation) and 430 nm (emission)
wavelengths. The sensitivity of the spectrophotometer was
adjusted to 140 fluorescence units with 1 μg mL−1 of quinine
sulfate in 0.05 M H2SO4. The results were expressed as
relative fluorescence units per milligram of protein.
Quantification of reactive oxygen species
ROS were measured by the formation of 2′7′-dichloro-
fluorescein (DCF) as we previously described (Cano-Europa
et al. 2008). Five microliters of the homogenates was added
to 1945 μL of Tris-HEPES (18:1) and incubated in the
presence of 50 μL of 2′7′-dichlorofluorescin diacetate for 1 h
at 37°C. The reaction was stopped by freezing. The
fluorescence was measured in a Perkin-Elmer LS55 lumi-
nescence spectrophotometer at 488 nm (excitation) and
525 nm (emission) wavelengths. The results were expressed
as pmoles DCF mg−1 protein h−1.
Quantification of GSSG
GSSG was quantified as previously described (Cano-Europa
et al. 2008). Briefly, 300-μL aliquots were treated with 30%
phosphoric acid and centrifuged at 10, 000×g for 30 min
at 4°C. To measure the GSSG, we took 130 μL of
supernatant and combined that with 60 μL of N-
ethylmaleimide. After 30 min, 60 μL of the mixture was
mixed into 1.84 mL of FEDTA, and 100 μL of α-
phthaldialdehyde was added. GSSG were measured in a
Perkin-Elmer LS55 luminescence spectrophotometer at
350 nm (excitation) and 420 nm (emission) wave-
lengths. The results were expressed as nmoles GSSG
mg−1 protein h−1.
GPX activity
Glutathione peroxidase (GPX) activity was determined by
a method based on the enzymatic use of reduced
glutathione (Hafeman et al. 1974) as modified. Sixty
497
microliters of the cell homogenate was added to 2 mL
100 mM of phosphate buffer containing 0.4 mM EDTA,
10 mM sodium azide (NaN3), and 2 mM GSH. The
reaction mixture was incubated for 5 min at 37°C, then
1 mL of 1.25 mM H2O2 was added. Two samples were
taken at 5 and 10 min and were added to 1.2 mL 1.6%
metaphosphoric acid. This was then diluted 1:2 with
400 mM phosphate buffer. Finally, 250 μL of 5.5-dinitro-
bis-2-nitrobenzoic acid was added and the absorbance
recorded at 412 nm. Results are expressed as μg GSH
used mg−1 protein h−1.
Catalase activity
Catalase activity was measured by monitoring the enzyme
catalyzed decomposition of H2O2 (Aebi 1984). Briefly,
5 μL of cell extract was added to 3 mL of 100 mM
phosphate buffer, pH 7.4, containing 30 mM of H2O2. The
absorbance was recorded at 240 nm after 10 min at 37°C.
The decomposition of H2O2 by catalase present in the
samples follows first-order kinetics according to the
equation k=2.3t log Ao/A, where k is the first-order reaction
rate constant, t is the time over which the decrease of H2O2
caused by catalase activity was measured (15 s), and Ao/A is
the ratio of optical densities at times 0 and 15 s. Catalase
activity is expressed as kg mg−1 protein.
Statistical analysis
The scores of the kidney damage were given as the median ±
percentile intervals. The relative weights of the kidneys,
LP, ROS quantification, GSSG content, GPX, and
catalase activities are given as the mean ± SE. The
scores of kidney damage and the relative weights of the
kidneys were analyzed using a one-way analysis of
variance by the Kruskal–Wallis method. The other
variables were analyzed using a one-way analysis of
variance and a post hoc Dunnett test. A value of P<0.05
was considered statistically significant.
Results
The groups treated with HgCl2 had a mortality of 25%. The
group with 50 mg kg−1 phycobiliproteins had a mortality of
25%, and the group treated with 100 mg kg−1 phycobili-
proteins had no deaths. There were no deaths in the control
group or of the animals treated with 100 mg kg−1 phyco-
biliproteins only (Fig. 1).
Figure 2 shows characteristic photomicrographs of all
treatments. The mercury-caused cellular damage, edema,
cellular atrophy of distal and proximal tubules, distortion of
cellular continuity, nucleus loss, and hyperchromatic nuclei
498 J Appl Phycol (2010) 22:495–501

0.140
were seen. The severity of cellular damage was quantified
employing the relative kidney weight and the damage grade
0.105 scale. Figure 3a shows that only animals treated with HgCl2
Absorbance

had an increased relative kidney weight (F=5.27, P<0.005).

0.070 Figure 3b shows that animals treated with HgCl2 had a grade
4 (marked damage), which showed at least >75% of
glomerulus and tubulus affected (F=23.19, P<0.0001).
0.035
The damage is prevented by the treatment containing
100 mg kg−1 of phycobiliproteins because the histological
0.000 study shows only a minimal or mild damage.
Figure 4 shows the lipid peroxidation (panel a), reactive
360 410 460 510 560 610 660 710 oxygen species (panel b), and GSSG content (panel C). The
Wavelength (nm) animals treated with HgCl2 had oxidative stress because
of enhanced lipid peroxidation (F=23.47, P<0.0001),
Fig. 1 Absorption spectra of phycobiliproteins, from P. tenuis, rich in
c-phycoerythrin ROS (F=14.94, P<0.0001), and GSSG (F=3.12, P<0.05).
The two phycobiliprotein doses did protect against HgCl2-
caused oxidative stress.

a b

c d

Fig. 2 Photomicrographs of renal cortex. a Control group. b Group chromatic nuclei, and edema (arrows in b). Histological alterations were
treated with HgCl2. c Phycobiliproteins 50 mg kg−1 + HgCl2. d partially ameliorated in groups treated with phycobiliproteins (arrows in
Phycobiliproteins 100 mg kg−1 plus HgCl2. The tissue was stained by c and d)
hematoxylin–eosin. Treatment with HgCl2 causes cell atrophy, hyper-
J Appl Phycol (2010) 22:495–501 499

a a
3
* 0.25

*
Kidney relative weight (%)

Lipid peroxidation
0.20

(RFU/mg protein)
2 0.15

0.10
1
0.05

0.00
0

kg

kg
ro

kg

kg
g/

g/
t

g/

g/
on
ol

kg

kg

m
kg

kg

m
C
tr

0
g/

g/

50

0
g/

g/
on

10

10
m

l2
m

gC
C

s
5

50

in
H
10

10
l2

te
gC

ro
in
H

p
te

ili
Phycobiliproteins + HgCl2 5 mg/kg
ro

ob
p

yc
Phycobiliproteins + HgCl2 5 mg/kg
ili

Ph
ob
yc
Ph

(pmol of DCF formed/mg protein/h)

b ROS quatification
60
*
4 * * 50

40
Score of kidney damage

3 30

20
2
10

0
1
l

kg

kg
ro

kg

kg
g/

g/
t

g/

g/
on

m
C

0
5

50

0
10

10
l2
gC

s
in
H

te
pro
ili
ol

kg

kg

kg

kg

Phycobiliproteins + HgCl2 5 mg/kg

ob
tr

g/

g/

g/

g/
on

yc
m

Ph
C

50

0
10

10
l2
gC

c
in
H

te
ro

*
i lip

12.5
ob

Phycobiliproteins+ HgCl2 5 mg/kg

(pmoles of GSSG/mg protein)
yc
Ph

10.0
Fig. 3 Quantification of relative kidney weight (a) and the score of
GSSG content

kidney damage (b) of mice treated with HgCl2. In a, each bar 7.5
represents the mean ± SEM. In b, each box represents the median ±
intercuartilic space. *P<0.05 vs. control 5.0

Figure 5 shows the GPX (panel A) and catalase (panel 2.5

B) activities. It was shown that HgCl2 caused a reduction of

0.0
GPX activity (F=3.06, P<0.05), whereas the phycobilipro-
teins increase catalase activity (F=7.62, P<0.005) in all
l

kg

kg
ro

kg

kg

treatments.
g/

g/
t

g/

g/
on

m
m
C

50

0
10

10
l2
gC

s
in
H

te

Fig. 4 Quantification of lipid peroxidation (a), reactive oxygen„

p ro
ili

Phycobiliproteins + HgCl2 5 mg/kg

ob

species (b), and oxidized glutathione in the kidneys of mice treated

yc

with HgCl2. Bar represents the mean ± SEM. *P<0.05 vs. control
Ph
500 J Appl Phycol (2010) 22:495–501

a We found that Hg2+ caused oxidative stress because it

increases lipid peroxidation and the ROS and GSSG
(µg GSH used/min/mg protein)
45
content. This is in accordance with other studies (Su et al.
2008; Zalups and Lash 1990; (Fouda et al. 2008; Sener et

* al. 2007). Chemically, at the physiological pH, GSH is

GPX activity

30
stabilized in the enolic form where the –C=N– bond
* increases the coordinating power of nitrogen and the GSH
could bind to Hg2+. Thus, the mercuric conjugates change
15
the REDOX environment, causing a more oxidizing
environment. The oxidizing environment promotes oxida-
0 tive reactions between ROS or reactive nitrogen species and
biomolecules as lipids, carbohydrates, or nucleic acids
(Halliwell and Gutteridge 2007).
ol

kg

kg

kg

kg
tr

g/

g/

The oxidized state is increased because Hg2+ interferes

g/

g/
on

m
C

50

0
10

10
l2

with the mitochondrial respiratory chain increasing H2O2

gC

ns
H

ei

production (Lund et al. 1993). Also, mercury-caused Ca2+

ot
pr
ili

Phycobiliproteins + HgCl2 5 mg/kg

ob

release from the mitochondria collapses the membrane

yc
Ph

potential, causing cellular death (Chavez and Holguin

1988). In addition, the decrease of catalase and GPX
b activities increases the H2O2-produced Fenton-like reaction.
0.8
* * This generates oxidants causing cell damage (Stohs and
Bagchi 1995; Valko et al. 2006).
*
0.7
Cyanobacterial phycobiliproteins were used as anti
Catalase activity

0.6
(k/mg proteins)

oxidants. Specifically, it was demonstrated that c-

0.5
phycocyanin (Bermejo-Bescos et al. 2008; Roy et al.
0.4
2007; Sathyasaikumar et al. 2007) and R-phycoerythrin
0.3 (Soni et al. 2008, 2009) protect against toxicant-caused
0.2 oxidative stress and cellular damage.
0.1 The phycobiliproteins of P. tenuis, rich in c-
0.0 phycoerythrin, decreased mercury chloride-caused oxida-
tive stress and cellular damage. They can possibly reduce
the oxidative stress by three different mechanisms. The first
l

kg

kg
ro

kg

kg
g/

g/
t

g/
g/
on

involves the reduction of oxidative markers because the

m

m
C

50

0
10

10
l2

phycobiliprotein chemical structures of tetrapyrroles are

gC

ns
H

ei

nucleophilic and can neutralize the reactive species derived

t
p ro
ili

Phycobiliproteins + HgCl2 5 mg/kg from oxygen and nitrogen, avoiding oxidative biomolecule
ob
yc

damage. This hypothesis was demonstrated both in vitro

Ph

(Bermejo-Bescos et al. 2008) and in vivo (Sathyasaikumar

Fig. 5 Evaluation of GPX (a) and catalase (b) activities in the
kidneys of mice treated with HgCl2. Bar represents the mean ± SEM.
*P<0.05 vs. control
Discussion
Our results demonstrated that phycobiliproteins maintain
the renal cytoarchitecture against mercury chloride-caused
oxidative stress and nephrotoxicity. Also, they increase the
catalase activity. Mercury as Hg2+ is accumulated primarily
in the cortex and outer strip of the medulla (Friberg et al.
1957). It is well established that mercury in the +2
oxidation state causes oxidative stress because it has a
great affinity for intracellular sulfhydryl-containing proteins
and compounds of low molecular weight (Zalups 2000).
et al. 2007; Roy et al. 2007).
The second protecting mechanism of phycobiliproteins
is their chelating properties. We propose that phycobilipro-
teins probably chelate Hg2+ because these pigments from
other cyanobacteria are used to concentrate heavy metals
(Vannela and Verma 2006) and to chelate ferric ions
(Bermenjo et al. 2008).
The third mechanism involves the increase of catalase
activity, which reduces the H2O2 concentration in cells and
avoids a mercury-generated Fenton-like reaction. From our
present results, we suggest that phycobiliproteins act like
the catalase and mimics its action because of their linear
tetrapyrroles.
In conclusion, phycobiliproteins are good antioxidants
that prevent oxidative stress because of their nucleophilic
J Appl Phycol (2010) 22:495–501
ability to neutralize oxidants, protecting against mercury-
caused oxidative stress and damage to the kidney.
Acknowledgment This study was partially supported by SIP-IPN
20080113, 2009485. O-B, R. O-R, R. are fellows of EDI and COFFA.
C-E, E. and L-G, G. E. are fellows of CONACyT. B-V, V. and G-C, C.
A. are fellows of CONACyT and PIFI-COFFA-IPN. P-R, M. is fellow
of PIFI-COFFA-IPN. Thanks to Dr. Ellis Glazier for editing this
English language text.
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