Journal of Hazardous Materials 164 (2009) 1304–1309

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Journal of Hazardous Materials

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Spirulina platensis feeding inhibited the anemia- and leucopenia-induced lead

and cadmium in rats
Nejdet Simsek a , Ali Karadeniz b,∗ , Yildiray Kalkan c , Osman N. Keles c , Bünyami Unal c
a
University of Atatürk, Faculty of Veterinary Medicine, Department of Histology and Embryology, 25700 Erzurum, Turkey
b
University of Atatürk, Faculty of Veterinary Medicine, Department of Physiology, 25700 Erzurum, Turkey
c
University of Atatürk, Faculty of Medicine, Department of Histology and Embryology, 25240 Erzurum, Turkey

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

Article history: In the present investigation, the effect of Spirulina platensis (Sp) was undertaken on rats fed with lead
Received 23 January 2008 and cadmium including diet by using physiological, enzymehistochemical and stereological methods. For
Received in revised form 27 June 2008 this aim, 50 rats were equally divided into five groups as control (C), lead (Pb), Spirulina + lead (Sp + Pb),
Accepted 12 September 2008
cadmium (Cd), and Spirulina + cadmium (Sp + Cd). Red blood cell (RBC) and white blood cell (WBC) counts,
Available online 20 September 2008
packed cell volume (PCV), and haemoglobine (Hb) concentrations were determined by haemocytomet-
ric methods in blood samples collected on 30th day. Population of T lymphocyte was counted by the
Keywords:
␣-naphthyl acetate esterase (ANAE) staining method, and reticulocytes were counted by stereological
Spirulina platensis
Lead
method. The counts of RBC, WBC, and ANAE positive T lymphocyte, and the values of Hb, PCV, and MCHC
Cadmium were decreased in the Pb and Cd groups compared to control group. Also, the number of reticulocytes
Anemia (polychromatofilic erythrocyte) increased in the Pb groups, whereas it decreased in the Cd group. On the
Leucopenia other hand, these values were ceased by S. platensis in the treated groups. These results suggest that S.
platensis supplementation may be useful in adjuvant treatment of leukemia and anemia caused by lead
and cadmium toxication.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction more, the anemia and eosinophilia associated with Cd toxicity

Environmental contamination by toxic metals is a serious prob-
lem worldwide due to their incremental accumulation in the food
chain and continued persistence in the ecosystem. Lead (Pb) and
cadmium (Cd) are non-essential metals with a wide distribution in
the environment. Intoxication with these metals adversely affects
a wide variety of live organisms. Both Pb and Cd primarily affect
the pulmonary, renal, cardiovascular, central, and peripheral ner-
vous systems [1]. Cadmium can cause cancers in combination with
hydroxyl radicals, superoxide anions, nitric oxide and peroxides in
various organs such as the lung, prostate, pancreas, liver and kid-
ney. The essential mechanisms concerned in carcinogenesis are
the inhibition of DNA repair, oxidative stress, and interference
with apoptosis [2]. This heavy metal has been associated with the
destruction of renal tubule epithelial cells, renal proximal tubular
dysfunction in kidney, osteomalacia with pains in bone [1], hepa-
tocellular vacuolisation, fatty infiltration and fibrosis by increasing
MDA level, and reducing GSH and SOD levels in liver [3]. Further-
∗ Corresponding author. Tel.: +90 442 631 41 93; fax: +90 442 631 41 88.
E-mail addresses: karadenizali@gmail.com, karadenizali@atauni.edu.tr
(A. Karadeniz).
have been largely studied in Itai–itai syndrome [1,4]. The acute
and chronic lead poisoning also causes pathophysiological changes
in tissues, and morphological changes in bone marrow cells, and
necrosis in proximal tubular cells, reduces glomerular filtration rate
and dysfunction in kidney [5], reduces semen quality, decreases
sperm count and motility in the reproductive systems [6]. Lead tox-
icity induces changes in the composition of red blood cell (RBC)
membrane proteins and lipids that inhibit haemoglobine synthe-
sis, insufficient erythrocyte production, and reduce red cell survival
[5].
In the recent years, various researchers have suggested the pro-
tective effects of melatonin, vitamin E + selenium combination [7],
garlic [8], and vitamin C + vitamin E + sodium selenate combination
[9] against cadmium and lead carcinogenicity in liver, kidney, heart,
spleen and blood tissues. Moreover, biotechnology has been inves-
tigated as an alternative method for treating the metal-containing
wastewater and live organisms. For instance, the metal-binding
capacities of several biological materials have been identified to be
very high, including marine algae, fungi and yeasts [10]. Spirulina
platensis, a blue-green alga (Oscillotoreaceae), is rich in proteins,
lipids and carbohydrates, some minerals such as selenium, magne-
sium, manganese and vitamins including alpha tochopherol, alpha
lipoic acid, and riboflavine [11,12]. Spirulina is known for its

0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2008.09.041
 N. Simsek et al. / Journal of Hazardous Materials 164 (2009) 1304–1309
wide-ranging biological activities and antioxidative, anti-
inflammatory, antimutagenic, antiviral, cardioprotective,
anticancer properties [13,14] and immune enhancing [15]. For
example, Zhang et al. [16] stated that Spirulina increased the
haemoglobine concentration, RBC and white blood cell (WBC)
counts, and erythropoiesis during chemotherapy. The aim of
this study was to determine the protective or inhibitor effect
of S. platensis on red blood cell and leucocyte parameters on
leukemia-induced lead and cadmium in rats.
2. Materials and methods
2.1. Animals
Fifty adult female Wistar albino rats (Rattus rattus norvegicus)
(n = 10 × 5) weighing about 200–250 g were used. The animals were
given standard rat pellets and tap water ad libitum. The rats were
housed in individual cages (360 mm × 200 mm × 190 mm), each
containing two animals from 15 days before the start of the exper-
iment. All animals were kept under standard laboratory conditions
(light period 07.00 a.m. to 8.00 p.m., 12 h, at 21 ± 2 ◦ C, relative
humidity 55%), and received humane care according to the criteria
outlined in the “Guide for the Care and Use of Laboratory Animals”
prepared by the National Academy of Sciences and published by the
National Institute of Health [17]. All experiments of this study were
approved by the Center of Experimental Research and Practice in
Atatürk University.
2.2. Experimental design
Rats were divided into five groups [control (C), lead-treated
(Pb), Spirulina + lead-treated (Sp + Pb), cadmium-treated (Cd), and
Spirulina + cadmium-treated (Sp + Cd)], each containing 10 ani-
mals. Control group received normal food and water during the
Fig. 1. Application principle of optical fractionator in blood smears.
1305
experiment. Pb group received normal food and lead acetate
(2 g/L) dissolved in water. Sp + Pb group received normal food
and Spirulina (300 mg/kg) + lead acetate (2 g/L) dissolved in water.
Cd group received normal food and cadmium (50 mg/L) dis-
solved in water. Sp + Cd group received normal food and Spirulina
(300 mg/kg) + cadmium (50 mg/L) dissolved in water. S. platensis, Pb
and Cd were purchased from the Sigma Chemical Co., and herbal
store (SGM, Ankara, Turkey).
2.3. Physiological and enzymehistochemical procedures
In both control and experimental groups, blood samples from
tail veins of the animals were taken into heparinized (100 IU hep-
arin/ml blood) tubes at 30 days of the experimental period. From
each group, 10 blood films were prepared for ANAE staining demon-
stration [18]. Erythrocytes and leucocytes were counted by the
haemocytometric method, the haemoglobine concentration was
determined spectrophotometrically, and packed cell volume was
determined by the microhematocrit tube method [19]. For erythro-
cytes, the mean corpuscular haemoglobine concentration (MCHC),
the mean corpuscular haemoglobine (MCH) and the mean corpus-
cular volume (MCV) were calculated with the following formulas
[19]:
MCV (␮3 ) = PCV (%)/RBC (106 /mm3 )
MCHC (%) = haemoglobine (g/dl)/PCV (%)
MCH (pg) = haemoglobine × 10/RBC (106 /mm3 )
The prepared blood smears were fixed in glutaraldehyde ace-
tone fixative (3 min at −20 ◦ C) for ANAE demonstration, and an
incubation solution constituted by phosphate buffer hexazotized
pararozaniline (pH 5.0, 67 mM) containing the enzyme substrate
(0.025% ␣-naphthyl acetate dissolved in acetone) was prepared
according to the method of Mueller et al. [18]. The incubation
1306 N. Simsek et al. / Journal of Hazardous Materials 164 (2009) 1304–1309
Fig. 2. Unbiased counting frames used for numerical estimation of reticulocyte number by optical disector method (40×). Reticulocytes hitting inclusion lines (arrows) were
estimated.
solution was adjusted to pH 5.8 and sample films were stained for
3 h. The films were then counterstained with 1% methylene green
for 10 min. The ANAE positive lymphocyte rates were determined
by counting 200 U in every smear (40× magnification).
2.4. Stereological procedures
Fifty blood smears were examined in each group by stereological
methods which are based on uniform random systematical sam-
pling process. Total area (500,000,000 ␮m2 ) of each smear samples
was estimated by the Cavalieri method. Also smears were evaluated
by optical fractionator and the reticulocytes in the stained smears
were counted by using unbiased counting frame (see Figs. 1 and 2).
Stereological analyses used a combination composed of a stere-
ology workstation, a color camera (Optronics MicroFire), personal
computer and computer controlled motorized specimen stage (Bio-
Precision MAC 5000 controller system), and a light microscope
(Leica DM4000 B).
2.5. Area estimation with the Cavalieri principle
Area estimation of a structure that has an arbitrary shape and
size may be efficiently obtained by the Cavalieri principle and using
a point-counting grid for the area estimation of section profiles.
The point density of the grid was designed to obtain an appropriate
coefficient of error for the smear samples of the study. The coeffi-
cient of error and coefficient of variation were estimated according
to the formula by Gundersen and Jensen [20].
The test grid with a systematic array of points is randomly placed
on the screen of a PC and an appropriate point per interested area is
carefully calculated for each smear. The area of the smear samples
was estimated with the following formula:
a 
m
A= × Pi
p WBC counts, Hb, PCV and MCHC values significantly decreased in
i=1
where
A is the area of the smear samples, a/p is the inter-point area,
and P is the total number of points hitting whole smear sample.
2.6. Numerical density and total number of reticulocytes
Reticulocytes were counted using a 40× Leica Plan Apo objective
(NA = 1.40) which allowed accurate recognition (see Fig. 2). Total
reticulocyte number was counted according to the unbiased count-
ing rules of optical dissector (see Fig. 1) and to the following formula
[21]:
TM
Nd = × NSS, Nt = At × Nd
CFA
where numerical density: Nd, counting frame area (XY) (␮m2 ): CFA,
number of sampling sites: NSS, total markers: TM, total number: Nt
and total area: At.
2.7. Error predictions for the optical dissector estimation
Coefficient of error (CE) is the last calculated value. The generally
accepted highest limit of CE is 5% [13]. In our study, the coefficient
of error was found to be 0.043.
2.8. Statistical analysis
Mean ± standard error values were calculated for each group to
determine the significance of inter-group differences. Each param-
eter was analysed separately by using one-way analysis of variance
(ANOVA). For determining differences between groups, the Duncan
test was used. All p values of <0.05 were considered to be significant.
3. Results
3.1. Haematological results
Erythrocyte and leucocyte parameters observed in the con-
trol and experimental groups are shown in Table 1. The RBC and
Pb and Cd groups compared to control group (p < 0.05). However,
these parameters were significantly increased in Sp + Pb and Sp + Cd
groups compared to Pb and Cd groups (p < 0.05). In the Pb and
 N. Simsek et al. / Journal of Hazardous Materials 164 (2009) 1304–1309 1307

Table 1
Effects of Spirulina platensis (300 mg kg−1 day−1 ) treatment for 30 days on erythrocyte and leucocyte parameters in female adult Wistar rats (n = 10 in each group). Results
are expressed as mean ± S.E.

Parameters Groups

C Pb Sp + Pb Cd Sp + Cd

RBC (mm3 /106 ) 9.41 ± 0.22 8.33 ± 0.19 9.14 ± 0.09a 8.10 ± 0.02 9.05 ± 0.23b
WBC (mm3 /103 ) 8.74 ± 0.14 8.23 ± 0.46 9.33 ± 0.27a 8.32 ± 0.39 9.22 ± 0.29b
Hb (g/dl) 12.25 ± 0.33 11.81 ± 0.24 12.11 ± 0.27a 11.55 ± 0.47 13.05 ± 0.10b
PCV (%) 41.5 ± 1.5 39 ± 0.5 41 ± 1.5a 38.5 ± 0.5 40.5 ± 0.5b
MCHC (%) 29.52 ± 0.22 28.28 ± 0.48 30.54 ± 0.18a 28.00 ± 0.24 32.22 ± 0.20b
MCH (pg) 13.02 ± 0.15 14.18 ± 0.13 13.25 ± 0.30 14.26 ± 0.10 14.42 ± 0.04
MCV (␮3 ) 44.10 ± 0.68 46.82 ± 0.26 44.86 ± 1.66 47.53 ± 0.11 44.75 ± 0.21
a,b
Statistically important compared to lead (Pb) (a) and cadmium (Cd) (b) groups p < 0.05. Control (C), Spirulina (Sp), red blood cells (RBC), white blood cells (WBC),
haemoglobine concentration (Hb), packed cell volume (PCV), mean corpuscular haemoglobine concentration (MCHC), mean corpuscular haemoglobine (MCH) and mean
corpuscular volume (MCV).

Table 2
ANAE positive lymphocytes counts, reticulocyte numerical density and reticulocyte total number of all groups. Results are expressed as mean ± S.E.

Control Pb Sp + Pb Cd Sp + Cd

ANAE+ lymphocyte (%) 67.02 ± 1.30 55.25 ± 2.34 70.00 ± 4.35a 58.50 ± 5.80 72.50 ± 4.92b
Reticulocyte numerical 49 × 10−5 ± 8 × 10−5 95 × 10−5 ± 4 × 10−5 37 × 10−5 ± 4 × 10−5a 33 × 10−5 ± 4 × 10−5 45 × 10−5 ± 6 × 10−5
density
Reticulocyte total 245,864 ± 20,750 475,339 ± 42,059 184,386 ± 41,144a 166,553 ± 40,011 226,926 ± 28,005
number (in
500,000,000 ␮m2 )
a,b
Statistically important compared to lead (Pb) (a) and cadmium (Cd) (b) groups, p < 0.05.

Cd groups significant changes were seen in peripheral blood that were decreased in the Pb (55.25 ± 2.34%) and Cd (58.50 ± 5.80%)
microcytic erythrocytes and basophilic erythrocytic granulation groups, whereas the counts increased in the Sp + Pb (70.00 ± 4.35%)
were abundant in the Pb group compared to Cd group. Furthermore, and Sp + Cd (72.50 ± 4.92%) groups according to the control group
these cells were seen seldom in Sp + Pb and Sp + Cd groups. (67.02 ± 1.30%) (p < 0.05).

3.2. Stereological and enzymehistochemical results 4. Discussion

Total reticulocyte number and mean numerical density of retic-
ulocyte experimental groups and the control group are shown in
Table 2, Figs. 3 and 4. All our findings were evaluated by comparing
two groups with each other according to the type of experimen-
tal groups (Pb, Sp + Pb, Cd, Sp + Cd). There were approximately
0.00049 reticulocyte/␮m2 in the blood of the control group. Signif-
icant differences were found in reticulocyte numerical density and
reticulocyte number between Pb and Pb + Sp, and between control
and Pb groups (p < 0.05). No significant differences were found in
reticulocyte numerical density and reticulocyte number between
other groups (p < 0.05). T lymphocytes contained 1–5 specific gran-
ules red brown ANAE positive (see Fig. 5a) and B lymphocytes
determined ANAE negative (see Fig. 5b) in all the groups. Gener-
ally, reticulocytes contained 1–2 ANAE positive granules in the Pb-
and Cd-treated groups, and its ANAE activity was lower than T lym-
phocytes (see Fig. 5c). ANAE positive T lymphocyte counts obtained
This study reveals that Spirulina showed a protective effect
against cadmium- and lead-induced alteration in the counts of
T lymphocyte, reticulocyte, RBC, and WBC, the values of MCHC,
Hb, and PCV in rats. In the peripheral blood, T and B lymphocytes
are classified according to their enzymehistochemical properties.
Many researchers distinguished B lymphocytes from T lympho-
cytes by determining non-specific esterase in T lymphocytes in
the classification of lymphocytes [15,22,23]. ANAE marker enzymes
were present in T lymphocytes, monocytes, neutrophils [18,22], and
promyelocyte cells in patients with acute myeloblastic leukemia
[24]. In this study, the presence of ANAE was determined in
T lymphocytes and not in B lymphocytes. Mature erythrocytes
and reticulocytes are negative for ANAE, whereas reticulocytes
ANAE positive reactivity has been demonstrated in leukaemic
myeloblasts [25] and healthy Asian wild dog as typical diffuse,
granular or focal reactions [26]. In our study, reticulocytes were

Fig. 3. Mean numerical density of reticulocytes in all groups ± S.E.M. Fig. 4. Total number of reticulocytes in all groups ± S.E.M.
1308 N. Simsek et al. / Journal of Hazardous Materials 164 (2009) 1304–1309
Fig. 5. ANAE positive lymphocyte (bold arrow), ANAE negative lymphocyte (arrow), ANAE positive granules (arrow head), reticulocytes (asterisk). ANAE stain. X bar: 11 ␮m
(Fig. 5a and b) and X bar: 10 ␮m (Fig. 5c).
determined larger than normal erythrocytes, and they have an
ANAE light positive reaction in Cd- and Pb-treated animals.
Lead and cadmium are heavy metals which have toxic effect
for living beings even in a very low amount, are electrical power
sources and are used frequently in dye, plastic and fertilizer indus-
try [27,28]. Cd and Pb cause damage to the erythrocyte membrane
resulting in hemolysis or a decrease of blood iron level which
may be the cause of decreased concentration of haemoglobine and
hematocrit value [5]. The decrease in ANAE+ lymphocyte, RBC, and
WBC counts, MCHC, Hb and PCV of Cd- and Pb-added groups was
observed at the end of the study (p < 0.05). Chronic oral cadmium
and lead administration cause the development of hypochromic
anemia and hemolytic anemia, respectively [5]. Reticulocytes were
quitely increased in the Pb group compared to control group, but
were decreased in Cd group (p < 0.05). According to the above find-
ing, the increased reticulocyte counts in the Pb-treated group may
be associated with the decrease of RBC production, hematocrit
value and haemoglobine concentration.
Blue-green algae have been reported to improve the effects on
the metabolism of iron and haemoglobine in rats that they absorb
in a high-degree heavy metals such as Pb, Cd, Zn, and Hg [28,29,30].
Nutritional supplementations such as S. platensis can exert a pro-
tective role against Cd- and Pb-induced destruction of RBCs [31]. In
this research, S. platensis administration decreased the toxic effects
of Cd and Pb on the hematological values and has a protective role
in anemia. These results are similar to those obtained in experi-
ments with mammalian blood [32], as well as in experiments on
carps [33]. Previous studies have already reported that S. platensis
may reduce the severity of anemia and the UV-induced damage
of bone marrow, and increase blood haemoglobine concentrations
[34].
There are several scientific studies about Spirulina’s ability
to inhibit viral replication, and strengthen both the cellular and
the humoral defenses of the immune system [35]. S. platensis
enhances the phagocytic activity of bronchoalveolar and abdomi-
nal macrophages [36], and increases the numbers of T lymphocytes
[15], and leucocytes in Spirulina-treated animals [28]. We agree
with these authors, and conclude that the leucocyte counts were
significantly increased in Sp-treated groups (p < 0.05). Cell death
or cellular damage has been reported in lead-intoxicated animals
in spite of the enhancement of iron-dependent lipid peroxidation
and the production of free oxygen radicals [11,37]. Spirulina con-
tained phycocyanin which stimulates hematopoiesis and emulates
the effect of the hormone erythropoetin. Also it regulates the pro-
duction of WBCs [29,34] and enhances the numbers of IgA, IgE, IgM
and IgG antibody production [28]. Already in our previous study we
showed that the dietary S. platensis treatment increased the leuco-
cyte counts and leucocyte subtypes [15]. In this study, ANAE positive
T lymphocyte count was decreased in Cd (58.50 ± 5.80%)- and Pb
(55.25 ± 2.34%)-treated groups, whereas the count was increased in
Sp + Pb (70.00 ± 4.35%)- and Sp + Cd (72.50 ± 4.92%)-treated groups
when compared to control groups (67.02 ± 1.30%). These effects
relate to metal-binding capacities of S. platensis. Also our stereo-
logical data showed that Spirulina increased the density and total
number of reticulocyte in Pb group and it led to a decrease in these
values in Cd group. So we indicated that Spirulina can balance den-
sity and total number of reticulocyte when it was compared to the
control.
Spirulina was sold widely in pharmacies, health food stores,
and mass-market outlets throughout the world and was used
as immunestimulative, antiaging, and loss weight diet. In this
study, it was determined that heavy metals such as Pb and Cd
applied to rats caused severe anemia, leucopenia and increase in
the number of reticulocyte. These effects reached the normal val-
ues by the addition of Spirulina. We expect that Sprirulina will
be contributed to various clinical applications, including Spirulina
supplemental treatment for patients with peripheral blood dys-
function, leukemia, anemia and disorders caused by heavy metals
such as Cd, Pb, Au, and Co intoxications, as well as to biological
investigation efforts.
Acknowledgements
The authors are indebted to two anonymous reviewers for valu-
able suggestions on this manuscript. The authors would like to
express their gratitude to the Center of Experimental Research and
 N. Simsek et al. / Journal of Hazardous Materials 164 (2009) 1304–1309
Practice at the Atatürk University for providing the animals, and for
the approval of the ethical rules.
References
[1] R.M. Ermentrout, M.E. Layon, C.J. Ackley, P. Venkatesan, C.H. Lowrey, The effects
of lead and cadmium on GATA-1 regulated erythroid gene expression, Blood
Cells Mol. Dis. 37 (2006) 164–172.
[2] M. Waisberg, P. Joseph, B. Hale, D. Beyersmann, Molecular and cellular mecha-
nisms of cadmium carcinogenesis, Toxicology 192 (2003) 95–117.
[3] A. Karadeniz, M. Cemek, N. Simsek, The effects of Panax ginseng and Spirulina
platensis on hepatotoxicity induced by cadmium in rats, Ecotoxicol. Environ.
Saf. (April 2008) [Epub ahead of print].
[4] T. Inaba, E. Kobayashi, Y. Suwazono, M. Uetani, M. Oishi, H. Nakagawa, K.
Nogawa, Estimation of cumulative cadmium intake causing Itai–itai disease,
Toxicol. Lett. 159 (2005) 192–201.
[5] M.T. Ancheva, R. Metcheva, S. Teodorova, Bioaccumulation and damaging action
of polymetal industrial dust on laboratory mice Mus musculus alba. II. Genetic,
cell, and metabolic disturbances, Environ. Res. 92 (2003) 152–160.
[6] A. Kasperczyk, S. Kasperczyk, S. Horak, A. Ostałowska, E. Grucka-Mamczar, E.
Romuk, A. Olejek, E. Birkner, Assessment of semen function and lipid perox-
idation among lead exposed men, Toxicol. Appl. Pharmacol. 228 (2008) 378–
384.
[7] H. Kara, A. Cevik, V. Konar, A. Dayangac, K. Servi, Effects of selenium with vita-
min E and melatonin on cadmium-induced oxidative damage in rat liver and
kidneys, Biol. Trace Elem. Res. 125 (2008) 236–244.
[8] A.M. Massadeh, S.A. Al-Safi, I.F. Momani, A.A. Alomary, Q.M. Jaradat, A.S. AlKo-
fahi, Garlic (Allium sativum L.) as a potential antidote for cadmium and lead
intoxication: cadmium and lead distribution and analysis in different mice
organs, Biol. Trace Elem. Res. 120 (2007) 227–234.
[9] M. Koyuturk, R. Yanardag, S. Bolkent, S. Tunali, The potential role of combined
anti-oxidants against cadmium toxicity on liver of rats, Toxicol. Ind. Health 23
(2007) 393–401.
[10] H. Chen, S. Pan, Bioremediation potential of Spirulina: toxicity and biosorption
studies of lead, J. Zhejiang Univ. Sci. 6B (2005) 171–174.
[11] C.D. Upasani, R. Balaraman, Protective effect of Spirulina on lead induced dele-
terious changes in the lipid peroxidation and endogenous antioxidants in rats,
Phytother. Res. 17 (2003) 330–334.
[12] R. Gong, Y. Ding, H. Liu, Q. Chen, Z. Liu, Lead biosorption and desorption by intact
and pretreated Spirulina maxima biomass, Chemosphere 58 (2005) 125–130.
[13] G. Chamorro, M. Salazar, L. Favila, H. Bourges, Pharmacology and toxicology of
Spirulina alga, Rev. Invest. Clin. 48 (1996) 389–399.
[14] I.K. Mohan, M. Khan, J.C. Shobha, M.U. Naidu, A. Prayag, P. Kuppusamy, V.K.
Kutala, Protection against cisplatin-induced nephrotoxicity by Spirulina in rats,
Cancer Chemother. Pharm. 58 (2006) 802–808.
[15] N. Simsek, A. Karadeniz, T. Karaca, Effects of the Spirulina platensis and Panax
ginseng oral supplementation on peripheral blood cells in rats, Rev. Med. Vet.
158 (2007) 483–488.
[16] H.Q. Zhang, A.P. Lin, Y. Sun, Y.M. Deng, Chemo- and radio-protective effects of
polysaccharide of Spirulina platensis on hemopoietic system of mice and dogs,
Acta Pharmocol. Sin. 22 (2001) 1121–1124.
[17] NIH [National Institutes of Health], Guide for the Care and Use of Labora-
tory Animals, 7th ed., National Academy Press, 2101 Constitution Avenue, NW,
Washington, DC 20418, 1996.
1309
[18] J. Mueller, G. Brun Del Re, H. Buerki, H.U. Keller, M.W. Hess, H. Cottier, Nonspe-
cific acid esterase activity: a criterion for differentiation of T and B lymphocytes
in mouse lymph nodes, Eur. J. Immunol. 5 (1975) 270–274.
[19] R.G. Lee, J. Foerster, J. Jukens, F. Paraskevas, J.P. Greer, G.M. Rodgers, Wintrobe’s-
Clinical Hematology, 10th ed., Lippincott Williams & Wilkins, New York, USA,
1998.
[20] H.J. Gundersen, E.B. Jensen, The efficiency of systematic sampling in stereology
and its prediction, J. Microsc. 147 (1987) 229–263.
[21] C.V. Howard, M.G. Reed, Unbiased Stereology: Three Dimensional Measurement
in Microscopy, Bios Scientific Publishers, Oxford, 1998, pp. 1–37.
[22] M. Yörük, R.N. Aşti, N. Kurtdede, Z. Ağaoğlu, H. Altunay, Light and electron
microscopic studies on alpha-naphthyl acetate esterase activity of the periph-
eral blood T-lymphocytes in Van cat, Anat. Histol. Embryol. 27 (1998) 289–292.
[23] E. Ergün, L. Ergün, A. Özen, R.N. Aştı, Determination of alpha naphthyl acetate
esterase activity in the peripheral blood leukocytes of ostrich (Struthio camelus
masaicus), Rev. Med. Vet. 155 (2004) 147–150.
[24] L. Pegoraro, J. Abrahm, R.A. Cooper, A. Levis, B. Lange, P. Meo, G. Rovera,
Differentiation of human leukemias in response to 12-O-tetradecanoylphorbol-
13-acetate in vitro, Blood 55 (1980) 859–862.
[25] C.S. Scott, H.J. Limbert, A.G. Bynoe, B.E. Roberts, Cytochemical and elec-
trophoretic characterisation of alpha naphthyl acetate esterases (ANAE) in acute
myeloblastic leukaemia, Ann. Hematol. 49 (1984) 331–338.
[26] C. Salakij, J. Salakij, J. Rattanakunuprakarn, N. Tengchaisri, W. Tunwattana, S.
Apibal, Morphology and cytochemistry of blood cells from Asian wild dog (Cuon
alpinus), Kasetsat J. (Nat. Sci.). 34 (2000) 518–525.
[27] K.V. Sastry, K.M. Subhadra, In-vivo effects of cadmium on some enzyme activ-
ities in tissues of the freshwater catfish Heteropneustes fossilis, Environ. Res. 36
(1985) 32–45.
[28] G.S. Jensen, D.I. Ginsberg, C. Drapeau, Blue-green algae as an immuno-enhancer
and biomodulator, J. Am. Nutraceutical. Assoc. 3 (2001) 24–30.
[29] T.A. Davis, B. Voleskya, A. Muccib, A review of the biochemistry of heavy metal
biosorption by brown algae, Water Res. 37 (2003) 4311–4330.
[30] K. Chojnacka, A. Chojnacki, H. Górecka, Biosorption of Cr3+ , Cd2+ and Cu2+ ions
by blue-green algae Spirulina sp.: kinetics, equilibrium and the mechanism of
the process, Chemosphere 59 (2005) 75–84.
[31] K. Jeyaprakash, P. Chinnaswamy, Effect of Spirulina and Liv-52 on cadmium
induced toxicity in albino rats, Indian J. Exp. Biol. 43 (2005) 773–781.
[32] M.M. Kostić, B. Ognjanović, S. Dimitrijević, R.V. Žikić, A. Tajn, G.L. Rosić, R.V.
Ivković, Cadmium-induced changes of antioxidant and metabolic status in red
blood cells of rats: in vivo effects, Eur. J. Haematol. 51 (1993) 86–92.
[33] R.V. Žikić, A. Tajn, B.I. Ognjanović, S.Z. Pavlović, Z.S. Saičić, Activities of superox-
ide dismutase and catalase in erythrocytes and transaminases in the plasma of
carps (Cyprinus carpio L.) exposed to cadmium, Physiol. Res. 46 (1997) 391–396.
[34] Z. Cheng-Wu, T. Chao-Tsi, Z.Y. Zhen, The effects of polysaccharide and phyco-
cyanin from Spirulina platensis on peripheral blood and hematopoietic system
of bone marrow in mice, in: Proceeding of the 2nd Asia Pacific Conference on
Algal Biotechnology, National University of Singapore, 1994, p. 58.
[35] K. Hayashi, T. Hayashi, N. Morita, I. Kojima, An extract from Spirulina platensis
is a selective inhibitor of herpes simplex type I penetration into HeLa cells,
Phytother. Res. 7 (1993) 76–80.
[36] M.A. Qureshi, J.D. Garlich, M.T. Kidd, Dietary Spirulina platensis enhances
humoral and cell-mediated immune functions in chickens, Immunopharmacol.
Immunotoxicol. 18 (1996) 465–476.
[37] D. Shastri, M. Kumar, A. Kumar, Modulation of lead toxicity by Spirulina
fusiformis, Phytother. Res. 13 (1999) 258–260.