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Dietary Spirulina platensis alleviates
aluminum and aluminum fluoride
induced histopathological and
biochemical alterations in mice kidney
a
a
b
b
Nirmala Yadav , Anil Pandey , Shweta Sharma , Subhasini Sharma
a
& K.P. Sharma
a
Department of Botany, University of Rajasthan, Jaipur, India
b
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Department of Zoology, University of Rajasthan, Jaipur, India
Accepted author version posted online: 28 Jan 2015.
To cite this article: Nirmala Yadav, Anil Pandey, Shweta Sharma, Subhasini Sharma & K.P.
Sharma (2014) Dietary Spirulina platensis alleviates aluminum and aluminum fluoride induced
histopathological and biochemical alterations in mice kidney, Toxicological & Environmental
Chemistry, 96:7, 1106-1119, DOI: 10.1080/02772248.2015.1007987
To link to this article: http://dx.doi.org/10.1080/02772248.2015.1007987
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Toxicological & Environmental Chemistry, 2015
Vol. 96, No. 7, 1106 1119, http://dx.doi.org/10.1080/02772248.2015.1007987
Dietary Spirulina platensis alleviates aluminum and aluminum fluoride
induced histopathological and biochemical alterations in mice kidney
Nirmala Yadava, Anil Pandeya, Shweta Sharmab*, Subhasini Sharmab and K.P. Sharmaa
a
Department of Botany, University of Rajasthan, Jaipur, India; bDepartment of Zoology, University
of Rajasthan, Jaipur, India
Downloaded by [University of Rajasthan ] at 02:02 05 April 2015
(Received 14 May 2014; accepted 8 January 2015)
Aluminum and its salts widely used in our daily life have been reported nephrotoxic to
humans and animals following prolonged exposure. Therefore, the present study was
made to examine the renoprotective role of Spirulina platensis against Al3C and AlF3
in male Swiss albino mice. Exposure to these chemicals decreased feed and water
intake, and body and kidney weights. Histology of kidney and their biochemistry were
also markedly altered along with that of serum biochemistry. Spirulina not only
minimize toxic effects of test chemicals but also favored faster recovery of treated
mice after their withdrawal.
Keywords: aluminum and aluminum fluoride; sub-acute and sub-chronic toxicity;
male Swiss albino mice; kidney; histopathology; spirulina
1. Introduction
Aluminum (Al3C) and its salts are widely used in food additives, toothpaste, medicine,
cosmetics, and in the preparation of cooking utensils and containers from which it may
leach into foods, particularly those which are salty, acidic, or alkaline. It is also present in
corn, yellow cheese, or tea and is added to drinking water for purification (Al-Hashem
2009). Because of its greater affinity for fluoride, aluminum salts are used for defluoridation and contribute residual aluminum to the treated water (Gupta 1997). Aluminum fluoride complexes are also formed spontaneously in aqueous solution containing fluoride
and traces of aluminum ions (Strunecka and Patocka 2002). Defluoridation reduces
fluoride bioavailability;however, it increases exposure of humans to aluminum.
The kidney eliminates aluminum efficiently from the body but also accumulates the
metal to critical levels after long-term exposure (Kloppel, Fliedner, and Kordel 1997;
Graczyk et al. 2000; Sushma et al. 2011). AlF3 exposure may affect the activities of phosphatases, phosphorylases, and kinases (Bigay et al. 1987; Chabre 1990).
Spirulina platensis is a unicellular cynobacterium with high nutritional and medicinal
values. Its chlorophyll acts as a cleansing and detoxifying phytonutrient against toxic substances (Henrikson 1994), and its C-phycocyanin exhibits anti-inflammatory, neuroprotective, hepatoprotective, immunomodulatory, and anticancerous activities (Reddy et al.
2000; Khan, Bhadouria, and Bisen 2005). Spirulina ameliorates toxic effects of some metals (Jeyprakash and Chinnaswamy 2005; Kuhad et al. 2006; Simsek et al. 2009;
Karadeniz, Cemek, and Simsek 2009; Ponce-Canchihuaman et al. 2010; Paniagua-Castro
et al. 2011; El-Desoky et al. 2013) and hematological disorders due to fluoride, aluminum, and aluminum fluoride (Jain et al. 2012; Sharma et al. 2012). Since spirulina
*Corresponding author. Email: [email protected]
Ó 2015 Taylor & Francis
Toxicological & Environmental Chemistry
1107
protects against various toxicants, the aim of the present study was to explore its renoprotective role in mice exposed to aluminum and aluminum fluoride.
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2. Materials and methods
2.1. Experimental animals
Swiss albino mice (Mus musculus L.) were reared in a well-ventilated animal house under
controlled laboratory conditions of normal light dark cycle (12-h light:12-h dark), temperature (24 § 3 C), and humidity (40% 60%). All regulations of the Institutional Animal
Ethical Committee of the University (1678/GO/a/12/CPCSEA) were strictly followed.
Healthy young male mice (age D 30 40 days, weight D 12 15 g) acclimatized for a
week prior to entry into the experimental protocol were allotted randomly to six groups
of 30 mice each, housing 5 mice each in polypropylene cages (50 cm (length) £ 25 cm
(width) £ 15 cm (height)). Food and water (pH D 7.1; electrical resistance D 0.55
MV cm¡1; total hardness D 198 mg L¡1; chlorides D 30 mg L¡1; fluoride D 0.9 mg L¡1;
aluminum D nil) were provided ad libitum.
Groups 1, 3, and 5 were fed with standard laboratory diet (Ashirwad Ltd., Chandigarh,
India) throughout the study period, whereas groups 2, 4, and 6 were fed with spirulina (at
230 mg kg¡1 body weight) along with the standard diet 45 days prior to entry into the
experimental protocol. These groups are referred to as standard feed and spirulina groups,
respectively (Table 1).
Mice in each group were divided in to three subgroups, viz. control, aluminum, and
aluminum fluoride treatments.
Based on reported LD50 values of test chemicals (Al2 (SO4)3.16H2O (Merck Ltd. Mumbai, India) and AlF3 (Hi Media Laboratories Pvt. Ltd. Mumbai, India), their subacute and
subchronic doses were decided (Ondreicka, Ginter, and Kortus 1966; www.lucasmilhaupt.
com (accessed on 4 January 2009)). The test chemicals dissolved in the distilled water
were fed orally subacute (Al3C D 78.4 mg and AlF3 D 103 mg kg¡1 body weight per day)
and subchronic (Al3C D 7.8 mg and AlF3 D 21 mg kg¡1 body weight per day) doses
through gavage (0.5 mL per mouse per day) (Table 1). Because AlF3 forms a suspension
in distilled water, care was taken to ensure complete delivery of its calculated dose. The
suspension (3.5 mL) of salt was prepared in marked vials (1 10), and 0.5 mL of this was
administered to mice having the corresponding marking (1 10). Similar practice was followed during the subchronic exposure. Control mice received an equivalent volume of 0.5
mL distilled water for the exposure period. After termination of Al exposure, the mice of
groups 5 and 6 were allowed to recover under standard conditions; they are referred to as
post-treated mice.
A suspension of 500 mg spray-dried powder of S. platensis (Sunova capsule, Dabur
Ltd., India) in 30 mL distilled water was administered daily through gavage at 0.5 mL per
mouse.
Feed and water intake of mice were recorded every 24 h, while body weight at the termination of the study. Animals were sacrificed by cervical dislocation. Kidneys were
removed, cleaned, blotted free of blood, and fresh weights were recorded.
2.2. Serum biochemical estimation
A midline abdominal incision was performed and blood was collected through cardiac
puncture into marked vials. Serum was separated by centrifugation (at 5000 rpm) for 20
1108
N. Yadav et al.
Table 1. Detailed layout of the experiment.
Groups
Group 1 (subacute treatments)
Diet: standard feed
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Group 2 (subacute treatments)
Diet: standard feed C spirulina at
(230 mg kg¡1 body weight per day)
Group 3 (subchronic treatments)
Diet: standard feed
Group 4 (subchronic treatments)
Diet: standard feed C spirulina at
(230 mg kg¡1 body weight per day)
Group 5 (post-treatments)
Diet: standard feed
Group 6 (post-treatments)
Diet: standard feed C spirulina at
(230 mg kg¡1 body weight per day)
Treatments
Dose mg kg¡1
body weight per day
No. of
mice
Autopsy
AlC3
AlF3
Control
78.4
103
Vehicle
10
10
10
8th day
8th day
8th day
AlC3
AlF3
Control
78.4
103
Vehicle
10
10
10
8th day
8th day
8th day
AlC3
AlF3
Control
7.8
21
Vehicle
10
10
10
91st day
91st day
91st day
AlC3
AlF3
Control
7.8
21
Vehicle
10
10
10
91st day
91st day
91st day
AlC3
AlF3
Control
Withdrawal
Withdrawal
Withdrawal
10
10
10
181st day
181st day
181st day
AlC3
AlF3
Control
Withdrawal
Withdrawal
Withdrawal
10
10
10
181st day
181st day
181st day
min (Bain et al. 2012) and analyzed for acid phosphatase (ACP) and alkaline phosphatase
(ALP), total protein, urea, and creatinine (Varley 1969).
2.3. Tissue biochemistry
Kidney tissue was homogenized (G 20 Tissue Homogenizer, BR Biochem Company,
New Delhi, India) in ice cold 50 mmol L¡1 phosphate buffer for determination of protein
(Lowry, Rosebrough, and Ferry 1951) and ACP and ALP (Sadasivam and Manickan
1996).
2.4. Histological and morphometric studies
The kidney tissues fixed immediately in Bouin’s fluid were processed following standard
procedure for sections (6 mm thick), which were stained with hematoxylin eosin and
examined under light microscope (Humason 1972). Morphometric studies included measurements of Bowman’s capsule, glomeruli, Bowman’s space, and area of proximal and
distal convoluted tubules using oculometer standardized with stage micrometer. Further,
cell counting in tubules and of podocytes in glomerular tuft was also made.
Toxicological & Environmental Chemistry
1109
2.5 Data analysis
Results are expressed as mean § SEM. One-way ANOVA (SYSTAT computer program
version 5.0) was applied to find the significance difference between values of various
parameters recorded for control and treatments. Duncan’s multiple range test (http://shiny.
stat.tamu.edu:3838 (accessed on 30 September 2014)) was used to differentiate between
means (to determine differences between means of treatments at significance rates of 0.05).
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3. Results and discussion
3.1. General health
Visible toxicity symptoms observed only in treatments of standard feed groups were dullness and blackening of nails, and also tail and dyspnea in aluminum treatments. Further,
there was significant reduction in feed and water intake, and body and kidney weights in
treatments of standard feed groups in comparison to controls (Table 2). Although feed
intake and body and kidney weights in treatments of Spirulina groups were higher than
standard feed treatments, their water intake decreased. Aluminum exposure has been
reported to reduce the body weight of test animals (Kowalczyk, Kopff, and Kedzioraz
2004; Yousef, El-Morsy, and Hassan 2005; Sinha, Sharma, and Johri 2011) that improved
when their diet was supplemented with vitamins (Yousef, El-Morsy, and Hassan 2005),
garlic, and triphala (Sinha, Sharma, and Johri 2011).
3.2. Histopathological observations
3.2.1. Gross abnormalities of cortical region
Bowman’s capsule and tubules were intact and compactly arranged in the cortical region
of controls, particularly of spirulina groups (Figures 1 and 2). Histopathological lesions
in the intertubular region of treated mice were tissue necrosis, fibrosis (also calcification
in AlF3 treatment, Figure 1(H)), lysigenous cavities, mononuclear cell infiltration
(Figure 1(C)), and pyelonephritis (Figure 1(B)). Cellular infiltration and fibrosis are common characteristics in the cortex of virtually all progressive renal diseases with proteinuria (Eddy 2004; Karaoz et al. 2004).
3.2.2. Bowman’s capsule
Thickened parietal layer of Bowman’s capsules was fused with glomeruli in treatments of
standard feed groups (Figure 1(D) and 1(G)). Al-Kahtani (2010) also reported adhesion
of parietal layer with visceral in few Bowman’s capsules of AlCl3-treated Swiss albino
mice. The dilated capsules (" 10%–36%) recovered after the withdrawal of chemicals
(Table 3).
3.2.3. Glomerular alterations
The extensive lysis of capillaries in glomeruli led to their sclerotization in treatments of
standard feed groups (Figure 1(C), 1(F), and 1(G)), which did not revert after the withdrawal of chemicals (Figure 1(J) and 1(K)). Often these sclerotic regions were adherent
to and disrupt Bowman’s capsule (Figure 1(D) and 1(G)). Such sclerotic regions may provide an additional site for direct leakage of the glomerular ultrafiltration into the peritubular space, a misdirectional ultrafiltration as described by Kriz et al. (2001). Chemical
1110
N. Yadav et al.
Table 2. Feed intake (g per animal per day), water intake (mL per animal per day), body weight (g
per animal), and kidney weight (mg per animal) of controls, AlC3, and AlF3 treated and post-treatment mice of standard feed and spirulina groups.
Experimental groups
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Parameters
Control
S-Control
AlC3
S C AlC3
AlF3
S C AlF3
Feed intake (g per animal per day)
Subacute
4.8 § 0.8
4.9 § 0.3
Subchronic
6 § 0.3
5.6 § 0.3
Post-treatments
7 § 0.6 a 5.9 § 0.6 ab
3.4 § 0.4
3.8 § 0.7
5.8 § 0.3
5.9 § 0.3
4.9 § 0.4bc 3.4 § 0.4 d
3.5 § 0.4
5.3 § 0.1
4.4 § 0.3 cd
5.2 § 0.6
5.7 § 0.1
4.6 § 0.2 bd
Water intake (mL per animal per day)
Subacute
8 § 0.4 a 5.8 § 0.2 b
Subchronic
6.7 § 0.7
5.8 § 0.4
Post-treatments 8.5 § 0.7 a
6 § 0.3 bc
4.2 § 0.8c
6.5 § 0.4
7 § 0.5b
6.1 § 0.4 b
6.7 § 0.2
5.9 § 0.2 bc
3.2 § 0.2 c
7.5 § 0.1
4.8 § 0.2 cd
3.5 § 0.2 c
6.4 § 0.3
4.2 § 0.6 d
Body weight (g per animal)
Subacute
30.2 § 0.5 a 32.8 § 0.5 a 25.6 § 1.2b
30 § 1.7 a
23 § 1.4 b
a
a
a
a
Subchronic
34.8 § 1.9 32.4 § 0.9 32.8 § 1.6 35.2 § 1.6 26.8 § 1.5 b
Post-treatments 34.8 § 0.9 ab 32.3 § 0.7 ab 38.5 § 2.1a 28.7 § 2.2 b 38.7 § 2.9 a
29.6 § 1.4 a
33.4 § 1.5 a
34.7 § 2.9 ab
Kidney weight (mg per animal)
Subacute
183 § 8.9 a 195 § 7.6 a 155 § 9.8b 200 § 6.6 a 183 § 14.2 ab 194 § 9.1 a
Subchronic
253 § 8.6 ab 271 § 14.4 a 264 § 5.3ab 244 § 9.2 b 210 § 6.6 c
239 § 2.7 b
a
ab
b
a
b
Post-treatments 311 § 7.03 273 § 8.6
253 § 7.7 308 § 28.4 240 § 13
248 § 13.2 b
Notes: Data are means § SEM (n D 6) in a row followed by the same superscript letter are not significantly
different (p < 0.05) from each other according to Duncan’s multiple range test.
exposure also affected glomeruli size that reverted in only AlF3 post-treatment (Table 3).
Both fused and necrosed podocytes were present, particularly in treatments of standard
feed groups (Figure 1). The reduction in podocyte counts (about 50%) in 10% 30% glomeruli (subchronic treatments) suggests progressive renal disease which may exacerbate
the development of proteinuria (Nguyen 2006), whereas proliferation of mesangial cells,
especially in aluminum treatments, may be in response to clean dead cells to restore the
normal functioning of glomeruli (Yadav 2014).
3.2.4. Bowman’s space
Bowman’s space increased significantly (35% 106%) in treatments of standard feed
groups in comparison to controls (Table 3). Because of lesser dilation in treatments of spirulina groups, it decreased (Al3C D 8% 48%; AlF3 D 7%) in comparison to standard
feed treatments.
3.2.5. Tubular alterations
Chemical exposure increased the lumen size (22% 89%) of both proximal and distal
tubules (PCT and DCT) in treatments of standard feed groups in comparison to controls
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Toxicological & Environmental Chemistry
1111
Figure 1. Microphotographs (10 £ 40x) of standard feed groups.
(Table 4). Because of lower dilation of tubules in treatments of spirulina groups, their
lumen size decreased (9% 35%) in comparison to treatments of standard feed groups.
Chemical exposure also induced erosion of epithelial lining of tubules, and aberrations in
their nuclei such as condensation (pyknotic nuclei) and margination of chromatin material
(Figure 1(B), 1(C), 1(D), 1(F), 1(G), and 1(H)). Such stages of karyolysis were uncommon in treatments of spirulina groups (Figure 2(P)). The toxic effects of chemicals diminished after the withdrawal, but at a faster pace in the spirulina groups.
Epithelial cell counts decreased (19% 42%) in PCT and DCT of treatments of standard feed groups in comparison to controls (Table 4). Because of lower reduction in treatments of spirulina groups, their counts were higher (" 14%–57%) in comparison to
standard feed treatments. Epithelial cell counts recovered in the post-treated mice especially in the spirulina groups (Table 4). The reduction in epithelial cell counts ascribed to
necrosis (Chattopadhyay et al. 2010; Sushma et al. 2011) may adversely affect reabsorption process in tubules.
3.3.1. Tissue biochemistry
Protein content decreased significantly (33% 67%) in treatments of standard feed groups
in comparison to controls, possibly because of tissue lysis (Table 5). Conversely, it
increased (25% 129%) in treatments of spirulina groups, particularly of Al3C, in comparison to treatments of standard feed groups possibly on account of production of heat
shock protein (HSP) proteins imparting protection against oxidative stress (Stacchiotti et
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1112
N. Yadav et al.
Figure 2. Microphotographs (10 £ 40x) of spirulina groups.
al. 2006). Its production was perhaps delayed and observed in post-treated mice of standard feed groups (" 44% 94%). Spirulina groups, therefore, received protection when it
was needed most, i.e. during the period of chemical exposure.
ACP is an inducible enzyme because its activity goes up in response to toxic impact
that enzyme counteracts (Ramalingam et al. 2000). ACP content found almost similar to
controls in treatments of standard feed however increased (39% 49%) in post-treatments
suggesting delayed induction of ACP activities (Table 5). In contrast, its levels were significantly higher in treatments of spirulina groups (32% 64%) in comparison to standard
feed treatments possibly to counteract aluminum toxicity that was evident from the reduction in ACP levels in the post-treatment of spirulina group.
Compared with controls, ALP levels decreased (9% 14%) in the treatments of standard feed groups (Table 5). Its levels in treatments of spirulina groups were, however,
higher (" 24% 46%) than standard feed treatments. ALP levels became normal in the
post-treated mice, with the exception of spirulina (Al3C) having raised level (" 32%).
The reduction in ALP levels has been associated to negative or inhibitory disturbances in
Toxicological & Environmental Chemistry
1113
Table 3. Diameter of Bowman’s capsule (mm), glomeruli (mm), and Bowman’s space (mm) of
controls, AlC3 and AlF3 treated and post-treatment mice of standard feed and Spirulina groups.
Experimental groups
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Parameters
Control
S-Control
AlC3
S C AlC3
AlF3
S C AlF3
Diameter of Bowman’s capsule (mm)
Subacute
150 § 2 b
147 § 1.9 bc 168 § 5 a
d
Subchronic
150 § 2.5
163 § 2.1 c 204 § 4.5a
c
Post-treatments 167 § 3.3
162 § 1.5 c 145 § 2.9d
140 § 2.2c 154 § 3.7 b 154 § 2.5 b
171 § 2.9bc 164 § 4.2 c 178 § 2.7 b
145 § 2.2d 175 § 3 b
189 § 2.8 a
Diameter of glomeruli (mm)
Subacute
120 § 2.2 b 105 § 1.8 c 132 § 5.7a
Subchronic
114 § 2.4 c 126 § 2.2 ab 133 § 4.5a
Post-treatments 125 § 2.6 ab 129 § 2.2 a 102 § 2.8d
102 § 2.9c
130 § 2.8a
110 § 2.2c
107 § 3.9 c 112 § 3.3 bc
112 § 4.2 c 119 § 3.2 bc
119 § 2.7 b 124 § 3 ab
Diameter of Bowman’s space (mm)
Subacute
14.7 § 0.7 c 17.9 § 0.5 bc 19.8 § 1.4ab 18.2 § 1.1b 22.6 § 1.2 a 21.1 § 1.5 ab
Subchronic
17 § 0.6 c 15.8 § 0.6 c
35 § 1.8a 18.1 § 0.7c 25.6 § 1.6 b 25.3 § 1.3 b
bc
d
Post-treatments 20 § 1.2 15.4 § 0.9 21.3 § 0.9b 16.9 § 0.7cd 27.1 § 1.6 a 26.1 § 1.1 a
Notes: Values are means § SEM (n D 25). Means in a row without a common superscript letter differ (p < 0.05)
as analyzed by one-way ANOVA and the Duncan test.
the secretory activity or blockage in the transport of metabolites or other nephrotoxic condition (Edet et al. 2012), whereas increase in spirulina groups may have renoprotective
role.
3.3.2. Serum biochemistry
Protein content found almost similar to controls in subacute treatments of standard feed
groups decreased significantly in treated (23% 33%) and post-treated (15% 30%) mice
of subchronic exposure (Table 5). Its content was higher (41% 160%) in treated as well
as post-treated mice of spirulina groups in comparison to standard feed treatments.
ACP levels decreased in treated (5% 33%) and post-treated (13% 17%) mice of
standard feed groups when compared with controls (Table 5). Its levels were, however,
higher in treated (83% 131%) as well as post-treated (7% 11%) mice of spirulina
groups in comparison to standard feed treatments possibly to neutralize toxic effects of
chemicals.
Compared with controls, ALP levels decreased in treated (9% 20%) as well as posttreated mice (5% 35%) of standard feed groups possibly because of binding of aluminum salts to DNA and RNA governing its synthesis (Ochmanski and Barabasz 2000)
(Table 5). In treatments of spirulina groups, ALP activity increased significantly
(26% 49%) in comparison to standard feed treatments. The available data suggest that
spirulina feeding alleviated the ALP synthesis in the tissue from which it was released in
the serum. This may benefit spirulina groups since ALP is involved in the synthesis of
protein (Pilo, Asnani, and Shah 1972), glycogen metabolism (Gupta and Rao 1974), and
synthesis of certain enzymes (Sumner 1965) and transport of metabolites across cell
membranes (Denielli 1972).
10.6 § 0.4 c
12.4 § 0.4 d
15 § 0.5 b
5.9 § 0.2 de
5.2 § 0.2d
5.9 § 0.3 bc
12,800 § 696 a
19,900 § 1410a
11,400 § 778 b
7910 § 427 a
12,400 § 488 a
6050 § 212 a
Al
15.9 § 0.6b
14.6 § 0.6 bc
19.2 § 1 a
6.7 § 0.2 bc
6.1 § 0.2 c
6.5 § 0.2 ab
10,200 § 604 b
15,500 § 444 b
10,300 § 476 bc
5160 § 174 c
8590 § 535 bc
5210 § 322 bc
S C AlC3
Experimental groups
11.8 § 0.4 c
9.4 § 0.5e
13.8 § 0.7 b
5.4 § 0.3e
4.2 § 0.2e
5.4 § 0.3c
13,000 § 600 a
18,400 § 681 a
13,800 § 653 a
6680 § 383 b
12,600 § 623 a
6340 § 313 a
AlF3
Values are means § SEM (n D 25). Means in a row without a common superscript letter differ (p < 0.05) as analyzed by one-way ANOVA and the Duncan test.
20.2 § 1.1 a
17 § 0.6a
19.7 § 1 a
Cell counting in DCT (cells per tubule)
Subacute
16.2 § 0.7 b
Subchronic
16.2 § 0.8 ab
Post-treatments
17.6 § 0.6 a
8599 § 213 c
9897 § 378 c
8148 § 189 d
7.7 § 0.2 a
7.3 § 0.2 a
7.1 § 0.2 a
8490 § 248 c
10,500 § 520 c
9350 § 398 cd
Area of DCT (mm2)
Subacute
Subchronic
Post-treatments
4260 § 160 c
5960 § 265 d
4400 § 218 d
S-Control
Cell counting in PCT (cells per tubule)
Subacute
7.3 § 0.3ab
Subchronic
7 § 0.2ab
Post-treatments
6.9 § 0.2a
4810 § 123 c
7360 § 276 c
4780 § 202 cd
Control
Area of PCT (mm2)
Subacute
Subchronic
Post-treatments
Parameters
C3
14.9 § 0.7 b
13.6 § 0.6cd
17.8 § 0.8 a
6.4 § 0.2 cd
6.6 § 0.3 bc
6.5 § 0.3 ab
13300 § 760 a
15800 § 490 b
14100 § 430 a
6110 § 364 b
9560 § 389 b
5740 § 259 ab
S C AlF3
Table 4. Area (mm2) and cell counting (cells per tubules) in convoluted tubules of controls, AlC3 and AlF3 treated and post-treatment mice of standard feed and
spirulina groups.
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1114
N. Yadav et al.
Toxicological & Environmental Chemistry
1115
Table 5. Biochemistry of controls, AlC3 and AlF3 treated and post-treatment mice of standard feed
and Spirulina group.
Parameters
Control
S-Control
Experimental groups
AlC3
S C AlC3
AlF3
S C AlF3
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¡1
Protein content on kidney (mg g )
Subacute
22 § 1.4 a 22.2 § 1.1 a
Subchronic
6.1 § 0.5 c
4.4 § 0.5d
d
Post-treatments 5.4 § 0.1
6.3 § 0.3 cd
7.2 § 0.2 c 13.8 § 0.5 b 8.9 § 0.6 c
4.1 § 0.2 d 9.4 § 0.7 b 9.7 § 0.6b
7.8 § 1.4 bc 8 § 0.9 bc 10.5 § 0.5 a
12.1 § 0.2 b
12.1 § 0.5 a
9.8 § 0.5 ab
Total protein in serum (g dL¡1)
Subacute
4.4 § 0.2 c
8.2 § 1.2a
Subchronic
7.3 § 0.1
5.2 § 1.5
Post-treatments 6.6 § 0.6 b
8.2 § 0.8a
4.9 § 0.4 bc
5.6 § 0.3
4.6 § 0.4 c
7.5 § 0.8a
7.1 § 0.1
4.8 § 0.1c
ACP in kidney (m mole mg¡1)
Subacute
75 § 3.1 d
156 § 0.6 a
b
Subchronic
74.1 § 4.2 60.6 § 1.7 c
Post-treatments 85.5 § 1.5b 51.4 § 2.5 c
71.7 § 2.2d 94.8 § 1.6 c 75.3 § 4.1 d 123 § 10.1 b
52.9 § 5 c 79.2 § 3.4 b 80.6 § 1.5 b 91.1 § 3.7 a
119 § 3.8 a 52.5 § 6.5 c 127 § 16 a 126 § 2.5 a
ACP in serum(U L¡1)
Subacute
4.3 § 0.3 b
Subchronic
4.4 § 0.2 c
Post-treatments 7.1 § 0.8
5 § 0.2b
4.2 § 0.1 c
5.9 § 0.2
7.4 § 0.9a
4.3 § 0.2c
7.4 § 1.1
6.9 § 0.9ab 4.7 § 0.2 c
5.8 § 0.1
4.9 § 0.1
6 § 0.6 bc 5.6 § 0.1 bc
3.8 § 0.2bc
5.3 § 0.2 b
6.3 § 1.7
2.9 § 0.1 c
4.1 § 0.3 c
6.2 § 0.3
6.7 § 0.2a
7.5 § 0.3a
6.9 § 0.3
ALP in kidney (m mole mg¡1)
Subacute
459 § 0.5c
519 § 1 b
393 § 10.1 d 576 § 0.5 a 456 § 0.4c
a
d
Subchronic
515 § 6.5 432 § 13.4
453 § 0.3 c 473 § 1.1 b 468 § 0.2 bc
d
d
Post-treatments 455 § 0.4
460 § 0.5
477 § 0.4 c 629 § 10.4a 491 § 2 b
568 § 0.5 a
474 § 0.4 b
453 § 0.7 d
ALP in serum (U L¡1)
Subacute
234 § 10 c
Subchronic
200 § 5 a
Post-treatments 202 § 17 a
245 § 14 c
161 § 5 cd
132 § 10 b
365 § 13 a
148 § 4 d
156 § 10 ab
Urea (mg dL¡1)
subacute
24 § 3 b
56 § 5 a
30.3 § 4 b 52.8 § 6.1 a 35.1 § 6 b
a
d
subchronic
53.6 § 3
14.9 § 1.7
47.5 § 1.9a 34.5 § 1.9 b 40.5 § 3.1 b
a
a
Post-treatments 60.4 § 8.2 72.5 § 11.2
63 § 5.6 a 35.2 § 7.1 b 60 § 7.2 a
60 § 7 a
23.5 § 1.7 c
36 § 3.3b
Creatinine (mg dL¡1)
subacute
0.6 § 0.1 cd
subchronic
0.8 § 0.1
Post-treatments 0.9 § 0.1 b
1.7 § 0.2a
0.5 § 0.3
1.5 § 0.1b
344 § 17 ab
175 § 4 b
187 § 21 a
1.4 § 0.3 ab
0.6 § 0.03
0.7 § 0.1b
212 § 8 c
167 § 4 bc
192 § 13 a
316 § 19 b
210 § 5 a
112 § 18 b
0.5 § 0.01 d 1.1 § 0.1bc
0.4 § 0.1
0.5 § 0.03
1.1 § 0.1 b 5.9 § 1.4 a
0.5 § 0.2d
0.5 § 0.03
1.3 § 0.1b
Notes: Values are means § SEM (n D 25). Means in a row without a common superscript letter differ (p < 0.05)
as analyzed by one-way ANOVA and the Duncan test.
1116
N. Yadav et al.
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Urea levels were affected less in treatments of standard feed groups but these were
significantly higher (71% 74%) than the former in subacute treatments of spirulina
groups (Table 5). Interestingly, its levels decreased in its subchronic treatments
(27% 44%) suggesting spirulina alleviated aluminum toxicity only at higher doses.
Compared with controls, creatinine levels decreased (17% 49%) in treatments of
standard feed groups (Table 5). In comparison to standard feed treatments, creatinine levels were significantly higher in both treated (25% 240%) and post-treated mice (436%)
of spirulina groups (Table 5). Serum creatinine and urea are the most commonly used
parameters to assess renal function. An increase in their levels in treatments of spirulina
groups suggests renal damage but this is not revealed in our findings and requires further
study.
4. Conclusion
James et al. (2009) reported that spirulina feeding reduces copper toxicity in fish through
its release from the body tissue via feces. This may hold true for aluminum ions in the
present study. Our findings on the general health of mice and histology and biochemistry
(also serum) of their kidney suggest no toxic effects of spirulina in control mice when
compared with standard feed controls indicating safety of spirulina at the selected dose.
The dose and duration of pretreatment of spirulina varied in different publications (Khan,
Bhadouria, and Bisen 2005; Abdel-Daim, Abuzead, and Halawa 2013), but in our earlier
studies we reported its protective role on general health, hematology, and kidney of Swiss
albino mice at dose and duration of pretreatment used in the present study (Sharma et al.
2013; Yadav et al. 2015 forthcoming). Further, based on the findings we conclude that
dietary intervention of spirulina alleviates aluminum toxicities in the kidneys of Swiss
albino mice.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the CSIR for providing fellowship to N. Yadav [grant number 09/149(0547)/2009-EMR-I]; UGC [grant number F 15- 1 PDFWM 2013-14-GERAJ-17495] for awarding fellowships to Dr Shweta Sharma; ICMR [grant number 3/1/3/
jrf-2008/hrd-99(30293)] for providing fellowship to A. Pandey and the Heads, Botany
and Zoology Department, University of Rajasthan, Jaipur for laboratory facilities.
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