Badr E. El-Bialy*¹, Neveen G. El-Boraey², Ragaa A. Hamouda³ and Mohamed M. Abdel-Daim⁴  

¹Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, 32897, Egypt.

²Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat City, 32897, Egypt.

³Department of Biology, Faculty of sciences and Arts Khulais, University of Jeddah, Saudi Arabia Department of Microbial Biotechnology, Genetic Engineering and Research Institute, Sadat University, Sadat city, 32897, Egypt.

⁴Department of Pharmacology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt.

Corresponding Author E-mail: badr_elsaid10@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/1670

Abstract

Carbon tetrachloride (CCl₄) is used extensively as an industrial solvent and considered the best-characterized experimental animal model of xenobiotic-induced hepatic toxicity via reactive oxygen species (ROS) generation. This study was designed to evaluate the protective effects of Spirulina platensis (SP) versus Spirulina platensis supplemented with thiamine (SPt) against subacute CCl₄ toxicity in rats. Rats were divided into six equal groups; Control vehicle (0.5 ml/rat 1:1 olive oil in water), SP (800 mg/kg b.wt.), SPt (800 mg/kg b.wt.), CCl₄ (1ml/kg b.wt.), SP + CCl₄ and SPt + CCl₄. All treatments were orally and daily for a month except CCl₄ was given three times weekly. CCl₄ caused significant reduction in body weight gain, haemoglobin content and haematocrit percentage accompanied by leukocytosis, granulocytosis, monocytosis and lymphocytopenia. Moreover, there were significant increase in the levels of serum ALT, AST; total, direct and indirect bilirubin; urea and creatinine of CCL₄- intoxicated rats. CCL₄- induced significant increase of malondialdehyde levels with significant reduction of catalase activity in liver and kidney. In addition, hepatic and renal various histopathological alterations were recorded. SP and SPt ameliorated almost these changes while they couldn’t reverse the reduction of body weight gains and red blood indices. The more potent effects on measured parameters were elucidated by SPt. In conclusion SP and SPt could be used as natural antioxidant supplements to counteract the CCl₄ adverse effects.

Keywords

Antioxidants; Biomarkers; CCl₄; Haematology; Oxidative Stress; Spirulina

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Copy the following to cite this article: El-Bialy B. E, El-Boraey N. G, Hamouda R. A, Abdel-Daim M. M. Comparative Protective Effects of Spirulina and Spirulina Supplemented with Thiamine Against Subacute Carbon Tetrachloride Toxicity in Rats. Biomed Pharmacol J 2019;12(2).

Copy the following to cite this URL: El-Bialy B. E, El-Boraey N. G, Hamouda R. A, Abdel-Daim M. M. Comparative Protective Effects of Spirulina and Spirulina Supplemented with Thiamine Against Subacute Carbon Tetrachloride Toxicity in Rats. Biomed Pharmacol J 2019;12(2). Available from: https://bit.ly/2MivHWG

Introduction

Reactive oxygen species are among the main causes of almost degenerative diseases. Oxidative stress induced when generated ROS and free radicals overwhelm the antioxidant protective capacity¹.

Carbon tetrachloride is well known for causing oxidative stress, hepatotoxicity and nephrotoxicity in rats^2,3. CCl₄ intoxication augmented oxidative stress due to free radicals generation that is thought to be the reason behind tissue damage⁴.

Antioxidants possess protective and therapeutic values against various diseases by increasing the levels of body endogenous antioxidants and hence, decrease lipid peroxidation process⁵. Previous studies proved that natural substances from edible and medicinal plants exhibited strong antioxidant protective activity against CCl₄-induced oxidative damage, because they contain various free radical scavengers such as phenolic and flavonoid compounds⁶.

Spirulina platensis is a microscopic blue-green alga (cyanophytes /cyanobacteria).  Its nutritional values referred to its constituents of proteins of essential amino acids (55%-70%), carbohydrates (15%-25%), essential fatty acids (18%) minerals, vitamins and pigments like phycocyanin, carotenes, and chlorophyll⁷. The antioxidant properties of SP could be attributed mainly to polyunsaturated fatty acids, phycocyanin and phenolic contents⁸, in addition to some essential elements that have antioxidant effect like zinc and selenium⁹.

Previous studies cleared protective effects of SP against many toxicants. Simultaneous administration of SP to lead exposed animals significantly inhibited lipid peroxidation and restored the endogenous antioxidants to normal levels in liver, lung, heart, kidney and brain of rats¹⁰. SP has a protective effect against cardiotoxicity induced by doxorubicin in mice¹¹. It has antioxidant properties to scavenge free radicals and to inhibit lipid peroxidation induced by cadmium in liver of rats¹². SP supplementation could overcome deltamethrin induced hepatotoxicty, nephrotoxicity and neurotoxicity by abolishing oxidative tissue injuries¹³.

This study aimed to evaluate the potential protective effects of SP versus SPt against CCl₄ induced haematological changes, hepatotoxicity and nephrotoxicity in rats.

Materials and Method

Chemicals and Diagnostic kits

Chemicals of Zarrouk’s medium, phosphate buffer saline (PBS), diethyl ether, CCl₄ and chemicals of histopathology were purchased from El Nasr Chemical Company, Cairo, Egypt.

Diagnostic kits for assaying serum aminotransferases (alanine aminotransferase, ALT and aspartate aminotransferase, AST) were purchased from Spinreact Ctra. Sta. Coloma, Girona, Spain. Kits for assaying total bilirubin, direct bilirubin, urea and creatinine were purchased from the Diamond Diagnostic Company, Holliston, USA.

Diagnostic kits for assaying malondialdehyde (MDA) level as lipid peroxidation product and catalase (CAT) activity in hepatic and renal tissue homogenates were purchased from the Biodiagnostics Company, Dokki, Giza, Egypt

Constituents and Preparation of Zarrouk’s Medium

Zarrouk’s medium consisted of 1L distilled water, 1g/L NaCl, 0.2g/L MgSO4.7H2O, 0.01g/L FeSO4.7H2O, 0.04g/L CaCl₂.2H2O, 0.08g/L Na₂EDTA , 0.5g/L K₂HPO4, 2.5g/L NaNO3, 1g/L K₂SO4 and 16.8g/L NaHCO3 Plus A5 micronutrient 1ml micronutrients (H₃BO₃, MnCl₂.4H₂O, ZnSO₄.4H₂O, Na₂MoO₄, CuSO₄.5H₂O).

One liter of Zarrouk’s medium was prepared in a glass flask with adjusting PH of the medium to 9.5 (that suitable for growth of SP). Then this medium was sterilized in the autoclave under moist temperature at 121°C for 20 minutes after stoppering by aluminum foil. One liter is divided in flasks each contains 200 ml of the medium.

Spirulina collection and identification

Spirulina platensis (SP) was obtained from Al-Natron valley, Egypt. Its purification and identification were done according to methods described by Rippka¹⁴ and Vonshak¹⁵ respectively in microbiology lab, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat City Egypt. S. platensis was isolated after repeated light migrations on its medium described by Zarrouk¹⁶. It was grown in Erlenmeyer flasks containing 200 ml Zarrouk’s medium. at 25±2°C, pH 9.5  with continuous illumination using cool white fluorescent light (2500 Lux) and twice daily shaking by hand for 15 days. Cells were collected by filtration using filter paper 8 mm pore size (Screen printing paper) and washed with buffer solution (pH 7), diluted to known volume and processed for further inoculation (kept as stock solution).

The cultured S. platensis was recultivated by adding 50 ml of stock solution after its vigorous shaking to flasks contained 200 ml Zarrouk’s medium or Zarrouk’s medium supplemented with thiamine (0.01g/L) and incubated under the same mentioned conditions for 21 days.

Spirulina algae (SP and SPt) were collected by filtration and then dried in oven at 75°C for 2-6 hrs and then grinded by mortar until became powder.  The required daily dose from spirulina powder (SP or SPt) is dissolved in water to be in suspension form at the day of its administration to animals using Ultrasonic homogenizer sonicator (Biologics Inc. USA manufacturer and leading innovator).

Experimental animals

Sixty male Sprague-Dawley rats (120-150 g) were used in this study. They were purchased from AL-Zyade Experimental Animals Production Center, Giza, Egypt. All animals were placed in polypropylene cages with mesh wire tops. They kept under normal day and night cycle in a natural ventilated room with good hygienic conditions. Rats fed standard chow diet and clean tap water ad libitum. The rats were maintained for 2 weeks prior to the start of experiment for acclimatization. Animal rearing and handling guide lines were approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Egypt.

Experimental design

After acclimatization period, rats were divided into six equal groups and then weighed (Initial body weights) as following:

Group 1 (Control vehicle): Animals administered olive oil with water 1:1 orally (0.5 ml/rat/day) as vehicle of both Spirulina and CCl₄.

Group 2 (SP): Animals administered S. platensis (800 mg/kg body weight).

Group 3 (SPt): Animals administered S. platensis supplemented with thiamine (800 mg/kg body weight).

Group 4 (CCl₄): Animals administered CCl₄ (1ml/kg body weight) in olive oil (1:1).

Group 5 (SP + CCl₄): Animals were pretreated with SP an hour before CCl₄ administration.

Group 6 (SPt + CCl₄): Animals were pretreated with SPt an hour before CCl₄ administration.

All treatments were orally and daily for a month except CCl₄ was given three times weekly.

Samples Collection

At the end of the experiment (after one month), the animals were weighed (Final body weights) to calculate body weight gains, fasted overnight and then anaesthetized using diethyl ether and sacrificed for samples collection.

Blood samples were collected from the median canthus of the eye using heparinized capillary tube. Blood sample of each rat was received in two tubes (tube containing EDTA for haemogram and plain centrifuge tube for serum separation). Serum samples were stored at -20°C until biochemical assay.

After blood collection, rats were sacrificed. Liver and kidneys of each rat were collected. A part of each organ was homogenized in phosphate buffered saline and kept at -80°C after that rapid tissue biochemical investigations were performed (including lipid peroxidation and catalase activity). Another part was kept in 10% neutral buffer formalin solution for the histopathological examination.

Haematological Estimation

Haematological parameters were estimated automatically using H32 VET 3-Part Differential Hematology Analyzer (Avantor Performance Materials Inc. Company, Center Valley, USA)

Blood indices including red blood cells count (RBCs), haemoglobin concentration (HGB), hematocrit percentage (HCT%), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), total leukocytes (TLC), differential leukocyte percentages and platelets count.

Serum Biochemical Analysis

Serum biochemical parameters were estimated colourimetrically according to methods of Murray¹⁷ for ALT and AST activities respectively, Kaplan et al.¹⁸ for total and direct bilirubin, Fawcett and Soctt¹⁹ for urea level and Schirmeister²⁰ for creatinine level.

Determination of lipid peroxidation product and catalase activity in liver and kidney tissue homogenates

Malondialdehyde was estimated according to the procedure described by Ohkawa et al.²¹ and CAT activity was determined according to and Aebi²², following instructions of kits.

Histopathological Examination

Liver and kidney samples intended for histopathological investigations were fixed in 10 % neutral buffer formalin for 72 hrs. Samples were trimmed and processed by dehydration in serial grades of ethanol in 50–100%, cleared in xylene, and then embedded in paraffin. Sections (4 μm thick) were prepared by a microtome and then stained with hematoxylin and eosin as a general staining for tissue examination. The liver sections were examined for pathological changes by a light microscope.

Statistical Analysis

Values are presented as mean ± standard error of mean (SEM). Statistical significance of toxic effects of CCl₄ and the protective effects of SP and SPt were determined by one way ANOVA (Analysis of Variance) followed by Duncan’s multiple range test. All statistical analyses were performed using SPSS (Statistical Package for Social Sciences) Version 16 released on 2007. Statistical significances between different means were considered at P<0.05.

Results

Changes in Body Weight Gains

Changes in body weight gains are recorded in Table 1. Compared to control vehicle group, oral administration of SP caused significant reduction (P<0.05) in body weight gains, in contrary, oral SPt administration induced insignificant increase in body weight gains.

Table 1: Mean values of initial, final body weights (g) and body weight gains (g) of control and different treated groups.

	Initial body weights	Final body weights	Body weight gains
Control vehicle	161.6 ± 3.61a	201.4 ± 4.41a	39.8 ± 2.42a
SP	159.8 ± 2.42a	193.2 ± 3.31a	33.4 ± 1.96b
SPt	159.6 ± 3.69a	203.4 ± 4.24a	43.8 ± 1.02a
CCl4	161.4 ± 3.74a	169.2 ± 3.02b	7.80 ± 1.59c
SP + CCl4	159.6 ± 3.41a	170.2 ± 3.72b	10.60 ± 1.66c
SPt + CCl4	158 ± 2.02a	166.4 ± 1.47b	8.4 ± 0.93c

 

Data are presented as means ± SEM (n = 8). Spirulina platensis (SP), Spirulina platensis supplemented with thiamine (SPt), Carbon tetrachloride (CCl₄). Values having different superscripts within same column are significantly different (P<0.05).

Rats orally administered CCl₄ (1ml/kg body weight three times weekly for a month) without or with SP or SPt (800 mg/kg b.wt) showed significant reduction of body weight gains compared with that of control vehicle group at P<0.05.

Haematological Parameters

Red Blood Cell Indices

Red blood indices are recorded in Table 2. There were insignificant changes between control vehicle and all different groups in RBCs count and MCHC at P<0.05. In addition, there were insignificant changes between control vehicle group and groups administered SP and SPt in HGB concentration, HCT %, MCV and MCH. In the same manner, these parameters showed insignificant changes among the last three groups (CCl₄, SP+CCl₄ and SPt+CCl₄) but showed significant reduction in these groups compared with those of control vehicle group at P<0.05.

Table 2: Mean values of red blood cell indices of control and treated rats.

	RBCs × 106/µL	HGB (g/dl)	HCT %	MCV (fl/cell)	MCH (pg/cell)	MCHC (g/dl)
Control vehicle	6.49 ± 0.14a	12.5 ± 0.17a	30.88 ± 0.31a	47.68 ± 0.85a	19.32 ± 0.61a	40.5 ± 0.58a
SP	6.59 ± 0.19a	12.36 ± 0.22a	31.06 ±  0.35a	47.14 ±  0.57a	18.76 ± 0.34a	39.84 ±  0.75a
SPt	6.66 ± 0.07a	12.26 ± 0.11a	30.76 ± 0.46a	46.20 ±  0.56a	18.68 ± 0.28a	38.84 ± 0.19a
CCl4	6.47 ± 0.26a	11.34 ± 0.34b	28.4 ± 0.53b	43.92 ±  0.79b	17.48 ± 0.22b	40.08 ± 0.49a
SP + CCl4	6.66 ± 0.24a	11.26 ± 0.31b	29.20 ± 0.52b	43.9 ± 0.63b	17.40 ± 0.31b	39.02 ± 0.30a
SPt + CCl4	6.47 ± 0.34a	11.02 ± 0.46b	28.24 ±  0.85b	43.68 ± 0.69b	17.10 ± 0.34b	39.14 ± 0.48a

 

Data are presented as means ± SEM (n = 8). Spirulina platensis (SP), Spirulina platensis supplemented with thiamine (SPt), Carbon tetrachloride (CCl₄). RBCs (red blood cells), HGB (haemoglobin), HCT (hematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin) and MCHC (mean corpuscular haemoglobin concentration). Values having different superscripts within same column are significantly different (P<0.05).

Leukocytes Count and Differential Leukocyte Percentages

Total leukocytes and percentages of differential leukocytes are presented in Table 3. It was cleared that rats orally administered SP and SPt for a month caused elevation in TLC in relation to control vehicle group at P<0.05. Rats administered CCl₄ alone or with SP or SPt showed significant elevation in TLC in comparing with control vehicle rats at P<0.05.

In regarding lymphocyte percentage, SPt, CCl₄, SP+CCl₄ and SPt+CCl₄ induced significant reduction in lymphocyte percentage in comparing with that of control vehicle and SP groups.

At P<0.05, there were significant elevation in monocytes percentages at comparing SPt, CCl_4, SP+CCl₄, SPt+CCl₄ groups with control vehicle and SP groups.

Granulocyte percentages were significantly elevated in CCl₄, SP+CCl₄ and SPt+CCl₄ than those of control vehicle, SP and SPt groups at P<0.05.

Platelets Count

Table 3 cleared that, there were insignificant changes at P<0.05 in platelet count between control vehicle and other different groups.

Table 3: Mean values of leukocyte count, differential leukocyte percentages and platelets count of control and treated rats.

	TLC × 103/µL	Lymphocytes %	Monocytes %	Granulocytes %	Platelets × 103/µL
Control vehicle	12.31 ± 0.53c	79.45 ± 2.50a	6.36 ± 0.47c	14.19 ± 2.56b	618 ± 30.22a
SP	16.90 ± 1.01b	76.37 ± 2.98a	7.93 ± 0.61c	15.7 ± 2.98b	610 ± 31.41a
SPt	17.69 ± 0.79b	61.02 ± 3.72b	22.57 ± 1.57a	16.41 ± 3.38b	617± 30.85a
CCl4	24.27 ± 2.57a	59.87 ± 4.16b	12.52 ± 0.81b	27.61 ± 3.43a	594 ± 32.26a
SP + CCl4	25.27 ± 1.12a	60.33 ± 3.19b	12.77 ± 0.72b	26.9 ± 3.64a	551 ± 30.61a
SPt + CCl4	28.34 ± 1.36a	52.01 ± 2.43b	21.09 ± 0.91a	26.9 ± 1.92a	598 ± 42.98a

 

Data are presented as means ± SEM (n = 8 Spirulina platensis (SP), Spirulina platensis supplemented with thiamine (SPt), Carbon tetrachloride (CCl₄). TLC (Total leukocytes count) Values having different superscripts within same column are significantly different (P<0.05).

Serum Biochemical Findings

Serum Hepatic Biomarkers

The effects of CCl₄ exposure alone or with SP or SPt on serum ALT and AST activities in rats are recorded in Table 4.

Table 4: Mean values of serum hepatic biomarkers of control and different treated groups.

	ALT (U/L)	AST (U/L)	T Bilirubin (mg/dl)	D  Bilirubin (mg/dl)	IND Bilirubin (mg/dl)
Control vehicle	83.71 ± 1.61d	100.43 ± 1.09d	0.511 ± 0.008d	0.134 ± 0.003c	0.377 ± 0.009d
SP	82.00 ± 1.76d	98.71 ± 1.70d	0.569 ± 0.023d	0.147 ± 0.004c	0.421 ± 0.023d
SPt	81.71 ± 1.63d	97.43 ± 2.15d	0.549 ± 0.017d	0.139 ± 0.006c	0.410 ± 0.016d
CCl4	204.14 ± 3.28a	217.57 ± 2.26a	1.011±0.024a	0.183 ±0.004a	0.829 ± 0.023a
SP + CCl4	119.57 ± 0.57b	129.71 ± 1.08b	0.857 ± 0.029b	0.167 ± 0.005b	0.690 ± 0.027b
SPt + CCl4	101.71 ± 1.57c	113.29 ± 1.11c	0.708 ± 0.022c	0.166 ± 0.005b	0.543 ± 0.019c

 

Data are presented as means ± SEM (n = 8). Spirulina platensis (SP), Spirulina platensis supplemented with thiamine (SPt), Carbon tetrachloride (CCl₄), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T bilirubin), direct bilirubin (D bilirubin), indirect bilirubin (IND bilirubin). Values having different superscripts within same column are significantly different (P<0.05).

There were insignificant changes in serum ALT and AST activities at P<0.05 between control vehicle, SP and SPt groups. The activities of serum ALT and AST significantly elevated in CCl₄ intoxicated group compared to control vehicle one. Administration of either SP or SPt with CCl₄ in groups 5 and 6 induced significant reduction in ALT and AST activities compared with CCl₄ intoxicated rats at P<0.05 and these values were still significantly elevated than those of control vehicle.

From the recorded data, it was noticed that values of serum ALT and AST activities showed significant (P<0.05) reduction in SPt+CCl₄ group than those of SP+CCl₄ group.

Total, direct and indirect bilirubin of different groups were presented in Table 4. These parameters showed insignificant changes at P<0.05 between control vehicle, SP and SPt groups. Intoxicated rats with CCl₄ for one month revealed significant increase (P<0.05) in total, direct and indirect bilirubin in comparing to control vehicle rats. Daily administration of SP and SPt simultaneously with CCl₄ induced significant reduction in total, direct and indirect bilirubin but still significantly higher than those of control vehicle group. SPt+CCl₄ group showed significant reduction at P<0.05 in total and indirect bilirubin but insignificant reduction of direct bilirubin in relation to SP+CCl₄ group.

Serum Renal Biomarkers (Urea and Creatinine)

As recorded in Table 5, serum urea and creatinine showed no significant difference at P<0.05 between control vehicle, SP and SPt groups. In regarding CCl₄ intoxicated rats, the mean values of urea and creatinine significantly increased than those of control vehicle group. Coadministration of either SP or SPt with CCl₄ induced significant reduction in serum urea and creatinine levels compared with rats administered CCl₄ alone at P<0.05 and these values were still significantly elevated than those of control vehicle rats in SP+CCl₄ group only and returned around normal ranges in SPt+CCl₄ group.

Table 5: Mean values of serum urea and creatinine of control and treated rats.

	Urea (mg/dl)	Creatinine (mg/dl)
Control vehicle	25.43 ± 0.54c	0.689 ± 0.008c
SP	25.6 ± 1.21c	0.569 ± 0.01c
SPt	25.2 ± 0.86c	0.585 ± 0.01c
CCl4	38.1 ± 1.45a	1.123 ± 0.09a
SP + CCl4	31.4 ± 1.12b	0.864 ± 0.02b
SPt + CCl4	27.8 ± 1.39c	0.701 ± 0.03c

 

Data are presented as means ± SEM (n = 8). Spirulina platensis (SP), Spirulina platensis supplemented with thiamine (SPt), Carbon tetrachloride (CCl₄). Values having different superscripts within same column are significantly different (P<0.05).

From the recorded data, It was noticed that there were significant changes between SPt+CCl₄ and SP+CCl₄ groups in both serum urea and creatinine levels at P<0.05. These parameters showed reduction in SPt+CCl₄ than those of SP+CCl₄ group.

Tissue Biochemical Findings

Malondialdehyde (MDA) level in liver and kidney tissues homogenate

Malondialdehyde contents in liver and kidney tissue homogenates as one of lipid peroxide markers showed insignificant difference in control vehicle, SP and SPt groups at P<0.05 (Table 6). Also from the recorded data, there were significant elevation in MDA level of both liver and kidney in CCl₄ intoxicated rats versus those of control vehicle rats. Administration of either SP or SPt with CCl₄ caused significant reduction in MDA contents in hepatic and renal tissues in comparing with CCl₄ intoxicated group but still significantly elevated than those of control vehicle group at P<0.05.

Catalase activity of liver and kidney tissues homogenate

Table 6 showed CAT antioxidant enzyme activity of control vehicle and different treated groups. The control vehicle, SP and SPt groups showed insignificant variation in CAT activity of hepatic and renal tissues at P<0.05. Intoxicated rats with CCl₄ showed significant reduction in catalase activity of liver and kidney in comparing with control vehicle group. Daily administration of SP or SPt to intoxicated rats induced significant elevation in catalase activity in comparing with CCl₄ group but still significantly lower than those of control vehicle group in liver homogenate and around the normal ranges in kidney homogenate.

Table 6: Mean values of MDA levels (nmol/g tissue) and Catalase activities (U/g tissue) in liver and kidney tissues of control and treated rats.

	MDA (mmol/g tissue)		CAT (U/g tissue)
	Liver	Kidney	Liver	Kidney
Control vehicle	30.33 ± 0.79c	44.64 ± 2.15c	5.28 ±  0.15a	4.08 ± 0.18a
SP	29.78 ± 0.99c	42.54 ± 2.66c	5.39 ± 0.13a	4.31 ± 0.18a
SPt	29.03 ± 1.50c	43.20 ± 1.88c	5.24 ± 0.23a	4.24 ± 0.18a
CCl4	55.33 ± 2.22a	67.18 ± 1.90a	2.94 ± 0.19c	2.89 ± 0.11 b
SP + CCl4	42.63 ± 0.73b	52.58± 3.04b	4.09 ± 0.07b	3.86 ± 0.18a
SPt + CCl4	42.29 ± 1.55b	53.34 ± 2.99b	4.06 ± 0.17b	3.94 ± 0.04a

 

Data are presented as means ± SEM (n = 8). Spirulina platensis (SP), Spirulina platensis supplemented with thiamine (SPt), Carbon tetrachloride (CCl₄), MDA (malondialdehyde), CAT (catalase). Values having different superscripts within same column are significantly different (P<0.05).

Histopathological Findings

Liver histopathological changes of different groups (Figure 1) revealed no histopathological changes in control vehicle, SP and SPt groups (A, B and C). The livers of intoxicated rats with CCl₄ (D) showed severe peripheral fatty changes (+++), interlobular fibrosis (+++) and congestion. Leukocytes infiltration and necrosis were also recorded in intoxicated rats. Peripheral fatty changes and interlobular fibrosis became moderate (++) with congestion in group treated with SP with CCl₄ (E) and mild (+) in group treated with SPt with CCl₄ (F).

Figure 1: Liver histopathology, Rat.   Click here to view figure

 

A, B and C) represent control vehicle, Spirulina (SP) and Spirulina supplemented with thiamine (SPt) treated groups, respectively, which showing normal hepatic histoarchitectures. D, represent CCl₄ intoxicated group: showing extensive fatty change in peripheral hepatocytes (arrows) +++ and interlobular fibrosis (arrow head) +++ and congestion. E, represent SP+ CCl₄ group: showing moderate fatty change in peripheral hepatocytes (arrow) ++ and moderate interlobular fibrosis (arrowhead) ++ and congestion. F, represent SPt + CCl₄: showing mild fatty change in peripheral hepatocytes (arrow) + and mild interlobular fibrosis (arrowhead) +. H&E stain, X20.

Kidney histopathological changes of different groups (Figure 2) revealed normal renal histoarchitectures in control vehicle, SP and SPt groups (A, B and C). The kidneys of intoxicated rats with CCl₄ (D) showed severe hydropic degeneration. Mild congestion and mild intratubular edema in group treated with SP with CCl₄ (E) and mild intratubular edema in group treated with SPt with CCl₄ (F).

Figure 2: Kidney histopathology, Rat.   Click here to view figure

 

A, B and C) represent control vehicle, Spirulina (SP) and Spirulina supplemented with thiamine (SPt) treated groups, respectively, which showing normal renal histoarchitectures. D, represent CCl₄ intoxicated group: showing extensive hydropic degeneration in epithelial lining of renal tubules (arrows). E, represent SP+ CCl₄ group: showing mild congestion (thin arrow) and mild intratubular edema (thick arrow). F, represent SPt + CCl₄: showing mild intratubular edema (arrow). H&E stain, X20.

Discussion

This study was planned to investigate the comparative heptoprotective and nephroprotective effects of Spirulina (SP) and Spirulina supplemented with thiamine during growth (SPt) via their antioxidant mechanisms against CCl₄ subacute toxicity in rats.

The recorded results showed reduction in body weight gains in groups of rats intoxicated with CCl₄ alone or in combining with SP or SPt in comparing with control vehicle rats. This means that administration of SP or SPt to the intoxicated rats couldn’t counteract the lowering effects of CCl₄ on body weight gains.

Reduction in body weight gains of rats was recorded after one month of CCl₄ administration²³. CCl₄ caused loss of appetite due to its adverse effects on gastrointestinal tract including diarrhea, irritation, nausea, and abdominal pain²⁴. The reduction in body weight gains also could give indirect indication of disturbance in hepatic functions that affect food efficiency ratio²³.

Although administration of SP daily for 30 days caused significant reduction in body weight gains in G2, the administration of SPt showed increase in body weight gains in G3 in relation to control vehicle group at P<0.05.

Supplemented diet with SP resulted in a significant reduction of body weight in obese and ischaemic heart patients with significant improvement of lipid profil²⁵. However at feeding of rats on diet containing SP at 10, 20 and 30% (w/w) did not have adverse effect on body weight gains in male and female rats²⁶.

Spirulina is a highly nutritious microalga rich in protein, essential fatty acids, minerals, and vitamins and low in calories. It is used as food supplement to fight starvation and malnutrition and on the other hand to aid in weight loss. It is thought to be an appetite suppressant as it contains the amino acid L-phenylalanine that stimulates the secretion of cholecystokinin, an important satiety hormone in humans, helps in suppressing appetite center in the brain and reduction in feed intake^27,28. Also SP helps in fat mobilization and hypolipidemic effects that are critical in weight loss by activation of lipase enzymes²⁹.

In contrast SPt showed slight increase in body weight gains in relation to control rats. Thiamine has biotic and abiotic effects by acting as a coenzyme of the tricarboxylic acid cycle and catabolism of glucose with progressive generation of ATP that is used in other metabolic pathways and hence promoting growth rate^30,31.

At Reading the red blood cells indices, RBCs count and MCHC showed insignificant changes between different groups. While HGB concentration, HCT%, MCV and MCH showed significant reduction in the last three groups (CCl₄, SP+CCl₄ and SPt+CCl₄) compared to control vehicle group. From the listed data, it is cleared that CCl₄ caused microcytic hypochromic anaemia that was not corrected by daily concomitant administration of SP and SPt to rats.

Carbon tetrachloride caused anaemia in rats previously approved by^32,33. Saba et al. approved that CCl₄ increased erythrocyte fragility and caused microcytic hypochromic anaemia³². Abdel-Wahhab, et al. found that CCl₄ significantly affect the quantity and function of HGB molecule whereas total HGB and Oxy-HGB contents decreased significantly than that of control rats³⁴. It was found that CCl₄ induced insignificant changes in RBCs count, PCV% and MCV but caused significant decrease in HGB concentration in relation to control group².

In the present study the reduction in body weight gains and anaemia occurred concomitantly in CCl₄, SP+CCl₄ and SPt+CCl₄ groups; this means that SP and SPt couldn’t correct the depleting effects of CCl₄ frequent doses for a month.

The recorded data of leukocytes count and differential leukocyte percentages showed significant leukocytosis in CCl₄, SP+CCl₄ and SPt+CCl₄ groups in comparing with control vehicle group and this leukocytosis is associated with lymphocytopenia, monocytosis and granulocytosis (related to neutrophilia). Our results are in agree with^2,32,33,35.

The significant increase in WBCs recorded in this study in CCl₄ groups may refer to the release of marginal or peripheral neutrophils into the circulation which produced the recorded neutrophilia in those rats under the influence of stress hormones as cortisol and catecholamine³⁶. lymphopenia (through shifting haematopoiesis to granulocytic lineages) leading to increased ratio between neutrophils and lymphocytes under stress was recorded by Huff et al.³⁷ After CCl₄ intoxication, rats became under stress of adverse effects of the toxin that referred to its irritant effect during administration and/or its toxic effects after absorption.

Spirulina and SPt in all treated groups increased leukocytes count.  Spirulina had immuno-modulatory functions against lead acetate immunosuppression³⁸.

In this study, there were insignificant reduction in platelet counts in CCl₄ intoxicated groups in relation to control vehicle group as recorded by Essawy et al.³⁹

Obtained results of the present study revealed that administration of CCl₄ 1 ml/kg b.wt, three times a week for a month induced hepatotoxicity in albino rats that approved by significant increases in serum ALT and AST activities with increment in total, direct and indirect bilirubin in relation to those of control vehicle rats. The administration of SP and SPt concomitantly with CCl₄ induced significant reduction in these parameters in comparing with CCl₄ intoxicated rats and these reductions almost clear in SPt group.

Carbon tetrachloride proved to have hepatotoxic effects even after a single or repeated dose. It caused a significant increase in plasma AST, ALT, ALP and LDH activities and total bilirubin concentration^2,,23,40. Serum aminotransferases were increased as a result of hepatic cells injury and increased cellular membranes permeability and enzymes leakage⁴¹.

Carbon tetrachloride induced renal injury indicated by significant increase in serum levels of urea and creatinine in comparing with control vehicle group. This results in agree with Zahran et al.³⁵ but disagree with Fortea  et al.² SP and SPt administered with CCl₄ could ameliorate these changes as serum urea and creatinine levels significantly decreased than those of CCl₄ intoxicated rats and returned around control levels in SPt+CCl₄ group.

Nephrotoxicity induced by CC1₄ was attributed to its damage effect on nephron structural integrity in the form of degenerative changes in glomerulus and vacuolization of renal tubules⁴².

All toxic effects of CC1₄ are elucidated after its metabolic activation in the endoplasmic reticulum by cytochrome P450 enzymes (mainly by CYP2E1) to highly reactive trichloromethyl radical (CCl3^•), that rapidly reacts with oxygen to form the highly reactive trichloromethyl peroxyl radical (CCl3OO•). These radicals induce membrane lipid peroxidation and disturb calcium homeostasis to produce cellular injury⁴³.

Malondialdehyde (MDA) is among the many secondary products of lipid peroxidation process and used as biomarker for the assessment the degree of lipid peroxidation. From the recorded data, there were significant elevation in MDA level in both liver and kidney tissues homogenate in CCl₄ intoxicated rats in comparing with control vehicle rats and these findings agree with Laouar et al. and Suzuki et al.^40,44 CCl₄ can induce hepatic and renal injuries via lipid peroxidation by free radical metabolic products. Lipid peroxidation is in turn considered as an indicator of structural and functional alterations of cellular and organelles membranes with failure of antioxidant defense mechanisms to stop the formation of excessive free radicals⁴⁵. Whereas co-administration of either SP or SPt with CCl₄ significantly lowered MDA contents in hepatic and renal tissues compared to CCl₄-exposed rats. The inhibition of lipid peroxidation by S. platensis may be attributable to free radical scavenging activity of its antioxidant components¹².

Catalase (CAT) is an enzymatic antioxidant can catalyze the breakdown of hydrogen peroxide into oxygen and water⁴⁶. Our recorded findings revealed that intoxicated rats with CCl₄ showed significant reduction in catalase activity of both liver and kidney in comparing with control vehicle group. These results were in agreement with earlier findings of ^42,47. Daily treatment with SP or SPt induced significant elevation in catalase activity in comparing with CCl₄ group but still significantly lower than those of control vehicle group in liver tissue and around the normal ranges in kidney tissue.

Natural occurring plants and algae have medicinal and nutritional values referring to their content of many phytochemicals. In this study the improvement effects of SP and SPt against CCl₄ referred to Spirulina antioxidant properties. It has scavenging activity to free radicals and inhibition of lipid peroxidation due to its content of Phycocyanin⁴⁸. Spriulina acts as chemopreventive agent, which enhance the antioxidant detoxification system⁴⁹.

Addition of SP in basal diet improved lipids profile, liver and kidney functions altered by CCl₄²³. Also oral daily administration of SP resulted in significant decrease in the elevated liver enzymes, significant increase in the antioxidant enzymes, decrease in MDA and TNF-α levels and the lipid profile in hepatotoxicity induced by CCl₄ in rats⁴⁷.

Spirulina ameliorates methotrexate hepatotoxicity via antioxidant, immune stimulation, and proinflammatory cytokines and apoptotic proteins modulation⁵⁰.

Considering pathological alterations of rats’ livers in this study showed many degenerative changes of the hepatic tissue including fatty change, leucocytes infiltration, congestion of blood vessels, necrosis and fibrosis. Similar results were obtained by those of^51,52. Also CCl₄ induced pathological alterations in kidneys represented by congestion and severe hydropic degeneration as recorded by Venkatanarayana, et al.⁴²

The pathogenesis of various liver injuries including hepatic fibrosis and collagen deposition was associated with oxygen-derived free radicals and lipid peroxidation process⁵³. Moreover upon liver injury, hepatic stellate cells (HSCs) become activated, converting into myofibroblast-like cells and produce extracellular matrix (ECM), playing a major role in hepatic fibrosis⁵⁴.

In previous unpublished study to the authors of this paper, it was found that SPt contain higher concentrations of all pigments as (chlorophyll, carotenoids, phycocyanin, phycoerythrin, allophycocyanin, phycobiliprotein) and these phytopigments have antioxidative effect. Vadiraja et al. and liu et al. demonstrated that C. phycocyanin, one of the major biliproteins of S. platensis can significantly reduce CCl₄ induced acute liver injury in rats, possibly due to lower levels of reactive metabolites of CCl₄ by inhibiting some of the cytochrome P450 mediated reactions involved in the formation of CCl₄ reactive metabolites in addition to its free radical scavenging ability^55,56.

Conclusion

SP and SPt could mitigate the toxic effects of CCl₄ on liver and kidney indicated by improvement of hepatic markers, renal markers, tissue antioxidant status and pathological pictures. The more potent effects were referred to SPt than SP. From listed data we also concluded that in the time of counteracting some adverse effects of CCl₄, on blood leukogram some parameters didn’t reversed as body weight gains and red blood indices.

Conflict of Interest

The authors declare no conflict of interest associated with this work.

Acknowledgements

The authors gratefully acknowledge and thank Prof. Dr. Abd Ellatif  Elbaishy (Professor of Pathology, Faculty of Medicine, Banha University and Dr. Anis Zaid (Assistant Professor of Pathology Faculty of Veterinary Medicine, University of Sadat City) for their valuable help in finishing pathological work.

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