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Chitosan induced hepato-nephrotoxicity in mice with special reference to gender effect in glycolytic enzymes activities
Regulatory Toxicology and Pharmacology 62 (2012) 29–40
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Chitosan induced hepato-nephrotoxicity in mice with special reference to gender effect in glycolytic enzymes activities Enayat A. Omara a,⇑, Hanan F. Aly b, Somaia A. Nada c a
Pathology Dept., National Research Centre, P.O. 12622, Dokki, Cairo, Egypt Therapeutic Chemistry Dept., National Research Centre, P.O. 12622, Dokki, Cairo, Egypt c Pharmacology Dept., National Research Centre, P.O. 12622, Dokki, Cairo, Egypt b
a r t i c l e
i n f o
Article history: Received 25 June 2011 Available online 2 December 2011 Keywords: Chitosan Biochemical Histopathological Histochemical Mice
a b s t r a c t Chitosan is an antilipidemic dietary supplement used as a diet aide. The present study investigated the effect of sex–toxicity relationship between male and female mice orally given two dose levels (150 and 300 mg/kg) for 35 days. Chitosan treatment caused significant elevation in transaminases (ALT, AST) and alkaline phosphatase (ALP) in liver and in serum urea and creatinine in dose dependent manner; no sex differences between-treated groups. Lipid profile parameters significantly decreased and significant increase in glycolytic enzymes activities in all treatment groups. Female mice treated with chitosan (300 mg/kg) had significant reduction in lipid profile parameters than the same dose of male group. Phosphofructokinase (PFK) and lactate dehydrogenase (LDH) activities significantly enhanced without sex differences, while glucose phosphate isomerase (GPI) and hexokinase (HK) significantly elevated in the higher dose of females than male. Histopathological study of liver and kidney tissues showed moderate to severe histopathological changes depend on the dose and gender difference. Image analysis resulted significant depletion in glycogen and protein contents especially in female more than male. These results indicated that female mice were more susceptible to the toxic effect of chitosan than males when administered with the higher dose for a long period. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Chitosan is the deacetylated form of chitin, an aminopolysaccharide found in the exoskeletons and the fungal cell wall of various arthropods including insects, crabs and shrimp (Muzzarelli, 1977). Although it is not derived from plants, it shares the same characteristics as dietary fiber, which is an indigestible polysaccharide by mammalian digestive enzymes (van Bennekum et al., 2005). Special care should be taken in the clinical use of chitosan over a long time to avoid the adverse health effect (Tanaka et al., 1997). Chitosan has profound applications in the fields of clarification and purification, chromatography, paper and textiles, photography, food and nutrition, agriculture, pharmaceutical and medical, cosmetics, biodegradable membranes and biotechnology (Senel and McClure, 2004). Chitosan has been suggested to reduce fat absorption from gastrointestinal tract by binding with anionic carboxyl groups of fatty and bile acids, and it interferes with emulsification of neutral lipids (i.e., cholesterol, other sterols) by binding them with hydrophobic ⇑ Corresponding author. E-mail address: [email protected] (E.A. Omara). 0273-2300/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2011.11.010
bonds (Ylitalo et al., 2002). Inhibition of cholesterol absorption reduces absorption of dietary cholesterol, but more importantly, prevents reabsorption of biliary cholesterol, which is instead eliminated in the feces (Cohen, 2004). Orally administered chitosan binds fat in the intestine, blocking absorption, and has been shown to lower blood cholesterol in animals and humans (Ormrod et al., 1998). Chitosan acts by forming gels in the intestinal tract which entrap lipids but also other nutrients, including fat soluble vitamins and minerals, thus interfering with their absorption (Koide, 1998). The hypolipidemic influence of chitosan may also be due to interruption of the enterohepatic bile acid circulation (Razdan and Pettersson, 1996) and the reduction in duodenal bile acid concentration (Razdan et al., 1997). Chitosan has shown promise as a carrier in colon targeting, results suggesting that degradation by colonic bacterial enzymes might be one of the important properties of chitosan for its successful use in drugs colon targeting (Tozaki et al., 1997). Arsenic may concentrated in the shells as part of their normal development; this in turn may lead to arsenic-laced chitosan supplements. Chitosan cause loss of fat-soluble vitamins and block absorption of medicines such as birth control pills (Orr, 1999).This study was designed to investigate the hepato-renal toxic effect of chitosan on mice and who more susceptible to its toxicity.
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Animal Care and Use Committee and were in accordance with the guidelines of the International Association for the Study of Pain Committee for Research and Ethical Issues (Zimmermann, 1983).
2. Materials and methods 2.1. Animals Swiss albino mice of both sexes weighing 20–25 g were used throughout the experiments. Animals were housed under standard environmental conditions (23 ± 1 °C, 55 ± 5% humidity and a 12-h light:12-h dark cycle); maintained with free access to water and a standard laboratory diet ad libitum. Animal care and the experimental protocols were approved by the National Research Centre
(B)
15
Alkaline Phosphatase (ALP)in liver homogenate
4.50 4.25 4.00 3.75 3.50 3.25 3.00 2.75 2.50 2.25 2.00
kg
50
B
mg /dl A
45
B
40 35
B
A
30
A
kg
kg
20
(F)
Creatinine in serum C
C
700
15 0
g 30 0
m
m g/ k
g/ kg
l tro on
Female A
A
600
D mg /dl
B
A
C
Total Lipid Male
Female
Male
A
B
500
C
C
C
400 300
kg
kg
g/ m 0 30
0
m
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l 15
on tro C
kg g/ m 0 30
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C on tro l
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g/ kg m
l 0 15
C on tro
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m
g/
kg 30
g/ m 15 0
C on tro
kg
200 l
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
g/ kg m
15
30 0
0
C
m
on
g/ kg
tro l
m g/ 30 0
C
15 0
on t
m g/
ro l
kg m 30 0
15 0
m
g/
on tro C
g/
kg
25
(E)
mg /dl
C
C
55
B
A
Female
Male
60
C BC
g/
Urea in serum
(D)
Female
Male
l
µmole/mg protein
(C)
m
30 0
C on
m
m 15 0
C
tro l
g/ kg
g/ kg
l on t
ro
g/ kg m 30 0
15 0
C
m
on
g/
tro l
kg
20
A
A
kg
A
25
g/
A
30
B
30 0
35
B
m
B
15 0
B
m
40
C on tro l
45
C
C
g/ kg
µmole/mg protein
50
m
C
C
55
Female
Male
80 75 70 65 60 55 50 45 40 35 30
0
Female
60
0
Male
Aaspartate aminotransferase (AST) in liver homogenate
g/ kg
Alanine aminotransferase (ALT) in liver homogenate µmole/mg protein/min.
Alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), glucose, lipid profile: total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides and total lipid; kidney function: creatinine and urea; glucolytic enzymes in
30
(A)
2.2. Kits
Fig. 1. Effect of chitosan oral doses (150 and 300 mg/kg) on male and female mice in liver homogenate of ALT (A), AST (B) and ALP (C) and in serum urea (D), creatinine (E) and total lipid (F). ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
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Cholesterol Male
Male
80
A
C
D
mg /dl
B
B
Female A
A
70 60
B
BC
CD
50
D
40 30 20 10
g m g/ k
kg m g/
0
0
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15
Male
175
A
C on tro l
m g/ kg
kg
30 0
Triglycerides
Female
A
200 180 160 140 120 100 80 60 40 20
m g/
(D)
LDL - Cholesterol Male
15 0
C on tro l
30
15 0
0
m g/
m g/
kg
kg
l tro on C
m g/ kg 30
15 0
0
m g/
on C
kg
0
(C)
Female A
A
150 125
B
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C
D
mg /dl
100
B
75
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C
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50 25
g m g/ k 30 0
kg m g/ 15 0
tr o l on C
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kg 0
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tro C on
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g g/ k m 15 0
on tro l C
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kg
0 l
mg /dl
HDL -Cholesterol
(B)
Female
A
250 225 200 175 150 125 100 75 50 25
tro l
mg /dl
(A)
Fig. 2. Effect of chitosan oral doses (150 and 300 mg/kg) on male and female mice in serum cholesterol (A), HDL-cholesterol (B), LDL-cholesterol (C); and triglycerides (D). ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
liver; phosphofructokinase is measured (PFK), hexokinase (HK), glucose phosphate isomerase (GPI) and lactate dehydrogenase (LDH); protein and glycogen were purchased from Biodiagnostic, Cairo, Egypt. 2.3. Drug Chitosan was purchased from Sedico Pharmaceutical Co., Cairo. The studied doses were converted from human dose 2 g, two and threefolds to mice dose by using multiplication factors for dose conversion between different species by Paget and Barnes (1964). Mice were divided into six equal groups (seven mice each), the 1st three groups (1, 2 and 3) were males and the 2nd were females groups (4, 5 and 6) treated as follows: groups 1 and 4 was control groups and received 10 ml/kg distilled water; groups 2 and 5 orally administered 150 mg/kg; groups 3 and 6 were treated with 300 mg/kg for 35 successive days. At the end of the treatment period, all animals were fasted for 12 h. Blood samples were collected from the retro-orbital venous plexus under diethyl ether anesthesia and left to clot. The sera were separated using cooling centrifugation and stored at 20 °C until analysis. After blood samples were collected, all animals were sacrificed and the liver and kidney tissues of each animal were dissected. Livers were divided into two portions, 1st portion was homogenized and prepared for biochemical analysis, while the 2nd one for histopathological examination.
2.4. Enzyme assays Liver homogenates were performed to determine: PFK according to Zammit et al. (1978), HK (Uyeda and Racker, 1965), GPI (King, 1965), LDH (Babson and Babson, 1973), ALT and AST (Reitman and Frankel, 1957), ALP (Kind and King, 1954), glycogen (Hassid and Abraham, 1957) and protein (Bradford, 1976).
2.5. Serum biochemical analysis Serum was analyzed for determination of total cholesterol and HDL-cholesterol by methods of Stein et al. (1986), LDL-cholesterol (Friedewald, 1972), triglycerides (Wahlefeld, 1974), total lipid (Zollner and Kirsch, 1962), creatinine (Henry, 1974), urea (Fawcett and Soctt, 1960) and glucose (Trinder, 1969).
2.6. Histopathological examinations Liver and kidney specimens were fixed in 10% neutral buffered formalin, dehydrated in alcohol, cleared in xylol and embedded in paraffin. Five micrometer thickness sections were prepared and stained with hematoxilen and Eosin (H and E) for general histological investigations. In histochemical study, sections were stained with Periodic Acid Schiff (PAS) stain to demonstrate glycogen and bromophenol blue stains for determination of total proteins content in liver and kidney tissues (Bancroft and Gamble, 2002).
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Table 1 Chitosan effects (oral doses: 150 and 300 mg/kg) on serum glucose, protein and glycogen contents in liver and kidney homogenates of male and female mice. (n = 7, means ± standard error of means.) Parameters
Groups Male mice
Serum glucose (mg/dl) Protein content (mg/g tissue)
Liver homogenate Kidney homogenate
Glycogen content (lg/g tissue)
Liver homogenate Kidney homogenate
Female mice
Control
150 mg/kg
300 mg/kg
Control
150 mg/kg
300 mg/kg
A 91.7 ± 0.59 A 182.40 ± 1.55 A 150.31 ± 1.72 A 382.2 ± 0.77 A 156.30 ± 1.27
AB 90.32 ± 0.64 B 168.80 ± 2.08 B 135.2 ± 1.71 B 249.7 ± 0.98 B 142.70 ± 1.10
AB 89.50 ± 0.87 C 160.70 ± 1.43 C 127.0 ± 1.28 C 183.60 ± 1.58 C 129.40 ± 0.72
A 92.60 ± 0.95 A 180.12 ± 2.11 A 148.22 ± 1.94 A 378.10 ± 1.82 A 155.11 ± 1.48
AB 90.60 ± 0.77 D 151.20 ± 1.82 D 118.6 ± 1.39 C 179.50 ± 0.70 D 123.10 ± 1.10
B 88.20 ± 0.76 E 142.80 ± 1.52 E 107.32 ± 1.45 D 117.50 ± 1.65 E 104.66 ± 0.82
ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
(A)
(B)
Phosphofructokinase (PFK) in liver homogenate 15
Male
14
C
13 12
BC
B
B
11 10 9
A
A
8 7
C
85
CD
80 75
BD
B
70
A
A
65 60 55
6
m g/ kg
m g/ kg
30 0
0
30 0
30
15
0
C on tro
m g/ kg
m g/ kg
l
15
m g/
0
C on tro
kg
kg m g/
l tro
15 0
30 0
C
on
m g/
kg
kg
l
m g/
tro
15 0
on C
l
50
5
(C)
(D)
Glucose phosphate isomerase (GPI) in liver homogenate D C
B
BC
140 130
A
A
Female
Male
120 110
µ mole/mg protein/min.
Male 150
Hexokinase (HK) in liver homogenate 0.06
Female
160
µ mole/mg protein/min.
Female
Male
90
µ mole/mg protein/min.
µ mole/mg protein/min.
Lactate dehydrogenase (LDH) in liver homogenate
Female
E
0.05
C D
0.04
B 0.03 0.02
A
A
0.01
kg m g/ 30 0
15 0
m g/
kg
l C
on
tro
kg m g/ 30 0
m g/
C
15 0
tro
l
kg
0.00 on
m g/ kg 30 0
kg m g/ 15 0
C on tro l
kg m g/ 30 0
m g/ kg 15 0
C on tro l
100
Fig. 3. Effect of chitosan oral doses (150 and 300 mg/kg) on glycolytic enzymes in liver homogenate: PFK (A), LDH (B), GPI (C) and HK (D) of male and female mice. ANOVAone way, at P < 0.05. The different capital letters above columns are significantly different.
2.7. Quantitative analysis Quantitative analysis was achieved for the gray areas in histochemical stains using computerized image analyzer (Leica Qwin
500 image), Image Analyzer Unit, Pathology Department, National Research Center, Cairo, Egypt. The average means ± standard errors of the means were calculated from 10 fields per section at magnification 100.
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Fig. 4. (A) A photomicrograph of the control liver of male mice with central vein (CV) and surrounding hepatocytes (H), sinusoids (S) and nucleus (N). (B) Male mice administration chitosan (150 mg/kg, b.w.) showing inflammatory cellular infiltrations (thick arrow) around the portal tract, dilated blood sinusoids (S). (C) Male mice administered chitosan (300 mg/kg, b.w.) showing dilated and congested central vein (thick arrow), dilated blood sinusoids (S) and hemorrhage (thin arrow) vacuolar change in the cytoplasm of hepatocytes (head arrow). (D) The higher magnification showing vacuolar change in the cytoplasm of hepatocytes (thick arrow) with hemorrhage between hepatocytes (star) some pyknotic cells (head arrow) (H and E 400).
Fig. 5. (A) A photomicrograph of the control liver of female mice with central vein (CV) and surrounding hepatocytes (H), sinusoids (S) and nucleus (N). (B) female mice administration chitosan (150 mg/kg, b.w.) showing dilatation in central vein (long arrow) and inflammatory cellular infiltrations (arrow head) around the central vein (C) female mice administration chitosan (300 mg/kg, b.w.) showing congestion of portal tact (long arrow) and dilated blood sinusoids (S). (D) In the higher magnification showing vacuolar degeneration of hepatocytes (thin arrow), pyknotic cells (arrow head). Note binucleated hepatocytes (BN). (H and E 400).
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Fig. 6. (A and D) Section in the kidney of control male and female mice showing glomerulus (G), and renal tubules (T). (B) Male mice administration chitosan (150 mg/kg, b.w.) showing hypercellularity of glomerulus (long arrow) and degenerated of tubules (arrow head). (C) Male mice administration chitosan (300 mg/kg, b.w.) showing hypercellularity and highly degenerated of glomerulus (long arrow) and tubules (arrow head). (E) Female mice administration chitosan (150 mg/kg, b.w.) showing hypercellularity of glomerulus (long arrow) and degenerated of tubules with interstitial hemorrhage (star). (F) Female mice administration chitosan (300 mg/kg, b.w.) showing hypercellularity of glomerulus with degenerated (long arrow) of tubules (H and E 400.
2.7.1. Statistical analysis Data were statistically analyzed using ANOVA one-way test and least significant difference of the means (LSD) at P < 0.05 using Graph Pad Prism 5 software package.
3. Results 3.1. Biochemical results The present results revealed that chitosan treatment caused significant elevation in liver enzyme markers (ALT, AST and ALP) and kidney function tests (urea and creatinine) for both sex in dose dependent manner as shown in (Fig. 1A–E). The resultant effect of chitosan was similar in male and female mice (no sex differences were observed). Treatment with chitosan 300 mg/kg caused significant elevation in liver homogenate of ALT, AST, ALP, serum urea and creatinine in male and female mice, respectively, when compared with control or lower dose of chitosan-treated groups. Moreover, the resultant effect of chitosan treatment with 150 and 300 mg/kg on lipid profile generally caused significant decrease in its parameters for male and female treated groups. Total lipid values significantly decreased in both male and female
treated groups compared with their respective controls. This decrease is most abundant in females treated with chitosan (150 mg/kg) than male mice. Whereas, there were no significant differences in TL levels in female-treated groups (lower and higher doses of chitosan) and in male – administered the higher dose as shown in (Fig. 1F). Cholesterol levels decreased significantly in all treated groups with chitosan (150 and 300 mg/kg) when compared with the control groups (Fig. 2A). While, female mice treated with 300 mg/kg chitosan resulted significant reduction in cholesterol value more than any treated groups. HDL-cholesterol values showed moderate significant decrease in male and female treated groups in both doses of chitosan administration in dose dependent manner as illustrated in (Fig. 2B). LDL-cholesterol values had similar behavior to that of cholesterol levels in the treatment groups (Fig. 2C); in which, the higher dose of chitosan caused significant reduction in LDL-cholesterol especially in females more than male mice. Chitosan lower dose also caused significant decrease in cholesterol and LDL-cholesterol in male and female groups comparing with their respective controls without gender effect. Triglycerides level significantly decreased in chitosan treatment in higher and lower doses for both sexes. No-significant differences
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Fig. 7. (A and D) A photomicrograph of control liver of male and female mice shows the normal distribution of glycogen in hepatocytes with intense red color. (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing decrease in glycogen content in hepatocytes. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly decreases in glycogen content in hepatocytes. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing decrease in glycogen content in hepatocytes. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly decreases in glycogen content in hepatocytes (PAS 400).
between male treated with chitosan higher dose and female groups treated with 150 and 300 mg/kg. (Fig. 2D). However, chitosan lower dose (150 mg/kg) had gender effect on triglycerides level; significant elevation in triglycerides values were resulted in males when compared to female – treatment group. Fasting serum glucose level did not changed in all treatment groups (non-significant decrease) except for the female mice treated with chitosan (300 mg/kg) their glucose value decreased significantly than controls as shown in Table 1. Chitosan treatment caused significant decrease in protein content in liver and kidney homogenate (Table 1); particularly in females more in males (dose dependant manner) when compared to controls values. Generally, glycogen content in liver and kidney homogenate significantly decreased in all treated groups; gender effect is more obvious in females than in males treated with the higher dose of chitosan as illustrated in Table 1. Data obtained from glycolytic enzymes analysis showed significant enhancement of their activities. Significant elevation was obtained in PFK and LDH values in liver homogenate in all treated groups with the two doses of chitosan (150 and 300 mg/kg); whereas, the higher dose caused more
effect in male and female mice (no sex differences were observed in these two parameters) as shown in Fig. A and B. The resultant effect of chitosan administration on GPI (Fig. 3C) and HK (Fig. 3D) showed sex difference. Chitosan (two doses) caused significant elevation in GPI and HK values of female mice groups more than male groups treated with same doses of chitosan. Drastic effect of chitosan (300 mg/kg) was clearly obvious in the values of GPI and HK in female mice (significance at P < 0.05) when compared with the same dose in male mice treatment group.
3.2. Histopathological examination Histopathological results: in control male and female mice, liver showed normal histological features within the hepatic lobules, normal central vein, hepatic cords separated with blood sinusoids, portal tract and prominent nucleus (Figs. 4A and 5A). Chitosan treatment in male mice caused distortion of the normal hepatic of the normal hepatic architecture with mononuclear cell infiltration mainly in the portal tracts for 35 days at low dose (150 mg/kg, b.w.) (Fig. 4B). Degeneration, necrosis, eosinophilic substances were noticed in hepatic lobules, vacuolated cytoplasm,
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Fig. 8. (A and D) Photomicrograph of section of kidney of normal male and female mice showing normal distribution in glycogen content in glomerulus (long arrow) and tubules (arrow head). (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing decrease in distribution in glycogen content in glomerulus and tubules. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in distribution in glycogen content in glomerulus and tubules. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing decrease in distribution in glycogen content in glomerulus and tubules. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in distribution in glycogen content in glomerulus and tubules (PAS 400).
and presence of intracellular hemorrhage between hepatocytes in mice treated with chitosan 300 mg/kg, b.w. (Fig. 4C and D). Female mice treated with the lower dose of chitosan resulted dilatation of the central veins, encouraged with destructed red blood cells (Fig 5B). While the higher dose caused cytoplasmic vacuolation in the hepatocytes, fatty degeneration and leukocytic infiltration as shown in Fig. 5C and D. Severe pathological changes were observed in the liver of females comparing to males mice; particularly, the degree of degeneration, necrosis and mononuclear cell infiltration mainly in the portal tracts. Kidney: general histopathological examination of kidney in control male and female mice appeared normal glomerulus, bowman capsule, proximal and distal tubules as shown in (Fig. 6A and D). The most consistent finding in the histological section of kidney of male and female mice treated with chitosan at dose (150 mg/kg) showed hypercellularity and degeneration of glomeruli and tubules (Fig. 6B and E), respectively. The glomeruli and tubules became obvious and worthy in all treated groups with the higher dose of chitosan (Fig. 6C and F). From previous examinations, it is clearly to demonstrate that kidneys of female mice showed sever degeneration and hypercellularity of glomeruli and tubules when compared to males-kidney.
3.3. Histochemical studies Examination of control liver sections of mice stained with Periodic Acid Schiff’s (PAS) showed hepatocytes glycogen granules in the cytoplasm of hepatocytes (Fig. 7A and D). Daily treatment with chitosan at dose (150 mg/kg) for 35 days showed slight decreased in the glycogen content in male and female mice (Fig. 7B and E). Treatment with chitosan at dose (300 mg/kg) for 35 days induced marked decrease in glycogen content in female than male (Fig. 7C and F). The histochemical study of kidney sections of control mice revealed PAS positive material in the brush borders of the proximal tubules and the basement membrane of the renal tubules and glomeruli (Fig. 8A and D). PAS staining of kidney sections of mice treated with chitosan at dose (150 mg/kg) for 35 days showed slight decrease of the glycogen content in the renal tubules and glomeruli (Fig. 8B and E). Whereas examination of kidney sections of mice treated with chitosan at dose (300 mg/kg) for 35 days revealed marked decrease in glycogen content in female than male (Fig. 8C and F). Examination of control liver sections of mice showed protein content in the cytoplasm of hepatocytes. Some nuclei showed deep
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Fig. 9. (A and D) A photomicrograph of control liver of male and female mice showing normal distribution of protein content in hepatocytes. (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing mild decrease in protein content in hepatocytes. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in protein content in hepatocytes. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing mild decrease in protein content in hepatocytes. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in protein content in hepatocytes (Promophenol blue 400).
protein content (Fig. 9A and D). After daily treatment of mice with chitosan at dose (150 mg/kg) for 35 days, the protein inclusions showed slight in the cytoplasm of hepatocytes in male and female (Fig. 9B and E). Highly decrease in the protein inclusions after treatment with chitosan at dose with chitosan at dose (300 mg/ kg) for 35 days in the female mice than male was observed (Fig. 9C and F). Bromophenol blue stained kidney sections from control mice revealed strong protein deposits in the cytoplasm and nuclei of the renal tubules and glomeruli (Fig. 10A and D). The renal tubules and glomeruli of the kidney of mice treated with chitosan at dose (150 mg/kg) for 35 days showed decrease in the protein contents (Fig. 10B and E). There was a marked decreased of the protein content of the renal tubules and glomeruli treated with at dose (300 mg/kg) for 35 days. The decrease was obviously in female than male (Fig. 10C and F). 3.4. Quantitative analysis The optical density of glycogen and total protein contents were performed using image analysis system and revealed the histochemical investigation of glycogen and protein reaction in liver,
kidney of mice within different treatment groups showed a significantly decrease in glycogen and total protein content compared to controls (P < 0.05). The female was more effective than male (Fig. 11A–D).
4. Discussion The selected oral doses of chitosan 150 and 300 mg/kg treatment in male and female mice for 35 days caused significant (P < 0.05) elevation in the levels of liver enzyme markers (ALT, AST, ALP, LDH, PFK, GPI and HK) in liver homogenate as well as, there were significant increase in kidney functions (creatinine and urea) in dose dependent manner. Furthermore, there were significant decrease in protein and glycogen contents in liver and kidney homogenates due to gender and dose response effects. Significant hypoglycemia was observed only in females treated with chitosan 300 mg/kg. The activities of liver enzymes are sensitive indicators of hepatocellular damage; the release of these enzymes interacting with cellular structure and function resulting in tissue degeneration (Sudhahar et al., 2007).
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Fig. 10. (A and D) A photomicrograph of control kidney of male and female mice showing normal distribution of protein content in glomerulus (long arrow) and tubules (arrow head). (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing reduce in distribution of protein content in glomerulus and tubules. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly reduce in distribution of protein content in glomerulus and tubules. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing reduce in distribution of protein content in glomerulus and tubules. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly reduce in distribution of protein content in glomerulus and tubules (Promophenol blue 400).
In our study, it was noticed that the low dose levels of chitosantreatment altered the liver enzymes activities in both male and female mice treatment groups. This finding is in line with the earlier report of Imogen et al. (1995). Chitosan induced many histopathological changes in liver and kidney of rats and the magnitude of such changes were dose dependent. Examination of section of male and female mice showed distortion of the normal hepatic architecture with mononuclear cell infiltration mainly in the portal tracts at low dose. The cytoplasm of hepatocytes in the hepatic lobules appeared vacuolated and intracellular hemorrhage between hepatocytes at the higher dose. In kidney showed hypercellularity and degeneration of glomeruli and tubules. Serum creatinine and urea significantly (P < 0.05) elevated in all treatment groups with chitosan in dose related increase, whereas no gender effect was found in these two parameters. While, the treatment with the lower dose of chitosan showed significant increase in creatinine value comparing with male group treated with the same dose of chitosan. From previous findings it was proved that chitosan treatment caused nephrotoxicity and these results were supported by histopathological examination of the kidney, which revealed that hypercellularity of glomerulus and degenera-
tion in renal tubules with the interstitial hemorrhage. The severity increased by the higher dose. Urea level was significantly elevated mainly due to hepatic failure caused metabolic interruption in protein metabolism to convert amino acids and ammonia to urea (Santhosh et al., 2007). This fact explains the reduction in protein content in liver and kidney tissues of all chitosan treated groups. Our study resulted significant elevation in glycolytic enzymes activity (LDH, PFK, GPI and HK) in liver tissue of chitosan administration. This could be attributed to an increase in metabolic activity of chitosan which compensate the inhibition of Kreb’s cycle and mitochondrial oxidation which favors the conversion of pyruvate to lactate (Ahmed and Gad, 1995; Tielens, 1997). Landes and Bough (1976) stated that in an eight weeks feeding study, it has been suggested that chitosan is safe in rats up to 10% in the diet. At 15% level, enlargement of liver and kidneys with a few other changes have been observed. This is confirmed the histopathological observation in our study. There is significant evidence that long-term; high-dose chitosan supplementation can result in malabsorption of some crucial vitamins and minerals including calcium, magnesium, selenium, and vitamins A, D, E and K (Koide, 1998; Yao et al., 2010).
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Female
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Fig. 11. Effect of chitosan oral doses (150 and 300 mg/kg) on glycogen content of liver (A) and kidney (B); and on protein content of liver (C) and kidney (D). ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
In accordance to the present results, Tsung and Tsan (2002) and Hossain et al. (2007) reported that, chitosan intake was effective in lowering the plasma total cholesterol, VLDL-cholesterol and LDL-cholesterol levels in experimental animals. The hypocholesterolemic property of chitosan is probably related to its ability to inhibit the total lipid accumulation in serum and liver by its antilipidemic property, enhancing cholesterol 1-7-hydroxylase enzyme, regulate lipoprotein metabolism and alter VLDL particle size (Ausar et al., 2003; Xing et al., 2005; Hossain et al., 2007). In addition, the significant reduction in triglycerides in chitosan administered mice may related to chitosan exhibited inhibition of pancreatic lipase activity in liver and kidney that leading to increase in fecal fat and /or bile acid excretion beside, decrease in the absorption of dietary lipids (He and Xue, 2004; Sumiyoshi and Kimura, 2006). Xiao-hong et al. (2004) explained the decrease in rat serum free fatty acids, triglycerides, total cholesterol; fatty liver after chitosan –administration by inducing peroxisome proliferators activated receptor alpha mRNA expression and promoting liver intake and oxidation of fatty acid. Lee et al. (2008) indicate that estrogen induces the secretion of hormones that increase with stress .This in turn results in growth suppression. These hormones likely change the animal’s energy use towards a catabolic direction. Whereas, Lee et al. (1999) found the gain/food ratio was increased by gonadectomy and decreased by 17b-estradiol replacement in female rats.
Testosterone may function through regulation of the LDL-receptor and 7a-hydroxylase at the gene transcription level in liver. The effect of testosterone, a known anabolic, on cholesterol metabolism may also be partly mediated through secretion of growth hormone, which is shown to increase hepatic LDL receptor expression in rats (Rudling and Angelin, 1993). However, it could be suggested that females are less sensitive than males in feedback to control cholesterol synthesis and able to loss fats more than male (Fernandez et al., 1995). In the present study, glucose level significantly reduced only in females treated with chitosan (300 mg/kg) as well as non-significant decrease in other treated groups with chitosan (male or female mice). Few reports have claimed that chitosan has antidiabetic effects (Miura et al., 1995). Chitosan may be primarily absorbed after it has been transformed into oligosaccharides by chitosanase secreted from intestinal bacteria, or by lysozyme in intestinal fluid. Consequently, chitosan may exert its hypoglycemic effect with the oligosaccharides, but not with the monosaccharide (Hayashi and Ito, 2000).
5. Conclusion Therefore, the gender difference had a major role to induce hepato-renal toxicity in females more than males administered higher doses of chitosan for a long period.
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Conflict of interest There was no conflict of interest for any of the authors of this paper. References Ahmed, S.G., Gad, M.Z., 1995. Effect of schistosomal infection and its treatment on some key enzymes of glucose metabolism in mice livers. Arzneimittelforschung 45, 1324–1330. Ausar, S.F., Morcillo, M., Leon, A.E., Ribotta, P.D., Bianco, I.D., 2003. Improvement of HDL and LDL-cholesterol levels in diabetic subjects by feeding bread containing chitosan. J. Med. Food 6, 397–399. Babson, A.L., Babson, S.R., 1973. Kinetic colorimetric measurement of serum lactate dehydrogenase activity. Clin. Chem. 19, 766. Bancroft, J.D., Gamble, M., 2002. Theory and Practice of Histological Techniques, fifth ed. Churchill Livingstone Pub., Edinburgh (pp. 172–175, 593–620). Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cohen, D.E., 2004. Cholesterol absorption as a target for lipid-lowering therapy. Formulary 39, 3–7. Fawcett, J.K., Soctt, J.E., 1960. Urease–Berthelot method. J. Clin. Pathol. 13, 156–159. Fernandez, M.I., Vergara–Jimenez, M., Romero, A.L., Erickson, S.K., McNamara, D.J., 1995. Gender differences in response to dietary soluble fiber in guinea pig: effect of pectin, guar gum, and psyllium. J. Lipid Res. 36, 2191–2202. Friedewald, W.T., 1972. Total triglycerides, total cholesterol and high densitylipoprotein cholesterol assays. Clin. Chem. 18, 499. Hassid, W.Z., Abraham, S., 1957. Chemical procedures for analysis of polysaccharides. In: Colowick, S.P., Kaplen, N.O. (Eds.), Methods in Enzymology, vol. 3. Academic Press, New York, pp. 34–37. Hayashi, K., Ito, M., 2000. Antidiabetic action of low molecular weight chitosan in genetically obese diabetic KK-Ay mice. Biol. Pharm. Bull. 25, 188–192. He, X.B., Xue, C.K., 2004. Experimental study of the hepatoprotective effect of chitosan. Herald Med. 23, 45–65. Henry, R.J., 1974. Creatinine colorimetric method. In: Clinical Chemistry. Principles and Techniques, second ed. Harper and Row, p. 525. Hossain, S., Rahman, A., Kabir, Y., Shams, A.A., Afrose, F., Hashimoto, M., 2007. Effect of shrimp (Macrobracium rosenbergii) – derived chitosan on plasma lipid profile and liver lipid peroxide level in normo- and hypercholesterolaemic rats. Clin. Exp. Pharmacol. Physiol. 34, 170–176. Imogen, M., Julia, W., Sarah, F., Roger, W., 1995. Anti-tuberculous therapy and acute liver failure. Lancet 345, 555–556. Kind, P.R., King, E.J., 1954. Estimation of plasma phosphatase by determination of hydrolysed phenol (with-antipyrine). J. Clin. Phathol. 7, 322. King, J., 1965. Glucose phosphate isomerase. In: Practical Clinical Enzymology, D. Van Nostr and Co. Ltd., London, pp.1113–1117. Koide, S.S., 1998. Chitin-chitosan: properties, benefits and risks. Nutr. Res. 18, 1091–1101. Landes, D.R., Bough, W.A., 1976. Effects of chitosan – a coagulating agent for food processing wastes in the diets of rats – on growth and liver and blood composition. Bull. Environ. Contam. Toxicol. 15 (5), 555–563. Lee, C.E., Koh, D.H., Chang, Y.M., Kim, K.I., 1999. Effects of sex and diets containing alfalfa, laver or pine-needle meals on the plasma cholesterol level in rats. Nutr. Sci. 2, 46–50. Lee, C.E., Kang, J., Klim, K., 2008. Effect of gender, gonadectomy and sex hormones on growth and plasma cholesterol level in rats. Ann. Nutr. Metab. 53, 1–5. Miura, T., Usami, M., Tsuura, Y., Ishida, H., Seino, Y., 1995. Hypoglycemic and hypolipidemic effect of chitosan in normal and neonatal streptozotocininduced diabetic mice. Biol. Pharm. Bull. 18, 1623–1625. Muzzarelli, R.A.A., 1977. Enzymatic synthesis of chitin and chitosan. Occurrence of chitin. In: Chitin. Pergamon Press, New York, NY, pp. 5–17. Ormrod, D.J., Holmes, C.C., Miller, T.E., 1998. Dietary chitosan inhibits hypercholesterolaemia and atherogenesis in the apolipoprotein E deficient mouse model of atherosclerosis. Atherosclerosis 138, 329–334. Orr, A., 1999. Another New Diet Craze? [].
Paget, G.E., Barnes, J.M., 1964. Interspecies dosage conversion scheme in evaluation of results and quantitative application in different species. In: Laurence, D.R., Bacharach, A.L. (Eds.) ‘‘Evaluation of Drug Activities: Pharmacometrics’’, vol. 1. Academic press, London and New York, pp. 160–162. Razdan, A., Pettersson, D., 1996. Hypolipidaemic, gastrointestinal and related responses of broiler chickens to chitosans of different viscosity. Br. J. Nutr. 76, 387–397. Razdan, A., Pettersson, D., Pettersson, J., 1997. Broiler chicken body weights, feed intakes, plasma lipid and small-intestinal bile acid concentrations in response to feeding of chitosan and pectin. Br. J. Nutr. 78, 283–291. Reitman, S., Frankel, S., 1957. Determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56–60. Rudling, M., Angelin, B., 1993. Loss of resistance to dietary cholesterol in the rat after hypophysectomy: importance for hepatic low density lipoprotein– receptor expression . Proc. Natl. Acad. Sci. USA 90, 8851–8855. Santhosh, S., Sini, T.K., Anandan, R., Mathew, P.T., 2007. Hepatoprotective activity of chitosan against experimental rats. Eur. J. Pharmacol. 572, 69–73. Senel, S., McClure, S.J., 2004. Potential applications of chitosan in veterinary medicine. Adv. Drug. Deliver. Rev. 56, 1467–1480. Stein, E.A., DiPersio, L., Peace, A.J., Kashyap, M., Kao, J., Srivastava, L., McNemey, C., 1986. Enzyme-linked immunoabsorbant assay of apolipoprotein all in plasma, with use of a monoclonal antibody. Clin.Chem. 32 (6), 967–971. Sudhahar, V.S.A., Kumar, P.T., Sudharsan, S., Varalakshmi, P., 2007. Protective effect of lupeol and its ester on cardiac abnormalities in experimental hypercholesterolemia. Vascul. Pharmacol. 46, 412–418. Sumiyoshi, M., Kimura, Y., 2006. Low molecular weight chitosan inhibits obesity induced by feeding a high-fat diet long-term in mice. J. Pharm. Pharmacol. 58, 201–207. Tanaka, Y., Tanioka, S., Tanaka, M., Tanigawa, T., Kitamura, Y., Minami, S., Okamoto, Y., Miyashita, M., Nanno, M., 1997. Effects of chitin and chitosan particles on BALB/c mice by oral and parenteral administration. Biomaterials 18 (8), 591– 595. Tielens, A.G., 1997. Biochemistry of trematode. In: Fried, B., Graczyk, T.K. (Eds.), Advances in Trematode Biology. CRC Press, Bocaraton, pp. 309–343. Tozaki, H., Komoie, J., Tada, C., Maruyama, T., Terabe, A., Suzuki, T., Yamamoto, A., Muranishi, S., 1997. Chitosan capsules for colon specific drug delivery: improvement of insulin absorption from of rat colon. J. Pharm. Sci. 86, 1016– 1021. Trinder, P., 1969. Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen. J. Clin. Pathol. 22, 158–161. Tsung, Y.H., Tsan, C.M., 2002. Plasma lipoprotein cholesterol in rats fed a diet enriched in chitosan and cholesterol. J. Nutr. Sci. Vitaminol. 48, 379–383. Uyeda, K., Racker, E., 1965. Regulatory mechanisms in carbohydrate metabolism. VII. Hexokinase and phosphofructokinase. J. Biol. Chem. 240, 4682. van Bennekum, A.M., Nguyen, D.V., Schulthess, G., Hauser, H., Phillips, M.C., 2005. Mechanisms of cholesterol-lowering effects of dietary insoluble fibres: relationships with intestinal and hepatic cholesterol parameters. Br. J. Nutr. 94, 331–337. Wahlefeld, A.W., 1974. In: Bergmeyer, H.U. (Ed.), Method of Enzymatic Analysis, vol. 5, Academic Press, New York, pp. 1831–1835. Xiao-hong, Q., Zhang, Z.P., Li, X.Y., Que, L.L., Wu, C.Z., 2004. Therapeutic effect and mechanisms of chitosan compound on rats of experimental fatty liver. Zhongguo Xinyao Yu Linchuang Zazhi 23, 283–286. Xing, R., Liu, S., Guo, Z., Yu, H., Wang, P., Li, C., Li, Z., Li, P., 2005. Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro. Bioorg. Med. Chem. 13, 1573–1577. Yao, H.T., Lii, C.K., Chou, R.H., Lin, J.H., Yang, H.T., 2010. Effect of chitosan on hepatic drug-metabolizing enzymes and oxidative stress in rats fed low- and high-fat diets. Agric. Food Chem. 58 (8), 5187–5193. Ylitalo, R., Lehtinen, S., Wuollijoki, E., Ylitalo, P., Lehtimaki, T., 2002. Cholesterollowering properties and safety of chitosan. Arzneimittel-Forschung-Drug Res. 52, 1–7. Zammit, V.A., Beis, I., Newsholme, E.A., 1978. Maximum activities and effects of fructose biphosphate on pyruvate kinase from muscles of vertebrates and invertebrates in relation to the control of glycolysis. Biochem. J. 174, 989. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109–110. Zollner, N., Kirsch, K., 1962. Total lipids colorimetric method. Z. Ges. Exp. Med. 135, 545.
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Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Chitosan induced hepato-nephrotoxicity in mice with special reference to gender effect in glycolytic enzymes activities Enayat A. Omara a,⇑, Hanan F. Aly b, Somaia A. Nada c a
Pathology Dept., National Research Centre, P.O. 12622, Dokki, Cairo, Egypt Therapeutic Chemistry Dept., National Research Centre, P.O. 12622, Dokki, Cairo, Egypt c Pharmacology Dept., National Research Centre, P.O. 12622, Dokki, Cairo, Egypt b
a r t i c l e
i n f o
Article history: Received 25 June 2011 Available online 2 December 2011 Keywords: Chitosan Biochemical Histopathological Histochemical Mice
a b s t r a c t Chitosan is an antilipidemic dietary supplement used as a diet aide. The present study investigated the effect of sex–toxicity relationship between male and female mice orally given two dose levels (150 and 300 mg/kg) for 35 days. Chitosan treatment caused significant elevation in transaminases (ALT, AST) and alkaline phosphatase (ALP) in liver and in serum urea and creatinine in dose dependent manner; no sex differences between-treated groups. Lipid profile parameters significantly decreased and significant increase in glycolytic enzymes activities in all treatment groups. Female mice treated with chitosan (300 mg/kg) had significant reduction in lipid profile parameters than the same dose of male group. Phosphofructokinase (PFK) and lactate dehydrogenase (LDH) activities significantly enhanced without sex differences, while glucose phosphate isomerase (GPI) and hexokinase (HK) significantly elevated in the higher dose of females than male. Histopathological study of liver and kidney tissues showed moderate to severe histopathological changes depend on the dose and gender difference. Image analysis resulted significant depletion in glycogen and protein contents especially in female more than male. These results indicated that female mice were more susceptible to the toxic effect of chitosan than males when administered with the higher dose for a long period. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Chitosan is the deacetylated form of chitin, an aminopolysaccharide found in the exoskeletons and the fungal cell wall of various arthropods including insects, crabs and shrimp (Muzzarelli, 1977). Although it is not derived from plants, it shares the same characteristics as dietary fiber, which is an indigestible polysaccharide by mammalian digestive enzymes (van Bennekum et al., 2005). Special care should be taken in the clinical use of chitosan over a long time to avoid the adverse health effect (Tanaka et al., 1997). Chitosan has profound applications in the fields of clarification and purification, chromatography, paper and textiles, photography, food and nutrition, agriculture, pharmaceutical and medical, cosmetics, biodegradable membranes and biotechnology (Senel and McClure, 2004). Chitosan has been suggested to reduce fat absorption from gastrointestinal tract by binding with anionic carboxyl groups of fatty and bile acids, and it interferes with emulsification of neutral lipids (i.e., cholesterol, other sterols) by binding them with hydrophobic ⇑ Corresponding author. E-mail address: [email protected] (E.A. Omara). 0273-2300/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2011.11.010
bonds (Ylitalo et al., 2002). Inhibition of cholesterol absorption reduces absorption of dietary cholesterol, but more importantly, prevents reabsorption of biliary cholesterol, which is instead eliminated in the feces (Cohen, 2004). Orally administered chitosan binds fat in the intestine, blocking absorption, and has been shown to lower blood cholesterol in animals and humans (Ormrod et al., 1998). Chitosan acts by forming gels in the intestinal tract which entrap lipids but also other nutrients, including fat soluble vitamins and minerals, thus interfering with their absorption (Koide, 1998). The hypolipidemic influence of chitosan may also be due to interruption of the enterohepatic bile acid circulation (Razdan and Pettersson, 1996) and the reduction in duodenal bile acid concentration (Razdan et al., 1997). Chitosan has shown promise as a carrier in colon targeting, results suggesting that degradation by colonic bacterial enzymes might be one of the important properties of chitosan for its successful use in drugs colon targeting (Tozaki et al., 1997). Arsenic may concentrated in the shells as part of their normal development; this in turn may lead to arsenic-laced chitosan supplements. Chitosan cause loss of fat-soluble vitamins and block absorption of medicines such as birth control pills (Orr, 1999).This study was designed to investigate the hepato-renal toxic effect of chitosan on mice and who more susceptible to its toxicity.
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Animal Care and Use Committee and were in accordance with the guidelines of the International Association for the Study of Pain Committee for Research and Ethical Issues (Zimmermann, 1983).
2. Materials and methods 2.1. Animals Swiss albino mice of both sexes weighing 20–25 g were used throughout the experiments. Animals were housed under standard environmental conditions (23 ± 1 °C, 55 ± 5% humidity and a 12-h light:12-h dark cycle); maintained with free access to water and a standard laboratory diet ad libitum. Animal care and the experimental protocols were approved by the National Research Centre
(B)
15
Alkaline Phosphatase (ALP)in liver homogenate
4.50 4.25 4.00 3.75 3.50 3.25 3.00 2.75 2.50 2.25 2.00
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mg /dl A
45
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40 35
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Aaspartate aminotransferase (AST) in liver homogenate
g/ kg
Alanine aminotransferase (ALT) in liver homogenate µmole/mg protein/min.
Alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), glucose, lipid profile: total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides and total lipid; kidney function: creatinine and urea; glucolytic enzymes in
30
(A)
2.2. Kits
Fig. 1. Effect of chitosan oral doses (150 and 300 mg/kg) on male and female mice in liver homogenate of ALT (A), AST (B) and ALP (C) and in serum urea (D), creatinine (E) and total lipid (F). ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
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E.A. Omara et al. / Regulatory Toxicology and Pharmacology 62 (2012) 29–40
Cholesterol Male
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Fig. 2. Effect of chitosan oral doses (150 and 300 mg/kg) on male and female mice in serum cholesterol (A), HDL-cholesterol (B), LDL-cholesterol (C); and triglycerides (D). ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
liver; phosphofructokinase is measured (PFK), hexokinase (HK), glucose phosphate isomerase (GPI) and lactate dehydrogenase (LDH); protein and glycogen were purchased from Biodiagnostic, Cairo, Egypt. 2.3. Drug Chitosan was purchased from Sedico Pharmaceutical Co., Cairo. The studied doses were converted from human dose 2 g, two and threefolds to mice dose by using multiplication factors for dose conversion between different species by Paget and Barnes (1964). Mice were divided into six equal groups (seven mice each), the 1st three groups (1, 2 and 3) were males and the 2nd were females groups (4, 5 and 6) treated as follows: groups 1 and 4 was control groups and received 10 ml/kg distilled water; groups 2 and 5 orally administered 150 mg/kg; groups 3 and 6 were treated with 300 mg/kg for 35 successive days. At the end of the treatment period, all animals were fasted for 12 h. Blood samples were collected from the retro-orbital venous plexus under diethyl ether anesthesia and left to clot. The sera were separated using cooling centrifugation and stored at 20 °C until analysis. After blood samples were collected, all animals were sacrificed and the liver and kidney tissues of each animal were dissected. Livers were divided into two portions, 1st portion was homogenized and prepared for biochemical analysis, while the 2nd one for histopathological examination.
2.4. Enzyme assays Liver homogenates were performed to determine: PFK according to Zammit et al. (1978), HK (Uyeda and Racker, 1965), GPI (King, 1965), LDH (Babson and Babson, 1973), ALT and AST (Reitman and Frankel, 1957), ALP (Kind and King, 1954), glycogen (Hassid and Abraham, 1957) and protein (Bradford, 1976).
2.5. Serum biochemical analysis Serum was analyzed for determination of total cholesterol and HDL-cholesterol by methods of Stein et al. (1986), LDL-cholesterol (Friedewald, 1972), triglycerides (Wahlefeld, 1974), total lipid (Zollner and Kirsch, 1962), creatinine (Henry, 1974), urea (Fawcett and Soctt, 1960) and glucose (Trinder, 1969).
2.6. Histopathological examinations Liver and kidney specimens were fixed in 10% neutral buffered formalin, dehydrated in alcohol, cleared in xylol and embedded in paraffin. Five micrometer thickness sections were prepared and stained with hematoxilen and Eosin (H and E) for general histological investigations. In histochemical study, sections were stained with Periodic Acid Schiff (PAS) stain to demonstrate glycogen and bromophenol blue stains for determination of total proteins content in liver and kidney tissues (Bancroft and Gamble, 2002).
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Table 1 Chitosan effects (oral doses: 150 and 300 mg/kg) on serum glucose, protein and glycogen contents in liver and kidney homogenates of male and female mice. (n = 7, means ± standard error of means.) Parameters
Groups Male mice
Serum glucose (mg/dl) Protein content (mg/g tissue)
Liver homogenate Kidney homogenate
Glycogen content (lg/g tissue)
Liver homogenate Kidney homogenate
Female mice
Control
150 mg/kg
300 mg/kg
Control
150 mg/kg
300 mg/kg
A 91.7 ± 0.59 A 182.40 ± 1.55 A 150.31 ± 1.72 A 382.2 ± 0.77 A 156.30 ± 1.27
AB 90.32 ± 0.64 B 168.80 ± 2.08 B 135.2 ± 1.71 B 249.7 ± 0.98 B 142.70 ± 1.10
AB 89.50 ± 0.87 C 160.70 ± 1.43 C 127.0 ± 1.28 C 183.60 ± 1.58 C 129.40 ± 0.72
A 92.60 ± 0.95 A 180.12 ± 2.11 A 148.22 ± 1.94 A 378.10 ± 1.82 A 155.11 ± 1.48
AB 90.60 ± 0.77 D 151.20 ± 1.82 D 118.6 ± 1.39 C 179.50 ± 0.70 D 123.10 ± 1.10
B 88.20 ± 0.76 E 142.80 ± 1.52 E 107.32 ± 1.45 D 117.50 ± 1.65 E 104.66 ± 0.82
ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
(A)
(B)
Phosphofructokinase (PFK) in liver homogenate 15
Male
14
C
13 12
BC
B
B
11 10 9
A
A
8 7
C
85
CD
80 75
BD
B
70
A
A
65 60 55
6
m g/ kg
m g/ kg
30 0
0
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30
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C
on
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kg
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tro
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on C
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5
(C)
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Glucose phosphate isomerase (GPI) in liver homogenate D C
B
BC
140 130
A
A
Female
Male
120 110
µ mole/mg protein/min.
Male 150
Hexokinase (HK) in liver homogenate 0.06
Female
160
µ mole/mg protein/min.
Female
Male
90
µ mole/mg protein/min.
µ mole/mg protein/min.
Lactate dehydrogenase (LDH) in liver homogenate
Female
E
0.05
C D
0.04
B 0.03 0.02
A
A
0.01
kg m g/ 30 0
15 0
m g/
kg
l C
on
tro
kg m g/ 30 0
m g/
C
15 0
tro
l
kg
0.00 on
m g/ kg 30 0
kg m g/ 15 0
C on tro l
kg m g/ 30 0
m g/ kg 15 0
C on tro l
100
Fig. 3. Effect of chitosan oral doses (150 and 300 mg/kg) on glycolytic enzymes in liver homogenate: PFK (A), LDH (B), GPI (C) and HK (D) of male and female mice. ANOVAone way, at P < 0.05. The different capital letters above columns are significantly different.
2.7. Quantitative analysis Quantitative analysis was achieved for the gray areas in histochemical stains using computerized image analyzer (Leica Qwin
500 image), Image Analyzer Unit, Pathology Department, National Research Center, Cairo, Egypt. The average means ± standard errors of the means were calculated from 10 fields per section at magnification 100.
E.A. Omara et al. / Regulatory Toxicology and Pharmacology 62 (2012) 29–40
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Fig. 4. (A) A photomicrograph of the control liver of male mice with central vein (CV) and surrounding hepatocytes (H), sinusoids (S) and nucleus (N). (B) Male mice administration chitosan (150 mg/kg, b.w.) showing inflammatory cellular infiltrations (thick arrow) around the portal tract, dilated blood sinusoids (S). (C) Male mice administered chitosan (300 mg/kg, b.w.) showing dilated and congested central vein (thick arrow), dilated blood sinusoids (S) and hemorrhage (thin arrow) vacuolar change in the cytoplasm of hepatocytes (head arrow). (D) The higher magnification showing vacuolar change in the cytoplasm of hepatocytes (thick arrow) with hemorrhage between hepatocytes (star) some pyknotic cells (head arrow) (H and E 400).
Fig. 5. (A) A photomicrograph of the control liver of female mice with central vein (CV) and surrounding hepatocytes (H), sinusoids (S) and nucleus (N). (B) female mice administration chitosan (150 mg/kg, b.w.) showing dilatation in central vein (long arrow) and inflammatory cellular infiltrations (arrow head) around the central vein (C) female mice administration chitosan (300 mg/kg, b.w.) showing congestion of portal tact (long arrow) and dilated blood sinusoids (S). (D) In the higher magnification showing vacuolar degeneration of hepatocytes (thin arrow), pyknotic cells (arrow head). Note binucleated hepatocytes (BN). (H and E 400).
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Fig. 6. (A and D) Section in the kidney of control male and female mice showing glomerulus (G), and renal tubules (T). (B) Male mice administration chitosan (150 mg/kg, b.w.) showing hypercellularity of glomerulus (long arrow) and degenerated of tubules (arrow head). (C) Male mice administration chitosan (300 mg/kg, b.w.) showing hypercellularity and highly degenerated of glomerulus (long arrow) and tubules (arrow head). (E) Female mice administration chitosan (150 mg/kg, b.w.) showing hypercellularity of glomerulus (long arrow) and degenerated of tubules with interstitial hemorrhage (star). (F) Female mice administration chitosan (300 mg/kg, b.w.) showing hypercellularity of glomerulus with degenerated (long arrow) of tubules (H and E 400.
2.7.1. Statistical analysis Data were statistically analyzed using ANOVA one-way test and least significant difference of the means (LSD) at P < 0.05 using Graph Pad Prism 5 software package.
3. Results 3.1. Biochemical results The present results revealed that chitosan treatment caused significant elevation in liver enzyme markers (ALT, AST and ALP) and kidney function tests (urea and creatinine) for both sex in dose dependent manner as shown in (Fig. 1A–E). The resultant effect of chitosan was similar in male and female mice (no sex differences were observed). Treatment with chitosan 300 mg/kg caused significant elevation in liver homogenate of ALT, AST, ALP, serum urea and creatinine in male and female mice, respectively, when compared with control or lower dose of chitosan-treated groups. Moreover, the resultant effect of chitosan treatment with 150 and 300 mg/kg on lipid profile generally caused significant decrease in its parameters for male and female treated groups. Total lipid values significantly decreased in both male and female
treated groups compared with their respective controls. This decrease is most abundant in females treated with chitosan (150 mg/kg) than male mice. Whereas, there were no significant differences in TL levels in female-treated groups (lower and higher doses of chitosan) and in male – administered the higher dose as shown in (Fig. 1F). Cholesterol levels decreased significantly in all treated groups with chitosan (150 and 300 mg/kg) when compared with the control groups (Fig. 2A). While, female mice treated with 300 mg/kg chitosan resulted significant reduction in cholesterol value more than any treated groups. HDL-cholesterol values showed moderate significant decrease in male and female treated groups in both doses of chitosan administration in dose dependent manner as illustrated in (Fig. 2B). LDL-cholesterol values had similar behavior to that of cholesterol levels in the treatment groups (Fig. 2C); in which, the higher dose of chitosan caused significant reduction in LDL-cholesterol especially in females more than male mice. Chitosan lower dose also caused significant decrease in cholesterol and LDL-cholesterol in male and female groups comparing with their respective controls without gender effect. Triglycerides level significantly decreased in chitosan treatment in higher and lower doses for both sexes. No-significant differences
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35
Fig. 7. (A and D) A photomicrograph of control liver of male and female mice shows the normal distribution of glycogen in hepatocytes with intense red color. (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing decrease in glycogen content in hepatocytes. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly decreases in glycogen content in hepatocytes. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing decrease in glycogen content in hepatocytes. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly decreases in glycogen content in hepatocytes (PAS 400).
between male treated with chitosan higher dose and female groups treated with 150 and 300 mg/kg. (Fig. 2D). However, chitosan lower dose (150 mg/kg) had gender effect on triglycerides level; significant elevation in triglycerides values were resulted in males when compared to female – treatment group. Fasting serum glucose level did not changed in all treatment groups (non-significant decrease) except for the female mice treated with chitosan (300 mg/kg) their glucose value decreased significantly than controls as shown in Table 1. Chitosan treatment caused significant decrease in protein content in liver and kidney homogenate (Table 1); particularly in females more in males (dose dependant manner) when compared to controls values. Generally, glycogen content in liver and kidney homogenate significantly decreased in all treated groups; gender effect is more obvious in females than in males treated with the higher dose of chitosan as illustrated in Table 1. Data obtained from glycolytic enzymes analysis showed significant enhancement of their activities. Significant elevation was obtained in PFK and LDH values in liver homogenate in all treated groups with the two doses of chitosan (150 and 300 mg/kg); whereas, the higher dose caused more
effect in male and female mice (no sex differences were observed in these two parameters) as shown in Fig. A and B. The resultant effect of chitosan administration on GPI (Fig. 3C) and HK (Fig. 3D) showed sex difference. Chitosan (two doses) caused significant elevation in GPI and HK values of female mice groups more than male groups treated with same doses of chitosan. Drastic effect of chitosan (300 mg/kg) was clearly obvious in the values of GPI and HK in female mice (significance at P < 0.05) when compared with the same dose in male mice treatment group.
3.2. Histopathological examination Histopathological results: in control male and female mice, liver showed normal histological features within the hepatic lobules, normal central vein, hepatic cords separated with blood sinusoids, portal tract and prominent nucleus (Figs. 4A and 5A). Chitosan treatment in male mice caused distortion of the normal hepatic of the normal hepatic architecture with mononuclear cell infiltration mainly in the portal tracts for 35 days at low dose (150 mg/kg, b.w.) (Fig. 4B). Degeneration, necrosis, eosinophilic substances were noticed in hepatic lobules, vacuolated cytoplasm,
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Fig. 8. (A and D) Photomicrograph of section of kidney of normal male and female mice showing normal distribution in glycogen content in glomerulus (long arrow) and tubules (arrow head). (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing decrease in distribution in glycogen content in glomerulus and tubules. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in distribution in glycogen content in glomerulus and tubules. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing decrease in distribution in glycogen content in glomerulus and tubules. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in distribution in glycogen content in glomerulus and tubules (PAS 400).
and presence of intracellular hemorrhage between hepatocytes in mice treated with chitosan 300 mg/kg, b.w. (Fig. 4C and D). Female mice treated with the lower dose of chitosan resulted dilatation of the central veins, encouraged with destructed red blood cells (Fig 5B). While the higher dose caused cytoplasmic vacuolation in the hepatocytes, fatty degeneration and leukocytic infiltration as shown in Fig. 5C and D. Severe pathological changes were observed in the liver of females comparing to males mice; particularly, the degree of degeneration, necrosis and mononuclear cell infiltration mainly in the portal tracts. Kidney: general histopathological examination of kidney in control male and female mice appeared normal glomerulus, bowman capsule, proximal and distal tubules as shown in (Fig. 6A and D). The most consistent finding in the histological section of kidney of male and female mice treated with chitosan at dose (150 mg/kg) showed hypercellularity and degeneration of glomeruli and tubules (Fig. 6B and E), respectively. The glomeruli and tubules became obvious and worthy in all treated groups with the higher dose of chitosan (Fig. 6C and F). From previous examinations, it is clearly to demonstrate that kidneys of female mice showed sever degeneration and hypercellularity of glomeruli and tubules when compared to males-kidney.
3.3. Histochemical studies Examination of control liver sections of mice stained with Periodic Acid Schiff’s (PAS) showed hepatocytes glycogen granules in the cytoplasm of hepatocytes (Fig. 7A and D). Daily treatment with chitosan at dose (150 mg/kg) for 35 days showed slight decreased in the glycogen content in male and female mice (Fig. 7B and E). Treatment with chitosan at dose (300 mg/kg) for 35 days induced marked decrease in glycogen content in female than male (Fig. 7C and F). The histochemical study of kidney sections of control mice revealed PAS positive material in the brush borders of the proximal tubules and the basement membrane of the renal tubules and glomeruli (Fig. 8A and D). PAS staining of kidney sections of mice treated with chitosan at dose (150 mg/kg) for 35 days showed slight decrease of the glycogen content in the renal tubules and glomeruli (Fig. 8B and E). Whereas examination of kidney sections of mice treated with chitosan at dose (300 mg/kg) for 35 days revealed marked decrease in glycogen content in female than male (Fig. 8C and F). Examination of control liver sections of mice showed protein content in the cytoplasm of hepatocytes. Some nuclei showed deep
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37
Fig. 9. (A and D) A photomicrograph of control liver of male and female mice showing normal distribution of protein content in hepatocytes. (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing mild decrease in protein content in hepatocytes. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in protein content in hepatocytes. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing mild decrease in protein content in hepatocytes. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly decrease in protein content in hepatocytes (Promophenol blue 400).
protein content (Fig. 9A and D). After daily treatment of mice with chitosan at dose (150 mg/kg) for 35 days, the protein inclusions showed slight in the cytoplasm of hepatocytes in male and female (Fig. 9B and E). Highly decrease in the protein inclusions after treatment with chitosan at dose with chitosan at dose (300 mg/ kg) for 35 days in the female mice than male was observed (Fig. 9C and F). Bromophenol blue stained kidney sections from control mice revealed strong protein deposits in the cytoplasm and nuclei of the renal tubules and glomeruli (Fig. 10A and D). The renal tubules and glomeruli of the kidney of mice treated with chitosan at dose (150 mg/kg) for 35 days showed decrease in the protein contents (Fig. 10B and E). There was a marked decreased of the protein content of the renal tubules and glomeruli treated with at dose (300 mg/kg) for 35 days. The decrease was obviously in female than male (Fig. 10C and F). 3.4. Quantitative analysis The optical density of glycogen and total protein contents were performed using image analysis system and revealed the histochemical investigation of glycogen and protein reaction in liver,
kidney of mice within different treatment groups showed a significantly decrease in glycogen and total protein content compared to controls (P < 0.05). The female was more effective than male (Fig. 11A–D).
4. Discussion The selected oral doses of chitosan 150 and 300 mg/kg treatment in male and female mice for 35 days caused significant (P < 0.05) elevation in the levels of liver enzyme markers (ALT, AST, ALP, LDH, PFK, GPI and HK) in liver homogenate as well as, there were significant increase in kidney functions (creatinine and urea) in dose dependent manner. Furthermore, there were significant decrease in protein and glycogen contents in liver and kidney homogenates due to gender and dose response effects. Significant hypoglycemia was observed only in females treated with chitosan 300 mg/kg. The activities of liver enzymes are sensitive indicators of hepatocellular damage; the release of these enzymes interacting with cellular structure and function resulting in tissue degeneration (Sudhahar et al., 2007).
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Fig. 10. (A and D) A photomicrograph of control kidney of male and female mice showing normal distribution of protein content in glomerulus (long arrow) and tubules (arrow head). (B) Male mice treated with chitosan (150 mg/kg, b.w.) showing reduce in distribution of protein content in glomerulus and tubules. (C) Male mice treated with chitosan (300 mg/kg, b.w.) showing highly reduce in distribution of protein content in glomerulus and tubules. (E) Female mice treated with chitosan (150 mg/kg, b.w.) showing reduce in distribution of protein content in glomerulus and tubules. (F) Female mice treated with chitosan (300 mg/kg, b.w.) showing highly reduce in distribution of protein content in glomerulus and tubules (Promophenol blue 400).
In our study, it was noticed that the low dose levels of chitosantreatment altered the liver enzymes activities in both male and female mice treatment groups. This finding is in line with the earlier report of Imogen et al. (1995). Chitosan induced many histopathological changes in liver and kidney of rats and the magnitude of such changes were dose dependent. Examination of section of male and female mice showed distortion of the normal hepatic architecture with mononuclear cell infiltration mainly in the portal tracts at low dose. The cytoplasm of hepatocytes in the hepatic lobules appeared vacuolated and intracellular hemorrhage between hepatocytes at the higher dose. In kidney showed hypercellularity and degeneration of glomeruli and tubules. Serum creatinine and urea significantly (P < 0.05) elevated in all treatment groups with chitosan in dose related increase, whereas no gender effect was found in these two parameters. While, the treatment with the lower dose of chitosan showed significant increase in creatinine value comparing with male group treated with the same dose of chitosan. From previous findings it was proved that chitosan treatment caused nephrotoxicity and these results were supported by histopathological examination of the kidney, which revealed that hypercellularity of glomerulus and degenera-
tion in renal tubules with the interstitial hemorrhage. The severity increased by the higher dose. Urea level was significantly elevated mainly due to hepatic failure caused metabolic interruption in protein metabolism to convert amino acids and ammonia to urea (Santhosh et al., 2007). This fact explains the reduction in protein content in liver and kidney tissues of all chitosan treated groups. Our study resulted significant elevation in glycolytic enzymes activity (LDH, PFK, GPI and HK) in liver tissue of chitosan administration. This could be attributed to an increase in metabolic activity of chitosan which compensate the inhibition of Kreb’s cycle and mitochondrial oxidation which favors the conversion of pyruvate to lactate (Ahmed and Gad, 1995; Tielens, 1997). Landes and Bough (1976) stated that in an eight weeks feeding study, it has been suggested that chitosan is safe in rats up to 10% in the diet. At 15% level, enlargement of liver and kidneys with a few other changes have been observed. This is confirmed the histopathological observation in our study. There is significant evidence that long-term; high-dose chitosan supplementation can result in malabsorption of some crucial vitamins and minerals including calcium, magnesium, selenium, and vitamins A, D, E and K (Koide, 1998; Yao et al., 2010).
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Female
Male
400
A
350
A B
300
C
C
250
D
200 150
Female
Male
400
A
350
A
300
B C
C
250
D
200 150
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B
250
D
C
225
E
200 175 150
kg
30
0
0
m
m
g/
g/
kg
l tro 15
30
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C
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on
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g/ m 0
15 300
A
A
275
kg
l on C
m 0
Female
A
Kidney protein content Male Female
(D)
Mean % of grey area
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tro
g/ kg
g/ kg
l on tro
m
30
15
30
15 0
0
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C
m g/ kg
g/ kg
l tro on C
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(C)
kg
100
100
A
275
B C
250
D
225
E 200
m 0 30
m 0
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g/ kg
l tro C on
kg g/ m 0
15
15
30
m
g/ kg
tro l
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15
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g/
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l tro on C
g/ kg m 0
30
m 0 15
C
on
tro
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l
175 C on
Mean % of grey area
Kidney glycogen content
0
Mean % of grey area
(B)
Liver gylcogen content
Mean % of grey area
(A)
Fig. 11. Effect of chitosan oral doses (150 and 300 mg/kg) on glycogen content of liver (A) and kidney (B); and on protein content of liver (C) and kidney (D). ANOVA-one way, at P < 0.05. The different capital letters above columns are significantly different.
In accordance to the present results, Tsung and Tsan (2002) and Hossain et al. (2007) reported that, chitosan intake was effective in lowering the plasma total cholesterol, VLDL-cholesterol and LDL-cholesterol levels in experimental animals. The hypocholesterolemic property of chitosan is probably related to its ability to inhibit the total lipid accumulation in serum and liver by its antilipidemic property, enhancing cholesterol 1-7-hydroxylase enzyme, regulate lipoprotein metabolism and alter VLDL particle size (Ausar et al., 2003; Xing et al., 2005; Hossain et al., 2007). In addition, the significant reduction in triglycerides in chitosan administered mice may related to chitosan exhibited inhibition of pancreatic lipase activity in liver and kidney that leading to increase in fecal fat and /or bile acid excretion beside, decrease in the absorption of dietary lipids (He and Xue, 2004; Sumiyoshi and Kimura, 2006). Xiao-hong et al. (2004) explained the decrease in rat serum free fatty acids, triglycerides, total cholesterol; fatty liver after chitosan –administration by inducing peroxisome proliferators activated receptor alpha mRNA expression and promoting liver intake and oxidation of fatty acid. Lee et al. (2008) indicate that estrogen induces the secretion of hormones that increase with stress .This in turn results in growth suppression. These hormones likely change the animal’s energy use towards a catabolic direction. Whereas, Lee et al. (1999) found the gain/food ratio was increased by gonadectomy and decreased by 17b-estradiol replacement in female rats.
Testosterone may function through regulation of the LDL-receptor and 7a-hydroxylase at the gene transcription level in liver. The effect of testosterone, a known anabolic, on cholesterol metabolism may also be partly mediated through secretion of growth hormone, which is shown to increase hepatic LDL receptor expression in rats (Rudling and Angelin, 1993). However, it could be suggested that females are less sensitive than males in feedback to control cholesterol synthesis and able to loss fats more than male (Fernandez et al., 1995). In the present study, glucose level significantly reduced only in females treated with chitosan (300 mg/kg) as well as non-significant decrease in other treated groups with chitosan (male or female mice). Few reports have claimed that chitosan has antidiabetic effects (Miura et al., 1995). Chitosan may be primarily absorbed after it has been transformed into oligosaccharides by chitosanase secreted from intestinal bacteria, or by lysozyme in intestinal fluid. Consequently, chitosan may exert its hypoglycemic effect with the oligosaccharides, but not with the monosaccharide (Hayashi and Ito, 2000).
5. Conclusion Therefore, the gender difference had a major role to induce hepato-renal toxicity in females more than males administered higher doses of chitosan for a long period.
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