Protective role of Chitosan against lead acetate induced liver damage in rats

J. of Biological Chem. Env. Res., Vol. 6 No. 2, 431 – 444 (2011).

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Protective role of Chitosan against lead acetate induced liver damage in rats

Hoda E. A., Farid; Medhat M., Abozid; S. M., EL-Sayed

Biochemistry Department, Faculty of Agriculture, Menofia University, Shibin El-Kom, Egypt

ABSTRACT Evaluation of protective role of chitosan against the toxicity induced by

lead acetate in liver of male rats was investigated. Eighteen male albino rats

were divided into three groups and treated as follow: Control, Lead (500mg/L

in drinking water), Lead + chitosan (500mg/L in drinking water and 3%

chitosan in diet). Treatments were expanded for six weeks. Biochemical

investigations revealed that, lead acetate induced significant increase of

triglycerides, total cholesterol, LDL-cholesterol levels in plasma, while HDL-

cholesterol level was decreased. On the other hand a significant elevation in

the activity of plasma alanine aminotransferase (ALT), aspartate

aminotransferase (AST), alkaline phosphatase (ALP) and gamma glutamyl

transferase (GGT) were recorded, but albumin level was decreased.

Histopathological examination revealed degeneration of hepatocytes of rat

liver treated with lead acetate. Chitosan supplement improve the detrimental

effects of lead acetate in rats. KEYWORDS: Chitosan, Lead acetate, Lipid profile, Liver damage.

1- INTRODUCTION

Heavy metal pollutants are of significant ecological concern because they

are not biodegradable and have long half-lives in the soil (Ram et al., 2000).

According to Okunola et al. (2007), these metals also get accumulated when

plants and crops cultivated along major roads are consumed by man and

animals.

Lead (Pb), a highly toxic heavy metal, is widely distributed in nature. Pb is

the most ancient poison known to man. Contamination of soil, water and air

has become increasingly widespread through mining, refining and smelting

operations. The concentrations of Pb in liver and bone of red deer and wild

boar were higher in the mining area than in the control area (Reglero et al.,

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2009). The inhibitory effect of dietary Pb on digestive enzyme activities was

dietary Pb concentration dependent (Dai et al., 2009). Lead also is a

ubiquitous environmental and industrial pollutant that has been detected in

almost all phases of environmental and biological systems. This heavy use

has caused local and global contamination of air, dust and soil (CDC, Atlanta, 2002).

Chitosan a polyglucosamine derived from chitin is a cellulose-like polymer

located mainly in the exoskeletons of arthropods, such as carbs, shrimps,

lobsters and insects (Razdan and Pettersson 1994) it can be defined both

chemically and physiologically as a dietary fiber since it is a polysaccharide,

which cannot be digested by digestive enzymes of humans (Razdan and

Pettersson 1996). Chitin and its derivatives (chitosan, chitin oligosaccharide,

chitooligosaccharides) have characteristics that set them apart from other

polysaccharides and are used in medicine, pharmaceutics, biochemistry,

sewage treatment, agriculture and other areas (Sandford and Hutchings,

1987).

Chitosan is economically attractive, since it can be obtained from the

deacetylation of chitin, and chitin is the second most abundant biopolymer in

nature, next to cellulose (Annadurai et al., 2007). In nature, the main sources

of chitin/chitosan are from the animal and plant kingdoms, including the shells

of crustaceans and mollusks, the algae commonly known as marine diatoms,

and the cell walls of fungal species.

It is natural and nontoxic and growing evidence indicate that it exhibits a

marked hypolipidemic activity that would reduce the risk of cardiovascular

diseases (Zhou et al., 2006). It has exhibited a potent hypocholesterolemic

activity in rats (Simunek and Bartonova, 2005 and Liu et al., 2008).

Various studies of metal ion adsorption by chitosan have been undertaken in

recent years, such as the removal of Cu2+ ions from aqueous solution onto

chitosan and cross-linked chitosan beads (Wan et al., 2002, and Wan et al.,

2004). In addition, chitosan can be used to achieve adsorption of chromium

(Sag and Aktay 2002), cadmium (Evans et al., 2002), iron (Wan et al.,

2005), nickel (Pradhan et al., 2005 and Paulino et al., 2007) and lead

(Paulino et al., 2007) ions from aqueous solution.





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Therefore, the objectives of this study are: (i) to preparation of two type of

chitosan from shrimp waste and (ii) to determine whether chitosan would

reduce/prevent the liver damage, serum hyperlipidemia and hyperlipidemic

atherosclerosis induced by lead acetate.

2. MATERIALS AND METHODS 2.1. Production of chitosan

The production of chitosan involved the demineralization (DM),

deproteinization (DP), DC, and DA steps (No et al., 2003). Shrimp shell was

demineralized with 1N HCl for 30 min at ambient temperature with a

solid/solvent ratio of 1:15 (w/v). Following the DM step, the demineralized

shell was collected on a 100-mesh sieve, washed to neutrality in running tap

water, rinsed with deionized water, and filtered to remove excess moisture.

The DP step was accomplished by treating the demineralized shell with 3%

NaOH for 15min at 15 psi/121 °C and a solid/solvent ratio of 1:10 (w/v). The

residue was then washed and filtered as mentioned above. For the DC step,

the resulting chitin residue was bleached with 10% sodium hypochlorite

solution for 5min with a solid/solvent ratio of 1:10 (w/v). The bleached chitin

was collected, washed as mentioned above, and dried at 60 °C for 4 h in a

forced-air oven or by sun drying (approximately at 23 °C) for 4 h. The DA step

was achieved by treating chitin under conditions of 15 psi/121 °C with 45%

NaOH for 30min and a solid/solvent ratio of 1:10 (w/v). The resulting chitosan

was collected, washed as mentioned above, and dried at 60 °C for 4 h in a

forced-air oven or by sun drying (approximately at 23 °C) for 4 h.

2.2. Detremination of deacetylation degree of chitosan

The deacetylation degree of chitosan was determined by the

potentiometric titration method described by Broussignac, reported by

(Tolaimate et al., 2000). Chitosan was dissolved in a known excess of

hydrochloric acid. From the titration of this solution with a 0.1 M sodium

hydroxide solution, a curve with two inflexion points was obtained. The

difference between the volumes of these two inflexion points corresponded to





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the acid consumption for the salification of amine groups and permitted the

determination of chitosan’s acetylation degree, through Eq.

% NH2 = 16.1 ( V2 - V1 ) x Mb /W

where (V1) and (V2) are the base volumes referred to first and second

inflexion points, respectively, in mL, (Mb) is the base molarity in g/mol, and

(W) is the original weight of the polymer in g.

2.3. Experimental diets

The experimental diets and the full composition of the experimental

diets are detailed in Table 1. While Table 2 presents the composition of

minerals and vitamins in diet used in this study.

Table 1

Chemical composition (g/kg) and calculated analysis of

experimental diets

Control

Lead acetate

Lead acetate + chitosan

Casein 150 150 150

Starch 650 650 620

Corn oil 100 100 100

Salt mixture 40 40 40

Vitamin mixture 10 10 10

Cellulose 50 50 50

Chitosan - - 30





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Table 2

Composition of vitamins and mineral mixture in diet*

Vitamin Quantity Mineral Quantity Vitamin A 2000 iu NaCl 0.5 %

Vitamin D 200 iu KI 0.013 %

Vitamin E 75 iu K2HPO4 1.62 %

Vitamin K 0.5 mg MgSO4 0.325%

Inositol 10 mg CaCO3 1.5%

Niacin 4.0 mg CaHPO4 0.30%

Ca pantothenate 4.0 mg FeSO4 0.125%

Riboflavin 0.8 mg CuSO4 0.0015%

Thiamin HCL 0.5 mg MnSO4 0.011%

Pyridoxine 0.5 mg ZnSO4 0.00916%

Folic acid 0.2 mg

Biotin 0.04 mg

Vitamin B12 0.003 mg

Choline chloride 200 mg

Para amino benzoic acid

10 mg

*starch was added to make 1000 mg.

2.4. Animal experiments

The work was carried out at the Biochemistry Department, Faculty of

Agriculture, Menofia University, Egypt. To study the protective effect of

chitosan on lead acetate induced liver damage of albino rats; eighteen male

albino rats (weighing between 180 and 200 g) were used for this investigation.

Rats were obtained from Research Institute of Ophthalmology, Giza, Egypt.

The rats were fed ad libitum on a basal diet (BD) and water for 15 days as an

adaptation period. There were housed individually in stainless steel cages and

divided into three groups of six. The control group and lead acetate group

were fed only the BD. The third group fed on diet contain 3% chitosan .Diet





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intake was monitored daily. The first group (C) was used as controls and

received tape water as drinking water. The other two groups; received tape

water containing lead acetate (C2H3O2)2Pb at a dose of 500 mg/L, in

drinking water, daily for six weeks. All rats fasted before blood sampling. The

blood samples were drawn from eye plexuses, after 6 weeks , the rats were

anesthetized using diethyl ether. The weight gain of the rats was recorded on

a weekly basis.

2.5. Blood sampling and analysis

Blood samples were collected after six weeks in tubes contain heparin

as an anticoagulant from the eye plexuses under diethyl ether anesthesia and

then centrifuged at 3000 rpm for 20 min. to obtain plasma, which was kept

frozen until analysis. The total cholesterol was analyzed calorimetrically

according Richmond, (1973). HDL - cholesterol was determined according to

Lopez et al. (1977). Acording to Demacker et al. (1984) LDL – cholesterol

was calculated as the difference between total cholesterol and HDL –

cholesterol. The triglycerides were analyzed according to Fossati and Prencipe (1982). The both of alanine-aminotransferase (ALT) and aspartate-

aminotransferase (AST) activities were measured according to the method

described by Reitman and Frankel (1957). Gamma glutamyl transferase

(GGT) activity was measured kinetic method of Szasz (1969). Alkaline

phosphatase (ALP) activity was measured by Hausamen et al., (1967). And

albumin was determined according Doumas et al. (1971).

2.6. Histopathology

Liver from the experimental groups were immediately fixed in 10%

formalin, then treated with conventional grades of alcohol and xylol,

embedded in paraffin and sectioned at 4–6 lm thickness. The sections were

stained with Hematoxylin and Eosin (H&E) stain for studying the

histopathological changes (Lillie, 1965).

2.7. Statistical analysis

The results of the animal experiments were expressed as the Mean ±

SD and they were analyzed statistically using the one-way analysis of





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variance ANOV A followed by Duncan’s test. In all cases p<0.05 was used as

the criterion of statistical significance.

3. RESULTS AND DISCUSSION

3.1. The degree of N-deacetylation of chitosan

The degree of N-deacetylation and the molecular weight of the

chitosan were determined by titration as described in our previous work. The

final deacetylation degree was 85%.

3.2. Effect of chitosan supplementation on lipid profile of rats intoxicated with lead acetate.

The expressed data in Table (3) revealed that intoxication with lead

acetate induced significant increases in triglycerides, total cholesterol, and

LDL-cholesterol levels in plasma of male albino rats. Meanwhile HDL-

cholesterol level was decreased. Our finding of elevated total cholesterol and lipoproteins supports previous

reports (El-Gazzar et al., 1989; and Newairy and Abdou 2009).

The association between lead exposure and high serum lipid levels is

biologically plausible and could be due to either increased synthesis or

decreased removal of lipoproteins. Lead nitrate-mediated development of

hepatic hypercholesterolaemia involves the activation of cholesterol

biosynthetic enzymes (i.e., 3-hydroxy- 3methyglutaryl-CoA reductase, farnesyl

diphosphate synthase, squalene synthase, lanosterol 14xdemethylase) and

the simultaneous suppression of cholesterol-catabolic enzymes such as 7a-

hydroxylase (Kojima et al., 2002).

However, supplementation with chitosan improved effect of lead acetate on

these parameters. This data are in line with Xu et al., (2007) ; and Osman et

al., (2010) who reported that chitosan could decrease levels of total

cholesterol, low density lipoprotein cholesterol in plasma. The protective

effects of chitosan could be attributed to its metal chelating properties. This

data is in line with (Qin et al., 2006) who reported chitosan can absorb a

significant amount of heavy metal ions.

Deuchi et al., (1994) reported that chitosan had potent ability to increase lipid

excretion into the feces. Some recent in vitro studies (Sugano et al., 1980;





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and Nauss et al., 1983) have demonstrated that chitosan could bind much

more bile acids than cholestyramine to the extent of several times its original

weight under optimal conditions. (Sugano et al., 1988) Described that

chitosan brought about a high hypocholesterolemic effect, probably due to the

increased excretion of bile acids into the feces.

Table 3

Effect of chitosan supplementation (3%) on lipid profiles in plasma

of rats treated with lead acetate (500mg/L) for six weeks.

LDL-

cholesterol

mg/dl)(

HDL-

cholesterol

mg/dl)(

Total

cholesterol

mg/dl)(

Triglycerides

mg/dl)(

Parameters

Treatments

23.38 ± 6.3 c 43 ± 2.96 a 66.38 ± 3.45 c 78.88 ± 1.5 c Control

86.19 ± 9.55 a 25.6 ± 3.63 c 111.8 ± 5.95 a 117.24 ± 3.49 a Lead acetate

54.17 ± 1.31 b 30.95 ± 1.34 b 85.12 ± 2.59 b 94.4 ± 2.86 b


11.34 4.68 7.45 5.14 LSD 0.05 (a, b, c) means in the same column followed by the same letters do not differ significantly, and when the means followed by different letters differ significantly at p≤ 0.05. Each value represents a mean of (6) samples ± standard deviation

3.3. Effect of chitosan supplementation on plasma liver functions of rats

intoxicated with lead acetate.

Data in Table (4) showed the effect of treatment with 500 mg/L of lead

acetate in water on liver functions. In rats which fed on normal diet and drink

water treated with lead acetate (500mg/L). The results revealed that lead

acetate induced liver toxicity through increase plasma enzymes activities and

decrease plasma albumin level. The elevation of serum AST, ALT, ALP, GGT

due to the possible release of these enzymes from the cytoplasm into the

blood circulation rapidly after rupture of the plasma membrane and cellular

damage. These findings are in accordance with Shalan et al. (2005) and

Suradkar et al., (2009).





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The other finding in the present study is that chitosan highly improved plasma

AST, ALT, ALP, GGT activities and albumin level compared with rats treated

with lead acetate (Table 4).

LeHoux and Grondin, (1993); and Zhang et al., (2008) reported that

Chitosan improvement of liver functions.

The mode of action of chitosan in improvement of liver functions compared

with lead acetate treated group may be due to its reactive with lead ions. The

deacetylated product, chitosan, has an amine functional group, which is

strongly reactive with metal ions. These groups are more reactive than the

acetamide groups present on chitin. These reactive amine groups interact

with the metal ions in different ways, such as by chelation or electrostatic

attraction (Guibal, 2004; and Juang and Shiau, 2000). Some other

experiments suggest that only one amino group is involved in the binding and

the metal ion is bound to the amino group like a pendant; called the pendant

model. (Vold et al., 2003).

Table 4 Effect of chitosan supplementation (3%) on plasma liver enzymes of

rats treated with lead acetate (500mg/L) for six weeks.

Albumin

(g/dl)

GGT

(U/L)

ALP

(U/L)

ALT

(U/L)

AST

U/L(

Parameters

Treatments 3.25 ±

0.08 a

7.817 ±

0.87 b

61.109 ±

1.69 c

19.69 ±

0.46 c

58.37 ±

2.63 c Control 2.65 ±

0.015 b 14.765 ±

2.6 a 98.43 ± 0.88 a

31.99 ± 0.93 a

82.21 ± 3.74 a Lead acetate

2.69 ± 0.023 b

7.672 ± 1.59 b

71.299 ± 1.46 b

23.04 ± 0.76 b

66.16 ± 1.84 b


0.096 3.66 2.77 1.35 5.61 LSD 0.05 (a, b, c) means in the same column followed by the same letters do not differ significantly, and when the means followed by different letters differ significantly at p≤ 0.05. Each value represents a mean of (6) samples ± standard Deviation





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3.4. Histopathological results

Microscopically liver of rat from control group revealed the normal

histological structure of hepatic lobule (Fig. 1). However, liver of rat treated

with lead acetate showed hydropic degeneration of hepatocytes, congestion

of hepatoportal blood vessel, pyknosis of hepatocytic nuclei (Figs. 2, 3), portal

hemorrhage and portal infiltration with leucocytes (Figs. 4, 5).

No histopathological changes were noticed in liver from lead acetate +

chitosan group (Fig. 6) except kupffer cells activation in some examined

sections (Fig. 7). The activation of kupffer cells may be due to effect of

supplementation with chitosan, which may indicate on enhancement against

toxicity.

Fig. 1

Fig. 2





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Fig. 3 Fig. 4

Fig. 5

Fig. 6

Fig. 7

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ةأثیر الوقائي للكیتوزان المضاد لتأثیر خالت الرصاص المسببالت

لتلف الكبد في الفئران ، مدحت مصطفي أبوزيد ، صالح منصور عبد الجواد السید أحمد فريدھدي

مصر– جامعة المنوفیة – كلیة الزراعة –قسم الكیمیاء الحیوية

الملخص العربي

لكیتوزان المضاد لتأثیر خالت الرصاص ل الوقائيتأثیرالتھدف ھذه الدراسة إلي معرفة

فأر تجارب إلي ثالث ١٨ تم تقسیم وقد .السام علي كبد ذكور فئران التجارب

المجموعة األولي وھي الكونترول أما المجموعة الثانیة فتم بمعاملتھا : مجموعات

لتر في ماء الشرب، في حین تم معاملة المجموعة / ملجم ٥٠٠بخالت الرصاص بتركیز

ن الثالثة بنفس التركیز من خالت الرصاص في ماء الشرب ولكنھا تم امدادھا بالكیتوزا

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Protective role of Chitosan against lead acetate induced liver damage in rats

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