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Histopathological and Histochemical investigations on the protective effects of Curcumin on Kidney and Bone of Rats induced by Cadmium Chloride

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Vol. 6, No. 1 (2016): 1348-1355

Research Article Open Access

I

ISSSSNN::22332200--66881100

Histopathological and Histochemical investigations

on the protective effects of Curcumin on Kidney and

Bone of Rats induced by Cadmium Chloride

Nahed A.Omar

1

*, Ali Hussein A. Almaaty

2

, Ahmed M. Abdeen

3

and Yassmin M. Abd

El-Aziz

2

1

Zoology Dep., Faculty of Science, Damietta University, Egypt.

2

Zoology Dep., Faculty of Science, Port-Said University, Egypt.

3

Zoology Dep., Faculty of Science, Mansoura University, Egypt.

* Corresponding author: Nahed A.Omar, e-mail: nahedomar2000@yahoo.com

ABSTRACT

The curcumin is one of the most important herbs on folk or preventive medicine. It is important for the treatment of many diseases and toxicity. The aim of this work was to study the effect of curcumin on cadmium-induced kidney and bone damage. A total of 50 adult male rats were divided into four groups. Group I served as control and fed daily on basal diet. Group II maintained on the basal diet and 1.5 mg /kg curcumin. Group III treated with 1.5 mg/kg cadmium chloride mixed to basal diet. Group IV were treated with 1.5 mg /kg cadmium chloride and 1.5 ml of curcumin mixed with diet concurrently for 8 weeks followed by a withdrawal period of 2 weeks.. All animal of the groups were sacrificed after 8 weeks to examine with light microscope for estimation of histological procedures stained with haematoxylin and eosin and histochemically stained for demonstrating carbohydrates and total proteins on the examined kidney and bone organs. The histological examination of kidney cortex of Cd treated group revealed swelling of the cells lining the convoluted tubules and vaculation of their cytoplasm. On the other hand, administration of curcumin with Cd resulted in improvement of the examined tissues. In bone, longitudinal section of femur diaphysis Cd treated group displayed hole cavities with degenerated osteiod matrix, activated osteoblasts cells. It could be concluded that curcumin has the ability to protect rats against cadmium induced nephrotoxicity and bone tissues damage.

Keywords:

CdCl2; histopathology; histochemistry; curcumin; kidney; bone.

1. INTRODUCTION

Cadmium (Cd) is considered a dangerous poison for humans and animals. Acute ingestion of cadmium produces severe gastrointestinal irritation, which is manifested as severe nausea, vomiting, abdominal cramps and diarrhea. A lethal dose of cadmium for ingestion is estimated to be between 350 and 8900 milligrams [1]. The chronic effects of cadmium are dose dependent and also depend on the route by which the metal enters the body. Chronic inhalation causes emphysema and obstructive airways disease, and these occur before kidney damage is seen [2,1,3]. Long term ingestion causes kidney damage, which is first seen as proteinuria and ß2 microglobulinuria. In prolonged

cadmium exposure, disorders of calcium metabolism occur, causing osteomalacia [1,4,5].

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variety of disorder, including inflammation, arthritis and other proposed uses of curcumin is in Alzheimer's Disease, cancer prevention, cataract prevention, chronic, high cholesterol, liver protection, menstrual pain, multiple sclerosis, osteoarthritis and rheumatoid arthritis. [8,9,10]. Curcumin is also reported to have antibacterial, antiamoebic, antifungal, antiviral and anti HIV activities [11, 12, 13, 14]. In some experiments it has been shown to be 300 times more potent than vitamin E. However no concrete evidence in scientific field is available pertaining to the colonic protective effects of curcumin on cadmium chloride induced toxicity. Changes in bone such as osteopenia, osteoporosis, and osteomalacia, with increased bone fragility and pathological fractures have been noted in humans and experimental animals as a result of exposure to Cd [15,16]. In general, bone toxicity of Cd can be modulated by both direct and indirect mechanisms [17,18].

The aim of the present study was conducted to fulfill this lacuna and to determine the efficiency of curcumin in protection against cadmium chloride toxicity.

2. MATERIALS AND METHODS

2.1 Animals

Sixty adult male newly weanling albino rats (Rattus norvegicus) in an average weighing 70gm. The animals were offered the basal diet [19] and water were daily provided ad libitum. Then, the animals were divided into 4 groups. The first group (10 rats), served as control and was given the basal diet only. The second group (10 rats) was fed on the basal diet containing 1.5 mg. curcumin. The third group ( 20 rats) was maintained on 1.5 gm of cadmium chloride in basal diet. The forth group (20 rats) was fed on the basal diet containing 1.5 mg. cadmium chloride and 1.5 mg. curcumin. All animals were sacrificed after 8 weeks to examine the effect of cadmium and the other 10 animals from groups three and four were left for 2 weeks feeding only on basal diet with curcumin to examine the effect of the withdrawal period.

At the end of the experiment, pieces of kidney were taken, fixed in 10% neutral formalin, dehydrated, cleared and embedded in paraplast. Sections (5 µm thickness) were subjected to haematoxylin and eosin stains for histopathological examination [20]. Also, histochemical demonstration of polysaccharides [PAS reaction, 21] and total proteins [bromophynol blue, 22)]. The rat bone femurs were dissected out the body, immersed in fixative fluid (10% neutral formalin) for 2 days then decalcified in EDTA solution during 4 weeks [23]. After decalcification specimens dehydrated, cleared and processed to form paraffin blocks to prepare sections for histological and histochemical studies.

2.2 Chemicals:

CdCl2 is supplied in this study from Sigma co., Louis st.,

USA.

Curcumin powdered was obtained from herbal market

3. RESULTS AND DISCUSSION

3.1 Histopathology of kidney with H & E stains:

Histological examination of the kidney showed the typical structure of rat kidney, normal Bowman's capsules containing glomerulus and mesangial cells with normal urinary pole. Normal renal tubules lined by normal cuboidal epithelium and lumen are empty. Also, normal interstitial tissue (Fig.1. a). In rats fed on curcumin, the kidney tissue showed normal structure as the control tissue, on the other hand, Bowman's capsules containing glomerulus and mesangial cells with normal urinary pole. In addition normal renal tubules lined by normal cuboidal epithelium (Fig.1.b). Rats fed on cadmium only, kidney tissue showed that, evoke severe congestion and hemorrhage in interstitial blood vessels with fibroblastic proliferation replacing renal tissue, with hyperplasia in the mesangial cells of renal glomeruli, lymphatic infiltration surrounding the Bowmans's capsule noticed (fig. 1. c.). In rats fed on diet contained cadmium and curcumin for 8 weeks, the kidney tissue illustrated some restoration of the renal glomeruli, cloudy swelling of the renal tubular epithelium leading to bulging into the lumen with star shaped lumen, damage of uriniferous tubules and few vacuolation were seen (Fig.1.d.). After 2 weeks withdrawal period from cadmium, the kidney section showed congestion in interstitial blood capillaries with mid degeneration of the renal glomeruli and hypercellularity of the mesangial cells, and mild degeneration of the renal tubular epithelium were noticed (fig.1. e.). But after 2 weeks withdrawal period from cadmium and curcumin, the kidney section illustrated clear signs of kidney repair (fig. 1.f.).

3.2 Histochemical alterations: 3.2.1 PAS reaction for carbohydrates

In control untreated rats, the kidney tissue showed the typical structure intense purple coloration in renal tubular epithelium lining renal tubules (fig.2.a). Also, a strong color of PAS stain in kidney tissue with curcumin (fig.2.b). Less purple coloration displayed in section treated with cadmium only (fig.2.c). In the mixture of cadmium and curcumin the tissue illustrated a limited reddish coloration (fig.2.d). After withdrawal period from cadmium only and the mixture of cadmium and curcumin the fig.2 e & f illustrated clear signs with PAS stain.

3.2.2 Bromophenol blue for total protein

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3.3 Histopathology of bone with H &E stains:

In control untreated rats for 8 weeks, the longitudinal section of femur diaphysis compact bone showed typical structure of bone (fig. 4. a). In section of curcumin the structure is near to that of the control rat (fig. 4.b). On the other hand, sections of cadmium only displayed large areas of bone refraction represented by hole cavities with degenerated osteoid matrix, activated osteoblasts cells present in their lacunae ( fig. 4.c). In the mixture of cadmium and curcumin the tissue illustrated some restoration of the normal osteoid matrix, normal osteoblasts and normal blood vessels with normal osteoprogenator cell (fig. 4. d). After withdrawal period from cadmium only and the mixture of cadmium and curcumin the fig. 4 e & f illustrated clear signs of bone repair.

3.4 Histochemical observations of bone: 3.4.1 PAS reaction for carbohydrates

In control untreated rats , the bone tissue results showed the typical structure intense purple coloration (fig. 5.a). Also, a strong color of PAS stain in bone tissue with curcumin (fig. 5.b). Less purple coloration

displayed in section treated with cadmium only (fig. 5. c). In the mixture of cadmium and curcumin the tissue illustrated a limited reddish coloration (fig. 5. d). After withdrawal period from cadmium only and the mixture of cadmium and curcumin the fig. 5 e & f illustrated clear signs with PAS stain.

3.4.2 Bromophenol blue for total protein

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In humans cadmium has a long half life period, which is reported to be of 10-30 years in kidney, and 4.7-9.7 years in liver [24]. On average, 5% of the total orally ingested cadmium is absorbed in the intestines, but individual values range from less than 1% to more than 20%.This reflects the fact that humans do not have effective pathways for cadmium elimination, hence cadmium shows the phenomenon of bioaccumulation. Once absorbed by an organism, it remains resident for many years. Cadmium is one of the known environment toxins that are detrimental to liver function on exposure [25].

The present study aimed to determine the toxic effects of cadmium chloride and the possible protective effects of curcumin in kidney and bone of rats on the basis of histopathological observations. The results of various studies on cadmium has been reported to cause variations in histoarctecture of different tissues. In contrast to the injurious activity of cadmium, curcumin serves as protective agent where it has been shown to decrease congestion and inflammation in stagnant mucus membranes and no negative side effects have been associated with curcumin supplementation [26, 27].

The present study agree with the results obtained by [28] in which histopathological studies revealed the toxic effect of Cd in the kidney and edema was usually found [28]. In addition, Cd affects the glomeruli especially glomerular capillaries in favour of Bowman’s space, atrophy of some glomerulus, and proximal tubular necrosis, apoptosis, and tubular degeneration

[29]. The nephrotoxicity especially glomerulus and tubular alterations as shrinking and degeneration of glomeruli, pyknosis and vacuolated cytoplasm of tubules were also studied after treatment with Cd [30]. Kidneys sections from CdCl2 group showed acute tubular necrosis and glomerular widening [31]. In the present study rats treated with Cadmium chloride exhibited histopathological alterations such as shrunking or degeneration of glomerular tuft, cytoplasmic degeneration in cells of renal tubules, pyknotic nuclei, some tubules are necrotic, multiple foci of haemorrhage, dilatation and congestion of blood vessels as reported previously by [32]. Acute Cd intoxication induce renal damages, measured by increased histopathological alterations. Curcumin partially protects against lipid peroxidation induced by Cd in the renal homogenate and also reduces Cd-induced architecture damages in renal cortex [33].

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bone thickness (CBT), and increased the number of osteoclast cells compared to that of control. These findings are considered osteoporotic alterations and micro-architectural deterioration of bone structure.The examined bone tissues of the red fox in a risk assessment of exposure to trace elements, as they are subject to slow transfer of some metals in the body. Due to its characteristics and long renewal time, this tissue may reflect levels of chronic exposure and could be the basis of indirect environmental assessment [38, 39, 40, 41, 42].

Few studies were focused on the roles of curcumin in treatment of the bone toxicity which was induced by cadmium however some authors have used other herbal plants for treatment such as Ginger (Mustafa et al., 2013) and other. So, the present study revealed the protective effects of curcumin against cadmium induced protective against femur bone toxicity.Available studies have focuced that curcumin may have bone-protective effects. In vitro studies reported that curcumin promotes apoptosis and inhibits bone resorption by rabbit osteoclasts [43].

The present study concluded that curcumin can protect kidney and bone from the toxic effect of cadmium.

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