Protective Effect of Zinc (Zn) on the Histology and Histochemistry of Liver and Kidney of Albino Rat Treated with Cadmium

1Department of Anatomy and Histology, Faculty of Medicine and Medical Sciences, Taif University, Taif 21974, Saudi Arabia 2Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt 3Department of Pharmacognosy, Faculty of Pharmacy, Taif University, Taif 21974, Saudi Arabia 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Taif University, Taif 21974, Saudi Arabia


Introduction
Cadmium (Cd) is known as a heavy and high toxic metal that widely distributed in the environment. It is present in trace levels in seawater and a broad range of animal and plant species [1][2][3][4][5]. It was reported that the maximum tolerance dietary Cd level for domestic animals was 0.5 ppm. Dietary concentrations of one ppm Cd results undesirable effects, while concentrations of 5 ppm leads to adverse health effects [6]. Gastrointestinal absorption of Cd is affected by the diet and nutritional status [7]. Absorption of ingested Cd is only about 5% and after absorption it accumulates in the liver and then in the kidney [8,9]. Therefore, one of the most important health effects of chronic Cd exposure is the liver and kidney damage [2,10,11].
Zinc is one of the important nutrients that can reduce the toxicity of orally administrated Cd and shows its effect by competing with Cd for some transport system as well as for the bending sites in the metalothionin [2,9,12,13]. The aim of the present study was to investigate the histological and histochemical changes in the liver and kidney of albino rat exposed to intraperitoneal injection of Cd, and the protective effect of intraperitoneal injection of zinc on Cd-induced changes in both.

Animals
The present study was based on materials obtained from 60 adult albino rats of both sexes with body weigh from 220-250 gm. The experiment was conducted to 8 weeks, and the animals were randomly designed into three groups, each of 20 rats: two experimental groups and one control group. Each group was isolated in a single cage and kept in the same conventional condition of diet and environment in the animal house.

Chemicals
The cadmium chloride (CdCl 2 ) and zinc chloride (ZnCl 2 ) of analytical grade or higher purity were the substances used in this study. Each was soluble in physiological saline and was injected intraperitonealy at dose levels of 0.16 mg CdCl 2 /kg of body weight and 0.53 mg ZnCl 2 /kg boy weight [9].

Experimental design
The experiment was conducted for 8 weeks. The animals were randomly allocated into three groups: two experimental groups and one control group each include 20 rats.
Rats of experimental group 2 were injected intraperitonealy with both zinc chloride solution (0.53 mg ZnCl 2 /kg of body weight) [9] after injection with cadmium chloride in an aforementioned dose.
Rats of control group were received injection of the same dose of normal saline. The injections were done into the peritoneum of used rats for eight weeks five times/week. The animals of each group were sacrificed with inhalation of over dose of ether. The kidney and liver were excised and divided into small pieces for histological examination.

Light microscopy
Small slices of kidney and liver tissue were taken and fixed in 10 % formalin for 24 hours, and were imbedded in paraffin. Five-micronthick sections were routinely stained with hematoxyline and eosin [14].

Histo chemical study
Some paraffin sections were stained with (a) periodic acid-Schiff's method to demonstrate carbo hydrates [15] and with (b) Mallory method to demonstrate the tissue fibroses [14].

Electron microscopy
Both liver and kidney were cut into small pieces 1 mm thick and fixed in 4 F,G in phosphate buffer solution (pH 7.2) for 3 h at 4°C, after which the tissues were removed and postfixed in buffered 2% OsO 4 for one hour at 4°C. Postfixed tissues were rinsed in the buffer and dehydrated at 4°C through a graded series of ethanol. Then they were embedded in epon-araldite mixture in labeled beam capsules. Ultrathin sections (50 nm thick) were cut, collected on naked copper-mesh grids and stained with uranyl acetate for 1/2 hour and lead citrate for 20-30 min [16].

Light microscopic observations
The Liver of a control rat should a normal structure where the liver appeared to de composed of hexagonal or pentagonal lobules with a central veins and peripheral hepatic triads or tetrads embedded in connective tissue. The hepatocytes are arranged in trabecules running radiantly from the central vein and the spaces between the cell cords called blood sinusoids which converged towards the central vein and lined by Kupffer cells. Also the hepatocytes are regular and contain a large spherical nucleus with a distinctly marked nucleolus and peripheral chromatin distribution. Some cells have two nuclei ( Figure  1 and 2).
The Kidney of control rats, had normal renal structure of both (a) cortex which showed a normal structure of; renal glomeruli.
The proximal convoluted tubules are lined with typical thick cubic epithelium. The distal convoluted tubules show considerably lower cubic epithelium. The tubules have a relatively regular distinct lumen. , the glomerular capsule are lined with a flat epithelium, and (b) medulla in which, the collecting tubules are lined with the relatively low simple cubic epithelium. The thick descending and ascending parts of Henle's loops are lined with simple cubical epithelium with small caliber, and a small amount of interstitial tissue can be seen normally in the crosssections (Figure 3 and 4).
Light microscopic examination in the liver of rats treated with CdCl 2 showed that there were degenerative changes in numerous hepatocytes; the cells were enlarged and had light and foamy cytoplasm filled with numerous vacuole-like spaces. The walls of the blood sinusoids were dilated and showed numerous Kupffer cells. In a few liver zones, the CdCl 2 induced also hepatocytes necrotic changes which appeared as; a small, pycnotic cellular nucleus with condensed chromatin, lack of nucleolus and strongly acidophilic cytoplasm. Mononuclear cell infiltrates were also noted in hepatic areas ( Figure 5 and 6).
Light microscopic examination in the kidney of rats treated with CdCl 2 only showed that there were many areas of tubular damages   ranged from mild to severe in the kidney were observed in all treatment animals. These renal damages appeared as hypertrophy and degeneration of epithelia of renal tubules with distinct of mononuclear cells infiltration. A few renal tubules showed single epithelial cells desquamated to their lumen. Also some vascular glomeruli were apparently enlarged, tightly filling the Bowman's capsule with absence of the capsular spaces was observed. Moreover, hyperaemia of the kidney vessels was observed (Figure 7 and 8).
ZnCl 2 in combination with CdCl 2 caused an reduction of toxic effects of CdCl 2 on the liver were an absence of nucleus fragmen tation and a decrease in the sinusoidal dilation, necrosis of some hepatocytes, mononuclear cell infiltrations; was observed.. In fact we noticed the presence of rare inflammatory sites in the sinusoids and some hepatocytes with light cytoplasm (Figure 9 and 10).
Light microscopic examination also revealed a positive correlation between ZnCl 2 and CdCl 2 in the kidney tissues with marked reduction      of the toxic effect on the kidney. However, some toxic effects of CdCl 2 , as mild hyperaemia in the kidney vessels, some degenerative changes in the tubular epithelium and cystic dilatation were observed ( Figure  11 and 12).

Histochemical observations
The light microscopic observations revealed that, the liver and kidney tissues of the control group should positive PAS reaction in the cells cytoplasm, more in the liver ( Figure 13) than in the kidney ( Figure  14) with PAS positive reaction in the brush borders of the prox imal convoluted tubules. While the glomeruli were intensely positive to PAS re action.
The Liver and kidney tissues of the rats exposed to CdCl 2 alone should, a marked reduction in PAS reaction in both liver ( Figure 15) and kidney ( Figure 16) tissues particularly in degenerative and necrotic areas. The kidney tissue appeared more affected than the liver. The reduction in PAS reaction was more intensive in the renal tubules and glomeruli.
The PAS reaction of both liver and kidney tissues of the rats exposed to CdCl 2 in combination with ZnCl 2 for eight weeks appeared      to have a moderate increased in intensity of PAS positive reaction in the liver ( Figure 17) and kidney ( Figure 18) tissues but not reach to the normal level.

Transmission electronmicroscopic observations
The Liver of a control rat showed that a normal ultrastructure; the cytoplasm of hepatocytes appeared to have a fine granular appearance due the presence of numerous free glycogen granules. The nuclei of the hepatocytes were oval or rounded and the nucleoplasm showed a fine granular component with a thin peripheral heterochromatin and euchromatin condensation. The mitochondria were numerous, usually round to oval in shape. The blood sinusoids appeared to be lined by   flat endothelial cells or their extensions which were separated from the adjacent hepatocytes by the subendothelial space or space of Disse ( Figure 19).
Transmission electron microscopic observations of kidney of the control rats showed the endothelial cells in the glomerular capillaries were very richly fenestrated with large pores which appeared to lack any trace of a closing diaphragm. The proximal renal tubular epithelium of control rat is characterized by a dense brush border, basal or central nucleus, apical endocytic vesicles, occasional lysosomes and elongate or round mitochondria. Cisternal profiles of rough ER located between the mitochondria (Figure 20).
In the experimental group 1 which exposed to CdCl 2 alone the transmission electron microscopic observation of the liver tissue revealed a histopathological changed in the hepatocytes, which appeared as thickening and enfolding of the cell membrane ,damage of the nuclear membrane ,regression of mitochondrial cisternae, deterioration of rough endoplasmic retinacula, losses of glycogen particles, and proliferation of smooth endoplasmic reticula with condensation of the nuclear chromatins. The hepatocytes also appeared to be contains many cytoplasmic fat droplets and many vacuoles ( Figure 21). The blood sinusoid appeared to be more dilated with many Kupffer cells seen in contact with its endothelial lining. The hepatocytes appeare to be contains many cytoplasmic fat droplets and many vacuoles (Figure 21).
The CdCl 2 induced a toxic ultrastructural changed in the kidney which observed as, enlargement of the glumeruli with narrowing of  the capillary lumen, swelling of the capillary endothelium, and loss of the fenesrtae. damage of the renal tubules s were noted as focal loss of the brush border of the epithelial lining of proximal renal tubules ,disturbance of the nuclear membrane ,chromatin condensation, swelling of several mitochondria with regression of their crestae. The protein synthesis rough endoplasmic reticulum was degenerated. Increased number of lysosomes and cell death were also noticed ( Figure 22 and 23).
Rats of experimental group 2 which exposed to CdCl 2 in combination with ZnCl 2 ; in this group, co-administration of ZnCl 2 practically prevented most of the ultrastructural pathological toxic effects of CdCl 2 on the liver tissues. In fact we observed the presence of rare sites of ultrastructural pathological changes in the blood sinusoids and hepatocytes (Figure 24). In the kidney, many of ultrastructural pathological changes caused by the toxic effect of CdCl 2 on the kidney tissues were noticed to be decreased in the kidney tissues of rats received ZnCl 2 in combination with CdCl 2 for eight weeks ( Figure 25).

Discussion
Cd is used worldwide in modern industries. Therefore, Cd pollution was internationally recognized as the most important environmental health hazard that remains to be eliminated [17]. When Cd is taken into the human body through the oral route by the ingestion of contaminated water or food, it accumulates in the liver and kidneys and its half-life is very long, exceeding 10 yr [10,18].
Once Cd is absorbed into the liver from the digestive tract, it stimulates the synthesis of metallothionin (MT) in the organs and the forms MT bound Cd (MT-Cd). The MT-Cd transfers to the kidneys via the blood stream. When MTCd reaches the kidney, it is filtered through the glomerular membrane and is reabsorbed in the proximal tubular cells. MT-Cd shows strong nephrotoxicity after pinocytosis and accumulation in the proximal tubular cells [19]. Chromatin condensation suggests progressive inactivation of the nuclear component, probably due to inhibition of DNA repair and DNA methylation [43,44]. Furthermore, proliferation in the number of lysosomes in cells of the proximal tubules is typical of heavy metal exposure. It has been suggested that this alteration may represent a cellular response to heavy metals such as cadmium. The increased number of lysosomes, a result of the attempt to digest these heavy metals or toxic substances, is considered a general manifestation of injury. The sequestration of damaged organelles in lysosomes is a mechanism of cellular repair and follows all types of sublethal injury [45].
In Zn co-treated animals, we noticed an improvement in the Cd-induced damage in the liver. Our results were in agreement with published data [46] which reported that the effect of co-treatment with Zn during Cd administration completely prevented the changes in renal function produced by the toxic metal in the rat, even though they did not find any significant difference in the renal Cd content. There is considerable information in the literature regarding the protective eff ect of Zn against the cellular toxicity caused by Cd. Previous studies [47][48][49][50] supposed that Zn protection is perhaps due to redistribution of Cd in the organism since Zn is able to induce synthesis of metalothionin in the liver and kidneys. However, another hypothesis must be proposed to explain the protective effect of Zn which appeared through our histological. Findings in spite of the further accumulation of Cd in the liver and kidney. In fact, in recent studies, Zn has been demon strated to play an important rule in preventing the oxidative stress, apoptosis and necrosis induced by Cd [9,[51][52][53]].
In the present study there is considerable improvement in Cd induced toxic damages on both liver and kidney tissues in Zn supplementation with Cd for 8 weeks. Our results were in agreement with those previously reported [9,24,46,51,53] which postulated that, Zn play an important rule in preventing the oxidative stress, apoptosis and the necrosis induced by Cd. Moreover, in a plant study. Zn and Se have been shown to have similar characteristics in their antagonism against Cd. Soil enrichment with the two trace elements was helpful in antagonizing Cd absorption by plant roots [54]. In contrast to the present findings, it has been reported that, no significant effect of Zn on Cd toxicity of liver and kidney tissues, and they attributed this lack of positive effect of Zn to the lack of time for metalothionien synthesis caused by administration of Zn and Cd simultaneously [2].

Conclusion
Our results demonstrate the ameliorative effect of Zn supply on the Cd induced toxic structural changes in the liver and kidney tissues of the rats. These results validate the hypothesis that the metabolism and toxic action of Cd may be modulated by Zn supplementation.
In agreement with a large number of studies who noted similar or more pronounced changes in the hepa tic and renal tissues under Cd effect [2,9,11,[20][21][22][23][24]. Our results indicated significant CdCl 2 toxic effects on the liver and kidney structures. As mentioned previously [20] early pathological changes in rat kidney obtained after 6 weeks of administration of 50 mg Cd/l in drinking water. After 12 weeks, they revealed signs of tubular necrosis, interstitial fibrosis and glomerular epithelial cell hypertrophy in small areas of the kidney cortex. In fact, it has been suggested that Cd disturbs membranes integrity [25,26] generates reactive oxygen species [22,27] and involves cytotoxic and inflammatory mediators [28,29] in the li ver and kidney.
In agreement with a large number studies [11,24,[30][31][32][33], our results indicated a significant ultastructural damages in CdCl 2 contaminated liver, which observed mainly in hepatocytes as chromatin condensation, swelling of mitochondria with regression of mitochondrial cristea, disintegration of protein synthesis structures such as rough endoplasmic reticulum and increased number of lysosmes and ultimately cell death. Also the hepatcytes cytoplasm showed some intra cellular vacuoles and reduced glycogen granules. These were evident in all animals of the Cd-contaminated group. Similar or more advanced changes in the liver and kidney structures under CdCl 2 influence have been reported by others [11,24,[30][31][32][33][34][35] who suggested that Cd inhibits protein synthesis and glycogen metabolism in the liver. Also reported data [36] postulated that, cell membranes were disintegrated and manifested by the occurrence of vacuoles in the cytoplasm, and in some cells the changes were lees evident and dense mitochondria with distinct membranes were found.
In the present study, some pathological changes in kidney ultrastructure as narrowing of the capillary lumen and swelling of the capillary endothelium of the glomeruli, injured brush-border microvilli and swollen mitochondria in the proximal convoluted tubular cells were observed. These results were in agreement with that of [11,22,23,37].
The narrowing of capillary lumina contributes to the hypertension reported previously [38] in rats following cadmium intoxication. Hypertension may result from cadmium-induced changes in vasculature, the rennin-angiotensin system, or renal ion transport process. The initial effect of cadmium administration is on the integrity and permeability of the vascular endothelium; other necrotic changes occur secondary to this effect have been declared [39].
In the present study, the effects of cadmium on proximal cell ultrastructure were, focal loss of brush border, nuclear membrane damage, chromatin condensation, swelling of the mitochondria with regression of mitochondrial crestae, degranulation and disintegration of protein-synthesizing structures such as rough endoplasmic reticulum, increased number of lysosomes and ultimately cell death. These changes were attributed to the reduction of the surface density of microvillus membrane per unit cell volume to 19% in cadmium contaminated rats [15,40]. Cadmium also inhibits the vacuolar H*-ATPase and endocytosis in proximal tubule brush border of rat kidney and this may inhibit endocytosis of filtered proteins and impair vesiclemediated recycling of some membrane [41].
The mitochondrial swelling and degeneration of the mitochondria cristae observed in our study may reflect the disturbances in oxyreduction processes taking place in the organelle [42]. Also, the changes in the nucleus, such as chromatin condensation indicate that this organelle is affected in a major way from cadmium exposure.