Protective Effect Of Melatonin, Methionine And Zink On Cadmium Nephrotoxicity: Histopathologically, Histochemically And AgNORs Quantitation

Cadmium (Cd) is a highly toxic heavy metal that is naturally present in the environment. Chronic exposure to Cd causes hepatotoxicity and nephrotoxicity. The present study aimed to study the protective effect of melatonin, methionine and zinc against histopathological, histochemical and proliferative effects of cadmium on the kidney of rats. A total of 80 female albino rats were included in this study and divided into 8 groups. They were injected intraperitonealy with cadmium chloride (CdCl2) (2 mg / kg b.w.), melatonin (10 mg / kg b.w.), methionine (42.8 mg / b.w.) or zinc (20 mg / kg b.w.) with or without CdCl2 daily for 10 days. Treatment with CdCl2 induced marked tubular cell degeneration with large areas of interstitial hemorrhage.There were marked destruction of the brush borders with decrease in glycogen and protein contents of the degenerated tubules. AgNORs count significantly increased. Injection of melatonin or methionine to CdCl2 treated rats resulted in improvement of Cd-induced histopathological and histochemical changes. AgNORs count significantly decreased. Zinc injection partially protected the kidney from Cd-induced effects. In conclusion, melatonin and methionine have a more protective effect than zink against Cd nephrotoxicity. Introduction Cadmium (Cd) is a highly toxic element that is naturally present in the environment, including food, water, and soil (Sherlock, 1984). It is a trace element which has no known metabolic function. However, it is toxic to cellular processes by disrupting mitochondrial function (Miccadei and Floridi, 1993 ; Koizumi etal., 1994) and can interfere with the transport and metabolism of many essential elements, such as iron and copper (Crowe and Morgan, 1997). Cd was recently designated as a human carcinogen where it has been linked to prostate, lung, and male reproductive tumours (IARC, 1993 ; Waalkes and Rchm, 1994). In animals Cd is associated with tumours of the prostate, testes, lung, and the injection site (Magos, 1991 ; Waalkes and Misra, 1996). The nucleolus plays a vital role in the control of cell proliferation and protein synthesis, as it is the only part of the nucleus where ribosomal ribonucleic acid (rRNA) is transcribed. The region of the nucleolus perform this function is nucleolar organizer regions (NORs) (Egan and Crocker, 1992).These NORs are defined as nucleolar components Refree : Prof ; Dr. Mona M.El-Tounsy. 141 Protective Effect Of Melatonin, Methionine 142 containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver staining, the NORs can be easily identified as black dots exclusively localized throughout the nucleolar area, and are called “Ag-NORs” (Trere, 2000). Silver staining of NORs and subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models as a marker of cellular proliferation (Duskova and Povysil, 1995; Orsea etal., 2001). Kidney is the main target organ of Cd toxicity. Cd induced nephrotoxicity is thought to be caused by the Cdmetallothionein complex (CdMT) that leaks out of the liver and is taken by the kidney (Lui etal., 1998). CdMT is freely filtered through the renal glomeruli and efficiently absorbed by the proximal tubular epithelial cells, where it is rapidly degraded by lysosomal enzymes (Cherian and Shaikh, 1975 ; Squibb etal., 1979). Cd selectively affects the proximal tubular cells without inducing changes in the glomerular or distal tubular cells (Friberg, 1984 ; Dudley etal., 1985). The morphological changes produced by Cd are mainly degene rative. There are only a few reports of morphological effects of tubular atrophy and interstitial fibrosis (Goyer, 1989). Previous studies showed that the oxidative stress is the primary mecha nism of chronic Cd-induced hepatox icity and nephrotoxicity (shaikh etal.1999). The pineal hormone melatonin MLT, N-acetyl methoxytryptamine, exhibits antioxidant and anti-inflammatory effects (Reiter etal., 1994 ; Cuzzocera and Caputi, 1999). Melatonin was also reported to be more effective than glutathion (GSH) in scavenging the highly toxic hydroxyl radical (Tan etal., 1993) and more efficient than vitamin E in neutralizing the peroxyl radical (Piere etal., 1994). Moreover, this indole was shown to be an efficient protector of nuclear DNA (Tan etal. 1994), protein (Abe etal. 1994), and lipids in cellular membranes against free radical attack (Melchirorri etal. 1995). Methionine is one of the essential amino acids which has a protective effect against many oxidant drugs (Sai etal. 1995). It is an important methyl donor for some of the reactions involved in protein and DNA synthesis. An in vitro cell culture study showed that the growth of most malignant cells was arrested when methionine was replaced by its immediate precusor, homo-cysteine (Hoffman,1984). A subsequent study by Guo etal., (1993) showed that proliferation of human cancer cells was arrested at the S/G2 cell cycle in methionine-free cell culture media. Methionine has been reported to reduce glutathione (GSH) levels in tumors, because methionine is degraded to cysteine, which is a precursor for GSH synthesis (Yoshido, 1999). Zinc has a protective role against heavy metals toxicity. Zinc pretreatment protects against CdMT nephrotoxicity (Squibb and Fowler,1984).The prop osed mechanism involves the induction of MT by zinc and sequestration of Cd2+ released from lysosomal degrad ation of exogenous CdMT by newly synthesized renal MT (Lui etal., 1996). Other factors, such as glutathione (GSH), may also be involved because protection is offered even in MT-null mice (Tang etal., 1998). This study aimed at studying Cd induced histological and histochemical changes in renal tissue. Also Cd effect on cellular proliferation was studied using Ag-NOR silver staining. The protecttive effect of melatonin, methionine, and zinc against Cd toxicity was evaluated. Sonia L. El Sharkawy et al 143 Material And Methods Chemicals: The chloride salts of cadmium and zinc as well as Dmelatonin were purchased from Sigma chemical Co. (St. Luis, Mo). Lmethionine was obtained from BDH Co., England. Animals: A total of 80 female albino rats weighing 150-200g b.w (purchased from animal house Colony, N. R. C., Dokki, Cairo) were used in this study. All animals were maintained on ad libitum concentrate ration (prtein:16.04%; fat: 3.63%; fibers: 4.1% and metabolic energy 2887 Kcal/Kg), and housed in a room free from any sources of chemical contamination, artificially illuminated and thermally controlled, at the animal house Lab. National Research Center, Dokki, Cairo, Egypt. Experimental Design: Rats were randomly divided into eight equal groups treated for 10 successive days with the following treatments: G1: control G2: intraperitoneally (i.p.) injected with cadmium chloride (CdCl2) (2 mg/kg b.w) dissolved in 0.1 ml saline according to yamano etal. (1998) G3: i.p. injected with D-melatonin (10 mg/kg b.w) according to Kim etal (1998) G4: i.p. injected with L-methionine (42.8 mg/kg b.w) dissolved in 0.1 ml saline according to Reynolds (1991). G5: i.p. injected with zinc chloride (20 mg/kg b.w) dissolved in 0.1 ml saline according to Poirer (1996). G6: i.p. injected with CdCl2 plus melatonin. G7: i.p. injected with CdCl2 plus methionine . G8: i.p. injected with CdCl2 plus zinc chloride. On the end of 10 th day, all animals were sacrificed by decapitation. The kidneys of each animal were removed and fixed in 10% neutral formalin and processed to prepare 4μm-thick paraffin sections. Sections were stained with: Hematoxylin and eosin stain for histological exam. Periodic acid Schiff (PAS) stain for demonstration of glycogen content (Pears 1980). Mercuric-Bromophenol blue stain for the total protein determination (Pears 1980). Argyrophilic silver stain for Ag-NORs staining and quantitation (Ploton etal. 1986). In brief, hydrated sections were put in a solution of ethanol and acetic acid in a proportion of 50% before the incubation with the solution formulated by dissolving 2g gelatin in 1% aqueous formic acid to two parts of 50% aque ous silver nitrate solution. The sections were incubated in this solution for 30 min at 45°C and then washed in distilled water, dehydrated in ethanol, cleared in xylene and mounted in Canada balsam. No counterstaining was performed. Using the CAS 200 Image Analyzer, AgNORs were counted in 100 nuclei from the most significant areas of each specimen under 1000X magnification. By careful focusing, only well-defined and sharply stained intraand extra-nucleolar AgNOR dots were included in the counting regime, as well as larger dots representing the total nucleolus where AgNOR dots were wholly aggregated. The means of the AgNORs counts in different groups were compared statistically using the student t-test. P value < 0.05 was considered statistically significant.