Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.6/Oct-2013/26.pdf RESEARCH ARTICLE Open Access

Aim: To investigate the nephrotoxicity induced by the combined exposure of lead (Pb) and cadmium (Cd) and its amelioration with N-acetyl-L-cysteine (NAC). Materials and Methods: To evaluate the nephrotoxicity of Pb and Cd, the present study was taken up with a total of 48 male wistar rats, divided uniformly into eight groups each with 6 rats. Groups I and II were kept as untreated control and NAC control, respectively. Groups III, IV and V served as toxic control groups viz., Pb, Cd and combination of Pb+ Cd @ 1000, 300 and 1000+300 ppm, respectively. Whereas groups VI, VII and VIII were co exposed with NAC and served as therapeutic groups (Pb, Cd and combination of Pb+ Cd @ 1000, 300 and 1000+300 ppm, respectively and NAC @ 300mg/ kg). The experiment was carried out for 3 months and kidney tissues were collected for histopathological and electron microscopic study at the end of the experiment. Results: Histopathological examination of kidney tissue revealed marked alterations and electron microscopic observations also showed marked ultrastructural changes and these changes were more pronounced in Pb and Cd co exposure group (group 5) than the Pb and Cd alone exposed groups (groups 3 and 4), respectively. We also observed a significant (p<0.05) elevation in the serum creatinine levels. Use of the NAC counteracted the all toxic effects by chelating these metals, thereby reducing their accumulation and NAC also demonstrated its antioxidant properties, as judged by a significant reduction in the serum creatinine levels. Conclusion: We conclude that the co-existence of these metals showed a positive pharmacodynamic interaction and significant improvement was noticed after NAC therapy.


Introduction
(GSH) [11], decreased activities of Super oxide dismutase (SOD), Glutathione peroxidase (GPx), Heavy metals are increasingly being recognized Catalase, and significant elevation of Thiobarbituric as potential environmental pollutants.Lead (Pb) and acid reactive substances (TBARS) in the kidney, thus Cadmium (Cd) are often released simultaneously into suggesting increased renal oxidative stress [12,13].the environment from a number of natural and Oxidative stress could be responsible for renal damage manmade sources.Adverse health effects caused by and there by increase serum creatinine concentration combined exposure to Pb and Cd have provoked a which is a sensitive indicator of renal damage.significant public health concern [1,2].Pb and Cd are N-acetyl-L-cysteine (NAC), a potent antioxidant, divalent cations with a high propensity to settle in the is a sulfur-based amino acid needed for GSH synthesis, proximal tubules of the nephron, leading to and is a natural antioxidant enzyme produced in the nephrotoxicity [3,4].Moreover, co existence of these body to fight free-radical activity and has been used metals exhibit synergism in their toxic effects [5,6].
clinically since decades for the treatment of many Long-term exposure to these metals will lead to renal diseases.GSH is a sulfur-containing nucleophilic tubular damage by accumulation in the kidney once the substance found in high concentrations in the kidney.critical tissue threshold level is reached [7].Many NAC has been also used as a chelator of heavy metals authors proposed that the formation of free radicals is to protect against oxidative stress and prevent damage the most important molecular mechanism of their to cells [14][15][16][17].toxicity [8][9][10].Excess intake/ accumulation of these The goal of our study was to determine the metals cause depletion of endogenous glutathione histopathological and ultrastructural changes in kidney due to the interaction of Pb and Cd and their amelioration with NAC. 0

Materials and Methods
-20 C for future use.Analysis of creatinine was done according to the instructions provided in the kit Chemical Reagents: Lead acetate, Cadmium chloride supplied by SRL Pvt. Ltd., Mumbai.and all the other chemicals used in our experiments were purchased from SRL Pvt. Ltd., Mumbai.
Histopathological study: Rat kidneys were collected at rd the end of 3 month for which rats were fasted Animals: Male albino rats of Wistar strain weighing overnight and sacrificed by cervical decapitation.about 200-250 g were procured from the National Kidneys were then excised, weighed, dissected out and Institute of Nutrition, Hyderabad, Andhra Pradesh.The washed with ice cold saline.Approximately 0.2g of use of animals in this study were approved by the renal tissue was preserved in 10% neutral buffer Committee for the Purpose of Control and Supervision formalin for histological studies and the fixed tissues of Experiments on Animals (CPCSEA) (approval no. were processed and stained with routine Hematoxylin 5/I/10).
and Eosin (H & E) stain [18] at the Department of Experimental design: The study was carried out for 3 Veterinary Pathology, College of Veterinary Science, months on 48 male Wistar rats randomly divided into 8 Rajendranagar, Hyderabad.groups with six rats in each group.(OECD 408 Electron microscopic study: Small pieces of kidney GIUDLINES (1998), Oral toxicity for 90 days).
tetroxide in the same buffer for 1 h.The sections were Group-3: Lead toxic control (Lead acetate @ 1000 ppm stained with saturated aqueous uranyl acetate and in feed).
counter stained with 4% lead citrate [19] and were Group-4: Cadmium toxic control (Cadmium chloride observed at various magnifications under a @ 300 ppm in feed).
transmission electron microscope (Model: Hitachi, H-Group-5: Lead and Cadmium toxic control @1000 and 7500) at RUSKA Laboratory, College of Veterinary 300 ppm, respectively, in feed.
Science, Hyderabad, India.Group-6: Lead and NAC @ 1000 ppm in feed and @ 300 mg / kg body weight respectively Estimation of heavy metals: The concentrations of Pb Group-7: Cadmium and NAC @ 300 ppm in feed and and Cd [20] were estimated by digesting the tissue @ 300 mg / kg body weight respectively samples in a reagent grade of nitric acid-perchloric acid Group-8: Lead, Cadmium and NAC @1000, 300 ppm (2:1) mixture and concentrations were measured by in feed and @ 300mg / kg body weight respectively.
Atomic Absorption Spectrophotometer (Model; F S Pb and Cd were used in the form of lead acetate 120), at the Department of Environmental pollutants and cadmium chloride and were given in the form of and micro nutrients, Agricultural Research Institute, mash feed, while NAC was administered via oral Rajendranagar, Hyderabad.gavage by diluting in distilled water.
Analysis of variance: Results were analyzed by one-Biochemical study: Blood collection was carried out at way analysis of variance (ANOVA) using SPSS (Ver.monthly intervals for creatinine estimation after 10.00) followed by Duncan test at 5% (p < 0.05) level initiation of the drug administration till the end of of significance.Results were expressed as mean ± experiment.Feed was withdrawn 12 h before the blood standard error.collection and blood was collected from retro-orbital

plexus into serum vacutainers and centrifuged at 3000
In the present study, the sections of kidney RPM for 15 min, and serum was separated and stored at revealed dilated, ruptured tubules, mild to severe fatty margination of chromatin and presence of electron change, reduced glomerular space and rupture of few dense granular material in neucleolemma and glomeruli in Pb exposed group (G-3) (Fig. 1), where as shrunken cell walls (Fig. 7).Cd + NAC co treated group Cd exposed group (G-4) showed severe changes in (G-7) showed nuclei with dense chromatin scattered tubules with dilation and severe fatty change and throughout nucleolemma, mild condensed reduced glomerular space (Fig. 2), while Pb and Cd co mitochondria of varied sizes and shapes, presence of exposed group (G-5) exhibited ruptured glomeruli, endoplasmic reticulum, chromatin granules, reduced severe fatty change, reduced glomerular space and loss margination of chromatin in nuclei (Fig. 8).Pb + Cd co of few glomeruli (Fig. 3).The NAC co treated groups 6, exposed group (G-5) revealed shrunken cell size, 7 and 8 showed mild degenerative changes in and condensed nuclei of varied size and shape, indistinct around glomeruli when compared with their respective cell organelles, thin cell wall and few condensed and toxic control groups 3, 4 & 5 (Fig. 4, 5 & 6 ).Kidney is vacuolated mitochondria (Fig. 9).In NAC co treated the primary target organ for accumulation of Pb and Cd (Pb + Cd + NAC) group (G-8), chromatin was widely and long-term exposure damages renal tubules by distributed and there was mild hyperplasia of causing massive local haemorrhage, vascular endoplasmic reticulum, tight cell junctions, mild congestion, focal necrosis, destruction of nucleus, and variation in the sizes and shapes of mitochondria oedema of renal tissues [21,22].This damage is due to without vacuolation and distinct nucleus and nucleolus the generation of free radicals as they are divalent indicated by electroluscent central area (Fig. 10).Our cations that readily bind with oxygen to form Reactive results are in agreement with previous studies dealing Oxygen Species (ROS).Toxic metals increase with Pb and Cd toxicity where ultrastructural changes production of free radicals and decrease the availability in the renal tubules and mitochondrial damage were of antioxidant reserves to respond to the resultant noticed [24][25][26].Mainly, the toxic effects involve damage [23].
disruption and loss of mitochondrial cristae together Furthermore, the other significant aspect of our with mitochondrial swelling -which might be due to study is to decipher the electron microscopic changes the formation of free radicals [12].induced by these heavy metals.Ultrastructurally, Histopathological examination revealed that, the kidney tissue in group 4 (Cd exposed) revealed mild effects of either metal alone is less severe when hyperplasia of smooth endoplasmic reticulum, mild to compared to the co exposure (Pb + Cd) and this was moderately sized vacuoles, condensed mitochondria, further confirmed by electron microscopic examination vesicles containing dense mass, condensed nuclei with of the degree of damage to the mitochondria (as their numbers were reduced) and endoplasmic reticulum.exposed groups.On the other hand, use of NAC With respect to the mitochondrial ultra structure, counteracted the adverse effects of Pb and Cd induced renal damage to a major extent suggesting its chelating severe damage was noticed in the combined exposed action on heavy metals.groups than that of single metal exposure groups.Pb and/or Cd exposure induces damage to the concentrations of these metals in the renal tissue.

Table - 1
. Concentrations of serum creatinine in different groups of rats Values are expressed as mean ± standard error (n=6) Means with different alphabets as superscripts differ significantly (p<0.05);Capital alphabets are used for horizontal comparison and small alphabets are used for vertical comparison Table-2.Concentrations of Pb and Cd in different groups of rats.Values are expressed as mean ± standard error (n=6) Means with different small alphabets as superscripts differ significantly (p<0.05) for vertical comparison