Repeated dose oral toxicity of inorganic mercury in wistar rats: biochemical and morphological alterations -

Aim: The study was conducted to find out the possible toxic effect of mercuric chloride (HgCl2 ) at the histological, biochemical, and haematological levels in the wistar rats for 28 days. 
 
Materials and Methods: The biochemical and hematological alteration were estimated in four groups of rat (each group contain ten animals), which were treated with 0 (control), 2, 4, and 8 mg/kg body weight of HgCl2 through oral gavage. At the end of study all rats were sacrificed and subjected for histopathology. 
 
Result: A significantly (P < 0.05) higher level of serum alanine amino transferase (ALT), gamma Glutamyle Transferase, and creatinine were recorded in treatment groups, while the level of alkaline phosphtase (ALP) was significantly decreased as compared to the control group. The toxic effect on hematoclogical parameter was characterized by significant decrease in hemoglobin, packed cell volume, total erythrocytes count, and total leukocyte count. Gross morphological changes include congestion, severe haemorrhage, necrosis, degenerative changes in kidneys, depletion of lymphocyte in spleen, decrease in concentration of mature spermatocyte, and edema in testis. It was notable that kidney was the most affected organ. 
 
Conclusion: Mercuric chloride (HgCl ) caused dose-dependent toxic effects on blood parameters and kidney.


Introduction
low molecular weight substance such as cysteine (Cys), CoA (Coenzyme-A) but importantly are located Health condition of an animal/human is in most proteins.For nephrotoxicants (particularly characterized by its metabolic activities of vital organs mercury) toxicity mitochondria plays specific role and such as kidney, liver and, brain, out of which liver and it can also provide the opportunity as the subcellular kidney are most important.The animals can be exposed target of nephrotoxicants [6,7,8].Exposure to any to mercury contamination from water, soil, air, and foreign substance can disturb the metabolic pathways sometimes from food sources like fish protein of that particular organ.Mercury toxicity is a concentrates or the accidental use of treated seed grain significant clinical entity [9] and as it is ubiquitous in [1].Having been absorbed from the digestive tract, the environment, it poses serious risks to large animal's mercury forms a long lasting suspension with the health, which consume drinking water contaminated thionin, forming metallothioneins which deserve an with large amount metals mostly mercury.important role in afterwards metabolism of this Hence, the present study was designed to observed inorganic compound.So it has been a great concern for the toxic effect on blood biochemistry, hematology and the eco-toxicologist that if chronic ingestion of histopathology in wistar rat after 28 days repeated oral inorganic mercury can produce different type of dose of inorganic mercury (mercuric chloride) adminicumulative toxicity or carcinogenicity in different vital stration.organs in animals.However, more intensive and systematical investigations are needed to evaluate

Materials and Methods
commutative organ toxicity to whole body.Kidneys -Male wistar rats of 5-7 weeks old were procured mainly renal -cortex and liver are considered to be the from the Zydus Research centre, Zydus pharmamost susceptible organs in the case of exposure to ceutical company (Ahmedabad -India).The animals mercury [2].As transport of organic substances is the were kept under controlled conditions of temperature hallmark of functional kidney [3].The toxicity of Hg (22±3ºC) and humidity (60±5%).They were given (considered as nephortoxicants) is largely due to the pellet food (Amrut feeds Ltd., Pune, India) and high affinity to sulfahydral (SH) groups [4,5].The drinking water ad libitum.A twelve hour day and night sulfahydral (SH) groups are seen in some diffusible cycle was maintained in the animal house. doi:10.5455/vetworld.2013.563-567

Results
experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and OECD-407 The effects of mercuric chloride on selected liver was followed.
and renal function biochemical tests (ALT, GGT, ALP and creatinine concentration in serum) in animals are Procurement of Metal: Mercuric chloride was obtained presented in bar diagram (Plate-1).After Administration from Merck India Ltd. (Mumbai India), and dissolved of mercuric chloride to rats, resulted in a statistically in the distilled water and administered per OS route.
significant increase of ALT, gamma glutamyl transferase Experimental design: Experiment was conducted for and creatinine concentration in serum in relation to their four weeks.The animals were divided into four groups, respective control groups.The above mentioned enzymes each group contain 10 animals (n = 10).In preliminary were increased as the dose increased.However, the study it was found that LD50 of HgCl was 40 mg/kg 2 activity of serum alkaline phosphatase was significantly th th body wt.Daily oral dosing was carried out as: decrease after 14 and 28 days of treatment when Group I -Controlled animals-vehicle (normal saline), compared to its controls.Group II -2.0 mg/Kg HgCl , 2 In Haematology, after 28 days post-treatment Group III -4.0 mg/Kg HgCl , 2 with HgCl there were significantly (P < 0.05) decrease 2 Group IV -8.0 mg/Kg HgCl 2 in haemoglobin (Hb), packed Cell Volume (PCV), total Blood samples were collected on 0, 14 and 28 day erythrocyte count (TEC) and Total leucocyte count of experiment.Blood was collected similar time of day (TLC) as shown in Table -1.In differential Leukocyte and by retro-orbital venous plexus puncture under Count there was significantly decrease in lymphocyte ethyl-ether anesthesia.count while neutrophils, monocyte and eosiniphils increased significantly as compare to control.Parameters tested: All the biochemical parameters There were not any gross lesions in any of the viz.Alanine amino trasferase (ALT), gamaglutamyle organs during postmortem.Histologiclly mutifocally traspeptidase (GGT), alkaline phosphatase (ALP) and with the kidney parenchyma there were areas of creatinine, were analyzed by using respective Accucare haemorrhage, necrosis, infiltration of mononuclear Diagnostic Kit (LAB-CARE Diagnostic India Pvt.cells, hypercellularity, degenerative prominent nuclei, Ltd., Mumbai).While Hematological parameters viz.
narrowing of lumen, low amount of proteineous cast in haemoglobin, hematocrit, total leukocyte count, total lumen of convoluted tubules (Figure -1).Brain slide erythrocyte count, and differential leukocyte count showed demyelination (Figure-2) of nerve fibres.were estimated by blood auto-analyzer (Beckman There was a depletion of lymphocytes in spleen.In coulter).After 28 days all remaining rats were scarified testis, the concentration of mature spermatocyte and tissues like kidneys, liver, spleen, lung, intestine, decrease significantly (Figure -3) and degeneration in heart and brain had obtained from necropsied animals, germinal cell layer with edematous fluid found inand fixed in neutral buffer formalin and processed for between semineferous tubule.Severe degree of fatty hematoxylin and eosin staining.
changes, vacuolization (Figure-4), karyorrhexis in hepatocytes, sinusoidal dilatation and mononuclear Statistical analysis: Data are expressed as Mean and cell infiltration were observed in liver.Sloughing of standard error (SE).Analysis of variance (ANOVA) villi was observed in intestine.The lesions in mercury was used to find out significance (P< 0.05 was considered) treated rats was in dose depended manner.variation in the different studied groups [10].

Discussion
concentration is one of the traditional screening indices for kidney function and renal structural integrity.It is In the present investigation marked increase in well known that the mercury accumulate more in renal liver-ALT under stress of mercury has been observed.
epithelium [18].The rise in creatinine level might be This elevation in above said parameters has been well due to damage produce in kidney tubules by mercury.supported by Toshiko et al., [11], Ejebe et al., [12], Significant (P < 0.05) decrease of Hb, TEC, PCV Madhu et al., [13] and El-Dermadash [14].A significant th observed in mercuric chloride treated groups on 28 (P<0.05)increase in serum Gamma glutamyl day.These finding findings were in consistent with transferase activity was observed in all treatment Madureira et al., [19], Hounkpatin et al., [20].The groups on 28th day post-treatment as compare to decreased in Hb, PCV and TEC might due to the control of that group.Hepatic gland issite for biooxidative damage.There might be increased generation transformation by which the toxic compound (metal) of free radicals can cause cell membrane damage has been transformed in less harmful form to reduce + + which in turn inactivate membrane Na -K ATPase, toxicity.However, this will damage the liver cells and +2 thereby allows entry of Ca into the cell.The sustained produce hepatotoxicity.ALT is an enzyme that helps increase in intracellular calcium leads to free radicals metabolize protein.When the liver (hepatocyte) is generations, which in turn produce further inhibition of damaged, alanine amino transferase enzymes released + + Na -K ATPase [21].Thus, the mercuric chloride from hepatocyte in the bloodstream.ALT is a mediated generation of free radicals and consequent cytoplasmic enzyme, which is more specific for the oxidative damage to erythrocytes, or hematolysis [22] hepatocyte of liver than aspartate amino transaminase can cause mechanical fragility of plasma membrane, for small animals.The decrease in serum ALP was there by shortened RBC life span and its removal from observed on 28th day in all treatment groups as compare to control of that groups.The decreased in the circulation.That favour lower level of RBCs.ALP was also observed by Merzoug et al.,[15].This Significant (P < 0.05) decrease in TLC value observed might because of changes in the liver, it is closely in both mercury chloride treated groups.The decrease connected with lipid membrane in the canalicular zone, in TLC might be due to the toxic effect of orally given so that any disturbance with the bile excretion, whether mercuric chloride.extra-hepatic or intra-hepatic leads to decrease in A significant (P < 0.05) increase in neutrophils serum alkaline phosphatase level [15].and decrease in lymphocyte was observed count in rats The mean values of serum creatinine was receiving mercuric chloride.While significant increase significantly increase as compare to control on 28th in monocytes and eosinophil count was also observed day.This elevation in creatinine level is in accordance on the 28 day.In the present study, decrease in to Toshiko et al., [11] in mice, Joshua et al., [16], lymphocyte count might be due to the toxic effect of Oriquat et al., [17] in rat.As we know serum creatinine mercury on lymphocytopoesis.The decrease in T -