Renal Toxicity Induced by Carbon Tetrachloride in Experimental Model

Carbon tetrachloride (CCl ) is largely used as a solvent in chemical industries. It is also well 4 known for hepatic and renal toxic actions. It imposes serious health threats. It is also one of the major causes that is toxic for the vital organs like lungs, kidney, liver, brain, etc. Objective: To check nephrotoxicity of Carbon Tetrachloride (CCl ) on Rat Kidneys. Methods: The experiment 4 was conducted at the animal house of the Department of Zoology, University of Okara. The targeted animal was Albino Rat. Two groups were designed control and experimental groups. The rats were fed with 30% diluted CCl to check the toxic effect on the kidneys and normal saline 4 to the control group for comparison. A trial for 12 days was conducted for this purpose. Sampling or dissection was done after 12 days to determine serum Urea, Creatinine, and Electrolytes Sodium (Na), and Potassium (K). Rats were dissected and the heart was punctured to take a blood sample and to collect organs. Results: We observed the increased values of Urea, Creatinine and Electrolytes, Sodium (Na), and Potassium (K) as compared to normal values, which have proved the renal toxicity was induced by CCl in Albino Rats. All the experimental data 4 were analyzed by using SPSS-19. The level of signi�cance among the various treatments was determined by LSD at a 0.05% level of probability. Conclusions: These �ndings underline the substantial health risks that CCl poses and emphasize the necessity of putting preventative 4 measures and safety regulations in place.

found to mostly harm the liver (swollen, painful liver, alterations in enzyme levels, and jaundice) and kidneys (nephritis, nephrosis, proteinuria) of humans during acute inhalation and oral exposures to high levels of carbon. There have also been reports of central nervous system depression [8]. Even at relatively large dosages, renal damage is rarely seen in animals exposed to carbon tetrachloride. Although the cause of animals' lower sensitivity to renal damage than humans is unknown, it may be related to how differently CCl is metabolized by their 4 kidneys. After 5 weeks of intermittent exposure to an anesthetizing dosage of carbon tetrachloride, the content of vitamin A in the kidneys doubled, and a 10% rise in wet organ weight was noted [9]. However, the concurrently Renal Toxicity Induced by CCl4 in Experimental Model

I N T R O D U C T I O N
Carbon tetrachloride (CCl ) is largely used as a solvent in chemical industries. It is also well 4 known for hepatic and renal toxic actions. It imposes serious health threats. It is also one of the major causes that is toxic for the vital organs like lungs, kidney, liver, brain, etc. Objective: To check nephrotoxicity of Carbon Tetrachloride (CCl ) on Rat Kidneys. Methods: The experiment 4 was conducted at the animal house of the Department of Zoology, University of Okara. The targeted animal was Albino Rat. Two groups were designed control and experimental groups. The rats were fed with 30% diluted CCl to check the toxic effect on the kidneys and normal saline 4 to the control group for comparison. A trial for 12 days was conducted for this purpose. Sampling or dissection was done after 12 days to determine serum Urea, Creatinine, and Electrolytes Sodium (Na), and Potassium (K). Rats were dissected and the heart was punctured to take a blood sample and to collect organs. Results: We observed the increased values of Urea, Creatinine and Electrolytes, Sodium (Na), and Potassium (K) as compared to normal values, which have proved the renal toxicity was induced by CCl in Albino Rats. All the experimental data 4 were analyzed by using SPSS-19. The level of signi cance among the various treatments was determined by LSD at a 0.05% level of probability. Conclusions: These ndings underline the substantial health risks that CCl poses and emphasize the necessity of putting preventative 4 measures and safety regulations in place.
. caused hepatotoxicity may have overshadowed this vitamin A effect. Rats subjected to 50 ppm for 5-10.5 months and monkeys exposed to 200 ppm for 10.5 months both showed a little renal edema. At an exposure level of 200 ppm, renal tubular degeneration became visible [10]. Through the production of free radicals, it has been noted that carbon tetrachloride (CCl ) induces renal damage in 4 rats [11,12] in addition to hepatic toxicity [13]. Another study found that the protein content of renal tissues signi cantly increased after treatment with CCl . When 4 CCl was administered, proteins suffered oxidative damage 4 and accumulated as a result of inadequate proteasomal and lysosomal breakdown, leading to metabolic ine ciency in the kidneys [14]. Similar to earlier investigations, the treatment of rats with the same substance reversed the shifts toward the control rats [12]. According to a histological analysis, CCl treatment caused 4 lipid peroxidation of the lipid structures in the renal tissues, which led to subcellular damages. The CCl -induced 4 vasoconstriction results in an ischemia local environment, and SA has the effect of reducing the morphological changes that CCl causes. Similar histological alterations 4 were seen in renal CCl -treated rats, and these alterations 4 resulted in a number of cellular impairments, including a decline in membrane integrity. The fact that the severe alterations were not seen in the groups given the same treatment suggests that rats given CCl plus Launaea 4 procumbens extracts had lost their protective effects. In other research [11,12], similar histological observations were described. Free radicals generate lipid peroxidation and are thought to be one of the main factors in cell membrane deterioration, which can result in acute and long-term renal injury and a number of clinical conditions [3][4][5]15]. In addition, ndings on several case studies with documentation demonstrated that CCl causes renal 4 problems in people [4]. Both the kidney's inner medullary area and outer cortex have noticeable histological alterations. Digera muricata extracts appeared to have protective bene ts in reducing the morphological alterations caused by CCl in the groups that were not 4 subjected to the severe modi cations. Ogeturk et al., (2005) found similar histological abnormalities in the kidneys of CCl -treated rats; however, these changes 4 vanished in rats treated with CCl + caffeic acid phenyl ester 4 (CAPE) [3,11]. In the kidneys of rats given CCl , tubular 4 epithelial cell changes, such as vacuolization, atrophy, and ultimately epithelial cell detachment, suggested tubular necrosis. After prolonged exposure to CCl , similar 4 histological changes were also discovered in other studies [16]. The capacity of tubular absorption may have been altered as a result of these histological changes, leading to the functional overload of nephrons and eventual renal

M E T H O D S
Carbon Tetrachloride (CCl ) was used for this study. The 4 tested substance, carbon tetrachloride (CCl4), was bought from a nearby market and stored in the zoology department's lab at the University of Okara in Pakistan. The chemical is diluted by 30%. distilled water was used to produce the stock solutions. Freshly manufactured stock solutions were used for all of the working solutions. The other substances that were used were all of pro-analysis quality and came from conventional commercial sources. The number of rats were randomly assigned into two groups, a control group (Co) having fteen (15) rats and an experimental group having the same number of rats as in the control group with exposure of 30% CCl mixed with 4 normal saline through oral gavage for 12 days. The experimental rats were anesthetized by putting in a desiccator exposed to chloroform and a small incision was made to cut the abdominal wall with sharp scissors. Then the muscular layer was cut on the sides to expose the internal organs. 0.9% pyrogen-free sodium saline solution was poured on the exposed organs of the animal to avoid drying. The dissections were done in completely aseptic conditions and tissues (heart, kidneys, liver, spleen & intestine) were excised. Normal feed was given to rats for 24 before dissection. Rats were euthanized with chloroform. Dissect the rats and by cardiac puncture, blood samples were collected in vacutainers by using 23 G1 syringes. Kidneys were dissected out, washed with icecold saline to remove debris. Organs were weighed and store at -20 ºC for tests. Centrifuge the blood samples at 10,000rpm for 15 minutes at 4 ºC. The serum was separated and store at -20 ºC. Urea, creatinine, and electrolytes (Na and K) in serum only write your parameters were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). For the analysis of serum samples In this investigation, adult male Albino Wistar rats weighing 180-200g were employed. The animals were bought from a nearby market and housed at the zoo's animal house at the University of Okara (Pakistan). Throughout the trial, they were kept in cages with four or ve rats each, at a temperature of 25 3 °C, with 12-hour light/dark cycles and a minimum relative humidity of 44%. Rat Chow (20% crude proteins) and water were available at all times. Every dose was administered in the morning ( Table 1). The animals were kept in these facilities for at least one week before the experiment.
of rats, the diagnostic kits were used to estimate urea, creatinine, and electrolytes levels in serum samples. Creatinine was determined using the Bartels & Böhmer (1971) technique [17]. The sample's creatinine combines with picrates in an alkaline solution to create a colorful complex at a wavelength of 500 nm. The amount of urea in the sample was determined using the Tabacco et al., (1979) method, which produced a colored complex that could be quanti ed by spectrophotometry (LKB, Sweden) at 600 nm [18]. Biochemical parameters measured were sodium (Na), potassium (K) using standard kits (Eve's Inn Diagnostics, Vadodara, India). Take blood in clot vile as a sample. Let it clot put it on the incubator for 5 to 6 minutes. Let it clot then centrifuge it 2,3 mint and then red blood cells came to the bottom and serum oat over the cells. Then take the serum to test RTF (Urea, Creatinine, and BUN) and Serum Electrolyte. Before performing any test, we have to give washings to the machine to clean it. Apparatus that was used during the serum sample tests are Micro -Lab 300, Reagent Hemans or Diesis Reagent 1 and Reagent 2, Yellow and Blue Tip, Pipette 100ml and 1000ml Take Glass Tube, before using the tip and glass tube clean it well with tissue paper. Reagent 1and Reagent 2 were used 400ml and 100ml respectively by using the yellow tip. The total was 500ml. 5 ml serum was taken and after shaking it well, was read in Micro-Lab 300 by setting it at Chapter Urea. After two or three minutes, trial results appeared on the screen of Micro-Lab. The sample was taken in Clot Vile. Incubated it by incubator for 5 to 6 minutes to clot the blood then centrifuge it for 2 to 3 minutes. After centrifugation, plasma and blood cells were separated. Micro -Lab 300, Reagent Hemans or Diesis Reagent 1 and Reagent 2, yellow and blue tips were used, Pipette 100 ml and 1000ml were used. Take Glass Tube. Before using tips and glass tubes cleaned it well with tissue paper. After taking the sample in a clot vile, incubated it to clot the sample and then centrifuged it. Sodium reagent was taken in the amount of o 1000ml and a 1000ml pipette was used. Incubate it at 35 C for 7 minutes. The sodium chapter was set at Micro-Lab 300. Samples were read through the machine and trial results were appeared on the screen. Samples were taken in clot vile. 500ml reagent was taken in the glass tube and 10-µl plasma was used. Incubated for three minutes and the potassium chapter was set at Micro-Lab 300. Results appeared on the screen. The computer program SPSS -19 conducted a one-way analysis of variance to evaluate the treatment effects. LSD was used to calculate the signi cance level for each of the treatments at a level of probability of 0.05%.

R E S U L T S
The analytical analysis showed that CCl exposure 4 alternates the normal physiology disturbing the normal values of urea, creatinine, Na, and K in the blood of the treated groups. Signi cant differences were observed in the hematology parameters between the control and treatment groups. We have found that those individuals who were exposed to CCl4 Urea levels are increased in them. Serum urea level was increased in treated groups (44.60 ± 1.68) in comparison to the control group by the value of (22.60 ± 0.95). Abnormally increased level of Creatinine was observed in CCl4 administered rats by the value of (1.386 ± 0.094) in comparison to normal control by the value of (0.88 ± 0.03) indicating the increased toxicity in rat' s kidneys. An abnormally increased level of Sodium (Na) was observed in CCl4 administered rats in treated groups (139.20 ±.685) in comparison to normal control by the value of (136 ± 0.392) indicating the increased toxicity in rat' s kidneys. An abnormally increased level of Potassium (K) was observed in CCl4 administered rats (4.722 ± 0.578) in comparison to normal control by the value of (3.68 ± 0.392) indicating the increased toxicity in rat' s kidneys (Table 2 and Figure 1). As we know Carbon Tetrachloride (CCl ) is a toxic compound 4 and this toxic compound also exerts toxicity on the kidney. One sample t-test was applied to determine the con dence interval of mean difference of Urea, Creatinine, Na, and K as shown in Table 3. of urea, creatinine, Na, and K. Upper and lower con dence interval for urea was 41.00 and 48.19 respectively. Upper and lower con dence interval for creatinine was 1.18 and 1.58 respectively. Upper and lower con dence interval for Na was 137.73 and 140.66 respectively. Upper and lower con dence interval for K was 4.59 and 4.84 respectively. Table 4: Mean difference of urea, creatinine, Na, and K

D I S C U S S I O N
disrupted, which results in kidney damage in albino rats. Similar ndings were noted by Ogeturk et al., Ozturk et al., Simerville and Bhattacharya and Lun, (2005) [3, 16, 19, 20]. They looked at high levels of urea and creatinine in the urine as the key signs of renal damage brought on by CCl 4 treatment. In their experiment, they discovered that the serum creatinine level doesn't increase until at least half of the kidney nephrons had been compromised or lost. Both Adewole et al., and Bhadauria et al., noted comparable outcomes. They claim that the CCl -treated rats had 4 signi cantly higher serum urea, creatinine, and BUN values, which is associated with decreased creatinine clearance [5,21]. Similar ndings and observations were made by Khan et al., and Xu et al., (2010) [11, 22] These ndings underline the substantial health risks that CCl poses and emphasize the necessity of putting 4 preventative measures and safety regulations in place. Descriptive statistics were done of the treatment group at the signi cant variation of (p > 0.05) to determine the mean and standard deviation of urea, creatinine, Na, and K (Table  5).