CARBON TETRACHLORIDE INDUCED HEPATORENAL FAILURE IN MALE ALBINO RATS: A DOSE RESPONSE STUDY

Barsha Dassarma1., Saptadip Samanta1*., Dilip K Nandi2 and Somnath Gangopadhyay3 1Department of Physiology, Midnapore College (Autonomous), Midnapore721101, W.B. India 2Department of Physiology and Nutrition, Raja N.L. Khan Women’s College, Midnapore721102, W.B. India 3Department of Physiology, Occupational Ergonomics Laboratory, University College of Science and Technology, University of Calcutta, 92 APC Road, Calcutta 700009, W.B. India


INTRODUCTION
Recently, numerous environmental toxicants from diverse chemical categories are suspected to cause acute or chronic life threatening toxicity in humans. Carbon tetrachloride (CCl 4 ) is a colorless, odorant solvent which makes its entry into environment as gaseous form because of improper disposal of industrial wastes. Exposure on CCl 4 leads to Headache, dizziness, vomiting, stomach pain, light headedness, tiredness, weakness and blurred vision. The principal target organs due to carbon tetrachloride exposure in humans are on liver, kidney. The mode of toxicity of CCl 4 mostly depends on lipid peroxidation of cell membrane and generation trichloromethyl radical (CCl3•), which then further due to low partial pressure of oxygen produces more toxic trichloromethyl peroxyl radical (CCl3O2•) and causes detrimental irreversible ill effects in exposed individual (Ritesh et al.,2015, Boll et al., 2001. This environmental toxin has also got importance because of its repeated use to induce hepatic dysfunction (especially liver fibrosis) in many animal experimental models (Elgazar A. F., 2013).
People exposed to CCl 4 often develop patchy, fibrotic, cirrhotic liver, which by time successfully develops a renal failure along with complete hepatic damage. The day by day incidence of hepatorenal dysfunction is becoming a serious threat to modern civilization. There is immense necessity for an affordable therapy for patients who cannot afford expensive liver or kidney transplant as the only effective remedy to keep them alive.
The present study was aimed to find out the threshold level (LD50) of CCl 4, which can initiate the hepatic injury in a dose dependent manner, by subcutaneous application in male rats as per the guidelines of Organization for Economic Cooperation and Development (OECD).

Selection of animals and care
The study was conducted on 18 healthy, Wister strain male albino rats (Supplied by Ghosh animal, animal foods and animal cages Supplier, Kolkata 54) having a body weight of 100 ± 15 g. These experimental animals were acclimatized in laboratory condition for period of 2 weeks prior to the experiment. Experimental animals were housed three rats/cage in a room with temperature 22 ± 2º C with 12-12 h dark-light cycles by the side of a humidity of 50 ± 10%. They were provided with standard diet and water ad libitum. The experimental animal care was provided according to the Guiding Principle for the Care and Use of Animals (Olert et al., 1993).

Acute toxicity study by limit test
An acute toxicity of Carbon tetrachloride was conducted by acute toxicity method as per Organization of Economic Cooperation and Development (OECD) guidelines 425 (OECD., 2001) where the used limit dose was of 2000 mg/ kg b.wt. Healthy Wister strain male rats (n = 6) were chosen for the acute study by random sampling method. Several wellness parameters of the experimental animals were made and recorded in durations of 30 min, 2hr, 4hr, 6hr, 8 hr, 10 hr, 12hr, 18 hr, 24 hr, 36 hr, and 48 h after the administration of said dose. Several changes with time, after application of dose for skin and fur, eyes, mucus membrane, behavioral pattern changes, tremor, convulsions, salivations, diarrhea, lethargy, sleep and mortality were recorded for 48 hr.

Main test
The main test was conducted on eighteen healthy male albino rats (n = 18). The doses were selected 115 mg and 230 mg/ kg b.wt., for 7 days continuously. The randomly selected experimental rats were divided into three equal groups as follows: Group 'A': control, Group 'B': CCl 4 at 115 mg/kg bw/rat/day; Group 'C': CCl 4 at 230 mg/kg bw/rat/day with basal diet and water ad libitum for 7 days.

Animal's sacrifice
This experimental design for threshold dose selection continued for 7 days. After 7 days, the animals were sacrificed & blood was collected from the aorta after which the liver & kidneys were collected for different biochemical and hematological analysis. Half of liver and kidney tissue were taken and wrapped with sterilized aluminum foil and stored into -20º c deep refrigerator until preparation of tissue homogenates. The other halfs of liver and kidney were preserved in 10% neutral formaldehyde solution till processed for histological examination.

Separation of serum and homogenization of liver and kidney
Serum was separated by centrifugation of blood samples at 1500× g for 15 min taken without anticoagulant. The isolated samples were kept at -80ºC for the biochemical estimation of different parameters (Tripathy et al., 2012) and washed tissues were immediately homogenized in the ice-cold buffer containing 0.25 M sucrose, 1 mM EDTA, and 1 mM Tris-Hcl, pH 7.4. The homogenate was first centrifuged at 600×g for 10 min at 4•C, and the supernatant was stored at -80°C for the biochemical estimation of different parameters (Tripathy et al., 2013).

Biochemical estimation Total cholesterol, (TC)
Total cholesterol level in serum assayed according to the method of Warnick et al., 1995 by test kit.

Triglycerides (TG)
The serum level of triglyceride estimated by using test kit according to the method of Stein et al., 1995.

Serum glutamic oxaloacetic transaminases (SGOT)
The level of aspartate aminotransferase in serum measured according to the Bergmeyer et al.,1974, 2,4-DNPH Method by using Test Kit.

Serum glutamic pyruvic transaminases (SGPT)
The serum level of alanine aminotransferase estimated according to the Bergmeyer et al., 1974, 2,4-DNPH Method by using Test Kit.

Alkaline phosphatase (ALP)
The activity of alkaline phosphatase estimated according to the method of King et al.,1978 by using ALP Test Kit.

Creatinin level in serum
Serum creatinin level measured according to bonses et al., 1945, Alkaline picrate method, by using test kit.

Urea level
Urea level in serum determined according to Coulambe et al., 1965, by using kit.

Determination of Lipid Peroxidation (MDA) Level
According to the method of Ohakawa et al. (1979), lipid peroxidation of liver and kidney homogenate was evaluated. In brief, the reaction mixture prepared with Tris-HCl buffer (50 mM, pH 7.4), tetra-butyl hydroperoxide (BHP) (500μM in ethanol), and 1 mM FeSO4. The samples were incubated at 37 • C for 90 min and the reaction was stopped by adding 0.2 mL of 8% sodium dodecyl sulfate (SDS) followed by 1.5 mL of 20% acetic acid (pH 3.5). The amount of malondialdehyde (MDA) formed during incubation was estimated by adding 1.5 mL of 0.8% TBA and further heating the mixture at 95•C for 45 min. After cooling, samples were centrifuged, and TBA reactive substances (TBARS) were measured in supernatants at 532 nm by using 1.53× 105M −1 cm −1 as extinction coefficient. The MDA levels were expressed in terms of nmol/mg protein.

Determination of Super Oxide Dismutase (SOD) Activity
SOD activity of liver and kidney homogenate was determined from its ability to inhibit the auto-oxidation of pyrogallol according to Mestro and McDonald et al. (1986). The samples were measure at 420 nm at 25 • C for 3 min. SOD activity was expressed as unit/mg protein.

Determination of Catalase (CAT) Activity
CAT activity of these tissue homogenate was measured by the method of Luck (1963). Catalase activity was calculated by using the molar extinction coefficient of 43.6 M−1cm−1for H2O2. The level of CAT was expressed as unit/mg protein.

Determination of Reduced Glutathione (GSH) Level
GSH estimation in tissue homogenate was estimated according to the method of Moron et al. (1979). The required amount of sample was mixed with 25% of TCA and then it was centrifuged at 2,000×g for 15 min. The supernatant was diluted to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6 mM DTNB was added. After 10 minutes the optical density of the yellow-colored complex formed by the reaction of GSH and DTNB (Ellman's reagent) was measured at 405 nm. The levels of GSH were expressed as μg of GSH/mg protein.

Determination of Glutathione Peroxidase (GPx) Activity
The GPx activity of liver and kidney homogenate was evaluated by the method of Paglia and Valentine (1967). Absorbance at 340 nm was recorded for 5 min. Values were expressed as nmol of NADPH oxidized to NADP by using the extinction coefficient of 6.2× 103M −1 cm −1 at 340 nm. The activity of GPx was expressed in terms of nmol NADPH consumed/min/mg protein.

Histological Study
Histological analysis of liver and kidney tissue of each experimental group was performed by the method of Iranloye and Bolarinwa (2009). The animals were sacrificed and the tissues were immediately perfused in saline and formalin and were fixed for 7 days in 10% formaldehyde. Next, dehydration was carried out in ascending grade of alcohol (70%-100%).   The tissues were kept in xylene overnight to remove the alcohol. Then, embedding and casting in paraffin wax with wooden block was done and sectioning of 5 μm thick carried out using a microtome. The sectioned tissues were mounted on slides using a thin film of egg albumen smeared on each side. After deparaffinization in xylene, the sections were passed through alcohol, stained with haematoxylin-eosin, and mounted in neutral DPX medium. The slides were then evaluated for pathological changes under microscope.

Data Analysis
The data were calculated and statistical analysis were done using a statistical package, Origin 6.1, Northampton, Mass, USA the software. The statistically calculated data were expressed as mean ± SEM, n = 6. Comparisons between the means of control and CCl 4 administered group at 115mg/kg/bw and another CCl 4 challenged group with 230 mg/kg bw group, by one way ANOVA, P<0.05, level of significance.

Wellness Parameter Study
Several parameters like skin and fur, eyes, mucus membrane, behavioral pattern changes, tremor, convulsions, salivations, diarrhea, lethargy, sleep and mortality were recorded. It has been observed that after 2hr of application of CCl 4 at 2000 mg/kg bw salivation begins, lathergy develops in some of experimental animals at 4hr hr from the administration of the said dose. At 6hr, eye colour of experimental animals became extensively red. The experimental animals started losing their skin fur at 10 hr from administration of the said dose. Convulsion and tremor appeared at 10 hr. After 24 hr from the time of administration of the dose one rat died, and after 36 hr three experimental rats were died. After completion of the duration of the experiment i.e. 48 hr four experimental rats were died and 2 were still alive as shown in Table. 1. Therefore, the approximate LD50 is less than 2000 mg/kg bw.   Main Test

Determination of TC, TG, MDA
Total cholesterol, total glyceride has been increased significantly (p<0.05) in serum in CCl 4 treated groups (at 115 and 230 mg/kg bw treatment). With the increment of CCl 4 administration at 230 mg/kg bw dose, the total cholesterol has also been increased by 73.71% (Fig. 1A), and total glyceride increased by 93.13% (Fig. 1A) significantly (p<0.05) compared to control. MDA level due to lipid peroxidation in tissue homogenate has been found to elevate by 128.45% and 132.35% in liver and kidney respectively (p<0.05), compared to control group (Fig. 1B).

Estimation of SGOT, SGPT and ALP
In serum, the SGOT, SGPT and ALP level been increased significantly (p<0.05) compared to control group. At 230 mg/kg bw CCl 4 treated group, SGOT has been increased by 120.29% ( Fig. 2A), SGPT has been increased by 139.95% ( Fig.  2A) and ALP has been increased by 106.99% (Fig. 2B) significantly (p<0.05) compared to control group.

Determination of creatinine and urea in serum
In this study, we measure the creatinine and urea level in the serum. It has been found that, creatinine concentration increased by 465.78% and 447.55% for urea significantly when compared to control (p<0.05) (Fig.3A, B).

Antioxidant enzyme level
Antioxidant enzymes like SOD and CAT decreased significantly (p<0.05) in tissue homogenate due to CCl 4 intoxication at 115 and 230 mg/kg bw. In group c the SOD and CAT has been decreased by 81.25% and 70.73% in hepatic cells and 68.33% and 71.31% in renal cells respectively (Fig.  4A, B).

Reduced glutathione (GSH) and glutathione peroxidase (GPx) activity
Fig. 5, shows that reduced glutathione level and glutathione peroxidase activity has been decreased in tissue homogenate.
The GSH activity has been decreased by 56.88%, compared to control in liver and 53.29% in kidney (p<0.05) and GPx activity has been decreased significantly (p<0.05) by 44.44% in hepatic cells and 42.47% in renal cells compared to control group.

Histological Examinations
Histological examination of liver and kidney sections of normal rats showed normal architecture of hepatic lobules with normal central vein, portal tract, hepatocytes and sinusoids and normal glomeruli with intact Bowman's Capsule as shown in Fig. 7A and 8A. Subcutaneous injection of CCl 4 (115 mg/kg body wt/day and 230 mg/kg body wt/day) to rats revealed fatty degeneration, coagulative necrosis, damaged hepatc sinusoids and damaged glomeruli, Bowman's Capsule as shown in Fig.  7B, 7C and 8B, 8C.

DISCUSSION
CCl 4 induced hepatorenal toxicity changes normal liver to fibrotic and cirrhotic one along with renal dysfunction due to vasoconstrictions in renal arteries (Ortega et al., 2002). However, due to lack of therapeutic agents with low expenses causes against hepatorenal dysfunction recently this disease came to an interest. The wellness study was done not only to find out behavioral changes happens in acute exposure, but also to gather an idea about mortality rate of the exposed animals. In this study, after 48 hr duration 2 experimental animals were alive. The result of the study is quite similar to Nandi et al.,  Time  30 min  2hr  4hr  6hr  8hr  10hr  12hr  18hr  24hr  36hr 48hr  where also the same wellness parameters observed. Wellness parameters actually reflect the in vivo changes after application of acute dose. By the study with these parameters the duration and degree of total systemic damage due can be observed. We observed that, it took only 2 hr to commence the toxic effect of CCl 4 and after 24 hr the mortality rate decreases (Table.1).
In male albino rats, 115 and 230 mg/kg bw CCl 4 for 7 days were charged. The level of total cholesterol and glyceride concentration in serum has been increased significantly (p<0.05) in liver and kidney compared to control (Fig. 1A).
The principle causes of carbon tetrachloride (CCl 4 ) induced hepatic damage are lipid peroxidation, generation of free radicals and decreased activities of antioxidant enzymes (Castro et al., 1974;Poli 1993). In this study, it has also been found that the MDA level has been elevated significantly (p<0.05) with the increase of CCl 4 concentration in the tissues (Fig.1B). Initially, lipid peroxidation amplify free radicals generation in the cells, and the reactive aldehydes, generated during lipid peroxidation may well act as "second toxic messengers" of the complex chain reactions and consequently, polyunsaturated fatty acids of the membrane bilayer are converted to lipid hydroperoxides (Esterbauer et al., 1990). The cytotoxic malonaldehyde is also chemically reactive, causes cell damage (Schmidt et al., 1996). MDA, reactive electrophile species, is a marker of oxidative stress as it originates stressed condition in tissues (Yoelii et al., 1975).
Here, we found that the CCl 4 exposed hepatorenal system go through the lipid peroxidation.
In this experiment, there was a marked increase in serum concentrations of SGOT, SGPT and ALP (Fig. 2) were actually sign of damaged hepatic function and impaired renal function were revealed by increased urea and creatinine concentration in serum (Fig.3).
Headed for confirmation about the oxidative stress and antioxidative function into the in vivo system, the SOD, CAT activity in the liver, kidney were measured. The results showed  International Journal of Recent Scientific Research Vol. 8, Issue, 3, pp. 15885-15892, March, 2017 15891 | P a g e that the SOD and CAT activity decreased in the liver and kidney significantly, compared to control (Fig. 4).
It may be due to the release of enzymes into blood from the tissues during the CCl 4 intoxification. SOD is a chain breaking antioxidant agent into the host. SOD protects cells from injury by catalyzing the superoxide into oxygen and hydrogen peroxide (Yoelii et al., 1975). From this study, the SOD generation in tissue, is decreased due to CCl 4 intoxication ( Fig.  4A) and another antioxidant enzyme, catalase, which is found in tissues (Beyer et al., 1989) also decreased due to the CCl 4 toxicity. CAT helps to defend the cell from the toxic property of hydrogen peroxide by catalyzing its disintegration into molecular oxygen and water without the production of free radicals and performs as a scavenger of H 2 O 2 produced by SOD and save from the harmful effects of H 2 O 2 (Iyawe et al., 2010). In the study, catalase activity also decreased significantly (P<0.05) in tissues of CCl 4 treated experimental groups (Fig. 4B).
In mamalian system, there are several enzymes, which are important for synthesis, turnover and maintenance of intracellular system (Tripathy et al., 2012). For the fulfillment of the purpose of study, reduced glutathione (GSH) and glutathione peroxides (GPx) were measured to understand the antioxidant enzyme status as well as glutathione system in liver. In our study, it has been evaluated that the GSH and GPx activity has been reduced significantly (p<0.05) in CCl 4 treated groups, compared to control (Fig. 5).
Reduced thiols (-SH) are the major antioxidants in the cell and Both GSH and protein bound thiols contributes maximum to the total thiols pool in the cell (Prakash et al., 2008). Low levels of total thiols pool have been shown to be associated with various disorders with increased generation of free radicals in liver and kidney due to the CCl 4 exposure. Throughout the study, it has been evaluated that CCl 4 causes damage in to the hepatorenal system gradually with the elevation of dose.
In between two concentration of CCl 4 , i.e 115 mg/kg bw and 230 mg/kg bw, maximum adverse effect has been found from 230 mg/kg bw treated group. The results obtained from different biochemical parameters totally supported by open eye examinations just after dissection (Fig. 6) as well as histological examinations (Fig. 7, 8).

CONCLUSION
From this study, it may be concluded that subcutaneous CCl 4 exposure, in albino male rats causes successful hepatorenal dysfunction by generation of liver fibrosis at 230 mg/kg bw after 7 days exposure. At this concentration, CCl 4 initiates fibrotic, cirrhotic liver and an impairment in kidney functions by creating an oxidative imbalance into cells which finally leads to hepatorenal dysfunction. This study suggest that this concentration may be followed as threshold dose of CCl 4 against male albino rats in laboratory research for further experiment. Furthermore, thorough study it is necessary to be confirm about the physiological changes and also to find out whether this dose is responsible to impair hepato-renal system.