AlternationofCCl4 -Induced Oxidative Stressand Nephrotoxicity by Omega-3 in Female Albino Rats

Hawzheen K. Othman. In the present study, twenty one female albino rats were used and they were divided in to three groups. This was carried out and designed to study the effect of omega 3 poly unsaturated fatty acid on carbon tetrachloride (CCl4) induced oxidative stress and kidney damage in female albino rats. However, the antioxidant activity of omega-3 were determined by assessing biochemical parameters (serum creatinine, urea, uric acid, and malondialdehyde(MDA)). Carbon tetrachloride caused significant increase inserumcreatinine, urea, uric acid, and MDA levelwhile, omega 3 significantly prevented the increase of the serum levels of creatinine, urea, uric acid, and MDA.


Introduction
Free radical is an atom or ionpossessing an unpairedelectron that can be considered as fragments of molecules, these unpaired electrons make free radicals very reactive towards other substances, or even towards themselves: (Chessman and Slater, 1993).It was generally accepted that free radical seek to pair with a new free electron, and free radicals would be generate andit is unstable and can react with another one to generate a new free radical. Thus, this chain reaction of free radical subsequently damage biological systems and tissues (Ozbek, 2012) However, reactive nitrogen species (RNS), reactive oxygen species(ROS)and their generation are two kinds of biologically active free radicals, their generation, chemical reactions, and in vivo effects are closely interrelated. Harman was the first person who recognized the pathogenic role of oxygen free radicals in diseased states (Jeng and Fan-fan, 2012). The studies of free radical and antioxidants in biology demonstrated that free radical played a role in chronic diseases including atherosclerosis, cancer (Lobo et al., 2010) neurodegenerative (e.g. Parkinson's disease, Alzheimer's disease, multiple sclerosis) (Aruoma, 2003), asthma and diabetes (Florence, 2014). Exposure to heavy metals, ultraviolet (UV) radiation,environmental pollutants, drugs, cigarette smoking, alcohol consumption (Sahreen. et al.,2012)and xenobiotics e.g., CCl4 (Kim.,et al 1990)lead to metabolic activation of ROS, which inturn causes cellular injuries (Khanet al., 2012).
Carbon tetrachloride intoxication in animals is an experimental model that generates oxidative stress in many pathophysiological states(Saber.,et al2012). The long term CCl4 exposure generates reactive oxygen species and different free radicalsin many tissues such as liver, heart, kidney, brain, lung and blood. Also, it defects antioxidant enzymes activities and increased thiobarbituric reactive substances concentrations (Théophileaet al., 2006). Inaddition, (Mohammedet al, 2013) reported that CCl4, distributes at greater concentrations in the kidney than in the liver and it has a high affinity to the cortex part of kidney which contains cytochrome P-450 predominantly.
Epidemiological studies investigate that certain that long chain highly unsaturated omega 3-fatty acids which derived from the tissues of oil fish are known to reduce the oxidant status of various tissues.Diet rich in long chain omega-3fatty acids has been shown to help in the development of healthy brain, heart and immune system (Meganathan., et al 2014). Omega-3 fatty acids are considered as strongantioxidant and have an anticancer role in most of the human malignancies (Abdou and Hassan, 2014). Furthermore, regular consumption of omega-3 fatty acids, particularly docosahexaenoic acid, has been reported to ameliorate cardiovascular disorders, including endothelial dysfunction, platelet aggregation, elevated blood pressure and triacylglycerols, and arrhythmia(Abdel-Moneim et al., 2014).
The present investigation was conducted to study the evaluatethe mechanism of omega-3 fatty acids on kidney function test parameters in CCl4 induced nephrotoxicity in female albino rats.

Experimental animals:-
Twenty one adult female albino rats (Rattusnorvegicus) were used in the present study. Animals bred in the animal house of Biology Department, College of Science, Salahaddin University. All rats were weighing about 150-250 gms and 9-12 weeks old. The experiments achieved under the standard laboratory conditions (12h light: 12h dark photoperiod) and 22 ± 4 Cº, the animals were supplied with standard rat diet and tap water ad libitum.

Experimental design:-
One experiment was carried out and designed to study the effects of omega-3 fatty acid on malondialdehyde (MDA) and some renal function test parameters including serum creatinine, urea and uric acids) in CCl4 induced nephrotoxicity in female albino rats. The experimental rats were divided into three groups, each with seven individuals and the treatments were continued for 4 weeks.
The group one received standard rat chaw and tap water (Maulood, 2005) and represents control rats. The rats in the second group received CCl 4 (0.25 mL/kg) body weight intraperitoneally and normal rat chaw with tap water. The rats in third group received standard rat chow supplemented with omega-3 (1gm/Kg) body weight and intraperitoneally administration of CCl4 (0.25 mL/kg) bodyweight.

Collection of blood samples:-
The rats were anesthetized using a combination of ketamine hydrochloride (35 mg/kg) and xylazine (5mg/kg) (Liard et al., 1996). Blood samples were taken by cardiac puncture into chilled tubes with EDTA (4.5 mM as an anticoagulant) and centrifuged at +4C o for 15 minutes; then plasma was stored at -80 o C (Sony, Ultra low, Japan).

Determination of serum malondialdehyde (MDA):-
Serum malondialdehyde (MDA) concentration, one of the end products of lipid peroxidation, was determined by the method of Wallinet al. (1993). The level of serum MDA was determined spectrophotometrically. In brief: To 150 µlserum sample the followings were added: 1ml of 17.5% trichloroacetic acid (TCA) and 1ml of 0.66% (TBA), mixed well by vortex, incubated in boiling water for 15 minutes, and then allowed to cool. One ml of 70% TCA was added and the mixture was allowed to stand at room temperature for 20 minutes, and then centrifuged at 2000 rpm for 15 minutes, and the supernatant was taken for scanning spectrophotometrically at 532nm. The concentration of MDA calculated as follow: Biochemical determination:-Determination of serum creatinine:-Serum creatinine level was determined by spectrophotometerCreatinine kit(Biolab, France). Creatinine in alkaline solution reacts with picrate to form a coloured complex; the intensity of the colour is proportional to the concentration of the creatinine in the serum and urine. The absorbance was measured at 500 nm using spectrophotometer.

Determination of serum uric acid:-
Serum uric acid was determined by uricase method, using colorometric test kit and the absorbance was measured at 492 nm using spectrophotometer (Biolab,France).
Determination of serum urea:-Serum urea was determined by using colorometric test kit and the absorbance was measured at 600 nm using spectrophotometer (Biolab,France).
Statistical analysis:-Analysis of data was performed by using {statistical package for social science (SPSS) version 24}. Results are expressed as mean ± standard error (mean ± SE). Statistical differences were determined by Ducan's test for multiple comparisons after analysis of variance (ANOVA).

Discussion:-
Treatment with CCl4 for days elevated serum MDA level. MDA has been reported to one of the secondary products produced following exposure toreactive oxygen species and free radicals, and it can be used to check out oxidative damage through measuring serum levels of thiobarbituric acid reactive substance (Lee et al., 2012). However our results confirm those of (Ganieet al., 2011) as they concluded that, CCl4 enhances lipid peroxidation through increases free radical formation. CCl4 is metabolize by cytochrome p-450 and produce highly reactive free radical which initiates lipid peroxidation of the endoplasmic reticulum s cell membrane and causes a chain reaction.
Our results were correlated with (Attia and Nasr 2009) who reported that omega-3 caused significant reduction in lipid peroxidation induced by CCl4 in different organs of rats. The decrease in the MDA level by fish oilmay be due to its antioxidant properties. Omega-3 could maintain normal levels of Superoxide dismutase andcatalase activities (Pauwels andKostkiewicz, 2008) Inaddition,the anti inflammatory and anti-oxidant properties of omega-3 may be through it is ability to inhibiting lipid peroxidation and scavenging of free radicals (Abdou and Hassan, 2014).
The present results reveal that, intoxicated rats with CCl4 showed a significant increase in serum creatinine, urea and uric acid levels as compared to normal control rats.These pathological changes indicate the potential damage to kidney cells induced with CCl4 treatment since the observed increase is indication of an impairment of glomerular function and renal disorder (El-bazet al., 2015). Our results are agree with earlier reports reported by (Lotfy, 2009) , who attributed these increases to the pathological changes observed in the kidney tissue and to the damage in kidney glomeruli as a result of CCl4 injection. Moreover (Diabet al., 2014) recorded that CCl4 induced elevation in serum urea could be related to the impairment of renal blood follow as well as reduction in glomerular filtration rate. However, creatinine production, infact, has a strong relationship with muscle mass and increased it is level indicates the muscular wasting occurred during CCl 4 intoxication (Banfi and Del Fabbro, 2006).Although, CCl4 causes depletion and deamination of muscle proteinsbut associated renal disorder prevents normal excretion process and therefore, causes accumulation and elevation serum levels of creatinine and urea (Rahmatet al., 2014). Oral administration of omega-3 produces significant nephroprotective effects in CCl4 intoxicated rats. Our results are in agreement with (Shaheenet al., 2016) who suggest thatOmega-3 may find clinical application in a different condition where cellular damage is a result of oxidative stress. The nephroprotective effects of omega-3 may be explained by the antioxidant effect of omega-3 fatty acids. The antioxidant or anti-inflammatory effects of omegathrough inhibiting lipid peroxidation and scavenging of free have been reported previously (Abdouand Hassan, 2014). However, omega-3 protects rat from CCl4 induced kidney injury as evident from significant decrease in serumcreatinine, urea and uric acid Changizet al., (2012) suggested thatthe ameliorative effects of omega-3 on kidney function may be due to the improvement of glomerular filtration. Moreover, it was generally accepted that fish oil reduce hyperlipidemia by decreasing serum total cholesterol and low density lipoproteincholesterol concentration, so, creatinine might be decreased due to glomerulosclerosis reduction (Lauretaniet al., 2009). In addition, in a study (Abdel-Moneim et al., 2014) demonstrated that elevated serum level of creatinine, urea and uric acid were improved in the hypothyroid treated rats with fish oil, they concluded that the protective effects of omega-3 in in the concentration of creatinine, uric acid and urea may due to that omega-3 ameliorated concentration of thyroid hormones and improved glomerular filteration.

Conclusion:-
In conclusion, from this study it was concluded that omega-3-fattyacids, protects the kidney against CCl4 induced nephrototoxicity in female albino rats. Omega-3(1gm/Kg)+ CCL4(0.25 mL/kg) 6.418 ± 0.5 a 9.61.96±0.71 a 23.57±0.64 a 65.64±6.1 a The same letters mean non significant differences while the different letters mean significant differences