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significant increase in liver MDA and decrease in its content of GSH and SOD activity. Contrarily, pre-treatment with CM and SM did not only decrease liver content of MDA but also increased hepatic GSH and SOD activity, suggesting that CM and SM attenuated CCl4-induced oxidative stress. In conclusion, CM and SM had a considerable prophylactic effectiveness against hematotoxicity, oxidative stress, lipid peroxidation, renal impairment and sexual hormones disturbances developed by intraperitoneal injection of rats withCCl4. 1 Introduction Liver is the largest body organ and plays a vital role in detoxification of deleterious materials. It regulates numerous metabolic functions and maintains body homeostasis (Mayuren et al., 2010). Liver disorders are one of the most common problems throughout the world. Liver injury can be induced by certain xenobiotics and microbialin filtration via ingestion or infection (Hai et al., 2011). Carbon tetrachloride (CCl4) is well-known as xenobiotic agent. Liver is not the only the target organ of CCl4 but it also affects several body organs such as lungs, heart, testes, kidneys and brain (Ozturk et al., 2003). Evidence demonstrated that CCl4 activated highly reactive trichloromethyl radical in liver which initiates free radical mediated lipid peroxidation of cell membrane phospholipids (rich with polyunsaturated fatty acids) which are vulnerable to peroxidation. Accordingly, various functional and morphological changes are developed in liver cell membrane which caused an accumulation of lipid-derived oxidants and finally liver injury encountered (Singh et al., 2008). CCl4 is rapidly absorbed by liver tissue in humans and animals. Once it absorbed, it is widely spread among tissues, especially those with high lipid content, reaching peak concentrations in <1–6 hours, depending on exposure dose or its duration time (U.S. EPA. IRIS, 2010). Oxidative stress and membrane damage in hepatocytes, mainly caused via CYP2E1 (Manibusan et al., 2007). Also, CCl4 alter the antioxidant profile of the liver by reacting with sulfhydryl groups of glutathione (GSH) and thiols group of protein, including the antioxidant enzymes as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione transferase (GST) (Knockaert et al., 2012; Yang et al., 2015). Camel milk (CM) is an excellent source of well-balanced nutrients. It exhibits a range of biological activities that influence digestion, metabolism, growth and development of specific organs. These biological properties are mainly due to the presence of certain peptides and proteins in milk (Yagil et al., 1984; Korhonen & Pihlanto, 2001). Camel milk is different from other ruminant milk; it has low cholesterol, sugar and protein but high minerals such as sodium, potassium, iron, copper, zinc and magnesium. Besides this, presence of vitamins A, B2, C and E was also reported in camel milk. The presence of high insulin concentration was also reported in camel milk (Knoess, 1979). Along with this it can be consumed by lactase-deficient individuals because of non-allergic properties of camel milk. A series of metabolic and autoimmune diseases can be successfully cured by camel's milk. In India, camel's milk widely used therapeutically against dropsy, jaundice, problems of the spleen, tuberculosis, asthma, anemia, piles and diabetes (Rao et al., 1970). Further, antibacterial and antiviral activities of CM were also studied by El-Agamy et al., (1992). Silymarin (SM), a polyphenolic flavonoid confined from milk and this is another antioxidant that has been found affective against liver injuries induced by various hepatotoxins including CCl4 (Shaker et al., 2011; Bektur et al., 2016). SM also prevents lipoperoxidation of membranes and scavenges ROS, thus increases GSH availability (Parveen et al., 2011; Vargas-Mendoza et al., 2014). The aim of this study was to investigate the prophylactic effect of camel milk and silymarin on hemotoxicity, oxidative stress, lipid peroxidation, renal function and sex hormone disturbances in CCl4-intoxicated rats. 2 MATERIALS AND METHODS 2.1 Chemicals Carbontetrachloride (CCl4) obtained from LobaChemie, India. Solution of CCl4 prepared by dissolving in 50% olive oil V:V and injected intraperitoneally in to the experimental rats at a dose of 1 ml/kgb.w, once daily, 3 times weekly for four weeks to induce toxicity as described by Abdel-Moneim et al. (2015). Raw silymarin was obtained from ElobourMedern Pharmaceutical Industries Co., Egypt. Rats were given silymarinorally at a dose of 150 mg/kgb.w suspended in distilled water (Chen et al., 2012). Recommended dose of silymarin were given once daily, 5 times in a week for 2 weeks and 3 times in a week for next 4 weeks. Early morning, hand milking camel milk (CM) was collected daily from western desert camel farm in sterile screw capped containers and transported to the laboratory in cool boxes. CM was given to rats in a dose of 5 ml/ rat according to El Miniawy et al. (2014), once daily, 5 times in a week for 2 weeks and 3 times in a week for next 4 weeks. 2.2 Experimental animals Total thirty six (36) adult male albino rats weighing about 180g in average were used for this study. They were selected among the animals bred in the small Animal House of the Nuclear Research Center, Atomic Energy Authority, Egypt. The animals were acclimatized for two weeks under ambient environmental conditions and housed in well aerated cages. 2.3 Animal grouping and treatment Animals were randomly assigned into six equal groups viz. Normal control animals, without any treatment (G1), SM ingested (G2), CM drenched (G3), CCl4 intoxicated (G4), SM prophylactic group (G5 – SM treatment for first six weeks + CCl4 treatment started from fifth week of SM treatment and continue for the next 4 weeks) and CM prophylactic group (CM treatment for first six weeks + CCl4 treatment started from fifth week of CM treatment and continue for the next 4 weeks) and they were fed on a balanced rodent diet. They had free access to feed and drinking water from the beginning of the experiment until its termination At the end of each treatment and after overnight fasting, animals were decapitated and trunk blood samples were collected in tubes with or without anticoagulant for assaying hematological indices, serum levels of renal function tests and sex hormones. Simultaneously liver was excised from each scarified rat. Promptly liver samples were rinsed in 0.1 M cold phosphate buffer (PH 7.4) and homogenized using a Teflon pestle to prepare 10% homogenates used for assessment of oxidative stress and peroxidation biomarkers. 2.4 Hematological Measurements Blood samples were collected in EDTA coated tubes and used for determination of hematological parameters included hemoglobin (Hb) level, erythrocytes (RBCs), platelets (PLT) and leucocytes (WBCs) counts which were evaluated according to Dacie & Lewis (1993). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated from the values of PCV, Hb and RBCs count as described by Jain (1986). For differential leucocyte counts, blood smear were stained with Giemsa. 2.5 TBARS and antioxidant enzymes assays Lipid peroxidation biomarkers were expressed as malondialdhyde (MDA) and determined according to Satoh (1978). Reduced glutathione (GSH) was assayed as described by Beutler et al. (1963) and superoxide dismutase was measured by the procedure given by Nishikimi et al. (1972). 2.6 Levels of serum testosterone and estradiol estimation Levels of serum testosterone and estradiol were assayed by RIA kits (10227-Czch Republic purchased from IMMUNOTECH Company) by following manufacturer’s instructions. 2.7 Renal function tests Renal function tests were determined by following the method of Fawcett & Scott, (1960) for urea, Barham &Trinder, (1972) for uric acid and Larsen, (1972) for creatinine. 2.8 Statistical Analysis: The obtained results were expressed as means ± standard errors. The data were subjected to F test one way analysis of variance (ANOVA) according to Snedecor & Cochran (1982) followed by Duncan’s multiple range test (Duncan, 1955) to determine the significance of difference (P≤ 0.01) between means of treated groups. 3 RESULTS Result of study presented in table 1, revealed that administration of CCl4 had negative effect on all studied hematological parameters. Significant reduction (P< 0.01) was reported in Hb level, RBCs numbers, PCV, MCHC, PLT and lymphocyte counts. While, an improvement (P< 0.01) was reported in MCV, MCH, WBCs and neutrophil counts in CM, SM treated animals as compared the control group. Similarly, when CM and SM were used as prophylactic agents against CCl4 toxicity, they nearly succeeded to bring back the values of the above mentioned parameters toward the basal figures of normal control groups. Regarding to lipid peroxidation and oxidative stress, data presented in table 2 denoted that IP injection of rats with CCl4 (G4) led to a significant increase (P< 0.01) in MDA content of liver. While, concentration of GSH and SOD activity were significantly (P< 0.01) reduced as compared with their corresponding values in control group. Pre- treatments of CM and SM remarkably repaired the negative effects of CCl4 on hepatic MDA and GSH whereas the activity of SOD was completely restored and the level of GSH was partially returned close to the normal values of control group.
Table 2 Effect of camel milk and silymarin treatment on hepatic lipid peroxides markers as (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) of normal rats and CCl4 intoxicated rats
Group Liver Functioning Parameters MDA (nmol/g.tissue) GSH (mg/g. issue) SOD (U/g. tissue) G1 11.89b±0.36 61.99a±0.56 12561.5a±367.7 G2 8.04bc±0.94 58.40a±1.38 12357.9a±190.5 G3 6.69c±1.40 59.20a±2.12 11735.2ab±263.0 G4 20.38a±2.19 22.87c±0.77 10517.9c±364.4 G5 22.31a±1.62 40.08b±1.23 10864.5bc±443.8 G6 19.75a±1.49 38.60b±1.38 11517.5abc±179.5 Data are expressed as Mean ± S.E, Mean values with different letters in the same column are significantly different at P< 0.01 Table 3 Effect of camel milk and silymarin treatment on serum urea, uric acid and creatinine of normal and CCl4 intoxicated rats
Group Urea (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl) G1 41.64c±1.60 3.13a±0.11 1.07bcd±0.05 G2 49.92b±1.81 2.38b±0.15 0.96cd±0.05 G3 48.92b±1.10 2.18b±0.06 0.92d±0.09 G4 56.76a±1.65 3.56a±0.25 1.47a±0.06 G5 48.03b±1.15 3.00a ±0.23