CCl4 induced genotoxicity and DNA oxidative damages in rats: hepatoprotective effect of Sonchus arvensis

Background Sonchus arvesis is traditionally reported in various human ailments including hepatotoxicity in Pakistan. Presently we designed to assess the protective effects of methanolic extract of Sonchus arvesis against carbon tetrachloride induced genotoxicity and DNA oxidative damages in hepatic tissues of experimental rats. Methods 36 male Sprague–Dawley rats were randomly divided into 6 groups to evaluate the hepatoprotective effects of Sonchus arvensis against CCl4 induced genotoxicity, DNA damages and antioxidant depletion. Rats of normal control group were given free access of food and water add labitum. Group II rats received 3 ml/kg of CCl4 (30% in olive oil v/v) via the intraperitoneal route twice a week for four weeks. Group III and IV received 1 ml of 100 mg/kg b.w. and 200 mg/kg b.w. SME via gavage after 48 h of CCl4 treatment whereas group V was given 1 ml of silymarin (100 mg/kg b.w.) after 48 h of CCl4 treatment. Group VI only received 200 mg/kg b.w. SME. Protective effects of SME were checked by measuring serum markers, activities of antioxidant enzymes, genotoxicity and DNA dmages. Results Results of the present study showed that treatment of SME reversed the activities of serum marker enzymes and cholesterol profile as depleted with CCl4 treatment. Activities of endogenous antioxidant enzymes of liver tissue homogenate; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHpx), glutathione-S-transferase (GST) and glutathione reductase (GSR) were reduced with administration of CCl4, which were returned to the control level with SME treatment. CCl4-induced hepatic cirrhosis decreased hepatic glutathione (GSH) and increased lipid peroxidative products (TBARS), were normalized by treatment with SME. Moreover, administration of CCl4 caused genotoxicity and DNA fragmentation which were significantly restored towards the normal level with SME. Conclusion These results reveal that treatment of SME may be useful in the prevention of hepatic stress.


Background
Carbon tetrachloride (CCl 4 ), a clear, colorless, volatile, heavy and nonflammable industrial liquid, widely used to inducede free radical toxicity in various tissues of experimental animals such as liver, kidneys, heart, lung, testis, brain and blood [1]. CCl 4 is converted through hepatic microsomal cytochrome P450 into trichloromethyl-free radical (•CCl 3 or •CCl 3 OO) [2] which in turn, initiate lipid peroxidation process [3,4]. The most widely accepted mechanism of CCl 4 induced hepatotoxicity is the formation of free radicals which is a rate limiting process in tissue peroxidative damage [5,6]. This free radical and related reactive species may cause oxidative stress, which produces major interconnected changes of cellular metabolism, increases the serum marker enzymes, DNA fragmentation, and destruction of the cells by lipid peroxidation [7]. The accumulation of lipid peroxides introduces hydrophophilic moieties and alters membrane permeability and cell function which causes the loss of hepatic integrity and depressed hepatic function resulting in hepatotoxicity and congestive hepatic failure [8]. To protect the body from such deleterious effects of free radicals, several endogenous enzymatic and non enzymatic systems are provided, but when the formation of free radicals is excessive, additional protective mechanisms of dietary antioxidants may be of a great importance [9]. Maintaining the balance between reactive oxygen species and natural antioxidants is therefore crucial, and could serve as a major mechanism in preventing damage by oxidative stress induced by toxic agents. Cooperative defense systems that protect the body from free radical damage include the antioxidant nutrients and enzymes [10]. Antioxidant and radical scavengers have been used to study the mechanism of CCl 4 toxicity as well as to protect tissue cells from CCl 4 induced damage by breaking the chain of lipid peroxidation [11]. Numerous studies have shown that horticultural crops and fruits are sources of diverse antioxidant properties, which can protect body against CCl 4, induced oxidative stress [12]. Sonchus arvensis is traditionally used in the treatment of kidney stone, gallstone, dysentri, haemorrhoid, gout arthritis, appendicitis, mastitis, hypertension, burn wound, and bruises. The present study was therefore designed to investigate the protective effect of Sonchus arvensis (SME) against CCl 4 induced hepatotoxicity in rats.

Animals and treatment
Six weeks old, 36 rats (200-210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25 ± 3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of University of Science and Technology Bannu, KPK, Pakistan. To study the hepatoprotective effects of SME, rats were equally divided into 6 groups (six rats). SME was administered after 48 h of CCl 4 treatment for four weeks.

Assessment of oxidative stress
Hepatic tissue were homogenized in 10 volume of 100 mmol KH 2 PO 4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected and used for the assessment of antioxidant enzymes. Protein concentration in the supernatant of liver tissue homogenate was determined using crystalline BSA as standard. The entire chemicals used in enzymatic analysis were purchased form sigma.

Catalase assay (CAT)
CAT activities were determined by the method of Chance and Maehly [13] with some modifications. The reaction solution of CAT activities contained: 2.5 ml of 50 mmol phosphate buffer (pH 5.0), 0.4 ml of 5.9 mmol H 2 O 2 and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at 240 nm were determined after one minute. One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.

Superoxide dismutase assay (SOD)
SOD activity of liver tissue was estimated by the method of Kakkar et al. [14]. Reaction mixture of this method contained: 0.1 ml of phenazine methosulphate (186 μmol), 1.2 ml of sodium pyrophosphate buffer (0.052 mmol; pH 7.0), 0.3 ml of supernatant after centrifugation (1500 × g for 10 min followed by 10000 × g for 15 min) of homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 ml of NADH (780 μmol) and stopped after 1 min by adding 1 ml of glacial acetic acid. Amount of chromogen formed was measured by recording color intensity at 560 nm. Results were expressed in units/mg protein.

Glutathione-S-transferase assay (GST)
Glutathione-S-transferase activity was assayed by the method of Habig et al. [15]. The reaction mixture consisted of 1.475 ml phosphate buffer (0.1 mol, pH 6.5), 0.2 ml reduced glutathione (1 mmol), 0.025 ml (CDNB) (1 mmol) and 0.3 ml of homogenate in a total volume of 2.0 ml. The changes in the absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 10 3 M −1 cm −1 .

Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. [18]. 1.0 ml sample of homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4°C for 1 h and then centrifuged at 1200 × g for 20 min at 4°C. The total volume of 3.0 ml assay mixture contained 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 mol; pH 7.4) and 0.2 ml DTNB (100 mmol). The yellow color developed was read immediately at 412 nm on a SmartSpecTM plus Spectrophotometer. It was expressed as μmol GSH/g tissue.

Estimation of lipid peroxidation assay (TBARS)
The assay for lipid peroxidation was carried out by the modified method of Iqbal et al. [19]. The reaction mixture  in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 mol; pH 7.4), 0.2 ml homogenate sample, 0.2 ml ascorbic acid (100 mmol), and 0.02 ml ferric chloride (100 mmol). The reaction mixture was incubated at 37°C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 ml 10% trichloroacetic acid. Following addition of 1.0 ml 0.67% thiobarbituric acid, all the tubes were placed in boiling water bath for 20 min and then shifted to crushed ice-bath before centrifuging at 2500 × g for 10 min. The amount of TBARS formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using spectrophotometer against a reagent blank. The results were expressed as nmol TBARS/min/mg tissue at 37°C using molar extinction coefficient of 1.56 × 10 5 M −1 cm −1 .

DNA fragmentation% assay
DNA fragmentation% assay was conducted using the procedure of Wu et al. [20] with some modifications. The tissue (50 mg) was homogenized in 10 volumes of a TE solution pH 8.0 (5 mmol Tris-HCl, 20 mmol EDTA) and 0.2% triton X-100. 1.0 ml aliquot of each sample was centrifuged at 27,000 × g for 20 min to separate the intact chromatin (pellet, B) from the fragmented DNA (supernatant, T). The pellet and supernatant fractions were assayed for DNA content using a freshly prepared DPA (Diphenylamine) solution for reaction. Optical density was read at 620 nm at (SmartSpecTM Plus Spectrophotometer catalog # 170-2525) spectrophotometer. The results were expressed as amount of % fragmented DNA by the following formula; DNA ladder assay DNA was isolated by using the methods of Wu et al. [20] to estimate DNA damages. 5 μg DNA of rats were separately loaded in 1.5% agarose gel containing 1.0 μg/ ml ethidium bromide including DNA standards (0.5 μg per well). Electrophoresis was performed for 45 min at 100 Volt. After electrophoresis gel was studied under gel doc system and was photographed through digital camera.

AgNORs count
Silver staining technique was used according to the Trere et al. [21]. The AgNORs technique was performed on dried slides as follows; unstained fixed slides were dewaxed by dipping for 3 minutes in xylene. After complete removal of wax the slides were hydrated in decrease ethanol concentration (90, 70 and 50%) and washed in distilled water for 10 min and dried in an oven. After drying slides were treated with one drop of colloidal solution (2% gelatin and 1% formic acid) and two drops of 50% AgNO 3 solution onto the slide and incubated at 35°C for about 8-12 min. The progressive staining was followed under microscope to get golden colored nuclei and brown/black NORs. Then, the slide was washed in distilled water, treated for 1 min with 1% sodium thiosulphate at room temperature to stop the reaction, and washed in tap water. The cells were examined under light microscope at 100 × magnification and number of AgNORs was counted per cell.

Statistical analysis
To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.
The results obtained with CCl 4 treatment and changes induced with SME are given below.

Body weight, liver weight
Treatment of CCl 4 caused significant reduction (P < 0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group; were significantly (P < 0.01) restored with treatment of 10 mg/kg b.w., and 200 mg/kg b.w., SME (Table 1).

Lipids profile
Administration of CCl 4 increased triglycerides, total cholesterol, LDL cholesterol while decreased the HDL cholesterol as shown in Table 2. Reduction of HDL cholesterol was significantly (P < 0.01) enhanced by SME while triglycerides, total cholesterol and LDL-cholesterol concentration was appreciably (P < 0.01) augmented to compensate the CCl 4 group.

Genotoxicity studies
Exposure of CCl 4 elicited the hepatic DNA damages (%fragmentation), number of AgNORs/cell. Treatment of rats with 100 mg/kg b.w. and 200 mg/kg b.w. SME restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).

Liver function profile
Administration of CCl 4 markedly increased (P < 0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P < 0.01) by 100 mg/kg b.w. and 200 mg/kg b.w. SME as compared with the CCl 4 group are shown in Table 4.

Assessment of oxidative stress
CCl 4 treatment in rats significantly decreased (P < 0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P < 0.01) by administration of 100 mg/kg and 200 mg/kg b.w. of SME in intoxicated rats (Table 5).

Discussion
Metabolism of various metabolites and exogenous toxic chemicals (pesticides, drugs, metals), are takes place inside the hepatic tissue causes the formation of free radicals which may be extensively toxic than the parent compound. CCl 4 , an extensively studied hepatotoxin is converted into its metabolites such as CCl 3 radicals which are involved in the liver pathogenesis including cirrhosis, genotoxicity of hepatic tissue and hepatic carcinoma [8].  Our present results showed that exposure of rats to CCl 4 caused significant increase in the secretion of ALT, AST, ALP, γ-GT and cholesterol profile due to hepatic injuries caused by their free radicals [22]. Co-administration of 100 mg/kg and 200 mg/kg b.wSME significantly improved the pathogenesis of liver, might be due to the presence of poly phenolic constituent as was reported by Xiao et al. [23]. SME might have the ability to chealat free radical which in turn lowering serum cholesterol, triglycerides and lipid peroxide were reported in other investigations while working on hepatoprotective effects of plant extract against CCl 4 induced hepatic injury in rats [24,25]. Super oxide dismutase and catalase are the main antioxidant enzymes which play an important role in oxidative dysfunction against free radicals induced oxidative stress. Results of our investigation showed that CCl 4 administration in rats result in depletion of antioxidant activities of SOD and CAT, which is in close relationship with other reports [26,27] and have an agreement with investigation following CCl 4 intoxication [28]. GSH is an important protein thiol which coordinates body defense system against oxidative stress. GSH effectively scavenge free radicals and other reactive oxygen species (e.g., hydroxyl radical, lipid peroxy radical, peroxy nitrite and H 2 O 2 ) directly or through GSHpx, GST and GSR [29]. Present study revealed that induction of CCl 4 caused significant reduction in GSH contents as well as significant depletion in the activity of phase II metabolizing enzymes; GSH-px, GST and GSR [30]. Co-treatment of SME in rats markedly improved the activity of metabolizing enzymes as mentioned in literature. TBARS is a major reactive aldehyde resulting during the peroxidation of polyunsaturated fatty acids (PUFA) a useful indicator of oxidative damages [31][32][33][34]. Results revealed that 100 mg/kg and 200 mg/kg b.w. SME significantly improved lipid peroxidation products as was altered by treatment of CCl 4 in rats, which has been well documented [35]. According to Marnett [36] the product of lipid peroxidation react with DNA to form adducts MIG, the mutagenic pirimedopurinone adduct of deoxyguanosine. Like other macromolecules such as lipids and proteins, nucleic acids are also attacked by free radicals to cause oxidative DNA damage.
In the present study, carbon tetrachloride degrades the DNA of liver tissue of rats by generating free radicals. On the other hand, co-treatment of SME appreciably reduced the DNA fragmentation% which also exposed by DNA ladder assay banding pattern. Similar results were reported by Murugesan et al. [37] while studying the protective effects of Kombucha tea against CCl 4 induced oxidative stress in kidneys of rats.