*2.4. Analysis of Liver-Damage Serum Markers*

Blood samples were obtained by cardiac puncture. After coagulation, the samples were centrifuged at 3000× *g* for 10 min and serum was stored at −20 ◦C for further analysis. Levels of alanine aminotransferase, aspartate aminotransferase, alcaline phosphatase and lactate dehydrogenase were determined by spectrophotometric assays according to the commercially available diagnostic kits (Biomaghreb, Ariana, Tunisia).

#### *2.5. Biological Determination of Liver Tissue*

#### 2.5.1. Preparation of Tissue Extracts

The liver tissues were homogenized into 2 mL of ice-cold lyses buffer (pH = 7.4) using a grinder (homogenizer ultra-turrax). Then, the homogenate was centrifuged (12,000 rpm, 4 ◦C) for 15 min. The obtained supernatant was frozen at −20 ◦C to determine the oxidative stress biomarkers: malondialdehyde (MDA), protein carbonyls (PC), SOD, CAT and GPX.

#### 2.5.2. Measurement of Lipid Peroxidation and Protein Oxidation in Hepatic Tissue

The lipid peroxidation was estimated in control and treated rats by the quantification of thiobarbituric acid-reactive substances (TBARS) using the method described previously [24]. Briefly, the solution containing 100 μL of liver tissue extract and 100 μL of trichloroacetic acid (TCA, 5%) was centrifuged at 4000× *g* for 10 min. Then, in a boiling water bath, 100 μL of the supernatant was incubated with 200 μL of thiobarbituric acid reagent (TBA, 0.67%) for 15 min. The absorbance was read at 530 nm, the degree of lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) and the result was expressed as nmol of MDA equivalents per gram of tissue (nmol MDA equivalents/g tissue).

The protein carbonyl (PC) level was determined using the method described in the literature [40]. In this method, the carbonyl group of proteins was measured in the resulting pellets by reaction with 2,4-dinitrophenylhydrazine to form protein hydrazone which was measured spectrophotometrically at 370 nm.

#### 2.5.3. Determination of Enzymatic Antioxidants Activities

Total SOD activity, expressed as units per milligram of protein, was determined by measuring the inhibition of pyrogallol activity [41]. One unit (U) corresponded to the enzyme activity necessary to inhibit the oxidation of half of the pyrogallol.

Catalase activity was determined in tissue supernatants using hydrogen peroxide (H2O2) as substrate according to the literature [42]. H2O2 degradation was accompanied by a decrease in absorbance and it was confirmed by measuring the absorbance at 240 nm for 1 min, and the enzyme activity was expressed as mmol H2O2 consumed per minute per milligram of protein.

Glutathione peroxidase activity was assayed by the subsequent oxidation of NADPH at 340 nm, using the method described by Flohé and Günzler [43], and the results were expressed as nmol of GSH oxidized per minute per milligram of protein.

Finally, protein concentration in liver homogenates was measured by the Bradford technique ((BCA) Protein Assay Kit, Pierce Biotechnology Inc., Rockford, USA) with bovine serum albumin (BSA) as standard.

#### *2.6. Histopathological Study*

The liver samples of the rats were fixed in 10% buffered formalin. After fixation, the tissues were dehydrated in a graded series of alcohol, cleared in xylene, embedded in paraffin, and cut into 5 μm thick slices. These serial tissue sections were stained with hematoxylin and eosin (HE) for routine histological examination. Sirius Red and Masson's trichrome staining were used to visualize the collagen deposit in hepatic tissue, which was coloured blue using Masson's trichrome and red by Sirius Red staining. The specimens were observed and photographed through a light microscope (Olympus CX31, Hamburg, Germany).

#### *2.7. DNA Fragmentation Assay*

Genomic DNA in the liver tissue of control and treated rats was isolated by phenol–chloroform DNA extraction method. The separation of intact and fragmented DNA fractions was visualized in an agarose gel by ethidium bromide staining, following the protocol described in the literature [44].

### *2.8. Statistical Analysis*

Statistical analysis was performed using GraphPad Prism 4.02 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) together with Tukey test were applied to observe significant differences between the tested treatments at a significance level (*p* < 0.05).

#### **3. Results**
