Materials and Methods This experimental study was performed under the supervision of the Animal Care Committee of Iran Veterinary Organization. A wild fox was hunted alive and bile was obtained from its gall bladder under aseptic conditions in the Comparative Medicine Research Center at Shiraz University of Medical Sciences, Shiraz, southern Iran. During the postoperative Inhibitors,research,lifescience,medical period, the animal was maintained under controlled environmental conditions

(ambient temperature of 21±2°C, relative humidity of 65-70%, and a balanced diet with free access to food and water). Cell Culture Two human cell lines, HepG2 (NCBI Code:  C158) and CCRF-CEM (NCBI Code:  C105), were purchased from the National Cell Bank of Pasteur Institute (Tehran, Iran). CCRF-CEM is a non-adherent Inhibitors,research,lifescience,medical lymphoblastoid cell line and HepG2 are adherent epithelial-like cells derived from liver tissue. Viability The cells (1×105) were seeded, in triplicate, 24 hours prior to treatment. A fresh two-fold serial dilution of complete bile is prepared in order to treat the cells. After treatment, the number of viable cells was estimated by trypan blue exclusion test. Cell Growth Inhibition Assay (MTT Assay) Cell growth inhibition was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay (Sigma, Germany) assay.12 Briefly, 1×105 cells/well were seeded in each well and exposed to

serial Inhibitors,research,lifescience,medical dilution of bile (in triplicate) and incubated at

37°C in a 5% CO2 incubator for 24 hours. Before the assay, the pH of the plates Inhibitors,research,lifescience,medical was checked. Then, MTT was added and incubation was continued for a further 4 hours at 37°C. The produced formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and the optical density (OD) was measured at 570 nm. The reference wavelength was 690 nm. The mean optical density (OD±SD) was calculated for each group. Non-treated cultures contained the solvent but not the bile. The values were the mean of three different experiments and the growth inhibition was estimated as the reduction of values from a Inhibitors,research,lifescience,medical DMSO control. The percentage of inhibition of cells exposed to the various treatments was obtained as follows: 1– (OD observed/OD control)×100=% inhibition Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay Individual apoptotic cell death was Astemizole observed using an in situ cell detection kit, AP (Roche, Mannheim, Germany) according to the manufacturer’s instructions. TUNEL preferentially labels apoptosis in comparison with necrosis. This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucleotidyl transferase (TdT) has been used for the incorporation of labeled nucleotides to DNA strand breaks in situ. After Adding substrate Mdm2 inhibitor solution, the samples were analyzed by light microscopy. Apoptotic cells were identified under a light microscope (Olympus, Japan) by dark blue nuclei.