Potential in vitro antioxidant and protective effects of sylimarin on carmustine-induced damage in HepG2 cells. Nesrine S. El Sayeda b, Saleh, S b c, Kenawy, S a; Gamal-Eldeen, A d and Emam, S c a German University in Cairo, b Faculty of Pharmacy Cairo University, c Faculty of Pharmacy 6 October University, d National Research Center. Methodology Preparation of Cell Culture of HepG2 cells Human hepatocellular carcinoma cell line (HepG2) were cultured in tissue culture flasks and maintained in RPMI medium supplemented with 10% fetal bovine serum, 2µmol/ml L-glutamine, 250 ng/ml fungizone, 100 units/ml penicillin G sodium, and 100 units/ml streptomycin sulphate at 37ºC in a humidified 5% CO2 incubator. The cultures were passaged every four days by trypsinization using 1.5 ml trypsin/EDTA solution for 5 min at 37ºC. Cells were used when confluence had reached 75%. To assess the cytotoxicity of BCNU & silymarin, HepG2 cells were treated with different concentrations of BCNU (50-200 µM) or silymarin (25-100 µg/ml) and incubated for 24 h. After incubation time the percentages of viable cells were measured using MTT assay. To assess the extent of silymarin protection, these parameters were measured Cytotoxicity and Cell cycle Analysis Apoptosis DNA fragmentation HDAC (Histone Deacetylase) Activity Assay Determination of lipid peroxides, GST activity, Glutathione content, GPx activity, SOD activity and Total protein content Discussion: Silymarin 25 µg/ml reduced lipid peroxidation induced by carmustine in HepG2 cells and normalized the activities of GST and GPx and the level of GSH, however did not recover the suppressed SOD activity. This is in agreement with the study Kiruthig et al. (2007), which was carried out to evaluate the in vitro antioxidant properties and protective effects of silymarin in human erythrocyte haemolysates against benzo(α)pyrene, and the protective effect was elucidated by significant reversal of the antioxidant enzymes and reduction in the levels of MDA, however SOD activities were not affected, suggesting that silymarin is involved in the removal of superoxide anion radicals and hence the available SOD were not changed by radicals. The hepatoprotective potential of silymarin could be due to its antioxidant property and inhibitory potential on liver enzymes or mitochondrial function. In this study silymarin increased cell viability through reducing the percentages of apoptotic cells, DNA fragmentation and inhibited the arrest in cell cycle induced by carmustine. It was reported that the mechanism of the hepatoprotective function of silymarin could be attributed to another pathway independant to its antioxidant properties, and the protection is mediated through inhibition of DNA fragmentation and suppressing caspase-3 activity (Song et al., 2007). In addition, it prevented cadmium-induced growth arrest and mitochondrial impairment in murin macrophages through decreasing caspase-3 activity and inhibiting DNA fragmentation and necrotic cell death (Kim and Sharma, 2006). L.-H Li et al. (2007) stated that the antiapoptotic effect of silymarin against UV irradiation is by activating SIRT1 (a human deacetylase that was reported to promote cell survival), followed by down regulation of Bax and decreased release of cytochrome c, moreover pretreatment of A375-S2 cells reversed the UV-induced S-phase arrest, resulting in G2/M phase allowing cell more time to repair damaged DNA before mitosis in M-phase. Abstract Carmustine, an important chemotherapeutic alkylating agent, used as either monotherapy or combination with other therapeutic agents for treatment of various cancers including brain tumors. Liver toxicity of nitrosoureas is well known particularly when used in high doses. They are highly cytotoxic, create some kind of cellular damage that leads to apoptosis. This study used human hepatoma cell line, HepG2, to investigate the possible protective effect of sylimarin against the cytotoxic effect induced by carmustine. Treatment of cells with various concentrations of carmustine (25 – 400 µM) resulted in a decrease in cell viability using MTT assay method. Moreover, The 100 µM concentration initiated the signal of apoptosis evidenced by increasing the % of DNA fragmentation, an early sign of apoptosis and the R4 region which represents the % of apoptotic cells. In addition, in cycle analysis by flow cytometry, the population of cells in the S-phase was elevated by 50 %. However, there was no change in histone deacetylase activity (HDAC). This indicates that carmustine-induced apoptosis might not be through inhibition of HDAC. Furthermore, MDA, GSH levels, GST and GPX activities were increased. Addition of sylimarin (25 µM) to the incubation medium protected HepG2 cells from carmustine toxicity. Sylimarin decreased the % of DNA fragmentation, apoptosis and the % of cell population in S-phase. Moreover, it reduced LPO and normalized GST and GPx activities and GSH level. This study shows that sylimarin, besides being an antioxidant, possesses antiapoptotic properties that might be important in protecting the liver from carmustine-induced toxicity. Cell cycle analysis in HepG2 cells after treatment with carmustine 100 µM alone or with antioxidant drugs to determine their effect on cell cycle progression using flow cytometry. R5: Apoptotic region, R10: G0/G1 Phase, R11: S-Phase and R12: G2/M Phase. Carmustine 200 µM +silymarin 25 µg/ml Carmustine 100 µM Carmustine 100 µM +silymarin 25 µg/ml Carmustine 200 µM cells). Background and Aim: Drug-induced liver injury is a major problem causing drug withdrawals. Hepatotoxicity results from the formation of reactive metabolites that can initiate a variety of chemical reactions including covalent binding, depletion of reduced glutathione, or oxidative stress leading to protein, lipid, and DNA damage. Carmustine is a chemotherapeutic agent that belongs to DNA-alkylating agents. It readily crosses the blood brain barrier and used in treatment of brain cancer and melanomas. Supportive nutritional therapy with antioxidants during chemotherapy reduces the generation of lipid peroxides that results from the treatment, therefore antioxidants may reduce several of the side effects induced by chemotherapy. In this study, the experiments were constructed to study the possible protective effects of silymarin against carmustine-induced hepatotoxicity using the hepatoma cell line HepG2 cells. This cell line was developed with the intention that they would replace fresh human hepatocytes in early toxicology screening. HepG2 cells have obvious advantage of their ready availability and assurance of a certain reproducibility of experiments as well as they retain many of the morphological and functional features of normal human hepatocytes, as well as cytochrome P450 activities. Results Conclusion Antioxidant supplementation may provide some benefit when combined with certain types of chemotherapy and provides protection against chemotherapeutic agents induced toxicity especially in liver. Carmustine is effective in treatment of brain tumor, and this because of its high lipid solubility; therefore water soluble antioxidant supplementation will be of great value by providing peripheral protection without affecting the central effects of carmustine on solid brain tumors as they can't pass the blood brain barrier. Antioxidants may provide protective effect results from activities other than their antioxidant properties Silymarin besides being an antioxidant, possesses an antiapoptotic properties. Carmustine100 µM Carmustine200 µM Carmustine 100 µM + silymarin 25 µg/ml Carmustine 200 µM + silymarin 25 µg/ml Recommendations 1- Investigation of novel mechanism of action of sylimarin other than its antioxidant and antiapoptotic effects on carmustine -induced hepatotoxicity 2- Large clinical trials should be focused on the use of sylimarin on the hepatotoxic damage- induced by carmustine Apoptosis analysis of HepG2 cells treated with 100 µM, 200 µM carmustine alone or in combination with sylimarin 25 µg/ml using flow cytometry to determine the different cellular events of cell death. The data represent percentage of the cell population (meanS.D) in different events of cell death (viable, necrotic early apoptotic and late apoptotic cells).