History Apigenin (4′ 5 7 AP) an active component of many medicinal Chinese herbs exhibits anticancer properties and 20 μΜ 50 μΜ 20 μΜ 50 μΜ 48 h 72 h 48 h 72 h 48 h 72 h var. HeLa cells [21] at 50 μΜ in human promyelocytic leukemia HL-60 cells [22] and at 25 μΜ in Chang liver cells [23]. AP induced apoptosis in Nutlin-3 the hepatic parenchyma [24-29] and exhibited antiproliferative and apoptotic properties in HepG2 Nutlin-3 Hep3B and PLC/PRF/5 human liver malignancy cell lines [25-28]. Its antiproliferative and apoptotic effects might be mediated through a p53-dependent pathway by p53 accumulation induction of p21 expression and downregulation of CDK4 expression [25 29 Generation of reactive oxygen species (ROS) might also play an important role in AP-induced apoptosis by transcriptionally downregulating catalase activity and increasing hydrogen peroxide levels [27 28 Cell death induction has also TGFB1 been associated with Bax/Bcl-2 ratio changes cytochrome c release and Apaf-1 induction leading to caspase activation and PARP-cleavage in leukemia prostate carcinoma lung cancer and cervical carcinoma cells [19 30 Although the properties of AP against various pro-oxidant and clastogenic brokers have been studied [11 34 there is little information around the genotoxic potential of this particular flavonoid. AP was highly clastogenic in Chinese hamster V79 cells and induced micronuclei formation in human peripheral lymphocytes in a dose-dependent manner [37 38 Other reports pointed out that AP could intercalate into both calf thymus DNA and RNA [39 40 The generation of DNA single-strand (SSBs) and double-strand breaks (DSBs) by DNA-crosslinking brokers [41 42 could lead to sister chromatid exchanges (SCEs) or chromosomal aberrations (CAs) [43]. An and study exhibited that AP can remodel chromatin by inhibiting class I histone deacetylases. This affects regulation expression and activation of various DNA damage Nutlin-3 response genes which results in cell cycle arrest and apoptosis. These affected genes include and and and correlates positively with the tumor’s response to these brokers [55 56 CAs analysis is usually another genotoxic endpoint [43 46 A high frequency of CAs can lead to cell death and it has been associated with increased overall malignancy risk [43 46 57 58 AP’s ability to intercalate into DNA remodel chromatin and upregulate p53 and p21 proteins [25 39 40 44 59 directed us to study the genotoxic potential of this flavonoid in HepG2 cells. We also investigated the proliferation rate index (PRI) and the mitotic index (MI) markers of the cytostatic and cytotoxic properties of chemical and physical brokers respectively [49]. The time course changes in the levels of anti- and pro-apoptotic proteins involved in the DNA damage response were also investigated. Methods Chemicals Apigenin (4′ 5 7 was purchased from Calbiochem (San Diego CA USA). Bovine serum albumin Bradford reagent dimethyl sulfoxide (DMSO) and 3-(4 5 5 tetrazolium bromide (MTT) were purchased from Sigma (St. Louis MO USA). 5-bromodeoxyuridine and bisbenzimide “type”:”entrez-nucleotide” attrs :”text”:”H33258″ term_id :”978675″ term_text :”H33258″H33258 were purchased from AppliChem (Darmstadt Germany). High glucose Dulbecco’s altered Eagle’s medium (DMEM) trypsin-EDTA answer colcemid fetal bovine serum (FBS) and penicillin/streptomycin answer (10 0 0 were purchased from GIBCO (Carlsbad CA USA). Cell death detection ΕLISAPlus kit was purchased from Roche (Mannheim Germany). Human sFas and human sFas ligand Nutlin-3 ELISA kits were purchased from R&D systems (Minneapolis MN USA). Human Bax ELISA kit was purchased from Assay Designs Inc. (Ann Arbor MI USA) and human Bcl-2 ELISA kit was purchased from Bender Medsystems (Vienna Austria). Cell cultures HepG2 cells were managed in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin answer in a 37°C humidified incubator under an atmosphere of 5% CO2. On attaining 75-80% confluency the cells were subcultured by trypsinization and then seeded in appropriate cell numbers depending on the type of the experiments. All experiments took place 24 h after seeding. Cytotoxicity assay The cytotoxic potential of AP was evaluated at 24 48 and 72 h by the MTT method. HepG2 cells were seeded in 96-well plates at a density of 104 cells per well in 100 μL of total culture medium. Cells were incubated with 0.1 1 5 10.