As what has been shown previously that mitochondrion is highly associated with cell viability, especially the MMP. Here, the mitochondria membrane potential based on JC-1 dye [40] was further analyzed. The ratio between red (high potential) and blue (lower potential) florescent intensity reflects the mitochondria functionality in HepG2 cells affected by AFB1 and ST (Fig. 5). Apparently, all the treatment led to a transition from red to blue florescent indicating decreased membrane potential in a dose-dependent manner. The fact that the combination of AFB1 and ST did not show significant difference with the other individual groups at the same toxicity

level showed additive effect of AFB1 and ST on the mitochondria membrane potential. The decreased mitochondria membrane potential, as the biomarker of oxidative stress [41], is a direct result of increased MMP[42], Cyclopamine supplier which is consistent with the cytotoxicity endpoint results of increased ROS and MMP. Mitochondria is the central player in cell apoptosis [43], and a decreased mitochondria membrane potential as well as increased membrane permeability would result in a release of proteins such as cytochrome c to activate caspase cascade and programmed cell death [44]. Thus, the apoptosis of HepG2 cell upon exposing

to AFB1 and ST was studied by FCM employing double staining reagents of propidium iodide (PI) and Annexin V labeled by fluorescein of isothiocyanatc (FITC)(green

fluorescence) that can discriminate intact cells (FITC-/PI −) from apoptotic (FITC+/PI −) or necrotic cells((FITC+/PI Panobinostat purchase +). The viable cell is present in the lower left quadrants (LLQ) of the panels while non-viable, necrotic cells are shown in the upper right quadrants(URQ), and the apoptotic cells are shown in the lower right quadrants(LRQ). The experimental results (Fig. 6) showed that most of the cells in the control sample (A) are present in the LLQ regions, and for samples treated by AFB1 (B-D) and the combinations of AFB1 and ST (H-J), the cell number in the LRQ regions increased in a dose-dependent manner. For ST treatment (E-G), the cell apoptosis occurs even at a very low concentration. With the trend of more cells present Y-27632 2HCl in the separation region between URQ and LRQ as the increase of cell number in LRQ regions (more evident in the group of AFB1 + ST), the cells in the separation region might be regarded as apoptotic cells in their late stages. Thus, the total apoptotic cells include the cells at LRQ and those in the separation region of URQ and LRQ, and when taking them together (Fig. 7), all the treatments induced apoptosis of HepG2 cells. Although the apoptosis rate is increased along the concentration of the mycotoxins (except ST), no significant difference was found among groups (paired t-test) with equivalent toxicity indicating an additive nature of AFB1 and ST on cell apoptosis.