d 100 X magnification, and the images were captured by a photographic camera Sony Cyber-Shot at 13.5 mega pixels. Hepatocytes were seeded at 1106 cells/plate in 60-mm and treated with compounds at 25 M up to 1824 h. After exposure, the morphology was analyzed by optical microscopy with a 20X objective. 2.7 DNA fragmentation The fragmented DNA content was determined by flow cytometry using PI. For these assays, 1×106 cells were dispensed in 60-mm plates, and incubated 
for 24 h for adhesion at 37C in 5% CO2. The culture medium was then replaced by fresh medium without or with 25 M derivatives, or the corresponding volumes of DMSO, and further incubated for 6 and 24 h. After incubation, the culture medium and cells were collected in a Falcon tube by tripsinization, and the samples were centrifuged at 2000 rpm for 5 min. The precipitate was suspended in phosphate buffered saline and centrifuged again under the same conditions. The cells were suspended in 0.3 mL of a solution composed of 50 g/mL of propidium iodide and 0.1% Triton X -100 in PBS. After labeling, the cells were kept in the dark and analyzed by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736355 flow cytometry with the FACSCalibur apparatus using Cell Quest program. The data acquisition was done using the FL2 filter, and analyzed as histograms. The number of cells in each phase of the cell cycle was expressed as a percentage of total events. Histograms were analyzed using the WinMDI 2.9 software. 2.8 Protein concentration assay Protein concentration was determined by the method of Bradford using bovine serum albumin BSA as a standard. 5 / 17 Selective Cytotoxicity of Mesoionic Derivatives on Hepatocarcinoma 2.9 Inhibition of drug efflux in multiple drug resistant cells HEK293ABCG2 cells were seeded at a density of 1.0105 cells/well into 24-well culture plates. After 72-h incubation, they were exposed to 5 M mitoxantrone for 30 min at 37C, in the presence or absence of each derivative, and then washed with PBS and trypsinized. The MedChemExpress DMXB-A intracellular fluorescence was monitored with a FACSCalibur cytometer, using the FL4 channel and at least 10,000 events were collected. The percentage of inhibition was calculated relatively to 1 M Ko143 which produced a complete inhibition. NIH-3T3ABCB1 were seeded at a density of 6 104 cells/well into 24-well culture plates and incubated for 48 h at 37C, whereas HEK293 cells transfected with ABCC1 were seeded at 2.5 105 cells/well for 72 h. The cells were respectively exposed to rhodamine 123 or calcein-AM for 30 min at 37C, in the presence or absence of each derivative, then washed with PBS and trypsinized. The intracellular fluorescence was monitored using the FL1 channel. The inhibition was measured relatively to 5 M GF120918 or 35 M verapamil, respectively, producing complete inhibitions. The percentage of inhibition was calculated by using the following equation: % inhibition C M = Cev Mx 100; where C corresponds to the intracellular fluorescence of resistant cells in the presence of compounds and fluorescent substrate, M to the intracellular fluorescence of resistant cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19737141 with the fluorescent substrate alone, and Cev corresponds to the intracellular fluorescence of cells inhibited with standard inhibitor in the presence of fluorescent substrate. 2.10 Statistical Analysis Results were expressed as mean standard deviation, and subjected to analysis of variance and Tukey test for comparison of means. A P-value lower than 0.05 was considered significant. All analyses and graphs were performed using
