3 μM of the copper-DEDTC complex added on cell medium (Viola-Rhenals et al., 2006 and Viola-Rhenals et al., 2007), and the copper-DEDTC complex was suggested to be the toxicological agent. When DEDTC was used without the presence of copper ions

in the same concentration (0.3 μM) in a cell medium complemented with fetal bovine serum (a source of copper ions) no effects on carcinoma cells were observed. Disulfiram (DSF) also have been show to facilitate the copper entrance in cells by the formation of copper-DEDTC complex (Cen et al., 2004), the active form of DSF in the presence of copper, which the induction of apoptosis in neuronal cells remains unclear. In order to contribute Lumacaftor to the elucidation of DEDTC toxicology in neuronal cells, we performed in vitro studies to elucidate the molecular effects of DEDTC and its correlation with copper chelation and concentration. Unless otherwise stated, the chemicals were obtained from Sigma–Aldrich and were of analytical grade. The solutions were prepared using Milli-Q water (Millipore, Bedford, MA, USA). The cell media were prepared with DNase- and RNase-free water and filtered through 0.22-μm filter membranes (Millex GV, Millipore) prior to use. The cell cultures were manipulated using sterile, disposable non-pyrogenic plastic ware and were maintained at 37 °C in an atmosphere of 5% CO2 in air at a relative humidity of 80%. Human neuroblastoma SH-SY5Y cells were purchased from

the American Type Culture Collection (ATCC) and grown in Dulbecco’s Modified Eagle F12 Medium (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine selleck inhibitor serum (Gibco), 100 U/ml penicillin and 10.0 μg/ml streptomycin. The cells were routinely trypsinized and seeded at a density of 4 × 104 cells/cm2.

Every month, the cells were cultivated in the absence of antibiotics for control purposes and subjected to a routine assay using a MycoAlert Mycoplasma selleck compound Detection kit (Lonza Rockland) to ensure that they had not become contaminated with mycoplasma. All SH-SY5Y cells used in this study were used at a low passage number (<15). To determine the levels of DEDTC that would promote maximum cell death, concentration-dependent cytotoxicity studies were performed. Typically, viability of neuroblastoma cells was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays, as previously reported (Mosmann, 1983). SH-SY5Y cells were inoculated in 96 well plates at a density of 1 × 105 cell/well and incubated for 24 h under the conditions described above. Aliquots of freshly prepared solutions of DEDTC (5.0 mM) were added to the culture medium to attain final concentrations in the 1.0–100.0 μM range, and the plates were then incubated for an additional 4, 12, 24, 48 and 96 h. The plates were also incubated in the presence of sodium bathocuproine (BCS, 2,9-Dimethyl-4,7-diphenyl-1,10-phenanthroline) and in copper free conditions.