The cells were cultured in a suspension using RPMI 1640 supplemented with 10% heat-inactivated horse serum, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL GSK 3 inhibitor streptomycin,
1 mM sodium pyruvate and 2.5 μg/mL of amphotericin B. The serum concentration was reduced to 5% during treatment and increased to 20% when the cells were dispensed into the microwells. Preliminary experiments were carried out to determine the solubility and cytotoxicity of the chemical compounds to be tested. The cytotoxicity was determined by way of the relative total growth (RTG) after 4, 24 and 48 h of treatment at concentrations from 0.1 to 500 μg/mL, without metabolic activation. MLA was carried out as previously described (Soriano et al., 2007). The Tk−/− mutants were selected adding 4 μg/mL of TFT to each culture.
TFT was added to the cultures (1 × 104 cells/mL) to a final concentration of 4 μg/mL. Each culture was treated with TFT, dispensing 0.2 mL/well onto plates containing 96 wells. The plates were incubated at 37 °C, 5% CO2 for 12 days and the colonies then visually scored using a qualitative assessment. To evaluate the TFT plates containing mutations, a thiazolyl blue tetrazolium bromide solution (MTT, 2.5 mg/mL) was added to each well, and the plates incubated for 4 h so that the cell colonies could acquire a black coloration. The colony size was estimated in a manner similar to that described by Honma et Cytidine deaminase al. (1999): a small colony was defined as one with a size ⩽one-fourth of the well diameter. The statistical approach used Selleckchem EPZ015666 was a one-way ANOVA followed by the Dunnett test, which was used to assess the significance of the difference in MF (mutant frequency) between the control and treated cultures. The dose–response was also calculated by testing for linear trend (Moore et al., 2003 and McClain et al., 2006). The level of statistical significance was set at 5%. Initially, a preliminary experiment was carried out to determine the best experimental conditions for the spectrophotometric analysis of DR1. Thus a spectrophotometric profile of the dye DR1
at different concentrations (2.5 × 10−5, 5.0 × 10−5, 7.5 × 10−5 and 1.0 × 10−4 mol L−1) dissolved in DMSO (data not shown) was carried out. After this initial analysis, the best working condition was established as being 1.0 × 10−4 mol L−1. DR1 showed a band at 510 nm corresponding to the chromophore group (azo group), i.e. the portion of the molecule responsible for the color of the dye. After fixing 1.0 × 10−4 mol L−1 as the best experimental condition, the dye was reacted with the S9 mixture. Fig. 2 (A and B) clearly shows that the chromophore group of DR1 is completely metabolized by the Cytochrome P450 isoenzymes, detected by suppression of the peak at 510 nm in the UV–Vis spectra and also by the removal of the peak attributed to the dye at tR = 5.5 min by HPLC/DAD.