The consumption of NADPH was followed by the decrease in absorbance at 340 nm for 3 minutes at 37 °C. Activity was corrected for protein content of the samples and expressed in nmol/mg protein per minute. 1.1E7 cells were incubated with 0.5 mM CML for 24 hours. After incubation, cells were find more washed with HBSS, harvested with trypsin-EDTA and centrifuged (1000xg, 5 minutes, 4 °C). Cell pellets were then resuspended in 100 mM potassium phosphate buffer pH 6.5 containing 6.3 mM EDTA. Next, cells

were sonicated in icy water for 10 minutes and centrifuged (15 minutes, 10.000xg, 4 °C). Final reaction mixture (1 ml) contained 1 mM GSH and 50 μl sample in buffer. The reaction was started by the addition of 1 mM CDNB (in ethanol). The production of GS-dinitrobenzene was followed by the increase in absorbance at 340 nm for 3 minutes at 37 °C. With each run a spontaneous reaction was included that contained buffer instead of a sample. Activity was corrected for spontaneous reaction and for protein content of the samples and expressed in μmol/mg protein per minute. 1.1E7 cells were incubated with 0.5 mM CML for 24 hours. After incubation, cells were washed with HBSS, harvested with trypsin-EDTA and centrifuged (1000xg, 5 minutes, 4 °C). Cell pellets were then resuspended

in 100 mM potassium phosphate buffer pH 7.0 containing 1 mM EDTA. Next, cells were sonicated in icy water for 10 minutes and centrifuged (15 minutes, 10.000xg, 4 °C). Final reaction mixture (1 ml) contained 0.5 mM GSH, 0.2 mM NADPH, 1

unit glutathione Selleckchem Rapamycin reductase and 50 μl sample in buffer. The reaction was started by the addition of 0.35 mM 2-hydroxyethyl disulfide (HED). The decrease in absorbance at 340 nm, which accompanies the oxidation of NADPH, was monitored for 3 minutes at 37 °C. Activity was corrected for protein content of the samples and expressed in μmol/mg protein per minute. Protein concentrations were determined spectrophotometrically using the DC protein assay kit (Biorad, Veenendaal, The Netherlands) according to manufacturer’s protocol. The effect of CML incubation was tested using Student’s t-test for independent samples or the Mann-Whitney U test when not normally distributed. P-values Dehydratase <0.05 were considered statistically significant and P-values <0.1 were considered statistical trends. We also include statistical trends because for bioactive molecules like GSH, even a small percentage change in the amount can be of biological relevance. Statistical analyses were analyzed with SPSS for Windows (version 20.0; SPSS Inc., Chicago, IL, USA). The effect of CML exposure on 1.1E7 cell viability was determined by MTT assay (Figure 1A) . At concentrations up to 0.125 mM a dose-dependent decrease in viability of 100% to 87% was observed, albeit with a high degree of variation between the different experiments. Above 0.125 mM the additional decrease in viability was only 4%. CML concentrations higher than 0.