The values are expressed as nanomoles of Wnt inhibitor GSH/106 cells using a standard curve. A blank with DTT was performed to eliminate its interference in the fluorescence intensity. Protein thiol groups were determined using Ellman’s reagent according to Sedlak and Lindsay (1968) with some modifications. A sample (0.5 mL) of cell suspension was centrifuged at 50 × g for 5 min and the supernatant was discarded. The cell pellet was treated with 1 mL of 5% trichloroacetic acid, 5 mM EDTA. The protein precipitate was washed twice with the same trichloroacetic acid-EDTA solution. When DTT was used, this procedure was repeated

four times. Protein was redissolved in 3 mL of 0.1 M Tris-HC1 buffer,

pH 7.4, containing 5 mM EDTA and 0.5% sodium dodecyl sulfate. Aliquots of this solution were reacted with 0.1 mM (final Adriamycin mw concentration) 5,5′-dithiobis(2-nitrobenzoic)acid (DTNB) in 2 mL of Tris-EDTA buffer, pH 8.6. Absorption was measured at 412 nm and subtracted from blank value obtained by treating sample aliquots with 5 mM N-ethylmaleimide before reaction with DTNB. The values are expressed as nanomoles of –SH equivalents/106 cells using GSH as a standard. Cell death by apoptosis was determined by observing morphological changes in the nuclei of cells incubated with the fluorescent dye Hoechst 33342 (Kurose et al., 1997). Samples (200 μL) were collected and centrifuged at 50 × g for 5 min, and the supernatants were discarded;

the pellet was suspended in Krebs/Henseleit medium, pH 7.4, and incubated with from 8 μg/mL of Hoechst 33342 for 15 min at room temperature. After incubation, the samples were centrifuged twice at 50 × g for 5 min to remove excess dye. After the washes, the cells were suspended in 100 μL of Krebs/Henseleit medium, pH 7.4. Cells were analyzed with a fluorescence microscope (DM 2500 type, Leica, Rueil-Malmaison, France), and the percentage of apoptotic cells was quantitated using QWin software. Comparisons of the several treated groups and the relevant controls were made by analysis of variance (ANOVA) followed by Dunnett’s test. Comparisons between multiple groups were made using Newman–Keuls’s test implemented in GraphPad Prism software, version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Values of P < 0.05 were considered significant. Fig. 1 shows the inhibitory effect of DHM on glutamate plus malate-supported state 3 (ADP-stimulated) respiration of mitochondria in digitonin-permeabilized hepatocytes. The effect was immediate and concentration-dependent, beginning at 50 μM DHM; the parent compound MCT did not inhibit state 3 respiration even at a concentration of 2 mM (Fig. 1). Neither MCT nor DHM stimulated state 4 (basal) respiration (results not shown).