Afterward, the Trametinib in vivo liver was cut into transverse slices 300 μm thick using a McIlwain tissue chopper (Campbell Instruments; The Mickle Laboratory Engineering Co). The slices were placed in Krebs–Ringer buffer (10 mM D-glucose, 129 mM NaCl, 1.25 mM NaHPO4, 22 mM NaHCO3, KCl 3 mM, CaCl2 1.8 mM, MgSO4 1.8 mM, Hepes 5 mM, pH 7.4), which was previously bubbled with O2 95% and CO2 5% for 30 min. Sixty slices (per group) were carefully selected, weighted (30 ± 2 μg each) and randomly placed in buffer (2 mL) for

the respective treatments. In the final step of each experiment the total protein content was determined (Peterson, 1977). The slices were subdivided to the following groups: (1) control; (2) MeHg (25 μM); (3) Cysteine (25 μM); (4) MeHg–Cys complex (25 μM each); (5) Methionine (250 μM); (6) Met (250 μM) + MeHg (25 μM); and (7) Met (250 μM) + MeHg–Cys complex (25 μM each). The slices were exposed to the different treatments for 30 min at 37 °C, in the presence of O2 (95%) and CO2 (5%). The molar ratio of cysteine to MeHg was 1, and the stoicheometric reaction between cysteine and MeHg Selleck BIBF 1120 was confirmed by Ellman’s reagent (Ellman, 1959).

The Methionine groups (250 μM) were pre-treated for 15 min with methionine before being exposed to MeHg or the MeHg–Cys complex. All reagents were dissolved in Krebs–Ringer buffer. Liver mitochondria were isolated as previously described by Brustovetsky and Dubinsky, 2000a and Brustovetsky and Dubinsky, 2000b, with some modifications. After treatment, the liver slices were washed three times and manually homogenized in cold buffer I (manitol 225 mM, sucrose 75 mM, K+ EGTA 1 mM, bovine serum albumin (BSA) 0.1%

and K+-HEPES 10 mM pH 7.2), using a potter MRIP glass (length: 10 cm; diameter: 1 cm). Next, the homogenized slices were centrifuged at 2000 ×g for 7 min at 4 °C. The pellet was discarded and the supernatant was centrifuged again at 12,000 ×g for 10 min at 4 °C. Then, the resultant supernatant was discarded, and the pellet was re-suspended in buffer II (manitol 225 mM, sucrose 75 mM, K+ EGTA 1 mM and K+-HEPES 10 mM pH 7.2) and re-centrifuged at 12,000 ×g for 10 min at 4 °C. Finally, the last supernatant was discarded, and the pellet was re-suspended and maintained in buffer III (sucrose 100 mM, KCl 65 mM, K+-HEPES 10 mM and EGTA 50 μM pH 7.2) for subsequent analyses. Both the aliquot of the homogenate of liver slices and the mitochondrial suspension isolated from liver slices were subjected to Hg analysis, which was carried out by Cold Vapor-Atomic Fluorescence Spectrometry according to the method described by Bergdahl et al., 1998. The total Hg content was determined after acid digestion with HNO3, H2O2, H2SO4 and perchloric acid (Bergdahl et al., 1998). RS levels were measured using the oxidant sensing fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCHF–DA) (Hempel et al., 1999).