2.9. Preparation and characterization of liver microsomes

Pooled liver tissues of 1.5‐2 g per dose group and species were transferred into ice‐cold 50 mmol/L TRIS‐HCl buffer (pH = 7.4) isotonized with 250 mmol/L sucrose, and homogenized with a tissue grinder (Potter S, Vitaris, Baar, Switzerland). Homogenates were initially centrifuged at 9000 g and 4°C for 20 minutes. The supernatants were collected and centrifuged again at 100 000 g and 4°C for 30 minutes (Sorvall Pro80, rotor T‐865, ThermoFischer Scientific). The microsomal pellet was suspended in 50 mL of a 100 mmol/L pyrophosphate buffer (pH 7.25) containing 1 mmol/L EDTA, centrifuged again at 100 000 g and 4°C for 30 minutes. The resulting pellet was re‐suspended in 100 mmol/L sodium phosphate buffer, aliquoted into 0.15 mL portions and stored at –80°C. Total microsomal protein was quantified using the BCA protein assay kit from Pierce (Perbio Science, Lausanne, Switzerland) on a FLUOstar OPTIMA (BMG Labtech GmbH, Offenburg, Germany) using bovine serum albumin as standard. Total P450 concentrations were determined on a PerkinElmer UV/VIS spectrophotometer (lambda 35, Perkin Elmer, Schwerzenbach, Switzerland) using the carbon monoxide assay with an extinction coefficient of 91 mmol/L⁻¹ cm⁻¹ 20 ).