Liver microsomal preparation and cytochrome P450 activity analysis

Liver samples (right, caudate, and quadrate lobes) from female offspring (F2) were shipped (on dry ice) to the Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden, for cytochrome P450 (CYP) activity analysis. Approximately 1 g tissue were used to prepare hepatic microsomes by a calcium aggregation method previously described [49]. Protein concentrations in the microsomes were measured with a commercially available kit (Bio-Rad Laboratories Inc., Hercules, USA) according to the manufacturer’s instructions and using bovine serum albumin as standard. Microsomes were diluted to a protein concentration of 4 mg/mL and stored at -80°C until use.

The activities of CYP1A and CYP2B10 were determined as a rate of 7-ethoxyresorufin (EROD, CYP1A1), 7-methoxyresorufin (MROD, CYP1A) and 7-pentoxyresorufin (PROD, CYP2B10) O-dealkylation [50]. CYP3A11 activity were established as a rate of and benzyloxyresorufin (BROD) O-dealkylation [51]. CYP2E1 and CYP2A5 activities were determined as a rate of p-nitrophenol (PNPH) and coumarin-7- hydroxylation (CoH), respectively [52]. These isoforms were selected because of their well-known role in xenobiotic metabolism. Experimental conditions for activity assays are reported in S1 File. Briefly, incubation mixtures were comprised of microsomal protein (0.2 mg for all enzymes except for PNPH with 0.5 mg), phosphate buffer (50 mM, pH 7.4) and the appropriate substrate (1 μM for EROD, 2 μM for MROD and BROD, 10 μM for PROD, and 200 μM for PNPH and CoH). Reactions were initiated by addition of 0.5 mM NADPH. The solutions were incubated in a water bath at 37°C for 5 (EROD), 7 (MROD and BROD), 15 (CoH), 20 (PROD), or 30 (PNPH) min. Reactions were terminated by adding 0.5 mL ice-cold methanol (40% TCA for PNPH) and the mixtures were centrifuged (7500 g at 4°C for 5 min). The amount of formed resorufin in the supernatants (for CYP1A, CYP2B10 and CYP3A11) was measured using HPLC with fluorescence detector (560 and 586 nm for excitation and emission wavelengths, respectively). The amount of formed p-nitrochatecol (for CYP2E1) was measured using HPLC with UV detector (345 nm). The amount of formed hydroxycoumarin (for CYP2A5) was measured using HPLC with fluorescence detector (338 and 458 nm for excitation and emission wavelengths, respectively). Formation of all metabolites were linear with microsomal protein concentrations and incubation times. All enzymatic activities were expressed as pmol of reaction product per mg protein and min.