Table 3

Effect of NADPH and various trapping and protecting agents on 8-MOP mediated microsomal P450 2A6 inactivation

Inactivation AssayRelative Percent Activity3-a
NADPH-GS100
8-MOP (2.5 μM)112
8-MOP (2.5 μM), NADPH-GS9
8-MOP (2.5 μM), NADPH-GS, 1 μM pilocarpine35
8-MOP (2.5 μM), NADPH-GS, 2 mM sodium cyanide13
8-MOP (2.5 μM), NADPH-GS, 70 μM K3Fe(CN)6 10
8-MOP (2.5 μM), NADPH-GS, 60 mM semicarbazide9
8-MOP (2.5 μM), NADPH-GS, 5 mM methoxylamine5
8-MOP (2.5 μM), NADPH-GS, 5 mMN-acetylcysteine9
8-MOP (2.5 μM), NADPH-GS, 5 mM glutathione10
8-MOP (2.5 μM), NADPH-GS, 5 mM deferoxamine10
8-MOP (2.5 μM), NADPH-GS, 750 units superoxide dismutase10
8-MOP (2.5 μM), NADPH-GS, 2000 units catalase10
8-MOP (2.5 μM), NADPH-GS, overnight dialysis3-b 10
  • 3-a Human liver microsomes (HL109, 50 pmol) were exposed to the indicated components for 3 min at 30°C. The remaining P450 2A6 activity was determined by the amount of 7-hydroxycoumarin formed relative to the control (+ NADPH-GS) using an HPLC fluorescence assay. The rate of turnover for the uninhibited reaction in microsomes (HL109) was 5.21 nmol/nmol P450/min.

  • 3-b Dialysis was carried out overnight against 1 liter (1000 × incubation volume) of 25 mM potassium phosphate buffer (pH 7.4) after exposure to 8-MOP in the absence (control) and presence of the NADPH-GS.