Diphenylhydantoin-induced difference spectra with rat-liver microsomes

Abstract

Addition of diphenylhydantoin (DPH) to rat-liver microsomes caused a type l substrate-induced difference spectrum with a peak at 390 nm and a trough at 425 nm. The magnitude of this substrate-induced change, δA max was relatively small (mean absorbance: 0.027 ± 0.003 S.E., N = 6 per 3 mg of microsomal protein per ml) as compared with that caused by benzphetamine or aniline. The apparent K s for the DPH-inicrosome interaction was low: 27 ± 2 μM(mean ± S.E., N = 6). The δA max of substrate-induced difference spectra caused by addition of DPH to microsomes was proportional to the microsomal protein concentration, but rapidly decayed during storage at 4° (80% reduction in 24 h). Treatment of animals with phenobarbital, DPH, chlordane, or DDT caused a reduction of δA max of the DPH-microsome interaction as compared with that obtained with hepatic microsomes from the control animals. Treatment of animals with 3-methylcholanthrene (3MC) either abolished the δA max of DPH-microsome interaction or caused a reversal to a modified type II response. Addition in vitro of phenobarbital, DPH, chlordane, DDT, or 3MC as modifiers to the microsomes from control animals, reduced or abolished the δA max of substrate-induced difference spectra with DPH as substrate. It may be concluded that DPH despite its low K s is a rather weakly binding substrate which is easily replaced by more strongly binding ligands.