Abstract
This study constitutes an intensive kinetic examination of the mechanistic basis of hamster hepatic microsomal metabolism and its induction. A concerted approach is made toward hydroxylation and spectrally apparent (Types 1 and 2) interaction, in order to elucidate relationships between these two facets of microsomal activity. A thorough investigation is concluded into the requirements and characteristics of normal and induced biphenyl hydroxylation, including its sensitivity to the substrate solubilising agent, Tween 80. A complicated pattern of in vitro [14C]biphenyl metabolism is divined. Biphenyl 2- and 4- hydroxylations are both cyt. P450-mediated'and probably are consumated by two similar, interacting systems which are, nevertheless, kinetically distinct, and which respond differently to inducers. Many apparent rate differences between biphenyl 2- and 4- hydroxylations may originate in the hitherto neglected further hydroxylations of these metabolites. Bimodal kinetics indicate two microsomal Type 1 spectrally apparent interaction sites for biphenyl, but these are not equatable with separate 2- and 4- hydroxylases. There is no simple kinetic relationship between biphenyl hydroxylation and spectrally apparent interaction. Induced kinetic changes suggest that both phenobarbitone and 3-methylcholanthrene stimulate synthesis of both novel and normal biphenyl metabolising systems. Systematically selected analogous compounds indicate some molecular attributes which predispose a substrate toward either Type 1 or Type 2 spectrally apparent interaction. Harmine and (-)warfarin are developed as metabolisable fluorescent probes, which emphasise the respective hydrophobic and hydrophilic natures of the Types 1 and 2 interaction sites. Silicic acid and sod. metasilicate show that spectral interactions are not confined to metabolisable compounds. The natures of microsomal hydroxylation and spectrally apparent interaction active sites are further investigated by monitoring the effects of microsomal storage on kinetic parameters and on various microsomal lipid constituents. Nycthemeral rhythms demonstrated in microsomal metabolism are partially accountable for by cyt. P450 rhythms, but kinetic behaviour indicates the more important involvement of other components of the mixed function oxygenase. Such rhythms are not regulated by plasma corticoids; rhythms in corticoid plasma levels arise partly from periodicity in their hepatic degradation.