A comparative study of cytochrome P450-dependent drug metabolism in man and rat
Cytochrome P450, the terminal oxidase of the mixed function oxidase system which metabolises a wide variety of drugs, occurs as a multigene superfamily of different forms. The role of P450 in drug metabolism has been extensively studied in several animal species and, to a lesser extent, in man. This has provided some insights into the catalytic functions, substrate specificities and multiplicity of P450. However, there are large species differences in drug metabolism and in most cases it is not known which forms of P450 specifically metabolise the drugs in the species involved, nor to what extent species differences in oxidative drug metabolism are due to species differences in P450 specificity. The broad aim of this project was to consider the extent to which differences in the functional specificities of rat and human P450 forms determine species diffferences in drug metabolism. The main objective was to study the metabolism of a drug, 2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone (58C80), manufactured by the Wellcome Foundation Ltd., Beckenham, Kent), which shows a large difference in metabolism between rat and man, and to identify and characterise the cytochrome P450 forms responsible for 58C80 metabolism in man and rat. The marked species differences which have been observed in the metabolism of 58C80 in vivo also occur in vitro , suggesting that there is a fundamental species-differences in the enzyme(s) which metabolise this drug. In man in vivo , more than 85&'37 of 58C80 adsorbed from a single oral dose is hydroxylated at the t-butyl moiety, whereas less than 2&'37 is metabolised by this route in the rat. The t-butyl-hydroxylation is typical of a P450-mediated reaction and was, therefore, assumed to be catalysed by P450, although there was no a priori direct evidence for this. By monitoring 58C80 hydroxylase activity throughout each stage of purification, a highly purified form of human P450 was isolated from human liver microsomes (designated P450hB) which catalysed the hydroxylation of 58C80. Based on its N-terminal sequence and its absorption spectrum, P450hB was identified as a member of the human IIC gene family. A polyclonal antibody was raised to P450hB and shown to extensively inhibit 58C80 hydroxylase activity in human liver microsomes, whereas the antibodies raised to other human or rat P450 forms were not inhibitory. P450hB was thereby identified as being the main form of P450 responsible for 58C80 hydroxylation in human liver microsomes. Polyclonal antibodies raised to human P450hB and to the various rat P450 forms purified in this project were used as inhibitors to identify the form of P450 responsible for 58C80 metabolism in the rat. The results indicated that in the rat, 58C80 hydroxylation was catalysed mainly by a form of P450 belonging to the steroid-inducible P450IIIA gene family. Thus, the difference in 58C80 metabolism between man and rat appears to be due to the involvement of members of entirely different P450 gene families in the two species respectively.