Human-liver cytochromes P-450 involved in polymorphisms of drug oxidation

Abstract

1. Nine forms of cytochrome P-450 have been purified to electrophoretic homogeneity from human-liver microsomes. These include the enzymes involved in debrisoquine 4-hydroxylation, phenacetin O-deethylation and mephenytoin 4-hydroxylation, three reactions which are characterized by genetic polymorphism in humans.

2. Evidence for the involvement of the above enzymes comes from reconstituted immunochemical inhibition studies with human-liver microsomes. These and other lines of evidence are consonant with the view that different forms of cytochrome P-450 are involved in the three reactions.

3. The debrisoquine 4-hydroxylase has been studied most extensively in terms of its substrate specificity. In addition, an analogous rat enzyme shows some homology and serves as a useful model. The use of antibodies raised to the rat-liver enzyme in immuno-inhibition studies with human-liver microsomes provides a means of determining the extent to which this enzyme participates in other reactions.

4. Translation of rat-liver mRNA in vitro yields the intact debrisoquine 4-hydroxylase; studies with human mRNA suggest a lower frequency than in rats. The basis for impaired catalytic activity in phenotypically poor human metabolizers appears to be an altered enzyme in all three cases, as opposed to a decreased level of a single enzyme. Using antibody screening of fusion proteins expressed in a cDNA library, it has been possible to isolate cDNA probes for all three of these cytochromes P-450 for use in screening individuals and ultimately determining the basis of these polymorphisms.