SUBSTRATE SPECIFICITIES OF CYP 2D SABFAMILY

Abstract

The Dark Agouti (DA) rat has been proposed as a poor metabolizer model for the human debrisoquine 4-hydroxylase polymorphism. Earlier studies suggested that the poor metabolizer phenotype in the DA rat is due to the absence of the expression of CYP2D1 mRNA. Although CYP2D 1 catalyzes debrisoquine 4-hydroxylation, we found that another P450 of CYP2D subfamily, presumably CYP2D2, which we have purified from SD rat livers by chasing bunitrolol (BTL) 4-hydroxylase activity as an indicator also has the ability to catalyze debrisoquine 4-hydroxylation. CYP 2D2 has previously been described as inactive species. Thus we obtained CYP2D1 and 2D2 cDNA from Dr. F. J. Gonzalez of NIH and using baculovirus expression system, these two isozymes were expressed in Sf9 insect cells. We found that both isozymes can catalyze debrisoquine 4-hydroxylation, but CYP 2D2 has about 10 fold higher activity than CYP 2D1. In addition, CYP 2D2 was able to catalyze BTL 4-hydroxylation and propranolol 4-, 5-, and 7-hydroxylations while CYP 2D1 was not able to catalyze BTL 4-hydroxylation and propranolol 7-hydroxylation. These results indicate that CYP isozyme responsible for PM phenotype in DA rats is not CYP2D1 as previously indicated but CYP2D2. Levels of expression of CYP proteins were assessed by Western blotting using isozyme specific peptide antibodies. The level of expression of CYP2D 1 in DA rats was about the same level as that in SD rats, but the level of expression of CYP2D2 was markedly lower in DA rats than that in SD rats. The levels of CYP2D 1 and 2D2 mRNAs were examined by Northern blotting to find that levels of both mRNAs were markedly lower in DA rats than those in SD rats. Although the discrepancy between expression levels of CYP 2D1 proteins and its mRNA remains unresolved, it is clear that the reduced level of expression of CYP2D2 is responsible for the PM phenotype of DA rats.