ESPE Abstracts

Background: Cytochromes P450 proteins metabolize several steroid hormones, drugs and xenobiotics. All cytochromes P450s in the endoplasmic reticulum require P450 oxidoreductase (POR) for their catalytic activities. Earlier it has been shown that mutations in POR cause metabolic disorders of steroid hormone biosynthesis and also affect some drug metabolizing P450 activities. We aimed to characterize the mutations identified in nonclinical samples to access their effects on steroid and drug metabolism.

Methods: WT and mutant POR proteins, as well as cytochrome P450s, were recombinantly expressed in bacteria and purified. We prepared liposomes embedding the P450 and POR proteins to create a functional P450 metabolic system. Metabolism of androstenedione, testosterone as well as small molecule dyes(MTT, ferricyanide) and tracer compounds was evaluated by radioactive ligand metabolism, fluorescent substrate metabolism and colorimetric assays. Enzyme kinetic analysis was performed using Prism.

Results: The variant P284T identified from normal subjects had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potential disease-causing mutation. As compared to WT, P284T showed 91% and 77% decrease in supporting CYP19A1 and CYP3A4 activity respectively. MTT reduction activity of P284T was also severely affected with only 15% residual activity as compared to WT.

Conclusions: These results suggest that likely pathogenic mutations may be found in non-clinical(apparently normal)population. Their combination as homozygous or compound heterozygous may have severe impact on steroid and drug metabolism by modification of its redox partner activities.