CYP1B1 Detection

Abstract

AbstractThis unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real‐time PCR (qRT‐PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7‐ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3′,5′‐Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC50 of 3 nM for EROD and ∼90 nM for E2 4‐hydroxylation. Binding studies with purified CYP1B1 suggests that TMS interferes in the proximity of the heme region of CYP1B1 with high affinity. Compared to other potent inhibitors such as α‐naphthoflavone, which is a known CYP1 family inhibitor with no selectivity between CYP1B1 and CYP1A2, TMS is ∼50‐ and 520‐fold selective for inhibition of CYP1B1 when compared to CYP1A1 and CYP1A2, respectively. Thus, TMS can serve as a helpful chemical scalpel for dissecting CYP1B1 activity from the overall activity of CYP1 family members against ethoxyresorufin. Curr. Protoc. Toxicol. 51:4.38.1‐4.38.26. © 2012 by John Wiley & Sons, Inc.