Effects of dibutyl phthalate and di(2-ethylhexyl) phthalate with their metabolites on CYP2C9*1 and CYP2C19*1 activities in vitro

The aim of this article is to assess the effect of phthalates on the activities of CYP2C9 and CYP2C19 in vitro. In this study, recombinant CYP2C9*1 and CYP2C19*1 microsomes were used to investigate the effects of phthalates and their metabolites on corresponding enzyme activities in vitro. 2-100 μM substrate of enzyme was incubated with series concentration of phthalates for 30 min at 37 °C. The metabolic products were detected using ultra-high performance liquid chromatography (UHPLC) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods. The results showed dibutyl phthalate (DBP) significantly inhibited CYP2C9*1 with an activity inhibition rate of 67.3% and half-maximal inhibitory concentration (IC50) of 29.63 μmol L^-1, but its metabolite monobutyl phthalate (MBP) had no significant effect. On the other hand, di(2-ethylhexyl)phthalate (DEHP) had no effect on CYP2C9*1, but its metabolite monoethylhexyl phthalate (MEHP) significantly inhibited the enzyme activity with an activity inhibition rate of 90.6% and IC50 of 6.37 μmol L^-1. With regards to CYP2C19*1, DBP completely inhibited the enzyme activity with an activity inhibition rate of 100% and IC50 of 2.63 μmol L^-1, but its metabolite MBP had no effect on it. DEHP and MEHP also inhibited the activity of CYP2C19*1. Further investigation showed MEHP was a competitive inhibitor of CYP2C9*1 (Ki = 7.063 μmol L^-1), and DBP was a competitive inhibitor of CYP2C19*1 (Ki = 7.013 μmol L^-1) against their substrates, both phthalates were non-competitive inhibitors of the cofactor NADPH. Our results suggested that DBP, DEHP, and their metabolites exhibited significant inhibitory effects toward either CYP2C9*1 or CYP2C19*1. These findings provided valuable information for a potentially toxic mechanism of DEHP and DBP on endocrine disruption and a useful guidance for safe and effective usage of drugs in patients with long-term DEHP and DBP exposure.