A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

Abstract

Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentrationduring the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2µM), estradiol (0.3µM), estriol (0.8µM), estrone (0.2µM), 17α-hydroxyprogesterone (0.1µM), and human growth hormone (0.005µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.