Elsevier

Biochemical Pharmacology

Volume 75, Issue 8, 15 April 2008, Pages 1677-1687
Biochemical Pharmacology

Changes in maternal liver Cyp2c and Cyp2d expression and activity during rat pregnancy

https://doi.org/10.1016/j.bcp.2008.01.012Get rights and content

Abstract

During human pregnancy, CYP2C9, CYP2C19, and CYP2D6 activities are altered. The aim of the current study was to determine if this phenomenon can be replicated in the rat, and to evaluate the mechanisms that contribute to the changes in Cyp2c and Cyp2d activity during pregnancy. The intrinsic clearance of dextromethorphan O-demethylation, a measure of Cyp2d2 activity, was decreased 80% at both days 9 and 19 of gestation when compared to non-pregnant controls. The decreased intrinsic clearance was a result of both decreased Vmax and increased Km-values at both days of gestation. Quantitative RT-PCR revealed that transcripts of Cyp2d2 and Cyp2d4 were significantly decreased at day 19 of pregnancy (p < 0.05) when compared to day 9 and non-pregnant controls. The decrease in Cyp2d mRNA levels correlated with a decrease in several nuclear receptor mRNA levels (RARα, RXRα, HNF1 and HNF3β) but not with the mRNA levels of nuclear receptors usually associated with regulation of P450 enzymes (PXR, CAR and HNF4α). In contrast, Cyp2c12 and Cyp2c6 transcription and protein expression were not significantly altered during rat pregnancy although the intrinsic clearance of Cyp2c6 mediated diclofenac 4′-hydroxylation was increased 2-fold on day 19 of gestation when compared to non-pregnant controls. The increase in intrinsic clearance was due to a decrease in the Km-value for 4′-hydroxydiclofenac formation. These data show that pregnancy significantly alters the expression and activity of drug metabolizing enzymes in an enzyme and gestational stage specific manner. These changes are likely to have toxicological and therapeutic implications.

Introduction

There is substantial evidence that pregnancy changes the disposition of many drugs and modifies the activity of drug metabolizing enzymes [1], [2], [3]. Increased clearance may lead to a lack of therapeutic effect or prolonged titration to achieve desired effect, while decreased clearance may lead to an increased occurrence of side-effects or toxicity due to elevated drug concentrations. Changes in drug clearance may also change the exposure of the developing fetus to the potentially harmful parent drug or its metabolites.

CYP2C9, CYP2C19, and CYP2D6 are major drug metabolizing enzymes and together account for approximately 40% of all Phase I drug metabolism [4]. CYP2C9 plays a major role in the metabolism of phenytoin and anti-inflammatory drugs, whereas CYP2D6 is important in the metabolism of almost all antidepressants, including fluoxetine, paroxetine, tricyclic antidepressants and several of the newer antidepressants [5]. Changes in CYP2C9 and CYP2D6 activity are important in the treatment of pregnant women, since many of their substrates are commonly administered to pregnant women. Increases in enzyme activity could result in significantly decreased plasma concentrations of their substrates during pregnancy, leading to therapeutic failure.

The available clinical data, although limited, suggests that CYP2C9 and CYP2D6 mediated metabolism is increased during pregnancy, while metabolism catalyzed by CYP2C19 is decreased. An increased clearance of phenytoin, an anti-epileptic drug predominantly metabolized by CYP2C9, was reported during pregnancy in two studies [6], [7] and phenytoin dosage had to be increased in 85% of pregnancies to maintain therapeutic efficacy [8]. In contrast, CYP2C19 mediated metabolism of proguanil to its active metabolite, cycloguanil, appears to decrease during pregnancy [9]. In a study of 44 women, the proguanil to cycloguanil plasma concentration ratio was 63% higher during the second and third trimesters of pregnancy compared to postpartum [9].

Two classical CYP2D6 probe substrates, dextromethorphan and metoprolol, have been used to assess CYP2D6 activity during human pregnancy. The data suggests that CYP2D6 activity increases significantly during pregnancy [3], [10], [11]. When evaluated at 26–30 weeks of gestation, the oral clearance of metoprolol increased 6-fold and the bioavailability decreased to half when compared to postpartum [10]. A significant decrease in the urinary dextromethorphan to dextrorphan metabolic ratio, indicative of an increase in CYP2D6 activity, was also observed at all trimesters of pregnancy [3]. Finally, in individuals genotyped for CYP2D6, a 53% decrease in dextromethorphan to dextrorphan plasma ratio was observed in extensive metabolizers whereas a 63% increase in the same ratio was observed in poor metabolizers during pregnancy [11].

The mechanisms underlying the indicated changes in CYP2C9, CYP2C19, and CYP2D6-mediated clearance during pregnancy are poorly understood. For this reason, the goal of the current study was to investigate changes in hepatic Cyp2c and Cyp2d expression and activity during pregnancy using the rat as a model and to investigate potential mechanisms behind any observed changes in Cyp2c or Cyp2d expression and activity. Hepatic Cyp2c and Cyp2d transcription and expression were measured by quantifying the mRNA and protein levels, as well as enzyme activity using isoform-specific probe substrates. Messenger RNA levels of nuclear hormone receptors known to regulate P450 expression were also measured and correlated to changes in P450 mRNA levels.

Section snippets

Animal studies

Animals were cared for in accordance to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication [12]. The experimental studies were approved by the Institutional Animal Care and Use Committee at the University of Washington. Virgin female Sprague–Dawley rats 8 weeks of age were mated and pregnancy was determined by the detection of a vaginal plug. Males were then removed and the females divided in random order into two

Characterization of pregnant rat liver microsomes

Liver weights, microsomal protein, cytochrome P450 content and reductase activity for non-pregnant (control), day 9 and day 19 pregnant animals are shown in Table 1. The liver weight increased significantly from 9.2 g in non-pregnant animals to 11.2 and 13.6 g at day 9 and 19 of gestation, respectively. Liver microsomal protein content (in mg protein/g liver) and microsomal P450 content were significantly lower at day 19 of pregnancy than in non-pregnant animals (Table 1). However, when scaled to

Discussion

The disposition of many drugs studied in pregnant women is altered during gestation and pharmacokinetic studies using probe drugs suggest that P450 and UGT activity is also altered during pregnancy [3], [16]. Increased P450 activity observed during pregnancy could be due to increased transcription (mRNA) of the P450 genes, increased protein translation, improved stability of mRNA or protein or allosteric modulation of enzyme activity. The goal of this study was to establish, using the rat as a

Acknowledgments

The authors would like to thank Professor Allan Rettie for the 3′-, 4′- and 5-hydroxydiclofenac standards and Professor Kent Kunze for use of his equipment. The help of Charles F Spiekerman in interpretation of statistical analyses is greatly appreciated.

This work was supported in part by the Obstetric-Fetal Pharmacology Research Network U10 HD047892 and by a pilot project grant from the University of Washington Specialized Center of Research (SCOR). This work was presented in part at the 14th

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