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

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 V(max) and increased K(m)-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 (RARalpha, RXRalpha, HNF1 and HNF3beta) but not with the mRNA levels of nuclear receptors usually associated with regulation of P450 enzymes (PXR, CAR and HNF4alpha). 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 K(m)-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.

Figure 1

Effects of pregnancy on rat hepatic Cyp2c6 and Cyp2c12 mRNA (A) and Cyp2c12 protein (B) levels, measured in non-pregnant (n=6), day 9 (n=6) and day 19 (n=10) pregnant rats. A) The fold difference in mRNA at days 9 and 19 were calculated relative to non-pregnant values. The bold x indicates mean values for that time point. Each symbol depicts an individual animal. Mean values for Cyp2c6 fold change in mRNA were 1.01 ± 0.14, non-pregnant; 1.30 ± 0.47, day 9; and 1.10 ± 0.24, day 19. Mean values for Cyp2c12 mRNA were 1.06 ± 0.38, non-pregnant; 0.78 ± 0.21, day 9; and 0.86 ± 0.52, day 19. No significant (p>0.05) differences were observed for both mRNA and protein at any of the gestational ages.

Figure 2

Effects of pregnancy on rat hepatic Cyp2d1, 2d2, 2d3, and 2d4 mRNA (A) and Cyp2d1 protein (B) levels measured in non-pregnant (n=6), day 9 (n=6), and day 19 (n=10) pregnant rats. A) The fold difference in mRNA at days 9 and 19 were calculated relative to non-pregnant values. The bold x indicates mean values for that time point. Mean values for fold change in Cyp2d1 mRNA are 1.07 ± 0.42, non-pregnant; 0.83 ± 0.46, day 9; and 0.52 ± 0.22, day 19. Mean values for Cyp2d2 mRNA are 1.04 ± 0.28, non-pregnant; 0.72 ± 0.24, day 9; and 0.38 ± 0.15, day 19. Mean values for Cyp2d3 mRNA are 1.11 ± 0.61, non-pregnant; 1.21 ± 0.49, day 9; 0.80 ± 0.72, day 19. Mean values for Cyp2d4 mRNA are 1.05 ± 0.38, non-pregnant; 1.17 ± 0.82, day 9; and 0.28 ± 0.14, day 19. Statistical significance (p<0.05) when compared to non-pregnant values is indicated by an asterisk and to day 9 values by a dagger.

Figure 3

Diclofenac hydroxylase and dextromethorphan N-demethylase activity in microsomes from non-pregnant, day 9, and day 19 pregnant rats. Individual microsomes from non-pregnant (n=6), day 9 (n=6), and day 19 (n=10) were incubated with either A) 1 μM dextromethorphan or B) 5 μM diclofenac as described in the Materials and Methods section. Data were compared using the student t-test and statistical significance (p<0.05) to non-pregnant values is indicated by an asterisk. Pooled microsomes from each time point were used to obtain velocity versus substrate concentration curves for C) dextrorphan and D) 4′-hydroxydiclofenac formation. Each data point represents the average of two separate values.

Figure 4

Correlation between the fold difference in Cyp2d2 and nuclear receptor mRNA levels for non-pregnant, day 9, and day 19 pregnant rats. The fold difference in Cyp2d2 mRNA levels was plotted versus the fold difference in A) HNF3β, B) RARα, C) CAR, and D) PXR. R-squared and p-values are indicated on the figure. P-values were calculated using linear regression.