Consequences of Phenytoin Exposure on Hepatic Cytochrome P450 Expression during Postnatal Liver Maturation in Mice

Abstract

The induction of cytochrome P450 (P450) enzymes in response to drug treatment is a significant contributing factor to drug-drug interactions, which may reduce therapeutic efficacy and/or cause toxicity. Since most studies on P450 induction are performed in adults, enzyme induction at neonatal, infant, and adolescent ages is not well understood. Previous work defined the postnatal ontogeny of drug-metabolizing P450s in human and mouse livers; however, there are limited data on the ontogeny of the induction potential of each enzyme in response to drug treatment. Induction of P450s at the neonatal age may also cause permanent alterations in P450 expression in adults. The goal of this study was to investigate the short- and long-term effects of phenytoin treatment on mRNA and protein expressions and enzyme activities of CYP2B10, 2C29, 3A11, and 3A16 at different ages during postnatal liver maturation in mice. Induction of mRNA immediately following phenytoin treatment appeared to depend on basal expression of the enzyme at a specific age. While neonatal mice showed the greatest fold changes in CYP2B10, 2C29, and 3A11 mRNA expression following treatment, the levels of induced protein expression and enzymatic activity were much lower than that of induced levels in adults. The expression of fetal CYP3A16 was repressed by phenytoin treatment. Neonatal treatment with phenytoin did not permanently induce enzyme expression in adulthood. Taken together, our data suggest that inducibility of drug-metabolizing P450s is much lower in neonatal mice than it is in adults and neonatal induction by phenytoin is not permanent.

Fig. 1.

Effects of phenytoin (PHY) treatment on the expression and activity of CYP2B10 at different ages during postnatal maturation. Male and female mice were treated with either vehicle control (PBS) or 100 mg/kg PHY at different ages following birth. The short-term effects of PHY treatment were measured 24 hours after treatment. Changes in gene expression were evaluated at the mRNA level using RT-PCR, and then fold changes were calculated using the 2^−ΔΔCt method. Data points represent fold changes in gene expression of individual mice treated with PBS or PHY compared with the average ∆Ct of mice that received PBS control (A). Ct values from RT-PCR experiments were also compared with the GAPDH Ct value in each individual mouse and the differences are expressed as 2^{−[Ct(P450) − Ct(GAPDH)]} (B). The dotted line represents GAPDH expression set to 1 (n = 4–6). Protein was quantified using a liquid chromatography–tandem MS method in males only on days 5 and 60 of age of PHY treatment (C) (n = 5). Enzyme activity was confirmed by measuring the amount of pentoxyresorufin metabolite produced by S9 fractions isolated from day 5 and 60 male liver samples (n = 5) treated with resorufin (D). The long-term effects of PHY treatment were investigated in a similar fashion. Mice were treated with 100 mg/kg PHY on day 5 after birth, and then livers were collected (n = 3–6) at different later time points for measurements of mRNA (E), and for protein and enzymatic activity at 60 days of age (F and G). The data are presented as mean ± S.E. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Effects of phenytoin (PHY) treatment on the expression of CYP2C29 at different ages during postnatal maturation. Male and female mice were treated with either vehicle control (PBS) or 100 mg/kg PHY at different ages following birth. The short-term effects of PHY treatment were measured 24 hours after treatment. Changes in gene expression were evaluated at the mRNA level using RT-PCR, and then fold changes were calculated using the 2^−ΔΔCt method. Data points represent fold changes in gene expression of individual mice treated with PBS or PHY compared with the average ∆Ct of mice that received PBS control (A). Ct values from RT-PCR experiments were also compared with the GAPDH Ct value in each individual mouse and the differences are expressed as 2^{−[Ct(P450) − Ct(GAPDH)]} (B). The dotted line represents GAPDH expression set to 1 (n = 4–6). Protein was quantified 24 hours after PHY treatment using a liquid chromatography–tandem MS method in males only on days 5 and 60 after birth (C) (n = 5). The long-term effects of PHY treatment were investigated in a similar fashion. Mice were treated with 100 mg/kg PHY 5 days after birth, and then livers were collected (n = 3–6) at different later time points for measurements of mRNA (D) and at 60 days of age for measurement of protein concentration (E). The data are presented as mean ± S.E. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Effects of phenytoin (PHY) treatment on the expression and activity of CYP3A11 at different ages during postnatal maturation. Male and female mice were treated with either vehicle control (PBS) or 100 mg/kg PHY at different ages following birth. The short-term effects of PHY treatment were measured 24 hours after treatment. Changes in gene expression were evaluated at the mRNA level using RT-PCR, and then fold changes were calculated using the 2^−ΔΔCt method. Data points represent fold changes in gene expression of individual mice treated with PBS or PHY compared with the average ∆Ct of mice that received PBS control (A). Ct values from RT-PCR experiments were also compared with the GAPDH Ct value in each individual mouse and the differences are expressed as 2^{−[Ct(P450) − Ct(GAPDH)]} (B). The dotted line represents GAPDH expression set to 1 (n = 4–6). Protein was quantified using a liquid chromatography–tandem MS method in males only on days 5 and 60 after birth of PHY treatment (C) (n = 5). Enzyme activity was confirmed by measuring the amount of 1′hydroxymidazolam metabolite produced by S9 fractions isolated from day 5 and 60 male liver samples (n = 5) treated with midazolam (D). The long-term effects of PHY treatment were investigated in a similar fashion. Mice were treated with 100 mg/kg PHY 5 days after birth, and then livers were collected (n = 3–6) at different later time points for measurements of mRNA (E), and for protein and enzymatic activity at 60 days of age (F and G). The data are presented as mean ± S.E. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Effects of phenytoin (PHY) treatment on the expression of CYP3A16 during postnatal maturation. Male and female mice were treated with either vehicle control (PBS) or 100 mg/kg PHY at different ages following birth. Twenty-four hours after treatment, livers were collected for quantification of mRNA by RT-PCR (n = 4–6). Changes in gene expression were evaluated at the mRNA level using RT-PCR, and then fold changes were calculated using the 2^−ΔΔCt method. Data points represent fold changes in gene expression of individual mice treated with PBS or PHY compared with the average ∆Ct of mice that received PBS control (A). Ct values from RT-PCR experiments were also compared with the GAPDH Ct value in each individual mouse and the differences are expressed as 2^{−[Ct(P450) − Ct(GAPDH)]} (B). The dotted line represents GAPDH expression set to 1 (n = 4–6). Protein was quantified using liquid chromatography–tandem MS 24 hours following PHY treatment in male neonates (day 5) and male adults (day 60) (C). The data are presented as mean ± S.E. *P < 0.05; **P < 0.01; ***P < 0.001.