Abstract
Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.
Highlights
Induction of cytochrome P450 2B (CYP2B)1 expression by xenobiotics, including clinically used drugs, is mediated by
Because primary human hepatocytes cultures but not hepatoma-immortalized cells maintain the expression of most nuclear receptors, these results suggest that transcriptional factors other than human PXR (hPXR) may be responsible for PHY induction of CYP2B6 within the range of tested treatment concentrations
PHY Induction of CYP2B6 Gene Expression in Primary Human Hepatocyte Cultures—To compare the extent of CYP2B6 induction by PHY and other prototypical inducers, primary human hepatocytes were treated with PHY, PB, RIF, and CLZ as described under “Experimental Procedures.”
Summary
Vol 279, No 28, Issue of July 9, pp. 29295–29301, 2004 Printed in U.S.A. Human Constitutive Androstane Receptor Mediates Induction of CYP2B6 Gene Expression by Phenytoin*. A notable exception is phenytoin (PHY), which exhibits efficacious induction of CYP2B6 protein and mRNA in primary cultured human hepatocytes at 50 M but relatively weak or nonactivation of hPXR in several human hepatoma cell lines (HepG2 and Huh7) at the same concentration [2] To our knowledge, this would represent the first clinically applied drug that might induce CYP2B6 through a PXR-independent pathway. Because primary human hepatocytes cultures but not hepatoma-immortalized cells maintain the expression of most nuclear receptors, these results suggest that transcriptional factors other than hPXR may be responsible for PHY induction of CYP2B6 within the range of tested treatment concentrations Based on these preliminary observations, we hypothesized that hCAR is the predominant regulator of increased CYP2B6 gene expression by PHY in human hepatocytes. Our results demonstrate for the first time that PHY induction of human CYP2B6 is mediated predominantly by CAR rather than PXR
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