Cross-talk between xenobiotic detoxication and other signalling pathways: clinical and toxicological consequences.

1. Recent investigations on nuclear receptors and other transcription factors involved in the regulation of genes encoding xenobiotic metabolizing and transport systems reveal that xenobiotic-dependent signalling pathways are embedded in, and establish functional interactions with, a tangle of regulatory networks involving the glucocorticoid and oestrogen receptors, the hypoxia-inducible factor, the vitamin D receptor and other transcription factors/nuclear receptors controlling cholesterol/bile salt homeostasis and liver differentiation. 2.

The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors.

Numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cytochromes P450 (CYPs), transferases and transporters. For example, natural and synthetic glucocorticoids (agonists and antagonists) as well as other clinically important drugs induce the hepatic CYP2B, CYP2C and CYP3A subfamilies in man, and these inductions might lead to clinically important drug-drug interactions. Only recently, the key cellular receptors that mediate such inductions have been identified.

Expression of CYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes.

The fully active dihydroxylated metabolite of vitamin D(3) induces the expression of CYP3A4 and, to a lesser extent, CYP2B6 and CYP2C9 genes in normal differentiated primary human hepatocytes. Electrophoretic mobility shift assays and cotransfection in HepG2 cells using wild-type and mutated oligonucleotides revealed that the vitamin D receptor (VDR) binds and transactivates those xenobiotic-responsive elements (ER6, DR3, and DR4) previously identified in CYP3A4, CYP2B6, and CYP2C9 promoters and shown to be targeted by the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR). Full VDR response of various CYP3A4 heterologous/homologous promoter-reporter constructs requires both the proximal ER6 and the distal DR3 motifs, as observed previously with rifampicin-activated PXR.

Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor.

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm-100 microm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low-dexamethasone component (nmol concentrations) of low amplitude (factor of 3-4) followed by a high-dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15-30).

Calcium channel modulators of the dihydropyridine family are human pregnane X receptor activators and inducers of CYP3A, CYP2B, and CYP2C in human hepatocytes.

The expression of three cytochromes P450 (CYP3A4, CYP2C9, and CYP2B6) was investigated in primary human hepatocyte cultures following treatment with four calcium channel modulators (CCM) of the dihydropyridine family, three antagonists (nifedipine, nicardipine, and isradipine), and one agonist (BK8644). Induction of CYP3A4 was studied by Northern blot, Western blot, and enzymatic activity. Induction began between 1 and 10 microM CCM and was dependent on the presence of dexamethasone (100 nM) in the medium.

Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators.

In this report we show that submicromolar concentrations of dexamethasone enhance pregnane X receptor (PXR) activator-mediated CYP3A4 gene expression in cultured human hepatocytes. Because this result is only observed after 24 h of cotreatment and is inhibited by pretreatment with cycloheximide, we further investigated which factor(s), induced by dexamethasone, might be responsible for this effect. We report that dexamethasone increases both retinoid X receptor-alpha (RXRalpha) and PXR mRNA expression in cultured human hepatocytes, whereas PXR activators such as rifampicin and clotrimazole do not.

Evidence for the presence of a functional pregnane X receptor response element in the CYP3A7 promoter gene.

Pregnane X Receptor (PXR) has been recently shown to regulate the inducible expression of CYP3A genes in response to xenobiotics and steroids. PXR forms a heterodimer with the retinoic acid receptor (RXR) and this complex binds to and transactivates an 18bp region containing two everted repeats TGA(A/C)CT separated by 6 nucleotides (ER6) and located at approximately -150 in the CYP3A4 promoter. In this work we have isolated and sequenced the proximal 5'-flanking region of CYP3A7 from two different human genomic libraries.