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. Such functional interferences provide new insight, first for understanding how xenobiotics might exert adverse effects, and second how physiopathological stimuli affect xenobiotic metabolism.

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. They include nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I2), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1). There is a wide promiscuity of these receptors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system appears now as a tangle of networks, where receptors share partners, ligands, DNA response elements and target genes. Moreover, they influence mutually their relative expression. This review is focused on these different pathways controlling human CYP2B6, CYP2C9 and CYP3A4 gene expression, and the cross-talk between these pathways.

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). We show that the low-dexamethasone component results from the glucocorticoid receptor-mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic-independent manner. At supramicromolar concentrations (>10 microm), dexamethasone binds to and activates pregnane X receptor thus producing the high-dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).

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. CYP3A4 mRNA accumulation started only after 16 h of treatment because pregnane X receptor (hPXR) synthesis was needed. Cotransfection experiments showed that the proximal and the distal PXR response elements of the CYP3A4 promoter and hPXR (HepG2 cells) or dexamethasone-induced hPXR (primary hepatocytes) were necessary to obtain full induction. Furthermore, glutathione S-transferase pull-down assays demonstrated that the CCM tested can act as hPXR ligands. In addition, cotransfection experiments in CV1 cells showed that these compounds failed to reverse CAR (constitutively activated receptor) inactivation by androstenol. Finally, 10 microM CCM induced both CYP2C9 and CYP2B6, strengthening the evidence that hPXR is involved in the regulation of these genes. All together, these results widen the field of hPXR activators to a new class of ligand, namely the CCM of the dihydropyridine family.

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. Accumulation of RXRalpha and PXR mRNA reaches a maximum at a concentration of 100 nM dexamethasone after treatment for 6 to 12 h and is greatly diminished by RU486. A similar pattern of expression is observed with tyrosine aminotransferase mRNA. Moreover, the effect of dexamethasone on PXR mRNA accumulation seems to be through direct action on the glucocorticoid receptor (GR) because the addition of cycloheximide has no effect, and dexamethasone does not affect the degradation of PXR mRNA. Furthermore, dexamethasone induces the accumulation of a RXRalpha-immunoreactive protein and increases the nuclear level of RXRalpha:PXR heterodimer as shown by gel shift assays with a CYP3A4 ER6 PXRE probe. This accumulation of latent PXR and RXRalpha in the nucleus of hepatocytes explains the synergistic effect observed with dexamethasone and PXR activators together on CYP3A4 induction. These results reveal the existence of functional cross talk between the GR and PXR, and may explain some controversial aspects of the role of the GR in CYP3A4 induction.

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. In contrast to a previously reported sequence (Itoh et al., 1992), we did not observe any mutation in the 3'-half of the CYP3A7 ER6 element. Using electrophoretic mobility shift assays and cotransfection experiments we show that this element is able to bind the PXR:RXR complex and transactivates the expression of a down stream promoter in response to rifampicin, clotrimazole, and RU-486, three compounds known to specifically activate the human PXR. This is consistent with the fact that CYP3A7 mRNA is inducible in several primary cultures of human hepatocytes from different patients, as well as in two hepatocarcinoma cell lines HuH7 and HepG2, in response to these compounds. In contrast to a previous report (Blumberg et al., 1998), based on the sequence published by Itoh et al., we conclude that CYP3A7, like CYP3A4, is inducible in response to xenobiotics and presumably in a large proportion of the population.