Dexamethasone controls aryl hydrocarbon receptor (AhR)-mediated CYP1A1 and CYP1A2 expression and activity in primary cultures of human hepatocytes

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Abstract

CYP1A1 and CYP1A2 genes encode members of the cytochrome P450 superfamily of enzymes primarily involved in xenobiotic and drug metabolism. In this paper we examined the effects of synthetic glucocorticoid dexamethasone (DEX) on aryl hydrocarbon receptor (AhR)-mediated regulation of CYP1A1 and CYP1A2 genes and their enzymatic activity in primary cultures of human hepatocytes obtained from 17 donors and prepared in 3 countries.

Dexamethasone significantly reduced both basal and inducible CYP1A1/2 ethoxyresorufin-O-deethylase (EROD) activities by more than 75 and 50%, respectively. Glucocorticoid receptor (GR) antagonist RU486 abolished this effect suggesting the involvement of GR in the process. In contrast, dexamethasone significantly augmented transcriptional activation of CYP1A2 mRNA but not CYP1A1 gene by prototype AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC). Dexamethasone had no effect on basal and TCDD-inducible levels of CYP1As proteins; however, it reduced the levels of AhR and GRα mRNAs and AhR protein levels. In addition, using RT2 Profiler™ PCR Array, we found the effect of dexamethasone on the expression of several co-activators of AhR and GR nuclear receptors in the primary human hepatocytes.

We conclude that dexamethasone controls CYP1A1 and CYP1A2 expression and activity in human hepatocytes via multiple mechanisms, which remain to be elucidated.

Introduction

CYP1A2 accounts for about 13% of the total cytochrome P450 content in the liver [1] and metabolises several clinically important xenobiotics and drugs, such as caffeine, clozapine, R-warfarin and verapamil as well as endogenous substrates melatonin and estradiol [2], [3]. In contrast, CYP1A1 is expressed at low levels but is highly inducible in the liver. CYP1A1 and CYP1A2 play critical roles in the metabolic activation of procarcinogenic polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines/amides (HAAs), respectively, to reactive intermediates, leading to toxicity and cancer [3]. Because of the critical role of CYP1A1/2 in chemical carcinogenesis, CYP1A1/2 induction remains a central focus of interest in cancer research, environmental toxicology, food safety, and drug development. Furthermore, since CYP1A1/2 can metabolize a range of drugs; induction of these enzymes may increase the metabolism of drugs, resulting in unexpected drug–drug interactions [2], [3], [4].

A master regulator of CYP1A1/2 enzymes is aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor. When discovered, it was believed that it regulated the responses to xenobiotic chemicals, i.e. induction of CYP1A1 and CYP1A2 enzymes and toxicity of dioxin-like compounds [5]. Now, it is clear that AhR plays multiple roles in fundamental cell biology and physiology, including development, cell differentiation, immune response, etc. [6]. Recent research has shown that AhR is a ligand-dependent ubiquitin E3 ligase [7], which demonstrates the complexity of AhR signalling since AhR itself is subject to degradation by the proteasome-ubiquitin system [8]. Collectively, given the role of AhR in xenobiotics metabolism by induction of CYP1As and multiple functions in cellular biology, there is urgent need to understand the mechanisms regulating the function of this important regulator [5], [9].

Although, there are a number of reports dealing with a cross-talk between AhR and GR signalling pathways in regulation of CYP1A/Cyp1a enzymes, the underlying mechanisms are not yet fully understood [5], [9], [10]. The majority of the research was performed in rodent [11], [12], [13], [14], [15], [16] or aquatic models [17]; thus, little information is available from human tissues [18], [19]. In addition, cancer cell lines are widely used in the studies, but their phenotype is substantially different from normal non-transformed cells.

Glucocorticoids are extensively used in pharmacotherapy, including topical applications (eczemas), inhalation (asthma) or systemic administration (chemotherapy, autoimmune diseases, etc.). Since AhR and GR are ubiquitously expressed in human body organs and tissues and human population is increasingly exposed to the environmental AhR activators, it is important to study AhR-GR cross-talk in human experimental models and in various tissues [9]. We have described elsewhere in the limited number of primary human hepatocyte cultures that DEX augments an inducibility of CYP1A2 mRNA by dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in primary human hepatocytes [20] but we did not observe this effect on CYP1A1 mRNA induction by 3-methylcholanthrene (3MC) [11].

In the current paper, we performed extensive examination of DEX effects on CYP1A1 and CYP1A2 inducibility by TCDD and 3MC in primary human hepatocytes. We used in total 17 primary cultures of human hepatocytes prepared from liver tissue obtained from 17 different donors that underwent surgery in three different countries (France, Hungary, Czech Republic). We employed the techniques of RT-PCR, western blots and measurement of EROD catalytic activity. In addition, using RT2 Profiler™ PCR Array, we screened the effects of dexamethasone (DEX) on the expression of mRNAs of 84 genes encoding human nuclear receptors, transcriptional factors, their co-activators and co-repressors.

We conclude that dexamethasone controls AhR-mediated CYP1A2 induction and CYP1A1/2 catalytic activity in human hepatocytes through complex alteration of homeostasis of many genes involved in AhR and GR transcriptional function. Even though we bring clear evidence for AhR–GR cross-talk in human hepatocytes, the exact molecular mechanism of the phenomenon remains unknown.

Section snippets

Materials

Collagen-coated culture dishes were from BD Biosciences (Le Pont de Claix, France). Hyperfilm™ ECL and chemiluminescence-developing reagents were from Amersham-Pharmacia Biotech (Amersham, United Kingdom). 2,3,7,8-tetrachlorodibenzo-p-dioxin was from Ultra Scientific (RI, USA). LightCycler FastStart DNA MasterPLUS SYBR Green I was from Roche Diagnostic Corporation (Meylan, France). Oligonucleotide primers used in RT-PCR reactions were from Invitrogen. Mifepristone (RU486) and dexamethasone were

Results

The data presented in the current paper were acquired from primary cultures of human hepatocytes obtained from 17 donors (see Table 1) and prepared in 3 countries (France, Czech Republic, Hungary).

Discussion

In the current paper, we demonstrate the existence of functional cross-talk between AhR and GR signalling pathways and provide convincing evidence that dexamethasone, a prototype synthetic agonist of GR, controls CYP1A2 expression and CYP1A1/2 activity in primary human hepatocytes. We show that dexamethasone dramatically decreases both basal and AhR ligand-induced CYP1A1/2 enzyme activity in primary human hepatocytes. We also show that dexamethasone significantly augments TCDD- and

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the grants from the Grant Agency of the Czech Republic GACR 303/07/0128 and GACR 305/08/P089, and by János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00413/05).

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