3 AROMATIC HYDROCARBON RECEPTOR STATUS IN THE METABOLISM OF XENOBIOTICS UNDER NORMAL AND PATHOPHYSIOLOGICAL CONDITIONS

The aryl hydrocarbon receptor (AhR), a cytosolic protein belonging to the family of nuclear receptors, controls transcription of a wide range of structurally unrelated genes. To date, the most potent AhR ligand is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Exposure to TCDD leads to a number of toxic effects, in particular to tumor promotion and immunosuppression. The function of AhR in cells and living organisms therefore seems to be of paramount importance. Its absence in AhR null mice, results in severe phenotypic abnormalities, such as liver half size with fibrosis, accumulation of retinoic acid and immune system insufficiency. An important role of AhR inheres in its transcriptional control of several biotransformation enzymes (CYP1A1/2,1B1). Hence AhR is the crucial factor in the regulation of xenobiotic metabolism. Under pathophysiological conditions, such as inflammation, the level and activity of the AhR target gene CYP1A is decreased. Thus it is likely, that mediators and/or products of inflammation affect AhR function. This review deals with the role of AhR in xenobiotic metabolism under normal and pathophysiological conditions, with respect to inflammation in particular.


INTRODUCTION
Human populations are exposed to innumerable chemical compounds of natural or synthetic origin, which are foreign to the human body and are called xenobiotics. These include food additives, environmental pollutants, drugs etc. Over the course of evolution, a detoxifi cation system for eliminating xenobiotics from the human organism has developed. This comprises a large group of enzymes, which participate in the excretion process. These, usually divided into two phases, modify the structure of the xenobiotic from a hydrophobic to hydrophilic form for easier excretion.
Biotransformation of xenobiotics is formally divided into three phases. In phase I of biotransformation, a polar moiety is mostly introduced into the molecule. A major representative of phase I enzymes is the enzyme superfamily of cytochrome P450 (CYP). Many CYP isoforms e.g. CYP3A4, CYP2B6, CYP1A1/2 are induced by xenobiotics and are under transcriptional control of the nuclear receptors PXR (pregnane X receptor), CAR (constitutive androstane receptor) and AhR (aromatic hydrocarbon receptor), respectively. Substantial variations between individuals with respect to CYP enzyme activity arises from a variety of modulating factors, including genetic polymorphisms, age, gender, disease status, pharmacotherapy, and dietary factors such as smoking and alcohol 1 .
In phase II of the biotransformation, the oxygenated products of phase I are conjugated to small hydrophilic endogenous molecules such as glucuronic acid, glutathione, sulfate, cysteine or acetate, yielding even more hydrophilic products that can be excreated easily. Examples of phase II enzymes are glutathione transferases, UDP-glucuronosyl-transferases and N-acetyltransferases.
A phase III has recently been described as transmembrane export. This includes transmembrane proteins acting as pumps, which transport non-metabolized xenobiotics or conjugates formed in phase II out of the cell thus decreasing the intracellular concentration of xenobiotics. One of the best-known pumps is P-glycoprotein.
Infl ammation, as a pathological state of the organism, aff ects metabolism of xenobiotics. Infl ammation mediators lead almost always, according to all available observations, to decreased levels of cytochromes P450. In this review we have focused on the CYP1A family and AhR, purine S-methyltransferase, and N-methyltransferases). UGTs, STs, and GSTs are gene families with multiple individual isoforms, each having its own preferred substrates, mode of regulation and tissue-specifi c patterns of expression 6 .
UDP-Glucuronosyltransferases (UGTs) 5 catalyse the transfer of glucuronic acid from UDP-glucuronic acid to phenols, hydroxylamines, carboxylic acid etc. UGTs are a gene family of integral proteins of the endoplasmic reticulum membranes and the nuclear envelope which are present in many tissues of vertebrates. Two UGT enzyme families termed UGT 1 (bilirubin, phenol) and UGT2 (steroids) have been described. Both families are in ducible enzymes, where enzymes of UGT1 family are induced by methylcholanthrene, while UGT2 enzymes are induced by phenobarbital 7 .
Glutathione-S-transferases (GSTs) 5 catalyse the conjugation of reduced glutathione (GSH) with compounds that contain an electrophilic centre through the formation of a thioether bond between the sulphur atom of GSH and the substrate. Two superfamilies of mammalian enzymes have evolved and on the basis of their sequence identity, they have been assigned to eight families. Sulphotransferases (STs) 5 transfer the sulpho moiety (SO 3 -), usually from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) as the donor, to a nucleophilic group of the acceptor molecule. The introduced negative charge aff ects important properties of the acceptor molecule, such as interaction with receptors and transport proteins, water solubility and penetration of the cell membranes.

Phase III -transport
For a long time, biotransformation was formally divided into two phases only. Recently, the process of drug export from the cell is considered as phase III of biotransformation. Thus, membrane proteins transporting xenobiotics across cell membranes are called "phase III enzymes". The most notorious example of membrane protein with toxicological signifi cance is P-glycoprotein (multidrug resistant protein, MDR1), which mediates the trans-epithelial effl ux of xenobiotics. It pumps hydrophobic neutral or positively charged molecules from the cells and plays a signifi cant role in drug absorption and fate 8 . Inhibition and induction of P-glycoprotein leads to drugdrug interactions and has always much greater impact on tissue distribution than on plasma concentrations. Owing to the overlapping substrate specifi city of CYP3A4 and P-glycoprotein, and because of similarities in P-glycoprotein and CYP3A4 inhibitors and inducers, many drug interactions aff ect both P-glycoprotein and CYP3A4 9 .
Conjugates with glucuronic acid, sulphate or glutathione are excreted from the cell by export pumps known as OATPs (Organic anion transporting polypeptides). OATPs are a gene superfamily and they transport amphipathic organic solutes 10 . Some members of this superfamily are selectively expressed in the liver where they are involved in hepatic clearance of albumin-bound compounds from the portal blood plasma, but most OATPs are expressed in multiple tissues including the blood-brain barrier, choroid plexus, lung, heart, intestine, kidney, placenta in terms of their regulation and interplay under normal and under pathophysiological conditions. In addition, we present a brief overview of drug metabolism, comprising main types of reactions, examples of substrates and enzymes participating in xenobiotics metabolism.
Cytochrome P450 (CYP) enzymes catalyze oxidation reactions. The total number of P450 substrates exceeds 200 000, including the majority of drugs, other xenobiotics, together with eubiotics such as steroids, fatty acids, eicosanoids, retinoids and prostaglandins 2 . The nomenclature of P450s is based on homology in amino acid sequences 3 . In man, the superfamily of P450s comprises four families of enzymes (CYP1, CYP2, CYP3 and CYP4) that are involved in xenobiotic metabolism 4 . Several CYPs are inducible, particularly by xenobiotics that bind to specifi c intracellular receptors and consequently modulate expression. These enzymes are expressed in many tissues, but the highest levels are found in the liver. Within the cell, CYPs are localized on the cytosolic side of the endoplasmic reticulum.
Flavin monooxygenases (FMO) 5 which catalyse the oxygenation of nitrogen-, sulphur-and other nucleophilic heteroatom-containing chemicals and xenobiotics found in foodstuff s. The nomenclature of this enzyme is based on the primary amino acid sequence identity.
Amine oxidases 5 may be distinguished by their cofactor requirements, substrate specifi cities and inhibitor sensitivities. Despite involvement of amino oxidases in the metabolism of some xenobiotics, the products of the reaction, ammonia, hydrogen peroxide and an aldehyde are themselves toxic.
Hydroxylases 5 are represented by aldehyde oxidase and xanthine oxidase, complex molybdofl avo-proteins with similar catalytic properties but with diff erences in their substrate/inhibitor specifi cities. They catalyze both oxidation and reduction reactions. Unlike P450, oxidative hydroxylation catalysed by the molybdenum hydroxylases generates reducing equivalents, and the source of the oxygen atom inserted into substrates is water and not O 2 . In most cases, these enzymes have complementary substrate specifi cities with microsomal monooxygenases.

Phase II -conjugation
The oxidized product of phase I is further "polarized" in phase II by conjugation with certain molecules. Phase II enzymes include arylamine N-acetyltransferase, UDP-glucuronosyltransferases (UGTs), glutathione S-transferases (GSTs), sulpho-transferases (STs), and methyl-transferases (catechol O-methyltransferases, thio-Aromatic hydrocarbon receptor status in the metabolism of xenobiotics under normal and pathophysiological conditions and testis 10 . Individual members of the OATP family represent polyspecifi c organic anion carriers with partially overlapping and partially distinct substrate preferences for a wide range of amphipathic organic solutes including bile salts, organic dyes, steroid conjugates, thyroid hormones, anionic oligopeptides, numerous drugs and other xenobiotic compounds. So far, 9 human OATPs have been identifi ed 10 . It was predicted that all OATPs would consist of 12 transmembrane domains. However, this has not been proven experimentally for any OATP 10 . OATP1 expression is controlled at transcriptional and post-transcriptional levels and it is, at least in part, tissue-specifi c. In the kidney, OATP1 expression is stimulated by testosterone and inhibited by estrogens, while hepatic OATP1 expression is not infl uenced by sex hormones 10 . Rat Oatp2 expression is induced by phenobarbital. Recently, it has been found that OATP2 expression is controlled by PXR 11 . At the protein level, functional down-regulation of rat Oatp1 occurs via serine phosphorylation 10 . Analogically to OATPs, there exists a transporter system called organic cation transporters (OCTs). Small organic cations like tetramethylammonium, tetraethylammonium, tetrabutylammoniun, N-methyl-nicotinamide, thiamine, choline, dopamine, cimetidine, serotonin, histamine, adrenalin and noradrenalin are transported by specialized organic cation transporters (OCT1 in humans and Oct1 in rats). In man, OCT1 is expressed mainly in the liver. The activity in rat Oct1 is modulated by phosphorylation 12 .

XENOBIOTICS METABOLIZING ENZYMES REGULATION
A common mode for XMEs regulation is transcriptional control by receptors. In the generalised model of transcriptional regulation of P450s, a receptor interacts with a low molecular weight ligand in the cytosol, consequently changes its conformation and binds to a cognate sequence in DNA where it triggers target gene transcription. Several receptors including PXR 13, 14 , CAR 13,14,15 , AhR, retinoid X receptor (RXR) 14 , vitamin D receptor (VDR) 13,14 , retinoic acid receptor (RAR) 14 and GR 13,14 are implicated in XMEs regulation. In this review, we have focused on the role of the AhR receptor in XMEs regulation, both under normal and under pathophysiological conditions.

Aryl hydrocarbon receptor (AhR)
The aryl hydrocarbon receptor is a transcription factor belonging to the PAS (Per-ARNT-Sim) family of transcription factors which controls the expression of human CYP1A1, CYP1A2 and CYP1B1, as well as several phase II metabolizing enzymes (NAD(P)H: quinoneoxidoreductase, GST, UGT) 16,17 . The human AhR gene is located on chromosome 7 18 . There are 12 exons, which encode 848 a.a. protein with a theoretical mass of 96,147 Da 18,19 . Typical AhR ligands are PAHs and TCDD. These trigger a number of toxic eff ects including cancer, immunosuppression and endocrine disruption in rodents 20 .

AhR functioning
The unliganded AhR exists in the cytosol in a multiprotein complex consisting of one AhR, two hsp90 chaperones, a small protein (p23) and an immunophilinlike protein termed XAP, AIP or ARA9, the latter being involved in the correct folding and stabilization of AhR. Hsp90 acts as chaperone protein system that prevents transcriptional activation of the AhR but keeps the receptor in a conformation that facilitates ligand binding ( Figure 1). Upon ligand binding, the chaperon proteins dissociate and AhR translocates to the nucleus, where it rapidly forms a 180-220 kDa 21 heterodimer with aryl hydrocarbon receptor nuclear traslocator (ARNT), known as the hypoxia inducible factor-1β (HIF-1β) 22 . This heterodimer binds to the specifi c DNA region termed dioxin or xenobiotic response element (DRE or XRE), which has a core sequence of 5'-TNGCGTG-3' 16 , and thereby activates gene expression.
The proteolytic degradation of transcription factors is an established mechanism for regulating signal transduction pathways. The AhR protein is degraded following ligand exposure and nuclear export both in vivo and in vitro. The magnitude of degradation appears to diff er among various tissues and cells and it is pro teasomeu biquitin dependent. The concentration of AhR and ARNT protein detected in nuclear lysates of culture cells is highest following 1-2 h of TCDD exposure 23 but then rapidly drops as AhR is degraded. Indeed, cells co-treated with 26S proteasome inhibitors (MG-132 or lactacystin) and TCDD exhibit high levels of AhR and ARNT protein in the nucleus up to 8 h after TCDD treatment 23 . When complexed with hsp90 and immunophilin-like proteins, AhR is inactive and these proteins participate in masking the nuclear localization signal (NLS) 16,24 , resulting in the cytoplasmic retention of AhR. Furthermore, AhR contains a nuclear export signal (NES) 16 , which is important for AhR removal from the nucleus.
The relationship of these proteins to the degradation of AhR seems to be relevant, since their association with AhR may mask key domains for nuclear import, nuclear export, ubiquitination, or phosphorylation. Recently a mouse protein has been identifi ed which is closely related to the AhR, designated aryl hydrocarbon receptor repressor (AhRR). AhRR is localized in the nuclei and does not bind TCDD. AhRR is able to interact with ARNT but AhRR-ARNT dimers are not transcriptionally active. The AhRR is induced by AhR, thus serving as negative feedback loop 16 (Fig. 1).

AhR family
PAS family is a gene family of a structurally related transcription factors with characteristic structural motifs designed as basic helix-loop-helix (bHLH). AhR, ARNT and AhRR are members of the PAS family. The bHLH motif is characteristic for a large number of proteins such as modulators of cell proliferation and diff erentiation, neurogenesis, myogenesis, B-cell diff erentiation, and sex determination 25 . The bHLH motif, localized in the N-terminal region, consists of two amphipathic α-helices separated by a non-helical loop. The HLH region medi-ates dimer formation, while the basic region is required for binding to DNA. The PAS domain, which has been identifi ed as a conserved sequence among Drosophila transcription factors PER (period), SIM (single-minded) and human protein ARNT, is involved in the formation of AhR-ARNT heterodimers, binds a ligand and interact with heat shock protein 90 kDa (hsp90). The ligand binding domain (LBD) of AhR is located in the sequence of 230-431 a.a. from the N-terminus, partly overlapping with the binding site for hsp90 which keeps AhR structurally competent to bind ligands 16 .

AhR -a member of the orphan receptor family
To date, no physiological endogenous ligand of AhR has been identifi ed. However, its existence is indirectly supported by numerous observations demonstrating AhRdependent responses in the absence of exogenous ligand 24 . Nevertheless, perhaps the best evidence for the existence of an endogenous AhR ligand comes from studies on AhR null mice. Growth rates of the AhR-null mice was retarded compared to wild type mice for the fi rst 3 weeks of life, and the mutant mice were defective in liver and immune system development 16 . Several phenotypic defects have been reported for adult AhR-null mice, such as retinoid accumulation in liver and abnormal hepatic and kidney vascular structures, decreased liver size and hepatic fi brosis 16 . Concerning xenobiotic metabolism, the absence of AhR abolished the inducible expression of CYP1A1 and 1A2 genes in response to TCDD or PAHs and resulted in a loss of teratogenesis caused by TCDD and susceptibility to chemical carcinogenesis by BP 16 . The loss of CYP1A1, 1A2 and 1B1 inducibility is considered a cause of resistance to chemical carcinogenesis in AhR-null mice.
The greatest exposure to AhR ligands comes from food. There are numerous reports of naturally-occurring dietary chemicals (e.g. fl avonoids, carotenoids, phenolics) that activate the AhR signaling pathway, although the majority of these chemicals appear to be relatively weak AhR ligands. Recently, the presence of AhR agonists/antagonists in extracts from a variety of diff erent fruits, vegetables, herbs and teas has been reported 24 . Certain fl avonoids, e.g. fl avone and diosmin, Aromatic hydrocarbon receptor status in the metabolism of xenobiotics under normal and pathophysiological conditions act as AhR agonists and induce CYP1A1, 1A2 and 1B1 activities 26 .
Classical exogenous AhR ligands such as TCDD, 3-MC (3-methylcholanthrene), BP or αnaphthofl avone 27 share several common structural features: they are hydrophobic, planar or co-planar molecules of polycyclic structure. Nevertheless several non-polycyclic and nonplanar compounds like omeprazole, thiabendazole or carotenoid canthaxanthin activate the CYP1A1 gene as well. However, they are very weak AhR ligands 27 .
The most studied AhR-mediated expression is that of CYP1A1 (benzpyrene hydroxylase, aryl hydrocarbon hydroxylase), however, other XMEs like paraoxygenase are induced as well 28 . It seems that there exists cross-talk between AhR and retinoid acid receptors (RARs) signaling pathways, the existence of which is supported by facts: i) AhR expression in keratinocytes is aff ected by retinoic acid and this depends on the state of diff erentiation, where an increased level of AhR transcript and activation of CYP1A1 have been observed 27 , ii) retinoid accumulation in liver has been observed in AhR null mice 14 , iii) CYP1A1 is induced through a retinoid acid responsive element (RARE). Indeed, functional interplay and transcriptional cross-talk between AhR and RAR, the vitamin D receptor (VDR) and peroxisome proliferator-activated receptors (PPARs) have recently been reported 29 , (PPARs are important factors in human health 30 ).

AhR and beyond
Post-transcriptional modifi cation of transcription factors is a common regulatory mechanism of gene expression in eukaryotic cells. Many AhR ligands stimulate signaling cascades similar to those initiated at the cell surface by growth factors, hormones, neurotransmitters and extracellular signals, and share with classical mitogenic factors the propagation of the signal including increased ion fl uxes across the membrane, elevation of intracellular Ca 2+ and cAMP levels, and the activation of protein ki nases (PKs) and protein phosphatases (PPs). AhR/ ARNT appears to be regulated via phosphorylation, because the binding activity of the AhR/ARNT heterodimer to the XRE is abolished by the phosphatase treatment 31 . These observations are consistant with the idea that the AhR and PK coexist in the cytosol as a part of a multimeric protein complex. TCDD causes an immediate increase in protein kinase C (PKC) and tyrosine kinase activities in hepatocyte membranes. Evidence for PKC involvement in AhR activation comes from the fi nding, that specifi c inhibition of PKC by staurosporine 27 prevents the ligand-induced DNA-binding of AhR/ARNT heterodimers and leads to suppression of CYP1 expression. Phosphorylation of AhR in vitro has been localized to the C-terminal part of the peptide 31 . AhR and ARNT are both phosphory lated on threonine residues 31 , suggesting that a serine/ threonine kinase may be directly or indirectly involved in the regulation of the functions of these proteins.

Cell cycle and AhR
The high degree of conservation in the primary structure of the AhR suggests that this protein is crucial for such life functions, as cell signaling, proliferation and apoptosis 19 . The AhR interacts with the retinoblastoma protein (RB1), and the AhR-RB1 complex hinders progression from the G1 to the S phase of the cell cycle by blocking E2F transcription factor-mediated expression of S phase genes. Thus, activated AhR obviously participates in cell cycle arrest at the G1/S boundary. The level of CYP1As in these cell types is critical, because CYP1As degrade AhR ligands and thus prevent the block in progression in G1/S. Constitutive CYP1A1 mRNA is detectable in the mature oocyte, in liver and many other mouse tissues 17 . It is likely that abundance of constitutive CYP1A1 mRNA in the fertilized ovum may be important for maintaining suffi cient amounts of the CYP1A1 enzyme during the transition from maternal to zygotic control 17 .

AhR and estrogen receptor
It has been reported that TCDD induced hepatocellular carcinogenesis in female but not male Sprague-Dawley rats and the tumorigenic response in females was estrogen-dependent 22 . Decreased formation of estrogen dependent uterine and mammary tumors in rats fed TCDD in the diet suggested that TCDD blocked estrogen receptor (ER) action. Indeed, existence of crosstalk between AhR and ER has been proposed. The AhR is expressed in the rodent uterus and several studies have shown that TCDD and other AhR agonists inhibited 17β-estradiol (E2)-induced uterine weight increase, cell proliferation and progesterone receptor binding (PR) 22 . E2 does not aff ect AhR protein, whereas TCDD induces degradation of ERα, and in cells co-treated with TCDD and E2, cellular ERα protein levels were considerably reduced 22 . Inhibitory AhR-ER crosstalk aff ects multiple estrogen-dependent pathways in breast cancer cell lines. For instance, CYP1A induction by AhR agonists results in rapid metabolism and depletion of E2 in the cell culture but not in vivo 22 .

AhR polymorphism
It is well known that there are marked strain and species diff erences in sensitivity to TCDD in mice, when diff erent responsiveness to TCDD depends on the AhR alleles. Convincing evidence comes from inbred mouse studies where allelic diff erences in the AhR gene resulted in striking inter-individual diff erences in susceptibility to cancer, mutagenesis, birth defects and cell type-specifi c toxicity. Heavy exposures of human populations to dioxin, halogenated hydrocarbons or cigarette smoke have been associated with increased rates of malignancies, birth defects and mental problems 32 . AhR shows polymorphism, but no dramatic eff ects of AhR polymorphisms on human health have yet been reported. The extraordinary toxicity of the potent AhR ligand, TCDD, in laboratory animals creates great concern that humans exposed to dioxin-like substances also may suff er from a variety of adverse eff ects 19 . Exposure to dioxin-like compounds has been clearly shown to result in the severe skin disorder chloracne. However, most people who have been exposed to TCDD, even at substantial levels, did not develop chloracne 19 . The basis of the large variation in human susceptibility to chloracne is not known but may be related to genotypic/phenotypic diff erences in the AhR. Only a few epidemiological studies have focused on association of AhR polymorphisms to cancer risk in men and to date, no study has revealed an increased cancer risk. The polymorphisms might reduce susceptibility to PAHs that are bioactivated by CYP1A1. There are little genetic variations in the AhR in human populations. However, more human AhR polymorphisms certainly remain to be discovered.

Polymorphism
Low-level exposure to carcinogenic chemicals is a frequent, if not ubiquitous, occurence in Western countries. Environmental tobacco smoke and vehicle exhaust are widespread sources of PAHs and other carcinogens 33 . Induction of P450 activities enhances the metabolic activation of substrates, which indirectly determines their toxic, mutagenic and carcinogenic eff ects. The induction level depends on genetic, dietary, physiological and environmental factors. There is considerable variability in human CYP activity levels due to the polymorphism of some human CYPs. A signifi cant percentage of the population is defi cient in a specifi c enzyme (e.g. CYP2D6) or have a functional enzyme with altered amino acid sequence (CYP2C9) 34 . Allelic variants of P450s exist due to point mutations in the coding region, but deletion of an entire gene can also occur. Polymorphism plays an important role in the homeostasis/protein level control of some P450s (e.g. CYP2D6), while others are regulated predominantly transcriptionaly (e.g. CYP3A4). However, the most P450s are both polymorphic and inducible 35 . Expression levels of specifi c CYP enzymes vary substantially among individuals. In men, polymorphisms can result in a population which is either highly sensitive or resistant to the biological eff ects of chemicals. This is well established in drug therapeutics where individuals lacking a particular P450 exhibit hypersensitivity to drugs having narrow therapeutic indexes. The role of polymorphisms in xenobiotic-metabolizing enzymes in cancer susceptibility is being actively investigated 33 .

Other regulatory mechanisms
Albeit transcriptional control is the predominant mean of gene regulation, post-transcriptional regulation plays a part as well. It is more diffi cult to obtain evidence for mRNA stabilization than for enhanced mRNA synthesis. However, there is evidence that certain proteins selectively bind the mRNA of some P450s and stabilise it 5,36 . Moreover, some chemicals inducing one P450 downregulate other P450s at the same time (e.g. CYP2C11 and PAHs) by a mechanism involving mRNA level lowering 5 . Another means for P450s regulation is posttranslational regulation, whereby the sensitivity of P450s to proteolytic degradation can be attenuated by ligand binding or enhanced by protein alkylation. Incorporation of heme, a post-translation event, plays a role in P450s stability. Evidence for destabilisation of P450s by serine phosphorylation has been demonstrated for rat CYP2B1 and 2E1 5 . The degradation of at least some P450s does involve ubiquitination 5 . Finally, gender-specifi c expression of CYP2C11 in male rats has been described, while in human mates, the gender eff ects of drug metabolism are rather small.

EXPRESSION AND XMEs ACTIVITY UNDER PATHOPHYSIOLOGICAL CONDITIONS
There are a large number of factors and conditions which modify CYPs levels and/or activities. It has been widely reported that the factors involved in host defense and infl ammatory responses interact with and alter the levels and activities of drug metabolizing enzymes. Since infl ammation is an accompaning feature of many pathological states, the alteration of drug biotransformation during any infl ammatory process should be considered in clinical therapeutics. Although the majority of reports describe a down-regulation of the enzymes, there are examples whereby a specifi c CYP isoform is induced during such responses. Cytokines which typically modulate P450s expression are interferon (IFN)-α,β,η and δ, interleukins 1 and 6 (IL-1, IL-6), and the tumor necrosis factor alpha (TNFα) 37 .
Infl ammatory processes are complex, and occur in response to a number of pathological disorders, including infection, tissue damage, burns, trauma, tumors, and autoimmune diseases. The process is characterized by the release of cytokines, mediators, acute phase proteins, and hormones. Some of these agents down-regulate (e.g. Staphylococcal enterotoxin B, TNF-α, Escherichia coli lipopolysacharide) or up-regulate (e.g. IL-1β vs. CYP3A1, hepatitis B or C infection vs. CYP2A6) XMEs 38 .

Cytokines
Defi nitive proof that IFN supresses hepatic drug metabolising systems has been provided using human IFNs recombinant preparations (IFN-α, IFN-β, IFN-γ) 38 . IFNs down-regulate the expression of constitutive enzymes, such as CYP3A2, CYP2C11, CYP2C12 and CYP2E1 in the rat, and CYP1A2 and CYP2C6 in the mouse. Inducible levels of CYP4A1 were depressed by IFN as well. However, the majority of inducible enzymes, e.g. rat CYP3A1 or mouse CYP1A1, CYP1A2, CYP26C, and CYP2B1, were resistant to IFN in the rat 38 . Recently, endogenous IFN-α/β has been shown to down-regulate all of the major inducible hepatic CYPs in rodents, including CYP1A1, CYP1A2, and CYP2E1, CYP2B and CYP3A1. Recombinant IFN-α administration to humans depresses a number of CYP-dependent drug biotransformation pathways. In addition, down-regulation of induced CYPs by recombinant IFNs also occurs in human and rat hepatocyte cultures 38 .
In primary rat and human hepatocytes cultures, the media obtained from activated Kupff er cells as well as IL-1 depressed CYP. Subsequently, recombinant cytokines, including IL-1α, IL-1β, IL-6, TNF, and TGF Aromatic hydrocarbon receptor status in the metabolism of xenobiotics under normal and pathophysiological conditions (tumor growth factor), were shown to suppress the expression of CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, and CYP3A3 in rodents and human cell lines (HepG2) 38 . IL-1 also induced CYP3A1 activity in female rats, while in male rats it depressed CYP3A2, CYP2C11, and CYP1A1. Selective depression of CYP1A1, CYP1A2, and CYP3A6 was attained, when IL-1 and IL-2 were added to cultured rabbit or human hepatocytes 38 . As compared to other cytokines, the eff ects of IL-6 on CYP expression are much less pronounced. IL-6 suppresses CYP2C11 and CYP2E1. It is only after dexamethasone pretreatment that IL-6 depresses CYP2C12 in female rats. In primary human hepatocytes, IL-6 specifi cally inhibited rifampicin and phenobarbital mediated CYP2B6, 2C8/9, and 3A4 expression by a mechanism involving impairement of CAR and PXR transcriptional regulation 38 . Since both CAR and PXR are under GR transcriptional control, and their transactivation in cell lines was not aff ected by IL-6, P450s suppression it is a logical consequence of combat between GR and antiglucocorticoid factor IL-6. By contrast, the same study showed that TCDD dependent CYP1A2 expression was only weakly disturbed by IL-6, suggesting role of GR in AhR functioning to be minor.

Cytokines versus AhR
Cytokines regulate the expression of a number of genes via diff erent transcription factors, such as nuclear factor kappa beta (NF-κB), signal transducer and activator of transcription (STA-T), and CCAAT-enhancerbinding protein alpha (CEBPα). It has been postulated that the endogenous ligand of AhR is aff ected by certain factors, several of them appart from AhR ligands, such as hormones, cytokines and chemicals have a modulation eff ect. Several studies have revealed that the cytokines TNF-α and IL-1β repress CYP1A1 38,40 . It has been demonstrated that the suppressive eff ects of TNF-α and lipopolysacharide (LPS) on the CYP1A1 promoter are at least in part due to direct NF-κB action 40 . NF-κB, a "pro-infl ammatory" transcription factor plays a role in a wide array of physiological and pathological processes including immune modulation, infl ammatory responses and apoptosis.
In the past few years, evidence has emerged to show that the AhR and NF-κB interact and transcriptionally modulate each other 40 . A few studies have suggested that CYP mRNA degradation may be accelerated following the infl ammatory process. The cytokine-mediated loss of CYP1A1 and CYP1A2 at accelerated rates in hepatocytes as compared with hepatocytes treated with a transcriptional inhibitor may also be explained by a more rapid turnover of mRNA 38 .
It has been shown, that dioxin causes chronic, sustained oxidative stress in animals, probably due to production of reactive oxygenated metabolites by CYP1 enzymes 14 . The production of reactive oxygen species (ROS) occurs during infl ammation or infections. ROS produced in vivo and in vitro have been implicated in the cascade of events, leading to a loss of CYP during infl ammation. Hydrogen peroxide and oxidative stress cause the loss of hepatic CYP1A1 and CYP1A2 mRNAs in isolated hepatocytes 38 . The role of oxidative stress on CYP2A5 expression has been examined 41 . These results indicated that antioxidants (e.g. vitamin E) prevent and pro-oxidants (buthioninesulfoximine, BSO) stimulate CYP2A5 induction.
Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria 42 , provokes an infl ammatory response in the infected host which sometimes leads to septic shock and multiple organ failure. Exposure to LPS rapidly activates the synthesis and release of several pro-infl ammatory cytokines including IL-1β, IL-6 and TNF-α by lymphocytes and macrophages and decreases drug clearence when administered to humans. The most frequently used model of infl ammation is the response to LPS, and almost universally, a loss of CYPs has been observed in a variety of species and cell culture systems, e.g. phenobarbital-inducible CYP2B 38 .
Proinfl ammatory cytokines such as IL-1, TNF-α, IL-6, interferons, stimulate the production of nitric oxide (NO) by increasing expression of inducible nitric oxide synthase (iNOS). NO depresses CYPs mRNAs levels and in parallel binds to the heme moiety of CYPs, which results in loss of CYP activity 43 . When NO biosynthesis was stimulated in cell lines derived from chinese hamster fibroblasts, benzo(a)pyrene turnover (CYP1A activity) was dramatically reduced to almost unmeasurable levels 43 .

CONCLUSIONS
Research on biotransformation enzymes regulation has been, is and will be an important and compelling issue. This is a problematique studied at a multidisciplinary level comprising chemistry, biochemistry, biophysics, pharmacology, toxicology, molecular biology, and clinical studies. The discovery and description of the complicated interplay and cross-talk between drug metabolizing enzymes and several nuclear receptors, in particular PXR, CAR, GR, and RXR, has led to the rapid development of current pharmacology. Recently, the role of receptors taking part in normal physiological processes such as farnesyl X receptor (FXR), lanosterol X receptor (LXR), and the vitamin D receptor (VDR), has been elucidated and this brings new insights into the classical concept of xenobiotic metabolism. In this review we have attempted to summarize and discuss current knowledge of the aromatic hydrocarbon receptor (AhR) function and its role in the organism under normal and pathophysiological conditions. As described above, AhR's role in XMEs regulation is undisputed. However, its physiological function remains to be unveiled.
They are hence defined as â€oexenobiotic receptorsâ€ . Recent studies have demonstrated that PXR, CAR and AhR also regulate the expression of key proteins involved in endobiotic responses such as the metabolic homeostasis of lipids, glucose, and bile acid, and inflammatory processes. It is suggested that the functions of PXR, CAR and AhR may be closely implicated in the pathogeneses of metabolic vascular diseases, such as hyperlipidemia, atherogenesis, and hypertension. Therefore, manipulation of the activities of these receptors may provide novel strategies for the treatment of vascular diseases.