Effects of rilpivirine, 17ß-estradiol and ß-naphthoflavone on the inflammatory status of release of adipocytokines in 3T3-L1 adipocytes in vitro.

Rilpivirine is a non-nucleoside reverse transcriptase inhibitor, recently developed as a drug of choice for initial anti-retroviral (ARV) treatment of HIV-1 infection, whereas estradiol is a major component of hormonal contraceptives. Both drugs have effects on lipid metabolism, impairment of adipocyte differentiation and alteration of adipose tissue distribution and function.This study investigated the effects of different concentrations of either rilpivirine or estradiol either alone or in combination on adipocyte differentiation and adipocytokines status in vitro in the absence and presence of ß-naphthoflavone, (BNF),a potent agonist of the aryl hydrocarbon receptor. 3T3-L1 human pre-adipocytes were cultured and differentiated with different concentrations of treatment drugs. After 10 days of differentiation procedure, cells were examined for their morphology and viability. Glycerol,adiponectin, leptin, resistin and interleukin-8 (IL-8) were quantified using commercially available kits. The results show that either rilpivirine or estradiol individually or during their combination can evoke significant increases in glycerol release and a concomitant significant decrease of adiponectin from adipocytes. These effects were dose-dependent. The effects of combined treatments were much larger than individual concentration for each drug. Both drugs had little of no effect on leptin levels, except for a small decrease with 10 µM rilpivirine alone or when combined with estradiol. In addition, both drugs evoked small increases in the release of resistin and interleukin-8 with significant values at higher doses compared to untreated adipocytes.When adipocytes were pretreated with BNF, either rilpivirine or, estradiol or when combined evoked a much larger release in glycerol and a much larger decrease in adiponectin compared to the absence of BNF. In contrast, BNF pretreatment had little of no effect on either leptin, resistin or IL-8 metabolism compared to the results obtained in the presence of either rilpivirine or estradiol alone or in combination.These results show that rilpivirine and estradiol either alone or when combined or pretreated with BNF can evoke marked effects on glycerol and cytokines levels from adipocytes. However, their mechanism (s) in inducing adipogenesis warrants further investigation of different transcription factors at gene expression levels.

Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1.

In vitro experimental systems based on continuous piscine cell lines can be used as an alternative to animal tests for obtaining qualitative and quantitative information on the possible fate and effect of chemicals in fish. However, their capability to reproduce complex metabolic processes and toxic responses as they occur in vivo is limited due to the lack of organ-specific tissue architecture and functions. Here we introduce a three-dimensional (3D) in vitro experimental system based on spheroidal aggregate cultures (spheroids) of the continuous rainbow trout liver cell line RTL-W1 and provide a first description of their structural and functional properties including growth, viability/longevity, metabolic activity, ultrastructure and cytochrome P450 1A (CYP1A) expression determined by bright-field, multi-photon fluorescence and transmission electron microscopy as well as RT-qPCR analysis. Our results show that RTL-W1 cells in 3D spheroids (ø ~ 150 µm) (including those in the interior) were viable, metabolically active and had higher basal and ß-naphthoflavone-induced CYP1A expression levels than conventional 2D cell cultures. Furthermore, they displayed ultrastructural characteristics similar to differentiated hepatocytes. The available evidence suggests that 3D RTL-W1 spheroids may have enhanced hepatotypic functions and be a superior in vitro model to assess hepatic biotransformation, bioaccumulation and chronic toxicity compared to conventional cell monolayer cultures.

Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1µM, but not 10µM, ß-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100µM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities.

Recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants.

Dioxin residues widely contaminate soil and agricultural products at low concentrations and may accumulate in organisms at the top of food chains owing to their physicochemical properties. In this study, we have developed novel, dioxin-inducible, reporter gene expression systems regulated by recombinant aryl hydrocarbon receptors (AhRs). The recombinant AhRs, referred to as XDVs, consist of the DNA-binding domain of the bacterial repressor protein LexA, a 90-kDa heat shock protein- and ligand-binding regulatory domain from mouse AhR, and the transactivation domain of herpes simplex virus regulatory protein VP16. Transgenic tobacco plants carrying XDVs absorb various AhR ligands, including 3-methylcholanthrene, beta-naphthoflavone and indigo from solid medium and vermiculite, and show dose- and time-dependent expression of the beta-glucuronidase reporter gene. The results clearly suggest that XDVs are functional transcription factors that respond to AhR ligands, and that the XDV-mediated reporter gene expression system is applicable to bioassays for dioxin residues in the environment.

Modulation of cytochrome P450 enzyme system by selected flavonoids.

OBJECTIVES: The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP). DESIGN: The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin. RESULTS: The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate. CONCLUSION: Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.

Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels.

In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/ or one parameter testing could produce misleading information.

Persistent binding of ligands to the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([(3)H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([(3)H] beta-naphthoflavone [betaNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [(3)H]TCDD and 26% to 85% for [(3)H] betaNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [(3)H]TCDD, shifted the apparent IC(50) of these compounds as competitive AhR ligands by approximately 10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs.

Agonist and chemopreventative ligands induce differential transcriptional cofactor recruitment by aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AHR) is a transcription factor whose activity is regulated by environmental agents, including several carcinogenic agonists. We measured recruitment of AHR and associated proteins to the human cytochrome P4501A1 gene promoter in vivo. Upon treatment with the agonist beta-naphthoflavone, AHR is rapidly associated with the promoter and recruits the three members of the p160 family of coactivators as well as the p300 histone acetyltransferase, leading to recruitment of RNA polymerase II (Pol II) and induction of gene transcription. AHR, coactivators, and Pol II cycle on and off the promoter, with a period of approximately 60 min. In contrast, the chemopreventative AHR ligand 3,3'-diindolylmethane promotes AHR nuclear translocation and p160 coactivator recruitment but, remarkably, fails to recruit Pol II or cause histone acetylation. This novel mechanism of receptor antagonism may account for the antitumor properties of chemopreventative compounds targeting the AHR.

Hepatic biomarker responses to organic contaminants in feral chub (Leuciscus cephalus)--laboratory characterization and field study in the Sava River, Croatia.

As a widely spread cyprinid fish species, the European chub (Leuciscus cephalus) has been used extensively in biomonitoring programs. However, no laboratory dose-response and/or time course studies related to applied biomarkers have been reported on chub yet. In order to address this issue, specimens of juvenile chub caught in September 2005 in the Sava River, Croatia, were laboratory exposed to various (0.25-50 mg/kg) doses of either model polycyclic aromatic hydrocarbons (PAH) premutagen benzo[a]pyrene (BaP), or beta-naphthoflavone (beta-NF), a well-known model cytochrome 1A (CYP1A) inducer, for 3 (BaP) or 5 d (beta-NF). The responses of several hepatic biomarkers were determined in the exposed fish: The hepatic 7-ethoxyresorufin-O-deethylase activity, CYP1A content, glutathione S-transferase (GST) activity, liver bioactivation potential, and the amount of hydroxylated polycyclic aromatic hydrocarbon bile metabolites determined by the fixed wavelength fluorescence and the high-performance liquid chromatography technique. The relevance of determined biomarker responses has been analyzed further and crosscorrelated with the same set of biomarkers, as well as with tissue concentrations of polycyclic aromatic hydrocarbons and polychlorinated biphenyls, determined in chub specimens collected in September 2005 at five different polluted locations along the Sava River. The species-specific upper and lower limits in responses of studied biomarkers were determined and the obtained ranges successfully evaluated in real field situation. With the exception of the GST activity, all other biomarkers determined in chub proved to be valuable indicators of environmental pollution. Finally, the results of the present study demonstrated that the same strategy of laboratory characterization in combination with field evaluation should be used regularly in the selection of optimal biomarkers and indicator species.

ß-Naphthoflavone-Induced Mitochondrial Respiratory Damage in Cyp1 Knockout Mouse and in Cell Culture Systems: Attenuation by Resveratrol Treatment.

A number of xenobiotic-inducible cytochrome P450s (CYPs) are now known to be localized in the mitochondrial compartment, though their pharmacological or toxicological roles remain unclear. Here, we show that BNF treatment markedly inhibits liver mitochondrial O2 consumption rate (OCR), ADP-dependent OCR, and also reserve OCR, in wild-type mice but not in Cyp1a1/1a2(-/-) double knockout mice. BNF treatment markedly affected mitochondrial complex I and complex IV activities and also attenuated mitochondrial gene expression. Furthermore, under in vitro conditions, BNF treatment induced cellular ROS production, which was inhibited by mitochondria-targeted antioxidant Mito-CP and CYP inhibitor proadefin, suggesting that most of the ROS production was intramitochondrial and probably involved the catalytic activity of mitochondrial CYP1 enzymes. Interestingly, our results also show that the AHR antagonist resveratrol, markedly attenuated BNF-induced liver mitochondrial defects in wild-type mice, confirming the role of AHR and AHR-regulated CYP1 genes in eliciting mitochondrial dysfunction. These results are consistent with reduced BNF-induced mitochondrial toxicity in Cyp1a1/1a2(-/-) mice and elevated ROS production in COS cells stably expressing CYP1A1. We propose that increased mitochondrial ROS production and respiratory dysfunction are part of xenobiotic toxicity. Resveratrol, a chemopreventive agent, renders protection against BNF-induced toxicity.

Enhancing bio-availability of ß-naphthoflavone by supramolecular complexation with 6,6'-thiobis(methylene)-ß-cyclodextrin dimer.

The aryl hydrocarbon receptor (AhR) is a ligand activated transcriptional regulator, which governs key biological processes including detoxification of carcinogens. ß-Naphthoflavone (ß-NF) is a non-toxic flavonoid, and a potent AhR agonist. Thus, ß-NF can induce the representative detoxifying enzyme cytochrome P4501A1, thereby enhancing the detoxification potential. However, its low water solubility hampers the use. We found that supramolecular complexation of ß-NF with the synthetic 6,6'-thiobis(methylene)-ß-cyclodextrin (ß-CD-S) dimer significantly enhanced ß-NF's role as an AhR agonist. The water solubility of ß-NF was increased to 469 fold by effective supramolecular complexation with the ß-CD-S dimer, and caused significant induction of cytochrome P4501A1. Stable formation of the supramolecular complex of ß-NF with ß-CD-S-dimer was verified by various analyses. In summary, supramolecular complexation of ß-NF with ß-CD-S dimer greatly enhanced bio-availability of ß-NF as an AhR agonist. Our findings provide an easy, non-destructive, and alternative approach to enhance the bio-availability of therapeutics.

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding.

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.

Upregulation of cytochromes P450 2B in rat liver by orphenadrine.

1 The alkylamine drug orphenadrine (ORPH) is an inducer and inhibitor of the microsomal cytochrome P450 (CYP) system in mammals. This study evaluated the selectivity of CYP induction by ORPH in rat liver. 2 Immunoblot analysis indicated that ORPH was a selective inducer of the phenobarbitone (PB)-inducible CYP2B in rat liver. CYP2B protein was increased to approximately 14-fold of levels in untreated rat liver. By comparison PB increased CYP2B expression 40-fold. Corresponding increases in the activity of CYP2B-dependent androstenedione 16beta-hydroxylation were measured in microsomes from ORPH and PB-induced rats. 3 Northern analysis indicated that CYP2B1/2 mRNA was increased in ORPH-induced rat liver. Consistent with this finding, ORPH was found to activate a PB-responsive enhancer module in constitutive androstane receptor (CAR)-transfected Hep G2 cells. 4 Other alkylamines like troleandomycin impair CYP turnover. We tested whether ORPH induction of CYP2B may include a post-translational component. In PB-pretreated animals ORPH administration delayed the loss of CYP2B after PB withdrawal, but no evidence for altered turnover was found. 5 These studies establish ORPH as a selective inducer of CYP2B in rat liver. Induction appears to be mediated pretranslationally by CAR activation of CYP2B gene transcription. Post-translational stabilisation by an ORPH metabolite does not elicit induction. Induction of CYP2B may influence pharmacokinetic interactions involving ORPH.

Naphthalene and beta-naphthoflavone effects on Anguilla anguilla L. hepatic metabolism and erythrocytic nuclear abnormalities.

The effects of a polycyclic aromatic hydrocarbon (PAH) such as naphthalene (NAP)--an environmental contaminant--and beta-naphthoflavone (BNF)--a model substance (PAH-like compound)--were investigated in European eel (Anguilla anguilla L.) over 3-, 6-, and 9-day exposure (0.1-2.7 microM). Both xenobiotics revealed to be strong biotransformation (phase I) inducers. After 3-day exposure, liver ethoxyresorufin O-deethylase (EROD) activity was significantly increased by all NAP and BNF tested concentrations. At 6 and 9 days, liver EROD activity was significantly induced mainly by the highest NAP and BNF concentrations. Liver cytochrome P450 content was significantly induced after 3-day exposure to 0.9 and 2.7 microM BNF and 9-day exposure to 0.1, 0.3 and 0.9 microM NAP. Liver alanine transaminase (ALT) activity was measured as an indicator of hepatic health condition, revealing a significant decrease after 6-day exposure to 0.9 microM BNF. Genotoxicity measured as erythrocytic nuclear abnormalities (ENA) was detected in all BNF treated fish on day 6, whereas on day 9, ENA frequencies returned to control levels, significantly decreasing at 0.9 microM BNF exposure. Immature erythrocytes (IE) frequency demonstrated a decreasing tendency along the BNF experiment and concomitantly with the above ENA response. The present experimental results elect EROD activity in A. anguilla as a useful short- to medium-term biomarker of exposure to both PAH and PAH-like compounds. However, some problems can emerge in the presence of high xenobiotic concentrations. Concerning genotoxicity, it is hypothesized that ENA response depends on different factors such as the exhaustion of the detoxification process, the balance erythropoiesis/erythrocytic catabolism and the DNA repairing capacity.

beta-Naphthoflavone disrupts cortisol production and liver glucocorticoid responsiveness in rainbow trout.

We investigated the impact of aryl hydrocarbon receptor (AhR) activation on cortisol production in rainbow trout (Oncorhynchus mykiss) using beta-naphthoflavone (BNF) and alpha-naphthoflavone (ANF) as agonist and antagonist of AhR, respectively. Fish were given a single intraperitoneal injection of either corn oil alone or corn oil containing ANF (50 mg/kg body mass), BNF (50 mg/kg) or a combination of both (ANF/BNF; 50 mg/kg each) and sampled at 2- and 6-day post-injection. Higher CYP1A protein expression in the liver and mRNA abundance in the head kidney confirmed AhR activation with BNF, but ANF did not abolish this response. BNF treatment did not significantly affect mRNA abundance of key steroidogenic enzymes (StAR protein, P450 side chain cleavage and 11beta-hydroxylase) in the head kidney of trout. However, BNF depressed the in vitro ACTH- or 8-br-cAMP-mediated cortisol production and this effect was also not modified by ANF. Plasma cortisol levels were significantly higher with BNF in the present study and this BNF-mediated cortisol response was completely abolished with ANF suggesting a role for AhR and/or CYP1A in the regulation of plasma cortisol concentration. However, despite elevated plasma cortisol levels, there was no significant effect of BNF on liver glucocorticoid receptor (GR) protein expression suggesting a lack of liver responsiveness to cortisol stimulation. Also, this was reflected in the absence of any change in plasma glucose concentration in the BNF group at 2 days, and a significant drop at 6 days compared to other treatments. Altogether, our results suggest that AhR activation disrupts interrenal corticosteroidogenesis and target tissue responsiveness to glucocorticoid stimulation in rainbow trout.

Evaluation of biomarker potential of cytochrome P450 1A (CYP1A) gene in the marine medaka, Oryzias melastigma exposed to water-accommodated fractions (WAFs) of Iranian crude oil.

CYP1A is involved in the metabolism of diverse chemicals, including polycyclic aromatic hydrocarbons and alkylated-PAHs, as a first line of detoxification mechanism. First, we identified and characterized the CYP1A gene from the marine medaka, Oryzias melastigma. O. melastigma CYP1A (Om-CYP1A) showed a high similarity of motifs/domains compared to those of vertebrates in their amino acid sequences. To check whether the Om-CYP1A would be inducible, we tested two strong CYP1A inducers, ß-naphthoflavone (ß-NF) and benzo[α]pyrene (B[α]P), and observed concentration-dependent transient expression on transcripts of Om-CYP1A for 96 h over a wide range of concentrations. Om-CYP1A mRNA level was significantly increased in exposure to different concentrations of ß-NF and B[α]P, and its expression was highly transcribed within 12 h upon the exposure to low concentrations of both chemicals. Inducible transcript profiles revealed that Om-CYP1A would be associated with the toxicant metabolism via AhREs/DREs/XREs in its promoter region. To uncover the effects of the water-accommodated fraction (WAF) of crude oil on transcripts of Om-CYP1A, we measured mRNA expression of Om-CYP1A towards different concentrations of WAF for 24h. As a result, WAF exposure significantly increased Om-CYP1A transcripts at all concentrations as well as during time-course experiments for 96 h. In this paper, we demonstrated that WAF would trigger up-regulation of the CYP1A gene that would be associated with the initiation of the cellular defense systems. This finding provides a better understanding of the molecular mechanism of cellular protection particularly that involved in the WAF-mediated cellular response in O. melastigma.

The effect of aryl hydrocarbon receptor ligands on the expression of polymerase (DNA directed) kappa (Polκ), polymerase RNA II (DNA directed) polypeptide A (PolR2a), CYP1B1 and CYP1A1 genes in rat liver.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. AhR is ligand activated transcription factor with high affinities for aromatic planar compounds such as ß-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding appropriate ligand, AhR trigger induction of expression of some phase I and phase II drug metabolizing genes together with numerous other genes. One of such gene appear to be polymerase (DNA directed) kappa (Polκ). Polκ gene encodes newly identified low fidelity DNA polymerase. The enzyme bypasses benzo[a]pyrene-N2-dG lesions in a mostly error free manner by incorporating predominantly dC opposite the bulky lesions. It was demonstrated that AhR activation increases expression of the mouse Polκ gene and probably human POLK gene. In this study we examined the effect of i.p. administration of different AhR ligands on the expression of Polκ, RNA polymerase II polypeptide A (PolR2a) and cytochrome P450 1B1 (CYP1B1), the genes controlled by AhR in Sprague-Dawley rat liver. Quantitative real-time RT-PCR analysis revealed significant induction in the mRNA expression levels of Polκ and PolR2a following BNF treatment. Time courses of mRNA expression after treatment with BNF were similar in both genes, with maximal increases at 8h after treatment. The maximal induction of CYP1B1 and CYP1A1 expression was observed after 24 and 8h after BNF injection, respectively. TCDD treatment caused the significant increase in the mRNA level of CYP1B1 at 72h after administration of the ligand but no effect on Polκ and PolR2a mRNA expression was observed. These results confirm connection between AhR and Polκ, and strongly suggest that AhR up-regulates the mRNA transcription of PolR2a as well. However physiological importance of AhR dependent regulation of PolR2a expression must be further elucidated.

CH223191 is a ligand-selective antagonist of the Ah (Dioxin) receptor.

The aryl hydrocarbon (dioxin) receptor (AhR) is a ligand-dependent transcription factor that produces a wide range of biological and toxic effects in many species and tissues. Whereas the best-characterized high-affinity ligands include structurally related halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs), the AhR is promiscuous and can also be activated by structurally diverse exogenous and endogenous chemicals. However, little is known about how these diverse ligands actually bind to and activate the AhR. Utilizing AhR ligand binding, DNA binding, and reporter gene expression assays, we have identified a novel ligand-selective antagonist (CH223191) that preferentially inhibits the ability of some classes of AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and related HAHs), but not others (PAHs, flavonoids, or indirubin), to bind to and/or activate the AhR and AhR signal transduction. HAH-specific antagonism of AhR-dependent reporter gene expression by CH223191 was observed with mouse, rat, human, and guinea pig cell lines. Ligand- and species-selective antagonism was also observed with the AhR antagonists 3'-methoxy-4'-nitroflavone and 6,2',4',-trimethoxyflavone. Our results suggest that the differences in the binding by various ligands to the AhR contribute to the observed structural diversity of AhR ligands and could contribute in ligand-specific variation in AhR functionality and the toxic and biological effects of various classes of AhR agonists.

Characterization of new metabolites from in vivo biotransformation of 2-amino-3-methylimidazo[4,5-f]quinoline in mouse by mass spectrometry.

In studying the metabolic pathways underlying the mechanism of carcinogenesis of the heterocyclic amine of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), we recently found a new metabolite which gave an [M + H](+) ion of m/z 217 when subjected to electrospray ionization (ESI) in positive-ion mode. Following i.p. injection of this metabolite of m/z 217 (designated as m/z 217) to beta-naphthoflavone-treated mice, 57% of the total radioactivity was recovered in a 24-h mouse urine sample. HPLC separation followed by MS analysis indicates that the urine sample contained m/z 217 (36 +/- 3% of total recovered radioactivity) and two other peaks that gave rise to the [M + H](+) ions of m/z 393 (31 +/- 4%, designated as m/z 393) and m/z 233 (14 +/- 1%, designated as m/z 233). Beta-glucuronidase treatment of m/z 393 resulted in a radioactive peak corresponding to m/z 217. ESI in combination with various mass spectrometry techniques, including multiple-stage mass spectrometry, exact mass measurements and H/D exchange followed by tandem mass spectrometry, was used for structural characterization. The urinary metabolites of m/z 217, 393 and 233 were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline, 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f]quinoline and 1,2-dihydro-2-amino-5,7-dihydroxy-3-methylimidazo[4,5-f]quinoline, respectively. Our results demonstrated that m/z 217 is biotransformed in vivo to m/z 393 by O-glucuronidation and to m/z 233 by oxidation. The observation of these more polar metabolites relative to IQ suggests that they may arise from a previously undescribed detoxification pathway.

Transcription factor Nrf2 regulates promoter activity of mouse aldose reductase (AKR1B3) gene.

Transcription factor Nrf2 regulates gene expression of drug metabolizing enzymes such as glutathione S-transferase via the antioxidant response element, ARE. Aldose reductase (AR), a member of the aldo-keto reductase (AKR) superfamily, metabolizes various endogenous and exogenous aldehydes. The AR gene 5'-flanking region contains a multiple stress response region (MSRR) composed of two putative AREs (ARE1 and ARE2), an AP1 site, and a tonicity response element (TonE). As this region is highly conserved among species, we examined the involvement of Nrf2 in transcriptional regulation of the AR gene. beta-Naphthoflavone, an Nrf2 activator, elevated the level of AR mRNA in HepG2 cells and increased the promoter activity of the mouse AR (AKR1B3) gene. The promoter activity of the AKR1B3 gene, containing MSRR, was also augmented by overexpression of Nrf2. Deletion and mutation analyses indicated that both ARE1 and the AP1 site were essential for the responsiveness to Nrf2, while ARE2 was nonfunctional. The presence of an ARE1 binding protein complex was revealed by electrophoretic mobility shift assay. These findings indicate that Nrf2 regulates the AKR1B3 promoter activity via ARE1 and the AP1 site.