Molecular characterization of the aryl hydrocarbon receptor 2 gene in black rockfish, Sebastes schlegelii, and its expression patterns upon exposure to benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of halogenated and polycyclic aromatic hydrocarbons in vertebrates. Thus, increased knowledge of AhR-mediated responses to xenobiotics is imperative. Sebastes schlegelii is increasingly being used as a model for studying environmental toxicology; hence, in this study, the presence of AhR2 was evaluated in S. schlegelii. The results showed that the predicted AhR2 amino acid sequence contained regions characteristic of other vertebrate AhRs, including the basic helix-loop-helix and PER-ARNT-SIM domains in the N-terminal half, but it had minor similarity with other vertebrate AhRs across the C-terminal half; it did not contain the distinct glutamine-rich domains found in mammalian AhR2. Phylogenetic analysis demonstrated that S. schlegelii AhR2 was clustered within the teleost AhR2 branch. Additionally, AhR2 mRNA was detectable in all 11 tissues tested, with the highest mRNA levels in the heart, pyloric ceca, and liver. Furthermore, exposure to the AhR agonists showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 µg/g body weight) induced a significantly higher increases in AhR2 expression in the gills, liver, kidneys, and spleen in 48 h than benzo[a]pyrene (2 µg/g body weight), and ß-naphthoflavone (50-µg/g body weight); AhR2 mRNA levels upon TCDD exposure were up-regulated by 16- and 10-fold in the gills and liver, respectively. These findings indicated that AhR was a highly sensitive receptor against TCDD. Thus, investigating AhR2 expression in the presence of other xenobiotics might offer further information for the elucidation of its crucial role in mediating toxicant metabolism in S. schlegelii.

Behavioural adaptations after antibiotic treatment in male mice are reversed by activation of the aryl hydrocarbon receptor.

The intestinal microbiota plays an important role in regulating brain functions and behaviour. Microbiota-dependent changes in host physiology have been suggested to be key contributors to psychiatric conditions. However, specific host pathways modulated by the microbiota involved in behavioural control are lacking. Here, we assessed the role of the aryl hydrocarbon receptor (Ahr) in modulating microbiota-related alterations in behaviour in male and female mice after antibiotic (Abx) treatment. Mice of both sexes were treated with Abx to induce bacterial depletion. Mice were then tested in a battery of behavioural tests, including the elevated plus maze and open field tests (anxiety-like behaviour), 3 chamber test (social preference), and the tail suspension and forced swim tests (despair behaviour). Behavioural measurements in the tail suspension test were also performed after microbiota reconstitution and after administration of an Ahr agonist, ß-naphthoflavone. Gene expression analyses were performed in the brain, liver, and colon by qPCR. Abx-induced bacterial depletion did not alter anxiety-like behaviour, locomotion, or social preference in either sex. A sex-dependent effect was observed in despair behaviour. Male mice had a reduction in despair behaviour after Abx treatment in both the tail suspension and forced swim tests. A similar alteration in despair behaviour was observed in Ahr knockout mice. Despair behaviour was normalized by either microbiota recolonization or Ahr activation in Abx-treated mice. Ahr activation by ß-naphthoflavone was confirmed by increased expression of the Ahr-target genes Cyp1a1, Cyp1b1, and Ahrr. Our results demonstrate a role for Ahr in mediating the behaviours that are regulated by the crosstalk between the intestinal microbiota and the host. Ahr represents a novel potential modulator of behavioural conditions influenced by the intestinal microbiota.

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.

The effects of ß-naphthoflavone on rat placental development.

The morphological effects of ß-naphthoflavone (ß-NF) on placental development in pregnant rats were examined. ß-NF, administered to pregnant rats intraperitoneally at 15 mg/kg bw from gestation day (GD) 9 to GD 14, had no effect on maternal body weight gain, mortality, or clinical sign. In the ß-NF-exposed rats, intrauterine growth retardation (IUGR) rates increased on GDs 17 and 21, although there was no effect on fetal mortality rate, fetal or placental weight, or external fetal abnormality. Histopathologically, ß-NF induced apoptosis and inhibition of cell proliferation of the trophoblastic septa in the labyrinth zone, resulting in its poor development. In the basal zone, ß-NF induced spongiotrophoblast apoptosis and delayed glycogen islet regression, resulting in their cystic degeneration. ß-NF-induced CYP1A1 expression was detected in the endothelial cells of the fetal capillaries in the labyrinth zone and in the endothelial cells of the spiral arteries in the metrial gland, but not in any trophoblasts. This indicates that CYP1A1 is inducible in the endothelial cells of the fetal capillaries in the labyrinth zone, and that these cells have an important role in metabolizing CYP1A1 inducers crossing the placental barrier.

Induction of expression of aryl hydrocarbon receptor-dependent genes in human HepaRG cell line modified by shRNA and treated with ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of ß-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.

Sulforaphane Alters ß-Naphthoflavone-Induced Changes in Activity and Expression of Drug-Metabolizing Enzymes in Rat Hepatocytes.

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, exerts many beneficial effects on human health such as antioxidant, anti-inflammatory, and anticancer effects. The effect of SFN alone on drug-metabolizing enzymes (DMEs) has been investigated in numerous in vitro and in vivo models, but little is known about the effect of SFN in combination with cytochrome P450 (CYP) inducer. The aim of our study was to evaluate the effect of SFN on the activity and gene expression of selected DMEs in primary cultures of rat hepatocytes treated or non-treated with ß-naphthoflavone (BNF), the model CYP1A inducer. In our study, SFN alone did not significantly alter the activity and expression of the studied DMEs, except for the glutathione S-transferase (GSTA1) mRNA level, which was significantly enhanced. Co-treatment of hepatocytes with SFN and BNF led to a substantial increase in sulfotransferase, aldoketoreductase 1C, carbonylreductase 1 and NAD(P)H:quinone oxidoreductase 1 activity and a marked decrease in cytochrome P450 (CYP) Cyp1a1, Cyp2b and Cyp3a4 expression in comparison to the treatment with BNF alone. Sulforaphane is able to modulate the activity and/or expression of DMEs, thus shifting the balance of carcinogen metabolism toward deactivation, which could represent an important mechanism of its chemopreventive activity.

Functional expressions of adenosine triphosphate-binding cassette transporters during the development of zebrafish embryos and their effects on the detoxification of cadmium chloride and ß-naphthoflavone.

Adenosine triphosphate-binding cassette (ABC) transporters, including ABCB, ABCC and ABCG families represent general biological defenses against environmental toxicants in varieties of marine and freshwater organisms, but their physiological functions at differential developmental stages of zebrafish embryos remain undefined. In this work, functional expressions of typical ABC transporters including P-glycoprotein (Pgp), multiresistance associated protein 1 (Mrp1) and Mrp2 were studied in zebrafish embryos at 4, 24, 48 and 72 h post-fertilization (hpf). As a result, both the gene expressions and activities of Pgp and Mrps increased with the development of embryos. Correspondingly, 4-72 hpf embryos exhibited an increased tolerance to the toxicity caused by cadmium chloride (CdCl2 ) and ß-naphthoflavone (BNF) with time. Such a correlation was assumed caused by the involvement of ABC transporters in the detoxification of chemicals. In addition, the assumption was supported by the fact that model efflux inhibitors of Pgp and Mrps such as reversine 205 and MK571 significantly inhibited the efflux of toxicants and increased the toxicity of Cd and BNF in zebrafish embryos. Moreover, exposure to CdCl2 and BNF induced the gene expressions of Pgp and Mrp1 in 72 hpf embryos. Thus, functional expressions of Pgp and Mrps increased with the development of zebrafish embryos, which could cause an increasing tolerance of zebrafish embryos to CdCl2 and BNF. Copyright © 2015 John Wiley & Sons, Ltd.

beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

ß-Naphthoflavone (ß-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of ß-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with ß-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, ß-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by ß-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by ß-NF treatment. Our findings show that ß-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that ß-NF might prevent TNF-α-induced inflammation.

Intact cell mass spectrometry as a rapid and specific tool for the differentiation of toxic effects in cell-based ecotoxicological test systems.

In the last few decades, MALDI-TOF MS has become a useful technique not only in proteomics, but also as a fast and specific tool for whole cell analysis through intact cell mass spectrometry (IC-MS). The present study evaluated IC-MS as a novel tool for the detection of distinct patterns that can be observed after exposure to a certain toxin or concentration by utilizing the eukaryotic fish cell line RTL-W1. Two different viability assays were performed to define the range for IC-MS investigations, each of which employing copper sulfate, acridine, and ß-naphthoflavone (BNF) as model compounds for several classes of environmental toxins. The IC-MS of RTL-W1 cells revealed not only specific spectral patterns for the various toxins, but also that the concentration used had an effect on RTL-W1 profiles. After the exposure with copper sulfate and acridine, the spectra of RTL-W1 showed a significant increase of certain peaks in the higher mass range (m/z >7000), which is probably attributed to the apoptosis of RTL-W1. On the contrary, exposure to BNF showed a distinct change of ion abundances only in the lower mass range (m/z <7000). Furthermore, a set of mass peaks could be identified as a specific biomarker for a single toxin treatment, so IC-MS demonstrates a new method for the distinction of toxic effects in fish cells. Due to fast sample preparation and high throughput, IC-MS offers great potential for ecotoxicological studies to investigate cellular effects of different substances and complex environmental samples.

ß-Naphthoflavone induces oxidative stress in the intertidal copepod, Tigriopus japonicus.

ß-Naphtoflavone (ß-NF) is a flavonoid and enhances oxidative stress in vertebrates with little information from aquatic invertebrates as yet. In this study, we investigated the effects of ß-NF on the antioxidant defense systems of the intertidal copepod Tigriopus japonicus. To measure the ß-NF-triggered changes in oxidative stress markers, such as intracellular reactive oxygen species (ROS), glutathione (GSH) concentration, residual glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) activity, T. japonicus were exposed to ß-NF (0.5 and 1 mg/L) for 72 h. Significant (P < 0.05) induction of the intracellular ROS content (%) was observed in 1 mg/L of ß-NF exposed T. japonicus, compared to the negative control and H2O2-exposed group. The GSH levels were significantly increased in the 0.5 mg/L of ß-NF-exposed group for 12 h and 1 mg/L of ß-NF-exposed groups for 12-24 h. GPx, GST, and GR activities showed a significant increase in the 1 mg/L ß-NF-exposed group, indicating that ß-NF induces oxidative stress in T. japonicus. To understand the effects of ß-NF at the level of transcript expression, a 6K microarray analysis was employed. Transcript profiles of selected antioxidant-related genes were modulated after 72 h exposure to 1 mg/L of ß-NF. From microarray data, 10 GST isoforms, GR, GPx, PH-GPx, and Se-GPx were chosen for a time-course test by real-time RT-PCR. T. japonicus GST-S, GST-O, GST-M, and GST-D1 were significantly increased in a 1 mg/L ß-NF-exposed group. T. japonicus GPx, GR, and Se-GPx mRNA levels were also significantly increased at both concentrations. Our results revealed that oxidative stress was induced by ß-NF exposure in T. japonicus.

EROD activity in the native fish Cnesterodon decemmaculatus as a biomarker for assessing aquatic pollution by AhR agonist chemicals within the Rio de la Plata Basin.

The 7-ethoxyresorufin-O-deethylase (EROD) activity was first time characterized in the neotropical fish Cnesterodon decemmaculatus as a biomarker for assessing environmental health in aquatic ecosystems of the Rio de la Plata Basin impacted by organic pollutants agonist of the aryl-hydrocarbon receptor (AhR). Both laboratory and field studies were conducted. Laboratory experiments were run using ß-naphthoflavone (BNF) as an AhR agonist model. A clear concentration-response relationship was found between 1 and 100 µg/L, with a NOEC and LOEC of 1 and 10 µg/L. A fast time-dependent response was observed with a significant induction after 24 h and a plateau from 24 to 48 h up to 264 h of exposure. Differences in basal activity were found between juveniles, females, and males, but induction levels were similar. Both basal activities and induction levels were distinct in the whole body, liver, gill, muscle, brain, and embryos. Fold-change inductions in the respective tissues were: 20, 114, 3, 5, 1, and 14. Maternal transfer and early cyp1a activation were unveiled by embryonic induction. Clear differences in EROD activity were found among juveniles collected in hydrocarbon-polluted streams, beside the La Plata Petrochemical hub, and a reference stream. Similar EROD activities were observed in laboratory and feral fish, usually with values below or above 1,000 pmol/min x mg protein for unexposed or exposed organisms. The study contributes with original information about EROD activity in C. decemmaculatus that encourages the use of both the response as a robust biomarker of exposure and the species as a good sentinel organism to be included in surveillant programs for assessing aquatic pollution by AhR agonist chemicals within the Rio de la Plata Basin within the One Health paradigm.

Assessment of energetic costs of AhR activation by ß-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis.

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist ß-naphthoflavone (ßNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na(+)/K(+)-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1µM ßNF for 48h did not alter hepatocyte AEC, protein synthesis, or Na(+)/K(+)-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies.

ß-naphthoflavone-induced upregulation of CYP1B1 expression is mediated by the preferential binding of aryl hydrocarbon receptor to unmethylated xenobiotic responsive elements.

Human cytochrome P450 1 (CYP1) enzymes are transcriptionally induced by specific xenobiotics through a mechanism that involves the binding of aryl hydrocarbon receptors (AhR) to target xenobiotic responsive element (XRE) sequences. To examine the effect of DNA methylation on the AhR-mediated pathway, reverse transcription-quantitative PCR analysis was performed. ß-naphthoflavone (ßNF)-induced CYP1B1 expression was found to be potentiated by pre-treatment of human HepG2 liver cancer cells with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, but not HuH7 cells. It was hypothesized that this increase is mediated by the demethylation of CpG sites within XRE2/XRE3 sequences, suggesting that methylation of these sequences inhibits gene expression by interfering with the binding of AhR to the target sequences. To test this hypothesis, a novel method combining the modified chromatin immunoprecipitation of AhR-XRE complexes with subsequent DNA methylation analysis of the XRE regions targeted by activated AhR was applied to both liver cancer cell lines treated with ßNF. XRE2/XRE3 methylation was found to be exclusively observed in the input DNA from HepG2 cells but not in the precipitated AhR-bound DNA. Furthermore, sub-cloning and sequencing analysis revealed that the two XRE sites were unmethylated in the samples from the AhR-bound DNA even though the neighboring CpG sites were frequently methylated. To the best of our knowledge, the present study provides the first direct evidence that ligand-activated AhR preferentially binds to unmethylated XRE sequences in the context of natural chromatin. In addition, this approach can also be applied to assess the effects of DNA methylation on target sequence binding by transcription factors other than AhR.

Temporal transcriptomic analysis of human primary keratinocytes exposed to ß-naphthoflavone highlights the protective efficacy of skin to environmental pollutants.

The skin covers almost the entire body and plays an important role in detoxification and elimination of xenobiotics. These processes are initiated following the binding of xenobiotics to the aryl hydrocarbon receptor (AhR), which leads to the expression of several detoxification enzymes. To gain some insights on their impacts on skin cells over time, a temporal transcriptional analysis using gene expression arrays was performed in human primary epidermal keratinocyte (HEK) cells exposed for 6, 24 and 48 h to ß-naphthoflavone (ßNF), a potent agonist of AhR. Our results demonstrated that expression of genes related to xenobiotic, inflammation, and extracellular matrix remodeling was increased upon ßNF treatment from 6 h onwards. In contrast, the anti-oxidative response was seen mainly starting at 24 h. While some of the genes controlled by the epidermal differentiation complex was induced as soon as 6 h, expression of most of the S100 related genes located within the same chromosomal locus and keratin genes was increased at later times (24 and 48 h). Altogether our transcriptomic data highlight that following ßNF exposure, HEK cells elicited a protective xenobiotic response together with the activation of inflammation and keratinocyte regeneration. Later on these processes were followed by the stimulation of anti-oxidant activity and terminal differentiation.

ß-Naphthoflavone Activation of the Ah Receptor Alleviates Irradiation-Induced Intestinal Injury in Mice.

Radiotherapy induced gastrointestinal syndrome results from the acute damage of intestinal stem cells, impaired crypts reconstruction, and subsequent breakdown of the mucosal barrier. The toxicity of ionizing radiation is associated with oxidative stress in the intestinal epithelial cells (IECs). Moreover, the rapid proliferation of IECs is a risk factor for radiation damage. ß-naphthoflavone (BNF) is an agonist of the aryl hydrocarbon receptor (AhR) and possesses potential antioxidative activity. We investigated BNF radioprotection in IECs experiencing γ-ray exposure, contributed to mitigation of radiation enteritis. BNF significantly enhanced cell viability and suppressed cell apoptosis in an AhR activation-dependent manner. The mechanism of BNF reducing the IECs radiosensitivity was associated with cell cycle arrest and suppression of cell proliferation. In contrast, AhR antagonist CH-223191 significantly blocked BNF-induced cell cycle arrest. Cyp1a1 mRNA levels are induced after irradiation in a dose-dependent manner, and CYP1A1 protein expression increased in the irradiated intestinal tract as well. BNF also reduces DNA strand breaks induced by irradiation. These studies demonstrate that BNF pretreatment prolonged median survival time of mice upon exposure to a lethal dose of radiation and alleviated irradiation-induced toxicity within the bowel.

Inducibility of cytochrome P450-mediated 7-methoxycoumarin-O-demethylase activity in zebrafish (Danio rerio) embryos.

The zebrafish (Danio rerio) embryo has increasingly been used as an alternative model in human and environmental toxicology. Since the cytochrome P450 (CYP) system is of fundamental importance for the understanding and correct interpretation of the outcome of toxicological studies, constitutive and xenobiotic-induced 7-methoxycoumarin-O-demethylase (MCOD), i.e. 'mammalian CYP2-like', activities were monitored in vivo in zebrafish embryos via confocal laser scanning microscopy. In order to elucidate molecular mechanisms underlying the MCOD induction, dose-dependent effects of the prototypical CYP inducers ß-naphthoflavone (aryl hydrocarbon receptor (AhR) agonist), rifampicin (pregnane X receptor (PXR) agonist), carbamazepine and phenobarbital (constitutive androstane receptor (CAR) agonists) were analyzed in zebrafish embryos of varying age. Starting from 36 h of age, all embryonic stages of zebrafish could be shown to have constitutive MCOD activity, albeit with spatial variation and at distinct levels. Whereas carbamazepine, phenobarbital and rifampicin had no effect on in vivo MCOD activity in 96 h old zebrafish embryos, the model aryl hydrocarbon receptor agonist ß-naphthoflavone significantly induced MCOD activity in 96 h old zebrafish embryos at 46-734 nM, however, without a clear concentration-effect relationship. Induction of MCOD activity by ß-naphthoflavone gradually decreased with progression of embryonic development. By in vivo characterization of constitutive and xenobiotic-induced MCOD activity patterns in 36, 60, 84 and 108 h old zebrafish embryos, this decrease could primarily be attributed to an age-related decline in the induction of MCOD activity in the cardiovascular system. Results of this study provide novel insights into the mechanism and extent, by which specific CYP activities in early life-stages of zebrafish can be influenced by exposure to xenobiotics. The study thus lends further support to the view that zebrafish embryos- at least from an age of 36 h - have an elaborate and inducible biotransformation system.

Induction of maxillary and mandibular squamous epithelial cell proliferation in mink (Neovison vison) by ß-naphthoflavone.

A jaw lesion reported in mink exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDD-like chemicals is considered a potential indicator of exposure to these chemicals. Many of the effects of TCDD-like chemicals are induced through interaction with the aryl hydrocarbon receptor. The present study indicates that mink dosed with ß-naphthoflavone, which is an aryl hydrocarbon receptor ligand but not a TCDD-like chemical, also develop the lesion. Environ Toxicol Chem 2019;38:460-463. © 2018 SETAC.

ß-Naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor, disrupts zinc homeostasis in human hepatoma HepG2 cells.

Recent studies have demonstrated a relationship between the disruption of zinc homeostasis and the onset of diseases. However, little is known about the factors that disrupt zinc homeostasis. Here, we investigated the effects of ß-naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor (AHR), on intracellular zinc levels. Human hepatoma HepG2 cells were treated with ß-naphthoflavone for 3 days, and intracellular labile and total zinc levels were assessed through flow cytometry and inductively coupled plasma atom emission spectroscopy, respectively. The mRNA levels of zinc transporters were determined by real-time PCR. Treatment of cells with ß-naphthoflavone induced a decrease in intracellular labile zinc in a dose-dependent manner, with significantly decreased levels observed at 1 µM compared with controls. Additionally, intracellular total zinc levels demonstrated a decreasing trend with 10 µM ß-naphthoflavone. Zinc pyrithione recovered the decrease in intracellular labile zinc levels induced by ß-naphthoflavone, while zinc sulfate had no effect. Moreover, significant decreases in the mRNA levels of zinc transporters ZnT10 and ZIP5 were observed in response to 10 µM ß-naphthoflavone. These results demonstrated that ß-naphthoflavone has the potential to disrupt zinc homeostasis in hepatocytes. Although the underlying mechanism remains to be determined, suppression of zinc transporter transcription through AHR activation may be involved in the ß-naphthoflavone-induced disruption of intracellular zinc levels.

Aryl hydrocarbon receptor is indispensable for ß-naphthoflavone-induced novel food avoidance and may be involved in LiCl-triggered conditioned taste aversion in rats.

Previous studies have shown that several aryl hydrocarbon receptor (AHR) agonists, including ß-naphthoflavone (BNF), elicit avoidance of novel food items in rodents, with this behavioral response displaying a similar dose-response to hepatic induction of CYP1A1. The avoidance has been found to bear substantial similarity to conditioned taste avoidance/aversion (CTA). The present study set out to confirm the indispensability of AHR in the avoidance response, to verify whether vagal afferent fibers are involved in it, and to see if AHR signaling might interfere with the effect of the classic trigger of CTA, LiCl. To this end, globally AHR deficient (AHRKO) or vagotomized wildtype rats were treated by gavage with 60 mg/kg BNF or ip with 0.15 M LiCl (4 ml/kg), and presented with chocolate which was either novel or familiar to them. Both the avoidance response and Cyp1a1 induction were missing in AHRKO rats. In contrast, Ahr+/- rats exhibited them in full, save for a single outlier. Total subdiaphragmatic vagotomy failed to interfere with the avoidance of novel or familiar chocolate or induction of Cyp1a1. After LiCl administration, male AHRKO rats showed a significantly mitigated suppression of chocolate consumption compared with wildtype animals (~60% vs. ~10% of control chocolate intake, respectively). A similar tendency was seen in females, but they were less responsive to LiCl. These findings corroborate AHR as a prerequisite of the BNF-induced novel food avoidance, prove vagal afferents unlikely mediators of this response, and imply an unforeseen involvement of AHR signaling in the thoroughly-characterized CTA instigated by LiCl.

A temporal high-resolution investigation of the Ah-receptor pathway during early development of zebrafish (Danio rerio).

In order to contribute to a comprehensive understanding of the regulating mechanisms of the aryl-hydrocarbon-receptor (AHR) in zebrafish embryos, we aimed to elucidate the interaction of proteins taking part in this signaling pathway during early development of the zebrafish (Danio rerio) after chemical exposure. We managed to illustrate initial transcription processes of the implemented proteins after exposure to two environmentally relevant chemicals: polychlorinated biphenyl 126 (PCB126) and ß-Naphthoflavone (BNF). Using qPCR, we quantified mRNA every 4 h until 118 h post fertilization and found the expression of biotransformation enzymes (cyp1 family) and the repressor of the AHR (ahr-r) to be dependent on the duration of chemical exposure and the biodegradability of the compounds. PCB126 induced persistently increased amounts of transcripts as it is not metabolized, whereas activation by BNF was limited to the initial period of exposure. We did not find a clear relation between the amount of transcripts and activity of the induced CYP-proteins, so posttranscriptional mechanisms are likely to regulate biotransformation of BNF. With regard to zebrafish embryos and their application in risk assessment of hazardous chemicals, our examination of the AHR pathway especially supports the relevance of the time point or period of exposure that is used for bioanalytical investigations and consideration of chemical properties determining biodegradability.