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Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-β-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species., (© 2024 John Wiley & Sons Ltd.)

Doxorubicin is one of the most effective chemotherapeutic agents available for treating various cancers, including lung cancer-the leading cause of cancer death in both men and women. However, its clinical application has been impeded by severe adverse effects, notably cardiotoxicity. Development of cellular resistance to doxorubicin is another major obstacle that must be overcome for broader application of the drug. In the present study, we examined the therapeutic potential of beta-naphthoflavone (BNF), a synthetic derivative of a naturally occurring flavonoid, in combination with doxorubicin for the treatment of lung cancer. Among our novel observations were that BNF enhances the efficacy of doxorubicin by inducing doxorubicin accumulation, mitochondrial ROS generation, and JNK pathway signaling in lung cancer cells. These combined effects were also evident in many other cancer cell types. BNF further exhibited synergistic induction of apoptosis in lung cancer cells when combined with several other cancer drugs, including irinotecan, cisplatin, and 5-fluorouracil. Our results suggest that BNF can be developed as a promising adjuvant agent for enhancing the efficacy of doxorubicin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H 2 O 2 , either directly or indirectly via superoxide anion, a reaction referred to as "NADPH oxidase" activity. H 2 O 2 production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched β-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 μM) for CYP1A2 and 4-methylpyrazole (32 μM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 μM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and H 2 O 2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Glutathione S-transferases (GSTs) are a key enzyme superfamily involved in the detoxification and cytoprotection of a wide variety of xenobiotics, such as carcinogens, anticancer drugs, environmental toxicants, and endogenously produced free radicals. In the liver, the hGSTA1 isoenzyme is the most abundant and catalyzes the glutathione conjugation of a wide range of electrophiles and has been the principal GST responsible for xenobiotic detoxification. Given the critical role of this enzyme in several cellular processes, particularly cell detoxification, understanding the molecular mechanisms underlying the regulation of hGSTA1 expression is critical. Therefore, the aim of the present study was to investigate whether AHR is involved in the modulation of hGSTA1 gene expression and to characterize the molecular mechanism through which AHR exerts this regulation. Two xenobiotic response elements (XREs) were located at -602 bp and -1030 bp from the transcription start site at the hGSTA1 gene promoter. After treatment of HepG2 cells with beta-naphthoflavone (β-NF), an AHR agonist, induction of hGSTA1 mRNA was observed. This effect was mediated by the recruitment of AHR to the hGSTA1 gene promoter and its transactivation, as indicated by the ChIP, EMSA and luciferase activity assays. The increase in hGSTA1 transcription regulated by AHR also resulted in enhanced levels of hGSTA1 protein and activity. Taken together, our data suggest that AHR ligands have the potential to modify xenobiotic and endobiotic metabolism mediated by hGSTA1, thereby altering the detoxification of xenobiotics, steroidogenesis and the efficacy of chemotherapeutic agents., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Numerous studies have shown that over-activation of microglia could cause neuroinflammation and release pro-inflammatory mediators, which could result in neurodegenerative diseases, like Parkinson's disease, Alzheimer's disease etc. Beta-naphthoflavone (BNF) has anti-oxidant and anti-inflammatory effects in borderline tissues, but BNF has not been reported the effect associated with neuroinflammation. Therefore, the purpose of this experiment is to inquiry the impact and mechanism of BNF on neuroinflammation. The results indicated that BNF significantly inhibited the production of pro-inflammatory mediators (inducible nitric-oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) andinterleukin-6 (IL-6)) in LPS-exposed BV-2 cells. Analysis of western blot results found that BNF accelerated the activation of AKT/Nrf-2/HO-1 signaling pathway and suppressed NF-κB pathway activation. Further study showed that BNF inhibited activation of NF-κB pathway via promoting HO-1, and SnPP IX (a HO-1 inhibitor) could inhibit anti-inflammatory function of BNF. We also found that BNF reduced the apoptosis rate of Human neuroblastoma cells (SHSY5Y) and mouse hippocampal neuron cell line (HT22) by inhibiting release of inflammatory mediators in LPS-exposed BV2 cells. In a word, our results suggested that BNF could inhibit inflammatory response via AKT/Nrf-2/HO-1-NF-κB signaling axis in BV-2 cells and exerts neuroprotective impact via inhibiting the activation of BV2 cells., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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 β-naphthoflavone (βNF), a potent agonist of AhR. Our results demonstrated that expression of genes related to xenobiotic, inflammation, and extracellular matrix remodeling was increased upon βNF 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 βNF 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The 1-methyl-4-phenylpyridinium (MPP + ) is a parkinsonian-inducing toxin that promotes neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it was reported that some Cytochrome P450 (CYP) isoforms, such as CYP 2D6 or 2E1, may be involved in the development of this neurodegenerative disease. In order to study a possible role for CYP induction in neurorepair, we designed an in vitro model where undifferentiated neuroblastoma SH-SY5Y cells were treated with the CYP inducers β-naphthoflavone (βNF) and ethanol (EtOH) before and during exposure to the parkinsonian neurotoxin, MPP + . The toxic effect of MPP + in cell viability was rescued with both βNF and EtOH treatments. We also report that this was due to a decrease in reactive oxygen species (ROS) production, restoration of mitochondrial fusion kinetics, and mitochondrial membrane potential. These treatments also protected complex I activity against the inhibitory effects caused by MPP + , suggesting a possible neuroprotective role for CYP inducers. These results bring new insights into the possible role of CYP isoenzymes in xenobiotic clearance and central nervous system homeostasis.

Ethnopharmacological Relevance: Inflammatory bowel disease (IBD) is one of the most common chronic inflammatory illnesses of the gastrointestinal tract due to the imbalance of immune homeostasis of T helper cells and/or regulatory T cells (Tregs). The Traditional Chinese medicine herb has been clinically proven for use in the treatment of IBD but its possible mechanism remains unknown. The study aims to assess the effect of Chinese medicinal herb decoction QRZSLXF (Qing Re Zao Shi Liang Xue receipt) for the treatment of TNBS-induced experimental colitis in mice and explore its relevant mechanism involved in Th17 and Tregs., Materials and Methods: Mice colitis was induced by 50% 2,4,6-Trinitrobenzenesulfonic Acid (TNBS) ethanol solution weekly manner. These established model mice were divided into model control (0.8% NaCl treatment), FICZ, naphthoflavone (NaFTV), dexamethasone (DXM), and QRZSLXF (QrLx) groups. The colonoscopy, H&E staining, and immune staining were used to analyze the disease severity, inflammatory condition and Th17 or Treg related factors expression. High-performance liquid chromatography-mass spectrometry (HPLC/MS) was used to assess the content of FICZ in the colon tissues. Western blot and ELISA were used to examine the expression of Th17 or Treg related factors protein levels. Flow cytometry analysis was performed to assess the number and ratio of Th17/Tregs in splenocytes, and mesenteric lymph node lymphocytes (MLNCs), and lamina propria mononuclear cells (LPMCs)., Results: NaFTV, DXM and QrLx groups intestinal inflammation scores were significantly lower than that in colitis model control and FICZ groups, while the IL-6, STAT3, and RORγt expression levels were significantly lower than those in the model control and FICZ groups. Mass spectrometry results showed FICZ that in both DXM and QrLx groups was lower than control model and FICZ groups. Flow cytometry results showed that DXM, NaFTV and QrLx could significantly reduce Th17 proportion and increase Treg proportion in splenocytes, MLNCs, and LPMCs., Conclusions: NaFTV and QrLx treatment could decrease symptoms and inflammatory colitis, by decreasing of FICZ concentration and AhR signaling in colon, resulting in reducing the expression of IL-6, STAT3, and RORγt, whereas increasing the expression of FOXP3, consequently reducing the proportion of Th17 cells and increasing the proportion of Treg cells, respectively., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microplastic particles (MPs) from lipophilic polymers have been shown to efficiently accumulate hydrophobic organic contaminants (HOCs) in aquatic environments. MPs have, therefore, frequently been discussed as vectors for contaminants, enhancing HOC uptake by various organisms after ingestion followed by pollutant release; however, integrative models of sorption argue against this mechanism and even predict cleansing of pollutants from biological systems under particular circumstances. In order to experimentally investigate such a depuration mechanism, RTL-W1 cells were dosed with three 7-ethoxyresorufin-O-deethylase (EROD) inducers of distinct lipophilicity via the medium before adding both native and hexane-purified polyethylene MPs (20-25 μm) to the medium surface. EROD activity was significantly reduced in the presence of MP, the extent of which correlated with the inducers' lipophilicity (K OW ) and thus affinity to MP. For hexane-purged MPs and TCDD (K OW  = 6.8), MPs reduce the bioavailability by up to 79%; the effect was marginally weaker with benzo[k]fluoranthene (K OW  = 6.11) and almost absent with β-Naphthoflavone (K OW  = 4.68). Compared to hexane-purged MPs, native particles possessed slightly less detoxification potential. These experimental results corroborate theoretically predicted mechanisms of detoxification via MPs. Yet, it is unclear if, under corresponding conditions in the environment, MPs can compete with organismal tissues for highly lipophilic compounds and, if so, to which degree they may act as a sink reducing the amount of bioavailable pollutants in situ. However, the present results suggest that in scenarios where pollutant-free MPs interact with organisms that accumulated HOCs via other routes of uptake, qualitatively the presence of such a mechanism is likely., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cytochrome P450 (CYP) gene superfamily catalyzes oxidative metabolism of a wide variety of drugs, carcinogens, and endogenous biomolecules in the liver and intestinal organs. In vitro assay platforms such as primary hepatocyte and immortalized liver-derived cell lines have been developed to evaluate drug effects. However, several limitations have been suggested regarding discrepancies between in vitro and in vivo assays. In this study, we aimed to investigate drug metabolism and toxicity based on mouse small intestinal and liver organoids derived from resident stem cells. At first, expressions and activities of CYP subfamilies (CYPs) in intestinal and liver organoids were investigated. Organoids treated with three CYPs-inducers dexamethasone (Dex), β-naphthoflavone (BNF), and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) were evaluated for CYPs activities. The CYPs-induced intestinal and liver organoids were confirmed to digest more docetaxel, as colon cancer cell-line survived more in CYPs-induced organoid's medium than in non-induced organoid's medium. Then, the activity of docetaxel in a co-culture platform of mouse liver organoids and human pancreatic tumoroids was measured. We obtained significant statistical values on CYPs-induced metabolic activities: cell survival rates of pancreatic tumoroids co-cultured with docetaxel-treated undifferentiated, differentiated, and CYPs-induced differentiated organoids were 66.05 ± 2.14%, 89.20 ± 2.67%, and 101.90 ± 0.94%, respectively. To sum up, gene expression modification and drug metabolism evaluation were able to be done with organoids as done with tissues. In vivo-like in vitro investigation on drug toxicity may potentially be done with organoids as a stepping bridge to the clinical trial., (Copyright © 2019. Published by Elsevier Inc.)

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with β-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and β-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M , while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

Many widespread and persistent organic pollutants, for example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and some polychlorinated biphenyls, activate the aryl hydrocarbon receptor (AhR) causing it to translocate to the cell nucleus where it transactivates target genes, increasing expression of a number of xenobiotic metabolizing enzymes as well as some transporters. AhR's ability to target transporters within the kidney is essentially unexplored. We show here that exposing isolated killifish (Fundulus heteroclitus) renal proximal tubules to micromolar β-naphthoflavone (BNF) or nanomolar TCDD roughly doubled the transport activity of Multidrug resistance-associated proteins Mrp2 and Mrp4, P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp), all ATP-driven xenobiotic efflux pumps and critical determinants of renal xenobiotic excretion. These effects were abolished by actinomycin D and cycloheximide and by the AhR antagonist, α-naphthoflavone, indicating that increased transport activity was dependent on transcription and translation as well as ligand binding to AhR. Quantitative immunostaining of renal tubules exposed to BNF and TCDD showed increased luminal membrane expression of Mrp2, Mrp4, P-gp and Bcrp. Thus, in these renal tubules, the four ABC transporters are targets of AhR action.

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as β-naphthoflavone (BNF), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding the ligand, AhR triggers induction of the expression of phase I and phase II drug-metabolizing genes, together with numerous other genes that are not directly involved in the metabolism of xenobiotics. Several studies have shown that AhR plays a role in tumor initiation, promotion and progression, but the molecular mechanisms involved in these processes are not fully understood. A previous study from our laboratory indicated that the SERPINB2 gene is presumably regulated by AhR. To prove that such induction is really AhR-dependent, in the present study we knocked down the expression of AhR by stable transfection of a laryngeal squamous cell carcinoma cell line (UT-SCC-34) with shRNA, resulting in 92% reduction of BNF-induced expression of SERPINB2. However, in silico analysis did not reveal AhR-dependent responsive elements in the promoter of the SERPINB2 gene. Therefore, to address this problem, we have used cycloheximide, an inhibitor of translation, and our results clearly indicate that an additional, newly synthesized protein is involved in AhR-dependent induction of SERPINB2 expression by BNF. So, to exclude that AhR binds to the putative xenobiotic-responsive elements (XREs) localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays. As expected, we found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene, further demonstrating the indirect SERPINB2 induction by AhR. However, the further analysis demonstrated that the expression of the enhancer RNA encoded by the region of DNA 20 kbp upstream from the SERPINB2 gene was AhR-dependent. Although AhR-mediated SERPINB2 induction clearly requires the synthesis of an additional protein, the kinetics of SERPINB2 induction is as fast as the kinetics of CYP1A1 and CYP1B1 induction (both genes directly regulated by AhR). Therefore, given previous studies regarding the induction of SERPINB2 expression by bacterial lipopolysaccharides (LPS), we think that, similarly, the interaction with pause-release proteins may be responsible for AhR-dependent regulation of SERPINB2 expression., (Copyright © 2019. Published by Elsevier B.V.)

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 β-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.

Previous studies have shown that several aryl hydrocarbon receptor (AHR) agonists, including β-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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

A high incidence of positive results is obtained with in vitro genotoxicity tests, which do not correlate with the in vivo negative results in many cases. To address this issue, the metabolic profile of rat liver 9000 × g supernatant fraction (S9) pretreated with phenobarbital (PB) and 5,6-benzoflavone (BNF) was characterized. Furthermore, the in vitro micronucleus tests of 10 compounds were performed with PB-BNF-induced rat S9. PB-BNF increased cytochrome P450 (CYP) activity and CYP1A1, CYP1A2, CYP2B1/2, CYP2C6, CYP3A1, and CYP3A2 expression in rat S9, whereas it decreased CYP2C11 and CYP2E1 expression. PB-BNF-induced S9 enhanced the micronucleus induction (MI) of benzo[a]pyrene (BaP), cyclophosphamide (CPA), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), which are metabolized by CYP1A1, CYP2C6, and CYP1A2, respectively. In contrast, coumarin and chlorpheniramine showed MI with PB-BNF-induced S9 despite the fact that they show negative results in the in vivo studies. Furthermore, diclofenac, piroxicam, lansoprazole, and caffeine showed MI regardless of the enzyme induction by PB-BNF, whereas phenacetin did not show MI. These results indicate that PB-BNF-induced rat S9 is effective in detecting the genotoxic potential of promutagens, such as BaP, CPA, and PhIP, but not of coumarin and chlorpheniramine, probably due to the differences in the in vitro and in vivo metabolic profile and its exposure levels of the drugs.

Cytochrome P450 (CYPs) enzymes are critical for the metabolism of exogenous and endogenous compounds. In mammals, the CYP3s are arguably the most important xenobiotic metabolizing enzymes and are all contained within the CYP3A subfamily. In fish, CYP3s include CYP3A and multiple subfamilies unique to the teleost lineage. The goal of this study was to provide insight on the regulation of genes in the CYP3C subfamily. Zebrafish, which have 4 CYP3C genes, were exposed to 17β-estradiol (E 2 ; 0.001-10 μM) or β-naphthoflavone (βNF; 0.005-1 μM), prototypical ligands of the estrogen receptor (ER) and the aryl hydrocarbon receptor (AhR), respectively. Gene expression was measured in the liver, intestine and gonads using quantitative PCR. CYP1A and vitellogenin (VTG) gene expression were used as positive controls for AhR and ER regulation, respectively. Exposure to βNF resulted in the dose-dependant induction of CYP1A and CYP3C genes in the female intestine but not in the liver. E 2 exposure resulted in the induction of all CYP3Cs in the male intestine and in the female liver. VTG was induced in both female and male livers. CYP3C3 and CYP3C4 were induced in the testis; CYP3C1 and CYP3C4 were slightly induced in the ovary. The time-course of gene induction was investigated in the liver and intestine after exposure to βNF (0.5 μM) and E 2 (0.1 μM). Inducible genes were up-regulated within 12 h after exposure. These data support a role for the AhR and ER in the regulation of CYP3Cs. Overall, the induction of CYP3Cs by AhR and ER ligands is different from mammalian CYP3A and may suggest a functional role for CYP3Cs that involves planar aromatic hydrocarbons and steroids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytochrome P450 (CYP) isozymes vary their expression depending on the brain area, the cell type, and the presence of drugs. Some isoforms are involved in detoxification and/or toxic activation of xenobiotics in central nervous system. However, their role in brain metabolism and neurodegeneration is still a subject of debate. We have studied the inducibility of CYP isozymes in human neuroblastoma SH-SY5Y cells, treated with β-naphtoflavone (β-NF) or ethanol (EtOH) as inducers, by qRT-PCR, Western blot (WB), and metabolic activity assays. Immunohistochemistry was used to localize the isoforms in mitochondria and/or endoplasmic reticulum (ER). Tetrazolium (MTT) assay was performed to study the role of CYPs during methylphenyl pyridine (MPP⁺) exposure. EtOH increased mRNA and protein levels of CYP2D6 by 73% and 60% respectively. Both β-NF and EtOH increased CYP2E1 mRNA (4- and 1.4-fold, respectively) and protein levels (64% both). The 7-ethoxycoumarin O -deethylation and dextromethorphan O -demethylation was greater in treatment samples than in controls. Furthermore, both treatments increased by 22% and 18%, respectively, the cell viability in MPP⁺-treated cells. Finally, CYP2D6 localized at mitochondria and ER. These data indicate that CYP is inducible in SH-SY5Y cells and underline this in vitro system for studying the role of CYPs in neurodegeneration.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6β-, 12β-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6β-, 9α-, 14α-hydroxy and 6β,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6β,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or β-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The smallest product of the Duchenne muscular dystrophy gene, dystrophin (Dp)71, is ubiquitously expressed in nonmuscle tissues. We previously showed that Dp71 expression in hepatic cells is modulated in part by stimulating factor 1 (Sp1), stimulating protein 3 (Sp3), and yin yang 1 (YY1) transcription factors, and that the polyaromatic hydrocarbon, β-naphthoflavone (β‑NF), downregulates Dp71 expression. The aim of the present study was to determine whether β‑NF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcription‑quantitative polymerase chain reaction was used to measure half‑life mRNA levels in β‑NF‑treated cells exposed to actinomycin D, an inhibitor of transcription, for 0, 4, 8, 12 and 16 h. Transient transfections with a plasmid carrying the Dp71 basal promoter fused to luciferase reporter gene were carried out in control and β‑NF‑treated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and β‑NF‑treated cells. To the best of our knowledge, the results demonstrated for the first time that this negative regulation takes place at the promoter level rather than the mRNA stability level. Interestingly, using EMSAs, β‑NF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that β‑NF may modulate the expression of other genes regulated by these transcription factors. In conclusion, β‑NF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor involved in the metabolism of physiological substances and xenobiotics, representing an interesting target in both toxicology and pharmacology. In this study, we investigated the ligand-dependent conjunction of nuclear import of the human AHR in living cells and target gene induction. Our findings strengthen the theory that the AHR triggers a precisely defined and rapid reaction upon binding to endogenous ligands, while the xenobiotic β-naphthoflavone only induces rather unspecific and slow effects. To better illuminate the ligand-mediated responses of the human AHR, we applied site-directed mutagenesis and identified histidine 291 as key residue for AHR functionality, essential for both nuclear import and target gene induction. Contrary, replacing histidine at position 291 by alanine did not affect nucleo-cytoplasmic shuttling, showing that permanent endogenous import and ligand-induced import of the AHR into the nucleus are two independent and differently regulated processes. Combining these observations with our structural investigations using a homology model of the AHR-PAS B domain, we suggest a dual role of histidine 291: (1) a major role for shaping the ligand binding site including direct interactions with ligands and, (2) an essential role for the conformational dynamics of a PAS B loop, which most likely influences the association of the AHR with the AHR nuclear translocator through interference with their protein-protein interface.

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 β-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., Competing Interests: The authors declare no conflict of interest.

Objective: Drug therapy is occasionally accompanied by an idiosyncratic severe toxicity, which occurs very rarely, but can lead to patient mortality. Methazolamide, an anti-glaucomatous agent, could cause severe skin eruptions called Stevens-Johnson syndrome/toxic epidermal necrolyis (SJS/TEN). Its precise etiology is still uncertain. In this study, the metabolism of methazolamide was investigated in immortalized human keratinocytes to reveal the possible mechanism which causes SJS/TEN., Methods: The metabolism of methazolamide was studied using immortalized human keratinocytes, HaCaT cells. HPLC was used to isolate a metabolite from the culture medium. Mass spectrometry (LCMS/ MS) was employed for its characterization. Three typical chemical inducers were assessed for the inducibility of cytochrome P450, and methimazole was used as the inhibitor of flavin-containing monooxygenase (FMO)., Results: A sulfonic acid, N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) was identified as the final metabolite. Dexamethasone and β-naphthoflavone behaved as an inducer of cytochrome P450 in the metabolism, but isoniazid did not. The effect of methimazole was not consistent. We did not detect any glucuronide nor any mercapturic acid (N-acetylcysteine conjugate)., Conclusion: N-[3-methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO) is not considered to be a direct product of an enzymatic reaction, but rather an auto-oxidation product of N-[3-methyl-5- sulfe-1,3,4-thiadiazol-2(3H)-ylidene]acetamide, a chemically unstable sulfenic acid, which is produced by cytochrome P450 from the β-lyase product of cysteine conjugate of methazolamide. MSO is considered to be susceptible to glutathione and to return to glutathione conjugate of methazolamide, forming a futile cycle. A hypothetical scenario is presented as to the onset of the disease., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of β-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., Competing Interests: The authors declare that there are no conflicts of interest.

Aim: To examine the effect of synthetic muscone on the expression of CYP1A2 and CYP3A4 enzymes in human liver L02 cells and in the liver tissue of Kunming mice., Methods: The L02 hepatic cell line was used to study the effect of low (10-4 μmol/L), middle (10-3 μmol/L), and high concentrations (10-2 μmol/L) of muscone on the expression of CYP1A2 and CYP3A4 enzymes. In addition, the cytochrome P450 (CYP) expression was investigated in Kunming mice after the administration of 10 mg/kg (low), 50 mg/kg (middle), and 100 mg/kg (high) dose of muscone for 6 days. A mixture of phenobarbital (30 mg/kg) and β-napthoflavone (80 mg/kg) was used as positive control and the effects of the compounds on CYP expression were investigated at the end of 6- and 12-day periods., Results: Muscone induced the expression of CYP1A2 (middle and low concentrations) and of CYP3A4 (high concentration) enzymes in L02 cells. In vivo, administration of muscone in Kunming mice revealed significant weight reduction at the end of 6- and 12-day periods (middle and high doses, respectively), compared to the control group (p < 0.05). Liver toxicity scores indicated that the liver injuries in the positive control and high doses of muscone group were significantly higher in the 6- and 12-day periods, compared to those in the blank control group (p < 0.05). Furthermore, muscone induced CYP1A2 and CYP3A11 expressions in Kunming mice at the middle dose and all doses during the 12-day period as demonstrated by immunoblotting experiments. A low dose of mucone induced the CYP enzyme expression more rapidly, whereas a high dose of muscone caused the longest inductive effect. The results were confirmed by immunohistochemistry experiments and real-time PCR studies, where similar patterns of muscone-mediated inductive effects were noted., Conclusions: Muscone induces CYP1A2 and CYP3A4 expression in liver cells in vitro and in vivo. In addition, it exhibits liver toxicity in Kunming mice at concentrations higher than 50 mg/kg. The CYP-inductive effect that is caused by muscone encompasses a 6- to 12-day period of activity after drug administration as demonstrated by follow-up in vivo studies., (© 2017 S. Karger AG, Basel.)

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of dioxins, but also plays important physiological roles, which are only beginning to unfold. Previous studies have surprisingly unveiled that low doses of the potent AHR agonist TCDD induce a strong and persistent avoidance of novel food items in rats. Here, we further examined the involvement of the AHR in the avoidance response in Sprague-Dawley rats with three established AHR agonists: 6-formylindolo(3,2-b)carbazole (FICZ), β-naphthoflavone (BNF) and benzo[a]pyrene (BaP); with a novel selective AHR modulator (C2); and with an activator of another nuclear receptor, CAR: 2,4,6-tryphenyldioxane-1,3 (TPD). As sensitive indices of AHR or CAR activity, we used Cyp1a1 and Cyp2b1 gene expression, as they are, respectively, the drug-metabolizing enzymes specifically regulated by them. We further attempted to address the roles played by enhanced neophobia and conditioned taste aversion (CTA) in the avoidance behaviour. All AHR agonists triggered practically total avoidance of novel chocolate, but the durations varied. Likewise, acutely subtoxic doses of C2, differing by 25-fold, all elicited a similar outcome. In contrast, TPD did not influence chocolate consumption at all. If rats were initially accustomed to chocolate for 6h after single FICZ or BNF exposure, avoidance was still clearly present two weeks later when chocolate was offered again. Hence, the avoidance response appears to specifically involve the AHR instead of being triggered by induction of intestinal or hepatic nuclear receptor signalling in general. It is also shared by both endogenous and exogenous AHR activators. Moreover, this behavioural change in rats seems to contain elements of both CTA and enhanced neophobia, but further clarification of this is still required., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In this study with crucian carp (Carassius auratus gibelio), the effect on enrofloxacin (EF) and its metabolite ciprofloxacin (CF) and on the activity of cytochrome P450 1A (CYP1A) and cytochrome P450 3A (CYP3A) was estimated following the oral administration of rifampicin (RIF) (12mg/kg) and β-naphthoflavone (BNF) (12mg/kg), respectively. First, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect the pharmacokinetics of EF with continual blood sampling. In RIF-treated, BNF-treated and control groups, the value of the CmaxCF/CmaxEF ratio was 4.41, 0.81 and 0.95, and the corresponding value of the AUC0-t-CF/AUC0-t-EF ratio was 3.69, 1.84 and 1.76, respectively. In the RIF-treated, BNF-treated and control groups, the MRT values of EF were 26.57, 27.45 and 30.88h, and the corresponding values for CF were 5.79, 35.18 and 38.11h, respectively. Based on these results for crucian carp, the accumulation and elimination of EF and CF in the RIF-treated group were more rapid than in BNF-treated and control groups. Second, liver microsomes were pretreated with the inducer of CYP1A for BNF and that of CYP3A for RIF, and then the enzymatic activities of CYP1A and CYP3A were measured, respectively. The activities of ethoxyresorufin-O-deethylation (EROD) and erythromycin-N-demethylation (ERND) increased significantly (P<0.05) for CYP1A and CYP3A, respectively. However, in further experiments on the formation of CF, the level of EF N-deethylation was significantly induced by RIF and inhibited by ketoconazole (KTZ) for CYP3A but had no influence for CYP1A, BNF and berberine chloride (BER). We concluded that CYP3A might be responsible for the N-deethylation of EF and because of this activity, could also serve as a toxicity biomarker in crucian carp., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: The effects of miroestrol (MR), an active phytoestrogen from Pueraria candollei var. mirifica, on expression of cancer-related genes were determined., Methods: Seven-week-old female ICR mice (n = 5 each) were subcutaneously administered estradiol (E2, 0.5 mg/kg/day) or MR (0.5 or 5 mg/kg/day) daily for 7 days. Some were given ER or MR in combination with β-naphthoflavone (BNF, 30 mg/kg/day) for the last 3 days. The expression of cancer-related genes including cytochrome P450 1A (Cyp1a), cytochrome P450 1B1 (Cyp1b1), aromatase P450 (Cyp19), NAD(P)H: quinone oxidoreductase 1 (Nqo1) and catechol-O-methyltransferase (Comt) were evaluated., Key Findings: In the presence of BNF, MR suppressed hepatic CYP1A1 activity and CYP1A2 activity, expression of CYP1B1 mRNA and expression of CYP1A1/2 and CYP1B1 protein. E2, by contrast, did not. MR restored expression levels of hepatic NQO1 and uterine COMT in BNF-treated mice. Furthermore, MR increased expression of uterine CYP19 to the same extent as E2., Conclusion: MR may be superior to E2 as it downregulates expression of CYP1. Moreover, MR normalized expression of both NQO1 and COMT, the protective enzymes, in murine liver and uteri. These results support the use of MR as an alternative supplement for menopausal women, MR having the extra benefit of reducing cancer risk., (© 2016 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology.)

In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage.

Uridine 5'-diphosphate-glucuronosyltransferase (UGT) catalyzes a major phase II reaction in a drug-metabolizing enzyme system. Although the UGT1A subfamily is expressed mainly in the liver, it is also expressed in the brain. The purpose of the present study was to elucidate the effect of β-naphthoflavone (BNF), one of the major inducers of drug-metabolizing enzymes, on Ugt1a6 and Ugt1a7 mRNA expression and their glucuronidation in the rat brain. Eight-week-old male Sprague-Dawley rats were treated intraperitoneally with BNF (80 mg/kg), once daily for 7 d. Ugt1a6 and Ugt1a7 mRNA expression increased in the cerebellum and hippocampus (Ugt1a6: 2.1- and 2.3-fold, respectively; Ugt1a7: 1.7- and 2.8-fold, respectively); acetaminophen glucuronidation also increased in the same regions by 4.1- and 2.7-fold, respectively. BNF induced Ugt1a6 and Ugt1a7 mRNA expression and their glucuronidation, and the degree of induction differed among 9 regions. BNF also upregulated CYP1A1, CYP1A2, and CYP1B1 mRNAs in the rat brain. Since the aryl hydrocarbon receptor signaling pathway was activated by BNF, it is indicated that Ugt1a6 and Ugt1a7 were induced via AhR in the rat brain. This study clarified that Ugt1a6 and Ugt1a7 mRNA expression and their enzyme activities were altered by BNF, suggesting that these changes may lead to alteration in the pharmacokinetics of UGT substrate in rat brain.

The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of cellular oxidative stress. Thus, assays that detect this pathway can be useful for identifying chemicals that induce or inhibit oxidative stress signaling. The focus of this chapter is to describe a cell-based ARE assay in a quantitative high-throughput screening (qHTS) format to test a large collection of compounds that induce nuclear factor erythroid 2-related factor (Nrf2)/ARE signaling. The assay is described through cell handling, assay preparation, and instrument usage.

Recurrent spontaneous abortion (RSA) is a common complication of pregnancy. Recent studies have demonstrated that the aryl hydrocarbon receptor (AhR) might play important roles in establishing and maintaining early pregnancy. In this study, we found that placental AhR protein levels were significantly lower and placental CYP1A1 mRNA levels were higher in unexplained RSA (URSA) patients than in control subjects. The results of immunohistochemical analyzes showed that placental AhR was expressed in syncytiotrophoblast cells and that the level of AhR was markedly lower in these cells in URSA subjects than in control subjects. β-Naphthoflavone (β-NF, an AhR ligand) at 5μM significantly inhibited proliferation and migration in HTR-8/SVneo cells and was associated with the activation of AhR. Moreover, overexpressing AhR in JAR cells significantly increased CYP1A1 mRNA levels and inhibited cell migration. These results indicate that AhR is highly activated in URSA placentas and that the activation of AhR in the placenta might impair trophoblast cell proliferation and migration, possibly leading to the occurrence of URSA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

β-Naphthoflavone (β-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 β-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with β-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, β-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 β-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 β-NF treatment. Our findings show that β-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 β-NF might prevent TNF-α-induced inflammation., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ubiquitin-proteasome system (UPS) is a specific, non-lysosomal pathway responsible for the controlled degradation of abnormal and short-half-life proteins. Despite its relevance in cell homeostasis, information regarding control of the UPS component gene expression is lacking. Data from a recent study suggest that the aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor, might control the expression of several genes encoding for UPS proteins. Here, we showed that activation of AHR by TCDD and β-naphthoflavone (β-NF) results in Ubcm4 gene induction accompanied by an increase in protein levels. UbcM4 is an ubiquitin-conjugating enzyme or E2 protein that in association with ubiquitin ligase enzymes or E3 ligases promotes the ubiquitination and 26S proteasome-mediated degradation of different proteins, including p53, c-Myc, and c-Fos. We also present data demonstrating increased c-Fos ubiquitination and proteasomal degradation through the AHR-mediated induction of UbcM4 expression. The present study shows that AHR modulates the degradation of proteins involved in cell cycle control, consistent with previous reports demonstrating an essential role of the AHR in cell cycle regulation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.

Aryl hydrocarbon receptor (AhR) agonists are known to cause lethal cardiovascular deformities in fish after developmental exposure. Acute adult fish toxicity of AhR agonists is thought to be minimal, but limited evidence suggests sublethal effects may also involve the cardiac system in fish. In the present study, adult zebrafish (Danio rerio) were aqueously exposed to solvent control or three nominal concentrations of the commonly used model AhR agonist, β-naphthoflavone (BNF), for 48 h. Following exposure, fish were subjected to echocardiography to determine cardiac function or swimming tests with concurrent oxygen consumption measurement. Critical swimming speed and standard metabolic rate were not significantly changed, while active metabolic rate decreased with increasing BNF exposure, reaching statistical significance at the highest BNF exposure. Factorial aerobic scope was the most sensitive end-point and was decreased at even lower BNF concentrations, indicating a reduced aerobic capacity after acute AhR agonist exposure in adult fish. The highest BNF concentration caused a significant decrease in cardiac output, while increasing the ratio of atrial to ventricular heart rate (indicating atrioventricular conduction blockade). In conclusion, the effect of acute BNF exposure on zebrafish metabolic capacity and cardiac function is likely to be physiologically important given that fish have a critical need for adequate oxygen to fuel essential survival behaviors such as swimming, growth, and reproduction. Future studies should be directed at examining the effects of other polycyclic aromatic hydrocarbons on fish cardiorespiratory function to determine whether their effects and modes of action are similar to BNF.

Acyl glucuronides are reactive electrophilic metabolites implicated in the toxicity of carboxylic acid drugs. Valproyl 1-O-β-acyl glucuronide (VPA-G), which is a major metabolite of valproic acid (VPA), has been linked to the development of oxidative stress in VPA-treated rats. However, relatively little is known about the toxicity of in situ generated VPA-G and its contribution to VPA hepatotoxicity. Therefore, we investigated the effects of modulating the in situ formation of VPA-G on lactate dehydrogenase (LDH) release (a marker of necrosis), BODIPY 558/568 C12 accumulation (a marker of steatosis), and cellular glutathione (GSH) content in VPA-treated sandwich-cultured rat hepatocytes. VPA increased LDH release and BODIPY 558/568 C12 accumulation, whereas it had little or no effect on total GSH content. Among the various uridine 5'-diphospho-glucuronosyltransferase inducers evaluated, β-naphthoflavone produced the greatest increase in VPA-G formation. This was accompanied by an attenuation of the increase in BODIPY 558/568 C12 accumulation, but did not affect the change in LDH release or total GSH content in VPA-treated hepatocytes. Inhibition of in situ formation of VPA-G by borneol was not accompanied by substantive changes in the effects of VPA on any of the toxicity markers. In a comparative study, in situ generated diclofenac glucuronide was not toxic to rat hepatocytes, as assessed using the same chemical modulators, thereby demonstrating the utility of the sandwich-cultured rat hepatocyte model. Overall, in situ generated VPA-G was not toxic to sandwich-cultured rat hepatocytes, suggesting that VPA glucuronidation per se is not expected to be a contributing mechanism for VPA hepatotoxicity., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Extended residual persistence of the pesticide dichlorodiphenyltrichloroethane (DDT) raises concerns about its long-term neurotoxic effects. Little is known, however, about DDT toxicity during the early stages of neural development. This study demonstrated that DDT-induced apoptosis of mouse embryonic neuronal cells is a caspase-9-, caspase-3-, and GSK-3β-dependent process, which involves p,p'-DDT-specific impairment of classical ERs. It also provided evidence for DDT-isomer-nonspecific alterations of AhR- and GPR30-mediated intracellular signaling, including changes in the levels of the receptor and receptor-regulated mRNAs, and also changes in the protein levels of the receptors. DDT-induced stimulation of AhR-signaling and reduction of GPR30-signaling were verified using selective ligands and specific siRNAs. Co-localization of the receptors was demonstrated with confocal microscopy, and the presence of functional GPR30 was detected by electrophysiology. This study demonstrates that stimulation of AhR-signaling and impairment of GPR30-signaling play important roles in the propagation of DDT-induced apoptosis during the early stages of neural development., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Beta-naphthoflavone (BNF, DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However, the mechanism by which BNF exerts this antitumor effect remains unclear. Thus, we explored mechanisms of BNF's antitumor effects in human breast cancer cells. This study showed that BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21(Cip1/Waf1), leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells, but not in ER-negative MDA-MB-231 cells. In addition, BNF inhibited PI3K/AKT signaling, and the PI3K inhibitor, LY294,002, exhibited the same inhibitory effects on cyclinD1/D3, CDK4 and the cell cycle as BNF. Interestingly, BNF activated mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling, and more notably, MEK inhibitor PD98059 significantly blocked the BNF-induced cell cycle arrest and upregulation of p21(Cip1/Waf1). Furthermore, specific ERα and AhR siRNA studies indicate that ERα is required in BNF-induced p21(Cip1/Waf1) expression, and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR-dependent. Taken together, AhR-dependent inhibition of the PI3K/AKT pathway, activation of MAPK/ERK and modulation of ERα is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer, which may represent a promising strategy to be exploited in future clinical trials.

Uridine 5'-diphosphate (UDP)-glucuronosyltransferase 1A (UGT1A), which catalyzes major phase II reactions, is regulated by endogenous and exogenous factors via nuclear receptors such as the aryl hydrocarbon receptor (AhR). Glucocorticoid, one of the adrenocortical hormones, regulates AhR and UGT1A expression. We examined the effects of adrenalectomy on the expression and induction of UGT1A via AhR in the rat liver and small intestine. Rats were adrenalectomized bilaterally (ADX) or sham-operated (SHAM) and received intraperitoneal treatment with β-naphthoflavone (BNF) for 4 d. Hepatic UGT1A6 and UGT1A7 mRNA levels were altered by ADX (0.1-fold and 1.6-fold, respectively). BNF treatment increased UGT1A6 and UGT1A7 mRNA expression and the intrinsic clearance of acetaminophen (APAP) glucuronidation, which is primarily catalyzed by UGT1A6 and UGT1A7, in both SHAM and ADX rats. Therefore, ADX rats maintained a functional AhR signaling pathway in the presence of BNF, expressed UGT1A6 and UGT1A7 mRNA, and showed APAP glucuronidation, namely induction by BNF via AhR was not abolished. Our results indicate that adrenal-dependent factors such as glucocorticoids are partially involved in the basal regulation of UGT1A6 and UGT1A7 transcription.

Oxidative stress is involved in the progression of several diseases such as diabetes, hypertension, and age-related diseases. Miroestrol (MR) is a potent phytoestrogen from the tuberous root of Pueraria mirifica, a plant used in traditional Thai medicine that is claimed to have rejuvenating effects. In this study, the effects of MR on the antioxidation system, including anti-lipid peroxidation; on the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase; and on glutathione content in the livers and uteri of β-naphthoflavone (BNF)-treated mice were determined. BNF-treated mice are a model of procarcinogen-exposed mice. The results showed that MR improved the antioxidant activities of SOD and CAT in the livers and uteri of both normal and BNF-treated mice, while estradiol (E2) increased SOD activity in the uteri of normal mice and CAT activity in the livers of both normal and BNF-treated mice. In the liver, MR increased the levels of several forms of glutathione, whereas in the uteri E2 and MR reduced the level of lipid peroxidation by decreasing the level of malondialdehyde. Therefore, the use of MR as an alternative hormone replacement therapy might be beneficial due to its ability to improve antioxidation systems.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and a wide variety of structurally diverse ligands through its ability to translocate into the nucleus and bind to a specific DNA recognition site (the dioxin-responsive element [DRE]) adjacent to responsive genes. Although the sequence of the DRE is well defined, several reports suggested that the nucleotide specificity of AhR DNA binding may vary depending on the structure of its bound ligand. Given the potential toxicological significance of this hypothesis, an unbiased DNA-selection-and-PCR-amplification approach was utilized to directly determine whether binding and activation of the AhR by structurally diverse agonists alter its nucleotide specificity of DNA binding. Guinea pig hepatic cytosolic AhR activated in vitro by equipotent concentrations of TCDD, 3-methylcholanthrene, β-naphthoflavone, indirubin, L-kynurenine, or YH439 was incubated with a pool of DNA oligonucleotides containing a 15-base pair variable region consisting of all possible nucleotides. The AhR-bound oligonucleotides isolated by immunoprecipitation were PCR amplified and used in subsequent rounds of selection. Sequence analysis of a total of 196 isolated oligonucleotides revealed that each ligand-activated AhR:ARNT complex only bound to DRE-containing DNA oligonucleotides; no non-DRE-containing DNA oligonucleotides were identified. These results demonstrate that the binding and activation of the AhR by structurally diverse agonists do not appear to alter its nucleotide specificity of DNA binding and suggest that stimulation of gene expression mediated by direct DNA binding of ligand-activated AhR:ARNT complexes is DRE dependent.

Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or β-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and β-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP-DNA adducts detected by (32)P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP-DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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 β-naphthoflavone (βNF) 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 βNF 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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Psoriasis is a T-cell-mediated autoimmune disease involving the skin. Two cytokines, interleukin-12 (IL-12) and IL-23 have been shown to play a pivotal role in the pathogenesis of the disease. Ustekinumab (Stelara) is a therapeutic monoclonal antibody (mAb) targeted against the p40 shared subunit of IL-12 and IL-23. Recently the ability of therapeutic proteins (TP) including mAbs that target either cytokines directly (e.g., Pegasys; peginterferon α-2a) or their respective cell surface receptors [e.g., tocilizumab (Actemra); anti IL-6R] to desuppress cytochrome P450 (P450) enzymes in vitro and in the clinic, has been demonstrated. In the present study the ability of IL-12 and IL-23 to suppress multiple P450 enzymes was investigated in vitro using six separate lots of cultured human hepatocytes. Following exposure of 10 ng/ml IL-12 and IL-23 for 48 hours, either alone or in combination, no change in CYP2B6, 2C9, 2C19, or 3A4 gene expression or functional activity was observed. None of the untreated hepatocyte donors showed appreciable expression of the IL-12 or IL-23 receptors. Similar results were seen with whole human liver samples. Exposure of hepatocytes to IL-12 and/or IL-23, known P450 suppressors (IL-6 and tumor necrosis factor-α) or known P450 inducers (β-naphthoflavone, phenobarbital, and rifampicin) did not appreciably alter the expression of the IL-12 and IL-23 receptors either. Finally, in contrast to the positive control IL-6, expression of the acute phase C-reactive protein was unaltered following IL-12 and/or IL-23 treatment. Together, these data suggest a negligible propensity for IL-12 or IL-23 to directly alter P450 enzymes in human hepatocytes.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most of the toxic effects of environmental contaminants. Among the multiple pleiotropic responses elicited by AHR agonists, the antiestrogenic and endocrine-disrupting action of the receptor activation is one of the most studied. It has been demonstrated that some AHR agonists disrupt estradiol-induced vitellogenin synthesis in the fish liver via a mechanism that involves crosstalk between the AHR and the estrogen receptor (ER). Chicken hepatocytes have become a model for the study of AHR action in birds and the induction of the signal and its effect in these cells are well established. However, the impact of AHR activation on estradiol-regulated responses in the chicken liver remains to be demonstrated. The aim of the present study was, therefore, to determine the effect of AHR action on ER-driven transcription in a convenient model of chicken liver cells. For this purpose, we designed a reporter construct bearing the 5' regulatory region of the chicken vitellogenin II gene and used it to transfect chicken hepatoma LMH cells. We found that β-naphthoflavone represses ER-driven vitellogenin promoter activity and that this action is mediated by the AHR. This inhibitory crosstalk between both pathways appears to be unidirectional, since estradiol did not alter the transcript levels of an AHR target gene. Besides, and highly relevant, we show that LMH cell line transfected with a reporter construct bearing the chicken vitellogenin promoter sequence is a useful and convenient model for the study of AHR-ER interaction in chicken liver-derived cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Examination of three retinoid X receptor (RXR) agonists [Targretin (TRG), UAB30, and 4-methyl-UAB30 (4-Me-UAB30)] showed that all inhibited mammary cancer in rodents and two (TRG and 4-Me-UAB30) strikingly increased serum triglyceride levels. Agents were administered in diets to female Sprague-Dawley rats. Liver RNA was isolated and microarrayed on the Affymetrix GeneChip Rat Exon 1.0 ST array. Statistical tests identified genes that exhibited differential expression and fell into groups, or modules, with differential expression among agonists. Genes in specific modules were changed by one, two, or all three agonists. An interactome analysis assessed the effects on genes that heterodimerize with known nuclear receptors. For proliferator-activated receptor α/RXR-activated genes, the strongest response was TRG > 4-Me-UAB30 > UAB30. Many liver X receptor/RXR-related genes (e.g., Scd-1 and Srebf1, which are associated with increased triglycerides) were highly expressed in TRG and 4-Me-UAB30- but not UAB30-treated livers. Minimal expression changes were associated with retinoic acid receptor or vitamin D receptor heterodimers by any of the agonists. UAB30 unexpectedly and uniquely activated genes associated with the aryl hydrocarbon hydroxylase (Ah) receptor (Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1). Based on the Ah receptor activation, UAB30 was tested for its ability to prevent dimethylbenzanthracene (DMBA)-induced mammary cancers, presumably by inhibiting DMBA activation, and was highly effective. Gene expression changes were determined by reverse transcriptase-polymerase chain reaction in rat livers treated with Targretin for 2.3, 7, and 21 days. These showed similar gene expression changes at all three time points, arguing some steady-state effect. Different patterns of gene expression among the agonists provided insight into molecular differences and allowed one to predict certain physiologic consequences of agonist treatment.

In this study, the constitutive and inducible expression of the CYP genes (1A1, 1A2, 1B1, 2B22, 3A22, 3A29 and 3A46), related transcriptional factors (AhR, CAR, PXR, and Nrf2) and the antioxidant enzymes SOD, catalase, GSSH-reductase and GSH-peroxidase were investigated in the liver, heart regions and coronary arteries of control pigs and pigs treated with β-naphthoflavone (βNF) or with rifampicin (RIF). Real-time PCR experiments and enzymatic or immunoblot assays showed that CYP1A1 was predominantly enhanced by βNF in a similar manner in all the heart regions, whereas antioxidant enzyme activity was not affected. The rifampicin treatment resulted in an induction of CYP2B22 and CYP3As, at the transcriptional, activity and protein level in liver but not in heart nor in the coronary arteries, despite the expression of CAR and PXR in the cardiac tissues. These results obtained in vivo suggest that pig cardiac tissues may represent a useful model for humans., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαβ(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαβ(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.