Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors.

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to ß-naphthoflavone (ß-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to ß-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Beta-naphthoflavone and doxorubicin synergistically enhance apoptosis in human lung cancer cells by inducing doxorubicin accumulation, mitochondrial ROS generation, and JNK pathway signaling.

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.

The interference of alpha- and beta-naphthoflavone with triclosan effects on viability, apoptosis and reactive oxygen species production in mouse neocortical neurons.

Triclosan (TCS) is commonly used worldwide in a range of personal care and sanitizing products. A number of studies have revealed the presence of TCS in human tissues. It has recently been shown that TCS can interact with AhR in mouse neurons and the one of its effects is the stimulation of reactive oxygen species (ROS) production. Reactive oxygen species perform a wide spectrum of functions in neuronal cells, where they are generated as by-products of cellular metabolism. Therefore the aim of the study was to investigate effects of two synthetic naphthoflavones, the beta-naphthoflavone (ßNF) and alpha-naphthoflavone (αNF), well known agonist and antagonist of AhR on TCS-stimulated cytotoxicity, apoptosis and ROS production in mouse primary cortical neurons in vitro cultures. The results showed that both agonist (ßNF) and antagonist (αNF) of AhR enhanced the LDH release and caspase-3 activity stimulated by TCS. Interestingly, both naphthoflavones decreased the TCS-stimulated ROS production, however, they showed no scavenging properties as revealed by ABTS•+ and DPPH• methods. What's more, both ßNF as well as αNF inhibited the activity of xanthine oxidase (XO) stimulated by TCS. Thus, we can assume that αNF or ßNF act in a competitive way over TCS and inhibit its effect on antioxidant enzyme activity.

ß-Naphthoflavone treatment attenuates neonatal hyperoxic lung injury in wild type and Cyp1a2-knockout mice.

Exposure to supraphysiological concentrations of oxygen (hyperoxia) leads to bronchopulmonary dysplasia (BPD), one of the most common pulmonary morbidities in preterm neonates, which is more prevalent in males than females. Beta-naphthoflavone (BNF) is protective against hyperoxic lung injury in adult and neonatal wild type (WT) mice and in and mice lacking Cyp1a1gene. In this investigation, we tested the hypothesis that BNF treatment will attenuate neonatal hyperoxic lung injury in WT and Cyp1a2-/- mice, and elucidated the effect of sex-specific differences. Newborn WT or Cyp1a2-/- mice were treated with BNF (10mg/kg) or the vehicle corn oil (CO) i.p., from postnatal day (PND) 2 to 8 once every other day, while being maintained in room air or hyperoxia (85% O2) for 14days. Hyperoxia exposure lead to alveolar simplification and arrest in angiogenesis in WT as well as Cyp1a2-/- mice No significant differences were seen between WT and Cyp1a2-/- mice. Cyp1a2-/- female mice had better preservation of pulmonary angiogenesis at PND15 compared to similarly exposed males. BNF treatment attenuated lung injury and inflammation in both genotypes, and this was accompanied by a significant induction of hepatic and pulmonary CYP1A1 in WT but not in Cyp1a2-/- mice. BNF treatment increased NADPH quinone oxidoreductase (NQO1) mRNA levels in Cyp1a2-/- mouse livers compared to WT mice. These results suggest that BNF is protective in neonatal mice exposed to hyperoxia independent of CYP1A2 and this may entail the protective effect of phase II enzymes like NQO1.

A new insight into the aryl hydrocarbon receptor/cytochrome 450 signaling pathway in MG63, HOS, SAOS2, and U2OS cell lines.

Osteosarcoma is the most common malignant tumor of bone, with rapid progressive growth, early distant metastases, and frequent recurrence after surgical treatment. Osteosarcoma is characterized by changes in the ratio and expression of different cytochrome P450 (CYP) isoforms that can affect the effectiveness of anticancer therapies. The inducible expression of CYP1 genes depends on the ligand-dependent functionality of the aryl hydrocarbon receptor (AHR). In this study, we examined the AHR/CYP1 signaling pathway in four osteosarcoma cell lines (MG63, HOS, SAOS2, and U2OS) induced by the known AHR ligands: indirubin, indole-3-carbinol, and beta-naphthoflavone. Using qPCR and Western blot analysis, we explored the effects of these ligands on the expression of the CYP1 genes and studied the correlation between these responses and the changes in the mRNA and protein levels of AHR and the AHR nuclear translocator (ARNT) in these osteosarcoma cell lines. The results show that the AHR/CYP1 signaling pathway retains its function only in MG63 and HOS cells, and is impaired in SAOS2 and U2OS cells. Our data should be taken into account when recommending new strategies for the treatment of osteosarcoma and when evaluating new drugs against osteosarcoma in vitro.

Beta-naphthoflavone inhibits LPS-induced inflammation in BV-2 cells via AKT/Nrf-2/HO-1-NF-κB signaling axis.

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.

Activation of the Aryl Hydrocarbon Receptor (AHR) induces human glutathione S transferase alpha 1 (hGSTA1) expression.

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 (ß-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.

Aryl hydrocarbon receptor (AhR) agonist ß-naphthoflavone regulated gene networks in human primary trophoblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in placenta. AhR belongs to a class of transcriptional regulators that control many developmental and physiological events (e.g. xenobiotic metabolism). Our study describes AhR regulated transcriptional responses in human primary trophoblast by using the AhR agonist, ß-naphthoflavone (BNF). Human primary trophoblast cells were isolated from full term placenta after delivery. The trophoblasts were exposed to 25 µM of AhR agonist, BNF, for 72 hours. Gene expression profiling was conducted with Illumina HT-12 expression beadchips. Expression of selected genes was confirmed with RT-qPCR. Ingenuity pathway analysis (IPA) was used to predict functional pathways and upstream regulators of differentially expressed genes in order to identify regulatory networks associated with AhR. In response to BNF exposure, 64 genes were upregulated, and 257 genes were downregulated compared to control trophoblasts (±1.5-fold, p < 0.05). BNF regulated genes included placental hormones and genes implicated in immune- and inflammatory responses in addition to their well-known effects on xenobiotic metabolism, oxidative stress, antioxidant defense. In conclusion, these results show that BNF has wide-ranging effects on placental gene expression beyond xenobiotic metabolism e.g. disruption of inflammatory processes and hormones in the placenta.

Nobiletin, sinensetin, and tangeretin are the main perpetrators in clementines provoking food-drug interactions in vitro.

For clementine juice, previous data indicate a possible food-drug interaction with substrates of key enzymes responsible for drug metabolism (i.e. cytochrome P450 [CYP] 3A4, CYP1A2). However, which compounds in clementine juice are responsible for these effects are unknown. Therefore, we aimed to identify the compounds in clementine juice provoking metabolic enzyme inhibition or induction. The results demonstrated that the flavonoid fraction of clementine juice provoked induction of several genes and inhibition of both CYP3A4 and CYP1A2, matching effects observed with whole clementine juice. CYP1A2 inhibition and induction can most likely be attributed to nobiletin, sinensetin, and tangeretin. Tangeretin was the only compound causing CYP3A4 induction while CYP3A4 inhibition was most likely the result of additive or synergistic effects caused by several compounds. Thus, whenever evaluating the clinical relevance of clementine interactions, flavonoid contents should be reported because these might explain differences between cultivars and harvests.

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

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

Identification and molecular characterization of cytochrome P450 (CYP450) family genes in the marine ciliate Euplotes crassus: The effect of benzo[a]pyrene and beta-naphthoflavone.

Marine ciliate Euplotes crassus, a single-cell eukaryote, and has been considered as a model organism for monitoring of environmental pollutions in sediments. Cytochrome P450 (CYP450) monooxygenase are phase I enzyme involved in detoxification of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). However, little information on CYP450 family genes in ciliate is available. In the present study, acute toxicity of PAH, benzo[a]pyrene (B[a]P) and PAH-like model compound, beta-naphthoflavone (ß-NF), was investigated; full-length cDNA sequences and genomic structure of five CYP450 genes (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) were analyzed; and finally their activities and transcriptional changes were measured after exposure to PAHs for 48h. According to the results, B[a]P exposure showed a negative effect on E. crassus survival, whereas ß-NF exposure showed no significant effect. The 8h-LC50 value of B[a]P was determined to be 2.449µM (95%-C.L., 7.726-3.619µM). Five genes belonging to the CYP450 family had conserved domains and clustered with those of ciliate group, as revealed in phylogenetic analysis. CYP activity did not change after exposure to B[a]P, whereas it was slightly, but significantly, induced after exposure to ß-NF. The mRNA expression of five CYP450 genes was significantly modulated in a concentration- and time-dependent manner after exposure to both the chemicals. Our findings suggest that CYP450 genes in E. crassus may be involved in detoxification of B[a]P and ß-NF. This study would give a better understanding about the mode of action of B[a]P and ß-NF in marine ciliates at the molecular level.

Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (Acipenser transmontanus) to dioxin-like compounds.

Sturgeons are ancient fishes, which are endangered in many parts of the world. Due to their benthic nature and longevity, sturgeon are at great risk of exposure to bioaccumulative contaminants such as dioxin-like compounds (DLCs). Despite their endangered status, little research has been conducted to characterize the relative sensitivity of sturgeons to DLCs. Proper assessment of risk of DLCs posed to these fishes therefore, requires a better understanding of this sensitivity and the factors that are driving it. Adverse effects associated with exposure to DLCs are mediated by the aryl hydrocarbon receptor (AhR). This study identified and characterized two distinct AhRs, AhR1 and AhR2, in white sturgeon (Acipenser transmontanus) for the first time as a first step in studying the relative sensitivities of sturgeons to DLCs. Furthermore, tissue-specific expression of both AhRs under basal conditions and in response to exposure to the model DLC, ß-naphthoflavone (ßNF), was determined. The sequence of amino acids of AhR1 of white sturgeon had greater similarity to AhRs of tetrapods, including amphibians, birds, and mammals, than to AhR1s of other fishes. The sequence of amino acids in the ligand binding domain of the AhR1 had greater than 80% similarity to AhRs known to bind DLCs and was less similar to AhRs not known to bind DLCs. AhR2 of white sturgeon had greatest similarity to AhR2 of other fishes. Profiles of expression of AhR1 and AhR2 in white sturgeon were distinct from those known in other fishes and appear more similar to profiles observed in birds. Expressions of both AhR1 and AhR2 of white sturgeon were greatest in liver and heart, which are target organs for DLCs. Furthermore, abundances of transcripts of AhR1 and AhR2 in all tissues from white sturgeon were greater than controls (up to 35-fold) following exposure to ßNF. Based upon both AhRs having similar abundances of transcript in target organs of DLC toxicity, both AhRs being up-regulated following exposure to ßNF, and both AhRs having greatest similarity to AhRs known to bind DLCs, it is hypothesized that both AhR1 and AhR2 of white sturgeon might mediate effects of DLCs in this species. Since current risk assessments are based on data derived largely from highly divergent fishes within the Salmonidae, presence of two functional AhRs in white sturgeon, one of which has greatest similarity to AhRs of birds, might have significant implications for the sensitivity of sturgeons to DLCs compared to other fishes.

The emerging roles of AhR in physiology and immunity.

The aryl hydrocarbon receptor (AhR) is traditionally defined as a transcriptional regulator involved in adaptive xenobiotic response, however, emerging evidence supports physiological functions of AhR in normal cell development and immune response. The role of AhR in immunomodulation is multi-dimensional. On the one hand, activation of AhR by TCDD and other ligands leads to profound immunosuppression, potentially via skewed Th1/Th2 cell balance toward Th1 dominance, and boosted Treg cell differentiation. On the other hand, activation of AhR can also induce Th17 cell polarization and increase the severity of autoimmune disease. In addition to T lymphocytes, the AhR also appears to play a vital role in B cell maturation, and regulates the activity of macrophages, dendritic cells and neutrophils following lipopolysaccharide challenge or influenza virus infection. In these scenarios, activation of AhR is associated with decreased host response and reduced survival. Furthermore, gene knock out studies suggest that AhR is indispensable for the postnatal maintenance of intestinal intraepithelial lymphocytes and skin-resident dendritic epidermal gamma delta T cells, providing a potential link between AhR and gut immunity and wound healing. It is well accepted that the magnitude and the type of immune response is dependent on the local cytokine milieu and the AhR appears to be one of the key factors involved in the fine turning of this cytokine balance.