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.

Distinct effects of form selective cytochrome P450 inhibitors on cytochrome P450-mediated monooxygenase and hydrogen peroxide generating NADPH oxidase.

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H2O2, either directly or indirectly via superoxide anion, a reaction referred to as "NADPH oxidase" activity. H2O2 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 ß-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 H2O2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.

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

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

Coupling high-performance thin-layer chromatography with a battery of cell-based assays reveals bioactive components in wastewater and landfill leachates.

Over the last two decades, effect-directed analysis (EDA) gained importance as a seminal screening tool for tracking biological effects of environmental organic micro-pollutants (MPs). As EDA using high-performance liquid chromatography and bioassays is costly and time consuming, recent implementations of this approach have combined high-performance thin-layer chromatography (HPTLC) with effect-based methods (EBMs) using cell-based bioassays, enabling the detection of estrogenic, androgenic, genotoxic, photosystem II (PSII)- inhibiting, and dioxin-like sample components on a HPTLC plate. In the present study, the developed methodologies were applied as a HPTLC-based bioassay battery, to investigate toxicant elimination efficiency of wastewater treatment plants (WWTPs), and to characterize the toxic potential of landfill leachates. Activity levels detected in untreated landfill leachates, expressed as reference compound equivalence (EQ) concentration, were up to 16.8 µg ß-naphthoflavone-EQ L-1 (indicating the degree of dioxin-like activity), 1.9 µg estradiol-EQ L-1 (estrogenicity) and 8.3 µg diuron-EQ L­1 (PSII-inhibition), dropping to maximal concentrations of 47 ng ß-naphthoflavone-EQ L-1, 0.7 µg estradiol-EQ L-1 and 53.1 ng diuron-EQ L-1 following treatment. Bisphenol A (BPA) is suggested to be the main contributor to estrogenic activity, with concentrations determined by the planar yeast estrogen screen corresponding well to results from chemical analysis. In the investigated WWTP samples, a decrease of estrogenic activity of 6-100% was observed following treatment for most of the active fractions, except of a 20% increase in one fraction (Rf = 0.568). In contrast, androgenicity with concentrations up to 640 ng dihydrotestosterone-EQ L-1 was completely removed by treatment. Interestingly, genotoxic activity increased over the WWTP processes, releasing genotoxic fractions into receiving waters. We propose this combined HPTLC and EBM battery to contribute to an efficient, cheap, fast and robust screening of environmental samples; such an assay panel would allow to gain an estimate of potential biological effects for prioritization prior to substance identification, and its routine application will support an inexpensive identification of the toxicity drivers as a first tier in an EDA strategy.

Serotonin Modulates AhR Activation by Interfering with CYP1A1-Mediated Clearance of AhR Ligands.

BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). METHODS: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. RESULTS: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of ß-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. CONCLUSION: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.

New Conceptual Toxicokinetic Model to Assess Synergistic Mixture Effects between the Aromatic Hydrocarbon ß-Naphthoflavone and the Azole Nocodazole on the CYP1A Biomarker in a Fish Cell Line.

Toxicokinetic interactions with catabolic cytochrome P450 (CYP) enzymes can inhibit chemical elimination pathways and cause synergistic mixture effects. We have created a mathematical bottom-up model for a synergistic mixture effect where we fit a multidimensional function to a given data set using an auxiliary nonadditive approach. The toxicokinetic model is based on the data from a previous study on a fish cell line, where the CYP1A enzyme activity was measured over time after exposure to various combinations of the aromatic hydrocarbon ß-naphthoflavone and the azole nocodazole. To describe the toxicokinetic mechanism in this pathway and how that affects the CYP1A biomarker, the model uses ordinary differential equations. Local sensitivity and identifiability analyses revealed that all the 10 parameters estimated in the model were identified uniquely while fitting the model to the data for measuring the CYP1A enzyme activity. The model has a good prediction power and is a promising tool to test the synergistic toxicokinetic interactions between different chemicals.

Microplastic particles reduce EROD-induction specifically by highly lipophilic compounds in RTL-W1 cells.

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 (KOW) and thus affinity to MP. For hexane-purged MPs and TCDD (KOW = 6.8), MPs reduce the bioavailability by up to 79%; the effect was marginally weaker with benzo[k]fluoranthene (KOW = 6.11) and almost absent with ß-Naphthoflavone (KOW = 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.

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 and Ethanol Reverse Mitochondrial Dysfunction in A Parkinsonian Model of Neurodegeneration.

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 ß-naphthoflavone (ßNF) and ethanol (EtOH) before and during exposure to the parkinsonian neurotoxin, MPP+. The toxic effect of MPP+ in cell viability was rescued with both ßNF 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.

Aryl hydrocarbon receptor ligands increase ABC transporter activity and protein expression in killifish (Fundulus heteroclitus) renal proximal tubules.

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 ß-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.

Development of organoid-based drug metabolism model.

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), ß-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 ±â€¯2.14%, 89.20 ±â€¯2.67%, and 101.90 ±â€¯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.

CYP3C gene regulation by the aryl hydrocarbon and estrogen receptors in zebrafish.

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 17ß-estradiol (E2; 0.001-10 µM) or ß-naphthoflavone (ßNF; 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 ßNF resulted in the dose-dependant induction of CYP1A and CYP3C genes in the female intestine but not in the liver. E2 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 ßNF (0.5 µM) and E2 (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.

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

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

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

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

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.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

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 6ß-, 12ß-, 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-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,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 ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.

Nuclear transport of the human aryl hydrocarbon receptor and subsequent gene induction relies on its residue histidine 291.

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 ß-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.

ß-Naphtoflavone and Ethanol Induce Cytochrome P450 and Protect towards MPP⁺ Toxicity in Human Neuroblastoma SH-SY5Y Cells.

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 ß-naphtoflavone (ß-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 ß-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.

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

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