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.

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.

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.

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.

The Chinese medicinal herb decoction QRZSLXF enhances anti-inflammatory effect in TNBS-induced colitis via balancing Th17/Tregs differentiation.

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.

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.

Expression of Serpin Peptidase Inhibitor B2 (SERPINB2) is regulated by Aryl hydrocarbon receptor (AhR).

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as ß-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.

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.

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.

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.

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.

The polyaromatic hydrocarbon ß-naphthoflavone alters binding of YY1, Sp1, and Sp3 transcription factors to the Dp71 promoter in hepatic cells.

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, ß-naphthoflavone (ß­NF), downregulates Dp71 expression. The aim of the present study was to determine whether ß­NF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcription­quantitative polymerase chain reaction was used to measure half­life mRNA levels in ß­NF­treated 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 ß­NF­treated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and ß­NF­treated 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, ß­NF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that ß­NF may modulate the expression of other genes regulated by these transcription factors. In conclusion, ß­NF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.

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.

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

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

The Metabolism of Methazolamide in Immortalized Human Keratinocytes, HaCaT Cells.

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 ß-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 ß-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.

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.

Cell-Based Assay for Identifying the Modulators of Antioxidant Response Element Signaling Pathway.

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.

Regulation of cancer-related genes - Cyp1a1, Cyp1b1, Cyp19, Nqo1 and Comt - expression in ß-naphthoflavone-treated mice by miroestrol.

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 ß-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.

Effects of ß-Naphthoflavone on Ugt1a6 and Ugt1a7 Expression in Rat Brain.

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 ß-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.

Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

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 (ßNF) 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 ßNF 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.