Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Perspective of structural flexibility on selective inhibition towards CYP1B1 over CYP1A1 by α-naphthoflavone analogs.

Research on action selectivity between CYP1A1 and CYP1B1 is particularly valuable for cancer chemoprevention and chemotherapy. However, they share a very close similarity in their ligand-binding pockets that α-naphthoflavone (ANF) is the co-crystal ligand for both isoforms, which poses a major challenge in revealing their selectivity mechanism. Therefore, three selective CYP1B1 inhibitors derived from ANF were selected to illustrate the structural basis for the selectivity between the two isoforms via a comprehensive computational strategy. It was found that the sustainability of the π-π stacking interactions with the phenylalanine residues of the two isoforms, namely, Phe123, Phe224, and Phe258 for CYP1A1, and Phe134, Phe231, and Phe268 for CYP1B1, played a crucial role in determining the selectivity of ligands with a classic aromatic conjugation system like ANF and its derivatives for CYP1B1 versus CYP1A1. Of note, the structural flexibility of the corresponding protein domains mainly orchestrated the sustainability of the corresponding π-π stacking interactions, thereby determining the binding selectivity. Therefore, the structure modification of naphthoflavone lead compounds into preferable binding configurations to satisfy the π-π stacking interactions of the key phenylalanine residues within CYP1B1 would be an inspiring strategy devised to improve the inhibitory selectivity towards CYP1B1. Collectively, this study revealed valuable insight into understanding the selective mechanism between CYP1A1 and CYP1B1 from the perspective of structural flexibility, which sheds light on the future rational design of CYP1B1 selective inhibitors.

α-naphthoflavone-derived cytochrome P450 (CYP)1B1 degraders specific for sensitizing CYP1B1-mediated drug resistance to prostate cancer DU145: Structure activity relationship.

We previously discovered extrahepatic cytochrome P450 1B1 (CYP1B1) degraders able to overcome drug resistance toward docetaxel using a PROTACs technology, however, the underexplored structure activity relationships and poor water solubility posed a major hurdle in the development of CYP1B1 degraders. Herein, continuous efforts are made to develop more promising α-naphthoflavone (ANF)-derived chimeras for degrading CYP1B1. Guided by the strongest ANF-derived CYP1B1 degrader 3a we ever reported, 17 ANF analogues are designed and synthesized to evaluate the CYP1B1 degradation and resultant resistance reversal. In degrading CYP1B1 and sensitizing drug resistance, 4d with a 1, 5-cis triazole coupling mode at (C3') of B ring of ANF exhibited the similar potency as 3a carrying a 1, 4-trans triazole fragment at (C4') of B ring, but more obvious selectivity of 4d toward CYP1B1 over CYP1A2 is observed. When an oxygen was inserted into the linker of 4d, 4f demonstrated better water solubility, a more potent ability in degrading CYP1B1 and reversing drug resistance, and a promising selectivity. Collectively, a substitution position, an alkyne-azide cyclization and a liker type significantly affect the ability of ANF-thalidomide conjugates in eliminating drug resistance of CYP1B1-expressing DU145 (DU145/CY) cells to docetaxel via targeted CYP1B1 degradation.

Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies.

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6ß-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for progesterone 6ß-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6ß-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6ß-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6ß-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6ß-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid-steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes.

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Resveratrol supports and alpha-naphthoflavone disrupts growth of human ovarian follicles in an in vitro tissue culture model.

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.

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.

Changes in the expression of genes involved in the ovarian function of rats caused by daily exposure to 3-methylcholanthrene and their prevention by α-naphthoflavone.

Daily exposure to low doses of 3-methylcholanthrene (3MC) during the pubertal period in rats disrupts both follicular growth and ovulation. Thus, to provide new insights into the toxicity mechanism of 3MC in the ovary, here we investigated the effect of daily exposure to 3MC on selected ovarian genes, the role of the aryl hydrocarbon receptor (AhR) and the level of epigenetic remodeling of histone post-transcriptional modifications. Immature rats were daily injected with 3MC (0.1 or 1 mg/kg) and mRNA expression of genes involved in different ovarian processes were evaluated. Of the 29 genes studied, 18 were up-regulated, five were down-regulated and six were not altered. To assess whether AhR was involved in these changes, we used the chromatin immunoprecipitation assay. 3MC increased AhR binding to promoter regions of genes involved in Notch signaling (Hes1, Jag1), activation of primordial follicles (Cdk2), cell adhesion (Icam1), stress and tumor progression (Dnajb6), apoptosis (Bax, Caspase-9) and expression of growth and transcription factors (Igf2, Sp1). Studying the trimethylation and acetylation of histone 3 (H3K4me3 and H3K9Ac, respectively) of these genes, we found that 3MC increased H3K4me3 in Cyp1a1, Jag1, Dnajb6, Igf2, Notch2, Adamts1, Bax and Caspase-9, and H3K9Ac in Cyp1a1, Jag1, Cdk2, Dnajb6, Igf2, Icam1, and Sp1. Co-treatment with α-naphthoflavone (αNF), a specific antagonist of AhR, prevented almost every 3MC-induced changes. Despite the low dose used in these experiments, daily exposure to 3MC induced changes in both gene expression and epigenomic remodeling, which may lead to premature ovarian failure.

Comparative Study of Folic Acid and α-Naphthoflavone on Reducing TCDD-Induced Cleft Palate in Fetal Mice.

OBJECTIVE: Tocompare the effect of folic acid (FA) and α-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice. DESIGN: Pregnant mice were randomly divided into seven groups. The mice treated with corn oil were used as a negative control. The mice in the other six groups were given a single dose of 28 µg/kg TCDD on GD 10 by gavage. For FA treatment, TCDD-treated mice were also dosed with 5, 10, and 15 mg/kg FA on GD 10, while for α-naphthoflavone treatment, the mice received a single dose of 50 µg/kg or 5 mg/kg α-naphthoflavone on GD 10. MAIN OUTCOME MEASURES: Fetal mice palates were imaged using light and scanning electron microscopy on GD 13.5, GD 14.5, and GD 15.5, and cleft palate were recorded on GD 17.5. The expression of guanosine diphosphate dissociation inhibitor (GDI) in fetal mice palate on GD 15.5 was examined by immunohistochemistry. RESULTS: TCDD successfully induced cleft palate. Ten mg/ml FA and 5 mg/ml α-naphthoflavone significantly reduced TCDD-induced cleft palate. FA and α-naphthoflavone partly reduced TCDD-induced cleft palate but did not affect the expression of Rho GDI. CONCLUSIONS: FA and α-naphthoflavone may reduce the generation of reactive oxygen species, inhibit MEE apoptosis through anti-oxidation, and increase filopodia and MEE movement. This may result in restoration of the ultrastructure of the palatal surface to a normal state, leading to the fusion and formation of complete palate in TCDD-treated fetal mice.

Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFß, and IDO1.

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor ß1 (TGFß1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFß1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications.

Expression of the aryl hydrocarbon receptor in growth plate cartilage and the impact of its local modulation on longitudinal bone growth.

Although dioxin has been reported to impair bone growth in both humans and animals, the underlying mechanisms have not been clarified. We conducted this study to rule out if dioxin may directly target the growth plate, via local modulation of the aryl hydrocarbon receptor (AhR). Initial studies in rare tissue samples of the human growth plate confirmed that the AhR protein is widely expressed in growth plate cartilage. To explore the local role of the AhR, mechanistic studies were performed in a well-established model of cultured fetal rat metatarsal bones. The longitudinal growth of these bones was monitored while being exposed to AhR modulators. The AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin, did not affect bone growth at any concentrations tested (1 pM-10 nM). In contrast, the AhR antagonist, alpha-naphthoflavone, suppressed bone growth and increased chondrocyte apoptosis, although only at a high, potentially cytotoxic concentration (50 µM). We conclude that although the AhR is widely expressed in the growth plate, bone growth is not modulated when locally activated, and therefore, dioxin-induced growth failure is likely mediated through systemic rather than local actions.

Microbial metabolites of omeprazole activate murine aryl hydrocarbon receptor in vitro and in vivo.

Omeprazole (OME), a proton pump inhibitor used to treat gastritis, is also an aryl hydrocarbon receptor (AhR) activator. OME activates AhR in human hepatocytes and hepatoma cells, but not in mice in vivo or in vitro. We recently discovered that this species-specific difference results from a difference in a few amino acids in the ligand-binding domain of AhR. However, OME activates both mouse and human AhRs in the yeast reporter assay system. Nevertheless, the cause of this discrepancy in OME responses remains unknown. Here, we report that CYP1A1 mRNA expression in mouse cecum was elevated after OME administration, although the mouse is regarded as an OME-unresponsive animal. Using the yeast reporter assay system with human and murine AhRs, we found AhR agonist-like activity in the cecal extracts of OME-treated mice. We speculated that OME metabolites produced by cecal bacteria might activate murine AhRs in vivo. In high-performance liquid chromatography (HPLC) analysis, AhR agonist-like activity of cecal bacterial culture and cecal extracts were detected at the same retention time. AhR agonist-like activity was also detected in the HPLC fractions of yeast culture media containing OME. This unknown substance could induce reporter gene expression via mouse and human AhRs. The agonist-like activity of the OME metabolite was reduced by concomitant α-naphthoflavone exposure. These results indicate that a yeast-generated OME metabolite elicited the response of mouse AhR to OME in the yeast system, and that bacterial OME metabolites may act as AhR ligands in human and mouse intestines.

AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells.

BACKGROUND: The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B). RESULTS: Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C. CONCLUSION: AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A.

The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.

Metabolism of chamaechromone in vitro with human liver microsomes and recombinant human drug-metabolizing enzymes.

Chamaechromone is a major component in the dried roots of Stellera chamaejasme with antihepatitis B virus and insecticidal activity. In this study, metabolic profiles of chamaechromone were investigated in human liver microsomes. One monohydroxide and two monoglucuronides of chamaechromone were identified. The enzyme kinetics for both hydroxylation and glucuronidation were fitted to the Michaelis-Menten equation. The hydroxylation of chamaechromone was inhibited by α-naphthoflavone, and predominantly catalyzed by recombinant human cytochrome P450 1A2, whereas the glucuronidation was inhibited by quercetin, 1-naphthol, and fluconazole, and mainly catalyzed by recombinant human UDP-glucuronosyltransferase 1A3, 1A7, 1A9, and 2B7.

CYP1A1 and Cnr nitroreductase bioactivated niclosamide in vitro.

Niclosamide produces genotoxic effects, such as point mutations in Salmonella sp., sperm-head abnormalities in mice and clastogenic effects in human lymphocytes in vitro and in vivo. As cytochrome P450 could be involved in the bioactivation of niclosamide, we investigated which subfamily was involved. We used liver microsomal fractions from rats treated with phenobarbital/ß-naphthoflavone (PB/ß-NF), benzo[a]pyrene (BaP) or cyclohexanol, which are known to induce different cytochrome P450 subfamilies, such as CYP2B, CYP1A1, CYP1A2 and CYP2E1. We also inhibited CYP1A and CYP2E using α-NF and diethyldithiocarbamate to identify the cytochrome P450 involved. Liver-S9 fractions obtained from PB/ß-NF- and BaP-treated rats significantly increased the number of revertants induced by niclosamide, while the CYP1A1 inhibitor α-NF decreased the number of revertants. The incubation of niclosamide with CYP1A1 Supersomes™ increased the number of revertants, suggesting that CYP1A1 is responsible for the bioactivation of niclosamide. Nitroreduction is also involved in niclosamide bioactivation, as the nitroreductase-deficient strain YG7132 did not respond to the niclosamide treatment. Our findings indicated that a metabolite, derived from the action of CYP1A1 and a nitroreduction-reaction process, has a key role in the bioactivation of niclosamide.

Classification of polycyclic aromatic hydrocarbons based on mutagenicity in lung tissue through DNA microarray.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. Exposure to PAHs raises the risk of lung cancer and inflammatory and allergic disorders such as asthma. DNA microarray technologies have been applied to research on toxicogenomics in the recent years. To evaluate the mutagenicity of PAHs and constituents of environmental pollutants in lung tissue, including metabolic activation, human alveolar epithelial type II cells (A549) were treated with nonmutagenic PAH pyrene and with the mutagenic PAHs benzo-[a]-pyrene, 1-nitropyrene, or 1,8-dinitropyrene. Comparison of genome-wide microarray expression profiles between a nonmutagenic and a mutagenic PAH-treated group revealed that xenobiotic response genes such as CYP1B1 were commonly upregulated in two groups and that DNA damage induced genes, especially p53-downstream genes such as p21 (CDKN1A) were upregulated only in the mutagenic PAH-treated group. Pretreatment with cytochrome P450 inhibitor α-naphthoflavone or p53 inhibitor pifithrin-α inhibited the benzo-[a]-pyrene-induced p21 expression. These data suggest that when PAHs enter the cells, lung epithelium induces PAH metabolic activating enzymes, and then the DNA damages-recognition signal is converged with p53 downstream genes. This metabolic activation and DNA damage is induced in lung epithelium, and the mutagenicity of PAHs can be classified by DNA microarray expression profiles.

Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer.

Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature, evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17ß-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Time course studies indicate that NQO1 levels tend to increase after 4 months of E2 treatment but decrease on chronic exposure to E2 for 8 months. Vitamin C and BHA significantly increased NQO1 levels after 120 days. 8-Hydroxydeoxyguanosine (8-OHdG) levels were higher in E2-exposed mammary tissue and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissue. In vitro studies using silencer RNA confirmed the role of NQO1 in prevention of oxidative DNA damage. Our studies further demonstrate that NQO1 upregulation by antioxidants is mediated through NRF2.

Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1 in multiple myeloma.

One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. Many pathways are involved including Notch signaling. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. In this study, we demonstrate that Dll1 can activate Notch signaling mostly through Notch2 receptor and can contribute to drug resistance to bortezomib, both in murine and human MM cells. Blocking the Notch pathway by DAPT (gamma secretase inhibitor) could reverse this effect and increased sensitivity to bortezomib. We describe the upregulation of CYP1A1, a Cytochrome P450 enzyme involved in drug metabolism, as a possible mechanism of Dll1/Notch induced bortezomib resistance. This was confirmed by inhibition experiments using α-Naphthoflavone or CYP1A1-siRNA that resulted in an increased sensitivity to bortezomib. In addition, in vivo data showed that combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model. Our data provide a potential strategy to overcome bortezomib resistance by Notch inhibition in MM therapy.

Selective aryl hydrocarbon receptor modulator-mediated repression of CD55 expression induced by cytokine exposure.

Modulation of aryl hydrocarbon receptor (AHR) activity by a class of ligands termed selective AHR modulators (SAhRMs) has been demonstrated to attenuate proinflammatory gene expression and signaling, including repression of cytokine-mediated induction of acute-phase genes (e.g., Saa1). These effects are observed to occur through an AHR-dependent mechanism that does not require canonical signaling through dioxin response elements. Previously, we have demonstrated that the SAhRM 3',4'-dimethoxy-α-naphthoflavone (DiMNF) can repress the cytokine-mediated induction of complement factor genes. Here, we report that the activation of the AHR with DiMNF can suppress cytokine-mediated induction of the membrane complement regulatory protein CD55. When CD55 is expressed on host cells, it facilitates the decay of the complement component 3 (C3) convertase, thereby protecting the cell from complement-mediated lysis. Tumor cells often exhibit elevated CD55 expression on the cell surface in the inflammatory microenvironment of the tumor, and such enhanced expression could represent a means of escaping immune surveillance. DiMNF can repress the cytokine-mediated induction of CD55 mRNA and protein. Luciferase reporter analysis has identified possible response elements on the CD55 promoter, which may be targets for this repression. A C3 deposition assay with [(125)I]C3 revealed that repression of cytokine-mediated CD55 expression by DiMNF led to an increase of C3 deposition on the surface of Huh7 cells, which would likely stimulate the formation of the membrane attack complex. These results suggest that SAhRMs such as DiMNF have therapeutic potential in regulating the immune response to tumor formation.