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Enzymes in the cytochrome P450 family 1 (CYP1) catalyze metabolic activation of procarcinogens and deactivation of certain anticancer drugs. Inhibition of these enzymes is a potential approach for cancer chemoprevention and treatment of CYP1‐mediated drug resistance. We characterized inhibition of human CYP1A1, CYP1A2, and CYP1B1 enzymes by the novel inhibitor N‐(3,5‐dichlorophenyl)cyclopropanecarboxamide (DCPCC) and α‐naphthoflavone (ANF). Depending on substrate, IC50 values of DCPCC for CYP1A1 or CYP1B1 were 10–95 times higher than for CYP1A2. IC50 of DCPCC for CYP1A2 was 100‐fold lower than for enzymes in CYP2 and CYP3 families. DCPCC IC50 values were 10–680 times higher than the ones of ANF. DCPCC was a mixed‐type inhibitor of CYP1A2. ANF was a competitive tight‐binding inhibitor of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 oxidized DCPCC more rapidly than CYP1A2 or CYP1B1 to the same metabolite. Molecular dynamics simulations and binding free energy calculations explained the differences of binding of DCPCC and ANF to the active sites of all three CYP1 enzymes. We conclude that DCPCC is a more selective inhibitor for CYP1A2 than ANF. DCPCC is a candidate structure to modulate CYP1A2‐mediated metabolism of procarcinogens and anticancer drugs. [ABSTRACT FROM AUTHOR]

Highlights • α-Naphthoflavone (αNF), an arylhydrocarbon receptor modulator (AhR), induces apoptosis in HT22 hippocampal neuronal cells. • ER stress plays a role in αNF-induced apoptosis. • The c-Src-, ROS-, MAPK-, and AhR-dependent pathways are implicated in αNF-induced ER stress and apoptosis. • The AhR nuclear translocator-dependent genomic pathway also mediates αNF-induced ER stress and apoptosis. Abstract α-Naphthoflavone (αNF) is a prototype flavone, also known as a modulator of aryl hydrocarbon receptor (AhR). In the present study, we investigated the molecular mechanisms of αNF-induced cytotoxic effects in HT22 mouse hippocampal neuronal cells. αNF induced apoptotic cell death via activation of caspase-12 and -3 and increased expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitor, salubrinal, or by CHOP siRNA transfection reduced αNF-induced cell death. αNF activated mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, and inhibition of MAPKs reduced αNF-induced CHOP expression and cell death. αNF also induced accumulation of reactive oxygen species (ROS) and an antioxidant, N-acetylcysteine, reduced αNF-induced MAPK phosphorylation, CHOP expression, and cell death. Furthermore, αNF activated c-Src kinase, and inhibition of c-Src by a kinase inhibitor, SU6656, or siRNA transfection reduced αNF-induced ROS accumulation, MAPK activation, CHOP expression, and cell death. Inhibition of AhR by an AhR antagonist, CH223191, and siRNA transfection of AhR and AhR nuclear translocator reduced αNF-induced AhR-responsive luciferase activity, CHOP expression, and cell death. Finally, we found that inhibition of c-Src and MAPKs reduced αNF-induced transcriptional activity of AhR. Taken together, these findings suggest that αNF induces apoptosis through ER stress via c-Src-, ROS-, MAPKs-, and AhR-dependent pathways in HT22 cells. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AhR) is a ligand-activated factor that regulates biological effects associated with obesity. The AhR agonists, such as environmental contaminants 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone (BNF), inhibit preadipocyte differentiation and interfere with the functions of adipose tissue, whereas the antagonist may have opposite or protective effects in obesity. This study investigated the effects of α-naphthoflavone (α-NF), an AhR antagonist, on adipogenesis- and angiogenesis-associated factors in mature adipocytes and on cross-talk of mature adipocytes with endothelial cells (ECs). Besides, the roles of the AhR on lipid accumulation and on secretion of vascular endothelial growth factor were also determined by introducing siRNA of AhR. Differentiated 3T3-L1 cells were treated with α-naphthoflavone (α-NF) (1–5 μM) for 16 h. Lipid accumulation and the expressions of AhR-associated factors in the cells were determined. The interaction between adipocytes and ECs was investigated by cultivating ECs with conditioned medium (CM) from α-NF-treated mature adipocytes, followed by the determination of endothelial tube formation. The results showed that α-NF significantly increased triglyceride (TG) accumulation in mature adipocytes, which was associated with increased expression of hormone-sensitive lipase (HSL), estrogen receptor (ER), as well as decreased expression of AhR, AhR nuclear translocator (ARNT), cytochrome P4501B1 (CYP1B1), and nuclear factor erythroid-2-related factor (NRF-2) proteins. In addition, CM stimulated formation of tube-like structures in ECs, and α-NF further enhanced such stimulation in association with modulated the secretions of various angiogenic mediators by mature adipocytes. Similarly, increased TG accumulation and vascular endothelial growth factor (VEGF) secretion were observed in AhR-knockout cells. In conclusion, α-NF increased TG accumulation in mature adipocytes and enhanced mature adipocyte-stimulated tube formation in ECs, suggesting that the AhR may suppress obesity-induced adverse effects, and α-NF abolished the protective effects of the AhR.

Graphical abstract Abstract Aflatoxin B 1 (AFB 1) is bioactivated by cytochrome P450 (CYP) 3A isoforms in humans to generate the highly reactive epoxide intermediate AFB 1 -8,9-epoxide (AFBO), causing hepatotoxicity and hepatocarcinoma. Due to the unavoidable contamination in their feed, pigs are more likely to be exposed to AFB 1 and indirectly harm human health. Therefore, identifying the porcine CYP3A isoforms involved in AFB 1 -8,9-epoxidation is critical. In this study, we used codon optimization and N-terminal coding sequence modification to modify a CYP3A46 recombinant protein that exhibits good structure and catalytic activities and revealed its strong AFB 1 -8,9-epoxidase activity for the first time. Site-directed mutagenesis, kinetics and docking analyses were performed and demonstrated that residues Phe-108, Ser-119, Phe-215, Phe-304 and Thr-309 play important roles in AFB 1 -8,9-epoxidation and its responsiveness to α-naphthoflavone. Interestingly, we uncovered the dual and reverse roles of Phe-304 in CYP3A46, CYP3A5 and CYP3A4 in AFB 1 oxidation. Unlike the π-π interaction between the Phe-304 phenyl of CYP3A4 and the AFB 1 aromatic ring, Phe-304 of CYP3A46 may function to provide steric hindrance to bind AFB 1. Phe-108 and Phe-215 could stabilize AFB 1 with a potentially productive orientation through van der Waals interactions with AFB 1. Ser-119 and Thr-309 are likely to function to form H-bonds with AFB 1. This study broadens our knowledge of AFB 1 bioactivation in pigs and may contribute to reduce the deleterious effects of AFB 1 in pigs and humans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human papilloma viruses 16 and 18 express E6 and E7 oncoproteins. E6 activates and redirects E6-associated protein (E6AP), an E3 ubiquitin ligase. E6AP interacts with Ube2l3, an E2 ubiquitin conjugating enzyme protein (also known as UbcH7), to promote p53 ubiquitination and degradation by the 26S proteasome. Therefore, blocking E6-mediated p53 degradation might be an alternative treatment for cervical cancer. In addition, activation of the aryl hydrocarbon receptor (AHR) induces Ube2l3 expression, resulting in p53 ubiquitination and degradation. The aim of the present study was to determine whether inhibition of AHR in HeLa cells resulted in an increase in p53 and apoptosis along with a decrease in cell proliferation. The results demonstrate that two AHR antagonists, α-naphthoflavone (α-NF) and resveratrol, decreased cell proliferation, arrested cells in the gap 1/synthesis (G1/S) phases, and increased p53 levels and apoptosis. However, knocking out the Ahr gene did not abrogate the effects of α-NF and resveratrol. Moreover, Ahr -null cells presented similar cell proliferation rates and apoptosis levels when compared to control HeLa cells. Taken together, the results indicate that α-NF's and resveratrol's cytostatic and cytotoxic actions, respectively, occur through an AHR-independent mechanism, and that AHR is not required for HeLa cell proliferation. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated an association between neurological diseases and oxidative stress (OS). Naphthoflavone is a synthetic derivative of naturally occurring flavonoids that serves an important role in the treatment and prevention of OS-related diseases. The current study was designed to apply α- and β-Naphthoflavone individually and in combination to counteract the detrimental effects of OS on neurons in vitro. Neuronal SH-SY5Y cells were subjected to 20 μM H2O2, followed by exposure to 20 μM α-Naphthoflavone and/or 10 μM β-Naphthoflavone. Results indicated that α- and β-Naphthoflavone effectively antagonized the apoptosis-promoting effect of H2O2 on neuronal SH-SY5Y cells, and that β-Naphthoflavone significantly (P<0.05) reversed H2O2-inhibited cell viability. Notably, co-treatment of α- and β-Naphthoflavone reversed the H2O2-induced apoptosis rate elevation and cell viability reduction. Further analysis demonstrated that H2O2 inhibited the activities of antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase, but this was reversed by the co-treatment with α- and β-Naphthoflavone and selectively enhanced by the treatment with α- or β-Naphthoflavone. H2O2-stimulated p38 mitogen-activated protein kinase activation was repressed following treatment with α- and/or β-Naphthoflavone, along with a decreased expression of the apoptosis-related factors and inhibited caspase-3 activation. In conclusion, co-treatment with α- and β-Naphthoflavone minimized H2O2-led neuron damage compared with treatment with α- or β-Naphthoflavone, suggesting a synergetic effect between α- and β-Naphthoflavone. This indicates that utilizing α- and β-Naphthoflavone together in the clinical setting may provide a novel therapeutic for neurological disease. [ABSTRACT FROM AUTHOR]

The article presents a comparative study of the effectiveness of folic acid and naphthoflavone on the reduction of the incidence and severity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice. Topics include an understanding of cleft lip or palate (CLP) which is a common developmental defect globally, the investigation of GDP disassociation inhibitor (GDI), and changes in the medial edge epithelia (MEE).

In the present study, we characterized the kinetic parameters of 7-ethoxy-resorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) in hepatic microsomes from entire and castrated male pigs. Validation parameters of an HPLC-based method to analyse EROD and MROD activities are also described. Eadie-Hofstee plot analysis demonstrated a biphasic kinetic of EROD, indicating that at least two forms of cytochrome P450 are involved in this reaction. MROD followed monophasic kinetic, suggesting that a single enzyme, or enzymes with similar affinities, is responsible for the reaction. Inhibitory effects of α-naphthoflavone (ANF), ellipticine and furafylline were studied using microsomes from entire and castrated male pigs. ANF is a known inhibitor of both cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2); the presence of ANF in the incubations resulted in the inhibition of both EROD and MROD activities in porcine liver microsomes. EROD activities in porcine liver microsomes were also inhibited by selective CYP1A1 inhibitor ellipticine, but not by CYP1A2 inhibitor furafylline. MROD activities were strongly inhibited by ellipticine and to a much lesser extent by furafylline. Further studies are needed to evaluate substrate specificities of porcine CYP1A1 and CYP1A2.

CYP1 B1 is a promising drug target for developing novel drugs against hormonal cancers and hypertension. The development of CYP1 B1-specific inhibitors is hindered mainly due to non-specific action of known CYP inhibitors. The active site of CYP1B1 is similar to other cytochromes with different substrate preferences rendering a scope to develop specific inhibitors. We have developed a novel in silico approach for design of selective CYP1 B1 inhibitors. The approach consists of deriving details of CYP1 B1-specific molecular interactions from prior studies, which is used to perform screening of CYP1 B1 with NCI compounds. The conventional compound screening is also complemented with the concept of cutoff distance between heme ( Fe) and compounds. The binding free energies and HB percentage occupancy calculations of 94 compounds of cluster 1 have verified the docking results using MD. The docking interactions in the active-site cavity of 7 clusters are also taken into account for optimal binding. Hence, we used knowledgebase filtering and MD simulations to enable discovery of selective CYP1 B1 inhibitors. The final filtered lead candidates consist of compounds sandwiched between phenylalanine π-π stacking and less than 6 Å from heme ( Fe) for enzymatic action. The findings in the study can help development of novel CYP1 B1 selective inhibitors. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the effect of 3-methylcholanthrene (3MC) on sexual maturity and the ability of α-naphthoflavone (αNF) to prevent this action. To this end, immature rats were daily injected intraperitoneally with 3MC (0.1 or 1 mg/kg) and/or αNF (80 mg/kg). Body weight, vaginal opening and estrous cycle were recorded and ovaries were obtained on the day of estrus. Ovarian weight, ovulation rate (measured by the number of oocytes within oviducts), and follicular development (determined by histology) were studied. No differences were found in body weight, ovarian weight, day of vaginal opening, or the establishment of the estrous cycle among the different groups of rats. However, animals treated with 3MC, at both doses, exhibited a lower number of primordial, primary, preantral and antral follicles than controls. Also, 3MC inhibited the ovulation rate and induced an overexpression of both the Cyp1a1 and Cyp1b1 genes, measured by chromatin immunoprecipitation assay. The daily treatment with αNF alone increased the number of follicles in most of the stages analyzed when compared with controls. Moreover, the αNF treatment prevented completely not only the 3MC-induced decrease in all types of follicles but also the 3MC-induced overexpression of Cyp enzymes and the genetic damage in bone marrow cells and oocytes. These results suggest that (i) daily exposure to 3MC during the pubertal period destroys the follicle reserve and alters the ovulation rate; (ii) the 3MC action seems to be mediated by an aryl hydrocarbon receptor-dependent mechanism; (iii) daily administration of αNF has a clear stimulatory action on the ovarian function; and (iv) αNF may prevent both the systemic and gonadal 3MC-induced toxicity. [ABSTRACT FROM AUTHOR]

Objective: The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6β-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6β-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily., Methods: 6β-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography., Results: Lower concentrations of DHEA and ANF increased cortisol 6β-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6β-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6β-hydroxylation. Although the Michaelis constant ( K m ) increased 2-fold in the presence of 20 μM DHEA compared to that of the control, the maximal velocity ( V max ) values gradually increased with increasing DHEA. For ANF, both K m and V max values increased, although the K m value decreased at 2.5 μM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6β-hydroxylation mediated by CYP3A4 with similar inhibition constants., Conclusion: The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Despite an initial positive response, breast cancer cells inevitably acquire resistance to doxorubicin (Dox). Alpha-naphthoflavone (ANF) is a well-known chemopreventive agent; however, its anti-cancer properties have not been established. We examined the therapeutic efficacy of ANF in doxorubicin-resistant MCF-7 (MCF-7/Dox) breast cancer cells and investigated its underlying molecular mechanisms of action. MCF-7/Dox cells expressed constitutively active forms of the tyrosine kinases: focal adhesion kinase (FAK-Y397) and protein tyrosine kinase 2 beta (Pyk2- Y579/580) compared with parental MCF-7 cells. ANF significantly enhanced the sensitivity of MCF-7/Dox cells to Dox cytotoxicity in vitro and when co-administered in vivo . This ANF-mediated chemosensitization has dual mechanisms of action: (a) intracellular Dox retention via suppression of P-glycoprotein pump activity, and (b) inhibition of clonogenic cell survival via de-phosphorylation of FAK, Pyk2, and EGF-induced Akt in MCF-7/Dox cells and tumor xenografts. Because of its strong chemosensitization action, broad safety profile, and health benefits, ANF is an attractive anti-cancer drug with therapeutic implications to circumvent drug resistance in breast cancer patients. [ABSTRACT FROM AUTHOR]

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Highlights: [•] Hydroxypropyl cyclosophoraoses make complexation with α-naphthoflavone(α-NF). [•] Complex formation was confirmed by various instrumental methods. [•] Hydroxypropyl cyclosophoraoses increased the solubility of α-NF up to 257-fold. [•] This is the first study about carbohydrates that can enhance the solubility of α-NF. [ABSTRACT FROM AUTHOR]

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes),but failed to produce activation in BD SupersomesTM or Baculosomes® containing recombinant CYP3A4 and NADPHCPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into SupersomesTM containing only recombinant CPR reproduced the behaviour observed with HLM. The activation in this systemwas dependent on the surface density of the enzyme. Although no activation was detectable at an L/P (lipid/P450) ratio ≥750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization, we probed P450-P450 interactions with a new technique that employs LRET (luminescence resonance energy transfer). The amplitude of LRET in mixed oligomers of the haem protein labelled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in SupersomesTM. The addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions. [ABSTRACT FROM AUTHOR]

The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W, and L211F/D214E was studied with fluorescence resonance energy transfer and absorbance spectroscopy. Although in the wild type enzyme, the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (nH) around 1.7 (S50 = 11.7 µM), the mutants showed no cooperativity (nH ≤ 1.1) with unchanged S50 values. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high-affinity interaction is characterized by a dissociation constant (KD) ≤ 1.0 µM, whereas the KD of the second binding has the same magnitude as the S50. Theoretical analysis of a two-step binding model suggests that nH values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event. Alteration of cooperativity in the mutants is caused by a partial displacement of the "spin-shifting" step. Although in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift on binding of the first substrate molecule. [ABSTRACT FROM AUTHOR]

Abstract: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and the first protein involved in a variety of physiological and toxicological processes, including those of xenobiotic metabolizing enzymes. AhR has been found in the ovary of many species and seems to mediate the ovarian toxicity of many environmental contaminants, which are AhR ligands. However, the role of AhR in the ovarian function is unknown. Therefore, the aim of this work was to study the action of α-naphthoflavone (αNF), known to be an AhR antagonist, on both follicular growth and ovulation. Immature Sprague–Dawley rats were daily injected intraperitoneally with αNF (0.1–80mg/kg) or vehicle for 12days, and primed with gonadotrophins (eCG/hCG) to induce follicular growth and ovulation. Ovaries were obtained 20h after hCG administration. By means of immunohistochemistry, we found that the numbers of primordial, primary and antral follicles were increased in rats treated with 80mg/kg αNF and that there were no differences with other doses. Likewise, the ovarian weight and the ovulation rate, measured by both number of oocytes within oviducts and corpora lutea in ovarian sections, were increased when the rats received either 1 or 10mg/kg daily. Although further studies are necessary to know the mechanism of action of αNF, it is possible that the different ovarian processes can be differentially responsive to the presence of different levels of αNF, and that the same or different endogenous AhR ligands can be involved in these ovarian processes in a cell type-dependent manner. [Copyright &y& Elsevier]

Lungs are permanently and simultaneously challenged by airborne microorganisms and airborne pollutants. Temporal increase of airborne particulate matter (APM), a potential carrier for extractable organic matter (EOM), degrades the situation of pulmonary patients. The Ah receptor (AhR) has been described as an important factor influencing the immunological challenge by viral infections. Molecular mechanisms underlying epidemiological observations are not well understood. Cytokine secretion (IL-6, IL-8, and TGF-β) from human bronchial epithelial cells (Beas2B) was determined as an indicator for immune responses upon co-stimulation with an artificial analog of viral dsRNA [polyinosinic/polycytidylic acid, (PIC)] and EOM of Standard Reference Material 1649a (SRM). Since polycyclic aromatic hydrocarbons are major components of APM usually acting via the AhR, particular focus was on AhR involvement. Cytokine secretion was demonstrated by enzyme-linked immunosorbent assay. To mimic the activation of organic matter during contact of particles with the human lung, Soxhlet extraction of SRM was performed. In some experiments, the AhR was blocked by α-naphthoflavone. Microbial stimulation (PIC) induced Beas2B cytokine release, whereas isolated exposure to EOM of APM did not. Co-stimulation with EOM and PIC increased IL-8 secretion, whereas neither IL-6 nor TGF-β was affected. Blocking of the AhR suppressed the release of IL-8. Organic compounds adsorbed on airborne particulate matter influence the cytokine secretion of lung epithelial cells induced by pathogen-associated molecular patterns. Further investigation of these observations is required to understand the molecular mechanisms underlying adverse health effects of APM reported in epidemiological studies. [ABSTRACT FROM AUTHOR]

Abstract: Heterotropic cooperative phenomena have been documented in studies with cytochrome P450 3A4, with few attempts to quantify this behavior other than to show the apparent stimulatory effect of certain CYP3A4 substrates on the enzyme’s catalytic activity for others. Here CYP3A4 solubilized in Nanodiscs is studied for its ability to interact with two substrates, α-naphthoflavone and testosterone, which produce transitions in the heme spin state with apparent spectral affinities (corrected for membrane partitioning) of 7 and 38μM, respectively. Simultaneous addition of both substrates at fixed molar ratios allows for the separation of specific heterotropic cooperative interactions from the simple additive affinities for the given substrate ratios. The absence of any changes in the normalized spectral dissociation constant due to changes in substrate ratio reveals that the observed stimulatory effect is largely due to differences in the relative substrate affinities and the presence of additional substrate in the system, rather than any specific positive heterotropic interactions between the two substrates. [Copyright &y& Elsevier]

In the present study, we characterized the kinetic parameters of 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) in hepatic microsomes from entire and castrated male pigs. Validation parameters of an HPLC-based method to analyse EROD and MROD activities are also described. Eadie- Hofstee plot analysis demonstrated a biphasic kinetic of EROD, indicating that at least two forms of cytochrome P450 are involved in this reaction. MROD followed monophasic kinetic, suggesting that a single enzyme, or enzymes with similar affinities, is responsible for the reaction. Inhibitory effects of α-naphthoflavone (ANF), ellipticine and furafylline were studied using microsomes from entire and castrated male pigs. ANF is a known inhibitor of both cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2); the presence of ANF in the incubations resulted in the inhibition of both EROD and MROD activities in porcine liver microsomes. EROD activities in porcine liver microsomes were also inhibited by selective CYP1A1 inhibitor ellipticine, but not by CYP1A2 inhibitor furafylline. MROD activities were strongly inhibited by ellipticine and to a much lesser extent by furafylline. Further studies are needed to evaluate substrate specificities of porcine CYP1A1 and CYP1A2. [ABSTRACT FROM AUTHOR]

Abstract: Each chromosome occupies its own-specific space called a ‘territory’ within the interphase nucleus, and the arrangement of chromosome territories (CTs) is important in epigenetic mechanisms. The molecular mechanism to determine the positioning of CTs, however, remains unknown. On the other hand, dioxin is known to be the typical environmental pollutant that affects a wide variety of biological events in many species. Here, we show that dioxin enlarges the minimum distance between chromosome 12 and chromosome 16 territories in human preadipocyte cells, and the alteration of chromosome positioning is canceled by an aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone. Thus, AhR may be a key molecule to regulate chromosome positioning. Our results suggest a novel effect of dioxin toxicity, and demonstrate a clue to reveal the novel molecular mechanism for the arrangement of CTs. [Copyright &y& Elsevier]

Abstract: Alkylated polycyclic aromatic hydrocarbons, such as retene (7-isopropyl-1-methylphenanthrene), induce cytochrome P450 1A (CYP1A) enzymes and produce dioxin-like toxicity in the embryo–larval stages of fish characterized by the signs of blue sac disease (BSD). The signs of toxicity are well characterized; however, the mechanism is not well understood. To elucidate the role of CYP1A in retene toxicity, larval rainbow trout (Oncorhynchus mykiss) were co-treated with a range of concentrations of α-naphthoflavone (ANF), a known CYP1A inhibitor. The co-treatment produced synergistic toxicity at 3.2–100μg/L ANF, after which toxicity at 180μg/L ANF dropped to levels typical of retene-only. At 320μg/L ANF, toxicity increased with or without retene, indicating that ANF alone was capable of inducing BSD. In addition, the additive toxicity of retene-only and 320μg/L ANF-only approximately equalled that of the co-exposed larvae (100μg/L retene+320μg/L ANF), indicating response addition. Thus, two mechanisms of action occurred in co-exposed larvae at different concentrations of ANF. In trout larvae, there was a correlation between toxicity and CYP1A protein concentrations, and in juvenile trout, ANF produced a concentration-dependent inhibition of ethoxyresorufin-O-deethylase (EROD) activity without a measurable drop in CYP1A protein. Taken together, the mechanism underlying the synergistic toxicity is EROD-independent and may be AhR-dependent. This study demonstrated that multiple, exposure-dependent mechanisms can occur in mixture toxicity, suggesting that current risk assessment models may drastically underestimate toxicity, particularly of mixtures containing both CYP1A inducers and inhibitors. [Copyright &y& Elsevier]

Abstract: Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1–4mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo. [Copyright &y& Elsevier]

Abstract: We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (K D =0.48μM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or α-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator. [Copyright &y& Elsevier]

Abstract: The effects of α-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, on the reproductive toxicity and teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated. Pregnant C57BL/6J mice were orally administered α-naphthoflavone either once on gestational day 12 (GD12; 50μg/kg) or for 6 days (GD8–GD13; 5mg/kg/day) followed by an oral challenge with TCDD (14μg/kg) on GD12. Cesarean section was performed on GD18 for the evaluation of maternal and fetal toxicities. TCDD caused severe fetal malformations including cleft palate (43.7%) and renal pelvic and ureteric dilatations (100%). The administration of α-naphthoflavone either in a single treatment or 6-days remarkably reduced the incidence of cleft palate to 27.6% and 26.5%, respectively. In addition, the degree of renal pelvic and ureteric dilatations caused by TCDD were significantly attenuated by repeated treatment of α-naphthoflavone. These results suggest that AhR antagonists such as α-naphthoflavone could be promising candidates for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero. [Copyright &y& Elsevier]

Abstract: Allelopathy as a mechanism of invasion in plant communities remains a debated topic, partly due to lack of techniques to measure allelochemical output and stability in the field. Evidence has arisen to support allelopathic mechanisms in several invasive knapweeds, including Russian knapweed. Previous studies have shown that a phytotoxin, 7,8-benzoflavone, is produced by Russian knapweed roots under soil-free conditions. Here we describe a high performance liquid chromatography (HPLC) method to detect this compound in soil and we present the first observations of this compound from Russian knapweed infested soils. [Copyright &y& Elsevier]

Abstract: Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, α-naphthoflavone (ANF) and the steroid, testosterone (TST). UV–vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: (1) a relatively tight spin-state insensitive binding site (CYP·ANF) and (2) a relatively low affinity spin-state sensitive binding site (CYP·ANF·ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s). The spin-state insensitive binding site of ANF appears to compete with the spin-state insensitive binding site of TST. The formation of the spin-state insensitive CYP·ANF complex is strongly temperature dependent, when compared to the formation of the CYP·TST complex, suggesting that the formation of the CYP3A4·ANF complex leads to long-range conformational changes within the protein. When the CYP·ANF complex is titrated with TST, the formation of CYP·ANF·TST is favored by 3:1 over the formation of CYP·TST·TST, suggesting that there is an allosteric interaction between ANF and TST. A model of heterotropic cooperativity of CYP3A4 is presented, where the spin-state insensitive binding of ANF occurs at the same peripheral binding site of CYP3A4 as TST. [Copyright &y& Elsevier]

CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity, but can differ in catalytic activity and regioselectivity. To investigate their characteristics further, the enzymatic reactions of the two CYP3A enzymes were compared using midazolam, nifedipine, testosterone and terfenadine as substrates. Both CYP3A5 and CYP3A4 showed sigmoid and substrate inhibition patterns for testosterone 6β-hydroxylation and terfenadine t -butylhydroxylation (TFDOH), respectively. In the other reactions, the kinetic model for CYP3A5 was not similar to that for CYP3A4. An inhibition study demonstrated that the interactions between α-naphthoflavone (αNF) and CYP3A substrates were different for the two CYP3A enzymes. αNF stimulated nifedipine oxidation catalysed by CYP3A5, but did not stimulate that catalysed by CYP3A4. αNF at less than 32&ThickSpace;µM inhibited TFDOH catalysed by CYP3A5, but did not inhibit that catalysed by CYP3A4. These results indicate that CYP3A5 has different enzymatic characteristics from CYP3A4 in some CYP3A catalysed reactions. [ABSTRACT FROM AUTHOR]

The objective of the study was to investigate the impact of aryl hydrocarbon receptor (AhR) activation on interrenal steroidogenesis in rainbow trout. To this end, fish were fed AhR agonist (β-naphthoflavone (BNF): 10mg/kg body mass/day) and antagonist (α-naphthoflavone (ANF): 10mg/kg body mass/day) either singly or in combination (ABNF) for 5 days to elucidate the mechanisms involved in AhR-mediated depression of cortisol production. Liver AhR protein expression was significantly elevated only with ABNF, but not with BNF and ANF compared to the control group. However, all three treatments (BNF, ANF, and ABNF) significantly elevated cytochrome P450 1A1 (CYP1A1) gene and protein expression in the kidney and liver, respectively. Also, these three treatment groups had significantly depressed ACTH-stimulated cortisol production in vitro compared to the control group. This attenuation of interrenal steroidogenesis corresponded with a lower mRNA abundance of steroidogenic acute regulatory (StAR) protein and cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), but not 11β-hydroxylase. Furthermore, in vitro incubation of head kidney pieces with 7-3H-pregnenolone failed to show any treatment effects on pathways downstream of P450scc, except for a significantly higher conversion to progesterone in the BNF and ANF groups. Plasma cortisol and glucose levels showed no significant change between the treated groups and control, but liver and brain glucocorticoid receptor (GR) protein expression was higher in the BNF group, and ANF abolished this response. Taken together, both BNF and ANF impaired cortisol production, and the mechanism may involve attenuation of StAR and P450scc, the rate limiting steps in steroidogenesis. Overall, endocrine disruption by xenobiotics acting via AhR includes impaired cortisol biosynthesis and abnormal cortisol target tissue GR responses in rainbow trout. [ABSTRACT FROM AUTHOR]

We investigated the impact of aryl hydrocarbon receptor (AhR) activation on cortisol production in rainbow trout (Oncorhynchus mykiss) using β-naphthoflavone (BNF) and α-naphthoflavone (ANF) as agonist and antagonist of AhR, respectively. Fish were given a single intraperitoneal injection of either corn oil alone or corn oil containing ANF (50 mg/kg body mass), BNF (50 mg/kg) or a combination of both (ANF/BNF; 50 mg/kg each) and sampled at 2- and 6-day post-injection. Higher CYP1A protein expression in the liver and mRNA abundance in the head kidney confirmed AhR activation with BNF, but ANF did not abolish this response. BNF treatment did not significantly affect mRNA abundance of key steroidogenic enzymes (StAR protein, P450 side chain cleavage and 11β-hydroxylase) in the head kidney of trout. However, BNF depressed the in vitro ACTH- or 8-br-cAMP-mediated cortisol production and this effect was also not modified by ANF. Plasma cortisol levels were significantly higher with BNF in the present study and this BNF-mediated cortisol response was completely abolished with ANF suggesting a role for AhR and/or CYP1A in the regulation of plasma cortisol concentration. However, despite elevated plasma cortisol levels, there was no significant effect of BNF on liver glucocorticoid receptor (GR) protein expression suggesting a lack of liver responsiveness to cortisol stimulation. Also, this was reflected in the absence of any change in plasma glucose concentration in the BNF group at 2 days, and a significant drop at 6 days compared to other treatments. Altogether, our results suggest that AhR activation disrupts interrenal corticosteroidogenesis and target tissue responsiveness to glucocorticoid stimulation in rainbow trout. [Copyright &y& Elsevier]

Human cytochrome P450 (P450) enzymes exhibit remarkable diversity in their substrate specificities, participating in oxidation reactions of a wide range of xenobiotic drugs. Previously, we reported that α-naphthoflavone (ANF) is bound to the recombinant P450 1A2 tightly and stabilizes an overall enzyme conformation. The present study is designed to determine the type of P450 1A2 inhibition exerted by ANF, using two different substrates of P450 1A2, 7-ethoxycoumarin (EOC) and 7-ethoxyresorufin (EOR). ANF is generally known as a competitive inhibitor of the enzyme. However, in our tight-binding enzyme kinetics study, ANF acts as noncompetitive inhibitor in 7-ethoxycoumarin O-deethylation (ECOD) (Ki=55.0 nM), but as competitive inhibitor in 7-ethoxyresorufin O-deethylation (EROD) (Ki=1.4 nM). Based on homology modeling studies, ANF is positioned to bind to a hydrophobic cavity next to the active site where it may cause a direct effect on substrate binding. It is agreed with the predicted binding site of ANF in P450 3A4, in which ANF is rather known as a stimulating modulator. Our results suggest that ANF binds near the active site of P450 1A2 and exhibits differential inhibition mechanisms, possibly depending on the molecular structure of the substrate. [Copyright &y& Elsevier]

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6β-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 (K m ) for progesterone 6β-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6β-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6β-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6β-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6β-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6β-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.

Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern-Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K d 1.8-2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K d 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K d 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K d 0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.

Previous studies have demonstrated an association between neurological diseases and oxidative stress (OS). Naphthoflavone is a synthetic derivative of naturally occurring flavonoids that serves an important role in the treatment and prevention of OS-related diseases. The current study was designed to apply α- and β-Naphthoflavone individually and in combination to counteract the detrimental effects of OS on neurons in vitro . Neuronal SH-SY5Y cells were subjected to 20 µM H 2 O 2 , followed by exposure to 20 µM α-Naphthoflavone and/or 10 µM β-Naphthoflavone. Results indicated that α- and β-Naphthoflavone effectively antagonized the apoptosis-promoting effect of H 2 O 2 on neuronal SH-SY5Y cells, and that β-Naphthoflavone significantly (P<0.05) reversed H 2 O 2 -inhibited cell viability. Notably, co-treatment of α- and β-Naphthoflavone reversed the H 2 O 2 -induced apoptosis rate elevation and cell viability reduction. Further analysis demonstrated that H 2 O 2 inhibited the activities of antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase, but this was reversed by the co-treatment with α- and β-Naphthoflavone and selectively enhanced by the treatment with α- or β-Naphthoflavone. H 2 O 2 -stimulated p38 mitogen-activated protein kinase activation was repressed following treatment with α- and/or β-Naphthoflavone, along with a decreased expression of the apoptosis-related factors and inhibited caspase-3 activation. In conclusion, co-treatment with α- and β-Naphthoflavone minimized H 2 O 2 -led neuron damage compared with treatment with α- or β-Naphthoflavone, suggesting a synergetic effect between α- and β-Naphthoflavone. This indicates that utilizing α- and β-Naphthoflavone together in the clinical setting may provide a novel therapeutic for neurological disease.

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.

Background: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood., Objective: To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers., Methods: Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E., Results: In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD>β-NF>ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype., Conclusion: Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

α-Naphthoflavone (α-NF) is a synthetic flavonone derivative and is well known as a potent inhibitor of aromatase in a variety of systems. However, its role in lipid metabolism remains far from understood. The aim of current study was to investigate the effects of α-NF on 3T3-L1 pre-adipocytes differentiation and the mechanism through which it acts. Treatment of 3T3-L1 cells with α-NF in conjunction with a hormone cocktail resulted in α-NF mediated suppression of adipocyte differentiation in a dose dependent manner. At the molecular level, our findings demonstrated that α-NF inhibited the mid and late phase, but not the early phase of adipogenic markers expression during 3T3-L1 adipogenesis. The phosphorylation of p38 was activated upon adipogenic stimulation, yet was substantially suppressed by α-NF treatment. α-NF also synergistically inhibited expression of the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) expression together with p38 selective inhibitor, SB203580. Our study demonstrated for the first time that α-NF is capable of suppressing 3T3-L1 adipocyte differentiation and that this effect likely occurs through repression of the p38MAPK signaling pathway.

Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b(5) (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b(5), and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b(5) in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b(5). The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro.