Development and Application of an Immunoaffinity Column Enzyme Immunoassay for Mycotoxin Zearalenone in Complicated Samples

The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC₅₀ against ZEA of 0.02 µg L⁻¹. The mAb 2D3 exhibited a high recognition of ZEA (100%) and β-zearalenol (β-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and β-zearalanol (β-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83–93%) and HPLC (94–108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize.     

Effects of fumonisin B1 alone and combined with deoxynivalenol or zearalenone on porcine granulosa cell proliferation and steroid production

Fumonisin B₁ (FB₁) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB₁, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B₁ had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB₁. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB₁. Regarding steroid production, FB₁ increased progesterone production, and FB₁ had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB₁ had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB₁ produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB₁. Fumonisin B₁ was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB₁ on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB₁ increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.     

Urinary zearalenone measured with ELISA as a biomarker of zearalenone exposure in pigs

The suitability to assess zearalenone (ZEA) exposure in pigs of a commercial ELISA kit for ZEA analysis in urine was tested. A daily dose of 0, 5, 10, 20 and 40 μg synthetic ZEA per kilogram BW was administered via the feed to four gilts per dose group, and after 3 and after 7 days of ZEA intake, urine samples were assayed with the ELISA which has a relative cross-reactivity of 42 % with α-zearalenol. The concentration of urinary ZEA equivalents (ZEA plus 42 % of α-zearalenol present) did not differ between day 4 and day 8 (P?=?0.50) within each dose group. The urinary ZEA equivalent/creatinine ratio was tightly correlated with ZEA intake (r?=?0.95). The urinary ZEA equivalent/creatinine values at 0 and 40 μg/kg BW were distinctly different from those of the intermediate dose levels, whereas there was some overlapping of the individual values at the dose levels 5, 10 and 20 μg/kg BW. The urinary ZEA equivalent/creatinine ratio can be used as a biomarker for ZEA exposure in pigs provided that urine samples of several animals receiving the same diet are assayed, either separately or after pooling.     

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone,deoxynivalenol and their metabolites in pig serum

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, β-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis? HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3–480 ng/ml) were prepared and high degree of linearity was achieved (r?≥?0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82–131 %. Limits of detection and quantification ranged 0.03–0.71 and 0.08–2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.     

On the distribution and metabolism of Fusarium-toxins along the gastrointestinal tract of endotoxaemic pigs

The aim of this study was to investigate the potential modulatory effect of E. coli lipopolysaccharides (LPS) on residues of deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) after pre- or post-hepatic administration along the gastrointestinal axis. Fifteen barrows were exposed to a naturally mycotoxin contaminated diet (4.59 mg DON/kg feed and 0.22 mg ZEN/kg feed) and equipped with jugular (ju) and portal (po) catheters. On sampling day (day 29), the barrows were infused with LPS or a control fluid (LPS, 7.5 µg/kg body weight; control, 0.9% NaCl) either pre- or post-hepatically, resulting in three infusion groups: CON_ju-CON_po, CON_ju-LPS_po and LPS_ju-CON_po. At 195 min relative to infusion start (210 min post-feeding), pigs were sacrificed and content of stomach and small intestine (proximal, medial and distal part) as well as faeces were collected. In all LPS-infused animals, higher amounts of dry matter were recovered irrespective of LPS entry site suggesting a reduced gastric emptying and a decreased gastrointestinal motility under endotoxaemic conditions. DON metabolism in the gastrointestinal tract (GIT) remained unaltered by treatments and included an increase in the proportion of DOM-1 along the GIT, particularly from distal small intestine to faeces. Variables describing ZEN metabolism suggest a stimulated biliary release of ZEN and its metabolites in LPS-infused groups, particularly in the LPS_ju-CON_po group. In conclusion, the GIT metabolism of ZEN was markedly influenced in endotoxaemic pigs whereby a jugular induction of an acute phase reaction was more effective than portal LPS infusion hinting at a strong hepatic first-pass effect.     

A rapid method for simultaneous determination of zearalenone, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol in traditional Chinese medicines by ultra-high-performance liquid chromatography-tandem mass spectrometry

A rapid and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of zearalenone (ZEN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) in traditional Chinese medicines (TCMs) was developed. The development of the method and investigations of the matrix influence were described in particular. After evaluation of the matrix effects of different TCMs, i.e., rhizomes and roots, seeds, flowers, grasses and leaves, by the post-extraction spiked method, a reliable sample clean-up method based on home-made clean-up cartridges, a suitable internal standard and the matrix calibration were combined using to minimize the matrix effects to ensure the accuracy of the method. The established method was further validated by determining the linearity (R(2)≥0.9990), sensitivity (limit of quantitation 0.11-0.99 ng mL(-1)), average recovery (86.6-113.5%) and precision (relative standard deviation ≤13.5%). It was shown to be a suitable method for simultaneous determination of ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL in different TCMs. Finally, the established method was successfully applied to the determination of the six mycotoxins in various TCMs and the results were presented to provide relevant insights to researchers in TCM analysis.     

The activities of mycotoxins derived from Fusarium and related substances in a short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells)

Cell transformation assays using BALB/3T3 cells can mimic the two-stage process of chemical carcinogenesis in experimental animals. A short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells), which was developed by Ohmori et al. and modified by Asada et al., has been reported to detect both tumor initiators and promoters as transformation initiators and promoters, respectively, with their differences based on their protocols. In this new short-term assay, we examined mycotoxins derived from Fusarium and related substances for the initiation and promotion activities of the transformation. The tested substances included deoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, fumonisin B₁, fumonisin B₂, zearalenone, α-zearalanol, β-zearalanol, α-zearalenol and β-zearalenol. Fumonisin B₁ and T-2 toxin were positive for promoting activity in the assay. Especially, T-2 toxin was active at concentrations as low as 0.001–0.002 μg/mL in the culture medium. From a comparison between the results of this study and published carcinogenicity assay data, it was expected that the Bhas 42 cell transformation assay had a good correlation with the two-stage carcinogenicity tests using experimental animals for estimation of the tumor-promoting activity.     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

A novel adhesion molecule in human breast cancer cells: Voltage-gated Na+ channel β1 subunit

Voltage-gated Na⁺ channels (VGSCs), predominantly the ‘neonatal’ splice form of Na_v1.5 (nNa_v1.5), are upregulated in metastatic breast cancer (BCa) and potentiate metastatic cell behaviours. VGSCs comprise one pore-forming α subunit and one or more β subunits. The latter modulate VGSC expression and gating, and can function as cell adhesion molecules of the immunoglobulin superfamily. The aims of this study were (1) to determine which β subunits were expressed in weakly metastatic MCF-7 and strongly metastatic MDA-MB-231 human BCa cells, and (2) to investigate the possible role of β subunits in adhesion and migration. In both cell lines, the β subunit mRNA expression profile was SCN1B (encoding β1) ? SCN4B (encoding β4) > SCN2B (encoding β2); SCN3B (encoding β3) was not detected. MCF-7 cells had much higher levels of all β subunit mRNAs than MDA-MB-231 cells, and β1 mRNA was the most abundant. Similarly, β1 protein was strongly expressed in MCF-7 and barely detectable in MDA-MB-231 cells. In MCF-7 cells transfected with siRNA targeting β1, adhesion was reduced by 35%, while migration was increased by 121%. The increase in migration was reversed by tetrodotoxin (TTX). In addition, levels of nNa_v1.5 mRNA and protein were increased following β1 down-regulation. Stable expression of β1 in MDA-MB-231 cells increased functional VGSC activity, process length and adhesion, and reduced lateral motility and proliferation. We conclude that β1 is a novel cell adhesion molecule in BCa cells and can control VGSC (nNa_v1.5) expression and, concomitantly, cellular migration.     

Antiproliferative constituents from Aphananthe aspera leaves

Extensive screening for the antiproliferative activity of different compounds found in trees was performed by extracting the leaves of Aphananthe aspera (Thunb.) Planch and then using chromatographic separation to afford 2 new compounds, (2S,4R)-2-carboxy-4-(E)-p-caffeoyl-1-methyl-hydroxyproline (1) and 5-O-caffeoyl quinic acid-(7′R,8′S,7′′E)-3′,4′,3′′-dihydroxy-4′′,7′-epoxy-8′,5′′-neolign-7′-ene-9- carboxyl (2). In addition, 6 known compounds were discovered from the leaves of this plant. The structural determination of all compounds, including their absolute configurations, was established by UV, IR, HRESIMS, 1D and 2D NMR, and CD spectroscopy. The novel compound 1 showed strong antiproliferative activity against human breast adenocarcinoma cells MCF-7 and MDA-MB-231.     

Crystal structure of the novel complex formed between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma

This is the first report on the formation of a complex between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an R_cryst of 0.199 (R_free = 0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven β-strands that are organized in the form of two antiparallel β-pleated sheets, resulting in the formation of a sandwiched β-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the β-structure of PIP is ideally aligned with the β-structure of domain α3 of ZAG to form a long interface between two proteins. The proximal β-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41-Asp233 and Lys68-Glu229. On the perpendicular interface involving α1-α2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 Å², which is considerably higher than the 660 Å² reported for the class I major histocompatibility complex structures.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

The effect of interleukin-1β, interleukin-6, and tumor necrosis factor-α on estradiol-17β release in the myometrium: The in vitro study on the pig model

Estradiol-17β (E₂) is a potent regulator of early pregnancy and the estrous cycle in pigs. Production of E₂ occurs in the porcine myometrium, but the factors involved in its regulation are unknown. In this in vitro study, it was investigated whether interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α affect the release of E₂ from the porcine myometrium on Days 10 to 11, 12 to 13, and 15 to 16 of pregnancy and the estrous cycle. The expression of the cytochrome P450 family 19 (CYP19) gene and the presence of the aromatase cytochrome P450 protein in the myometrium confirmed the ability of the tissue to produce E₂. In gravid pigs, the expression of IL1RI mRNA and IL6R mRNA was markedly increased on Days 15 to 16 of gestation, whereas TNFRI mRNA was increased on Days 10 to 11 of gestation. In cyclic pigs, the expression of myometrial IL1RI mRNA did not differ among the studied days, although the expression of IL6R and TNFRI mRNAs was increased on Days 15 to 16. In gravid pigs, IL-1β, IL-6, and TNF-α increased myometrial E₂ secretion on Days 15 to 16 but did not affect E₂ release on Days 10 to 11 and 12 to 13 of pregnancy. In cyclic pigs, IL-1β, IL-6, and TNF-α did not increase myometrial E₂ release. In conclusion, IL-1β, IL-6, and TNF-α affected myometrial E₂ release in a manner that is dependent on the physiologic status of the female. The porcine myometrium expresses IL1RI, IL6R, and TNFRI genes and is the target tissue for IL-1β, IL-6, and TNF-α. In gravid pigs, IL-1β, IL-6, and TNF-α may increase myometrial release of E₂in vitro specifically on Days 15 to 16 of pregnancy. These findings may be of interest to researchers using pigs as an animal model for fetal programming.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean (), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, α-zearalenol, and β-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.     

Estrogenic and anti-estrogenic compounds from the Thai medicinal plant, Smilax corbularia (Smilacaceae)

From the rhizomes of Smilax corbularia Kunth. (Smilacaceae), 11 compounds, (2R,3R)-2″-acetyl astilbin, (2R,3R)-3″-acetyl astilbin, (2R,3R)-4″-acetyl astilbin, (2R,3R)-3″-acetyl engeletin, (2R,3S)-4″-acetyl isoastilbin, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10R)-2H,8H-benzo [1,2-b:3,4-b′] dipyran-8-one, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10S)-2H, 8H-benzo [1,2-b:3,4-b′] dipyran-8-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxy-phenyl)-5-[(1E)-2-(3,4-dihydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, and 5,7,3′,4′-tetrahydroxy-3-phenylcoumarin along with 34 known compounds were isolated and characterized as 19 flavonoids, 14 catechin derivatives, 6 stilbene derivatives, and 6 miscellaneous substances. All isolates had their estrogenic and anti-estrogenic activities determined using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D. The major constituents were recognized as flavanonol rhamnosides by the suppressive effect on estradiol induced cell proliferation at a concentration of 1 μM. Meanwhile, flavanonol rhamnoside acetates demonstrated estrogenic activity in both MCF-7 and T47D cells at a concentration of 100 μM, and they enhanced the effects of co-treated E2 on T47D cell proliferation at concentrations of more than 0.1 μM.     

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β2-adrenergic receptor

The β₂-adrenergic receptor (β₂-AR) agonist [³H]-(R,R′)-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (K_i values) of the stereoisomers of a series of 4′-methoxyfenoterol analogs in which the length of the alkyl substituent at α′ position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [³H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α′ position. The results also indicate that the K_i values obtained using [³H]-(R,R′)-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [³H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [³H]-(R,R′)-4′-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [³H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.     

Synthesis,cytotoxic activity,and tubulin polymerization inhibitory activity of new pyrrol-2(3H)-ones and pyridazin-3(2H)-ones

A series of new pyrrol-2(3H)-ones 4a-f and pyridazin-3(2H)-ones 7a-f were synthesized and characterized using different spectroscopic tools. Some of the tested compounds revealed moderate activity against 60 cell lines. The E form of the pyrrolones 4 showed good cytotoxic activity than both the Z form and the corresponding open amide form. Furthermore, the in vitro cytotoxic activity against HepG2 and MCF-7 cell lines revealed that compounds (E)4b, 6f and 7f showed good cytotoxic activity against HepG2 with IC₅₀ values of 11.47, 7.11 and 14.80 μM, respectively. Compounds (E)4b, 6f, 7d and 7f showed a pronounced inhibitory effect against cellular localization of tubulin. Flow cytometric analysis indicated that HepG2 cells treated with (E)4b showed a predominated growth arrest at the S-phase compared to that of G2/M-phase. Molecular modeling study using MOE® program indicated that most of the target compounds showed good binding of β-subunit of tubulin with the binding free energy (dG) values about −10 kcal/mole.