Time-resolved fluoroimmunoassay of plasma and urine O-desmethylangolensin

We present a method for the determination of the phytoestrogen metabolite O-desmethylangolensin (O-DMA) in plasma (serum) and in urine. O-DMA is a metabolite of daidzein, which occurs in soybeans. It has been suggested that isoflavones may afford protection against breast and prostate cancer and therefore, also the metabolites are of interest. The method is based on time-resolved fluoroimmunoassay (TR–FIA) using a europium chelate as a label. After the synthesis of 4′′-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4′′-O-derivative of the -methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR–FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6′-OH-angolensin, trans-4-OH-equol, 6′-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. Plasma samples are hydrolyzed and extracted. Urine samples are analyzed directly after hydrolysis without extraction. The correlation coefficient between the plasma TR–FIA results and the GC–MS results was high; r value was 0.985. The correlation coefficient between the urine TR–FIA results and the GC–MS results was high over the entire range of concentrations (0–1500 nmol/l); r value was 0.976, but lower in the low concentration range (0–100 nmol/l), i.e. value was 0.631. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8–7.7 and 3.0–6.0%, respectively and the inter-assay CVs varied 3.8–8.9 and 4.4–6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5–512 nmol/l.     

Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.     

Design,synthesis, docking,ADMET studies,and anticancer evaluation of new 3-methylquinoxaline derivatives as VEGFR-2 inhibitors and apoptosis inducers

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in cancer angiogenesis. Inhibition of VEGFR-2 activity proved effective suppression of tumour propagation. Accordingly, two series of new 3-methylquinoxaline derivatives have been designed and synthesised as VEGFR-2 inhibitors. The synthesised derivatives were evaluated in vitro for their cytotoxic activities against MCF-7and HepG2 cell lines. In addition, the VEGFR-2 inhibitory activities of the target compounds were estimated to indicate the potential mechanism of their cytotoxicity. To a great extent, the results of VEGFR-2 inhibition were highly correlated with that of cytotoxicity. Compound 27a was the most potent VEGFR-2 inhibitor with IC₅₀ of 3.2 nM very close to positive control sorafenib (IC₅₀ = 3.12 nM). Such compound exhibited a strong cytotoxic effect against MCF-7 and HepG2, respectively with IC₅₀ of 7.7 and 4.5 µM in comparison to sorafenib (IC₅₀ = 3.51 and 2.17 µM). In addition, compounds 28, 30f, 30i, and 31b exhibited excellent VEGFR-2 inhibition activities (IC₅₀ range from 4.2 to 6.1 nM) with promising cytotoxic activity. Cell cycle progression and apoptosis induction were investigated for the most active member 27a. Also, the effect of 27a on the level of caspase-3, caspase-9, and BAX/Bcl-2 ratio was determined. Molecular docking studies were implemented to interpret the binding mode of the target compounds with the VEGFR-2 pocket. Furthermore, toxicity and ADMET calculations were performed for the synthesised compounds to study their pharmacokinetic profiles     

Potential cytotoxicity of silver nanoparticles: Stimulation of autophagy and mitochondrial dysfunction in cardiac cells

In the present study, we elucidated the potential cytotoxicity of AgNPs in H9c2 rat cardiomyoblasts and assessed the underlying toxicological manifestations responsible for their toxicity thereof. The results indicated that the exposure of AgNPs to H9c2 cardiac cells decreased cell viability in a dose-dependent manner and caused cell cycle arrest followed by induction of apoptosis. The AgNPs treated cardiac cells showed a generation of reactive oxygen species (ROS) and mitochondrial dysfunction where mitochondrial ATP was reduced and the expression of AMPK1α increased. AgNPs also induced ROS-mediated autophagy in H9c2 cells. There was a significant time-dependent increase in intracellular levels of Atg5, Beclin1, and LC3BII after exposure to AgNPs, signifying the autophagic response in H9c2 cells. More importantly, the addition of N-acetyl-L-cysteine (NAC) inhibited autophagy and significantly reduced the cytotoxicity of AgNPs in H9c2 cells. The study highlights the prospective toxicity of AgNPs on cardiac cells, collectively signifying a potential health risk.     

Living roots magnify the response of soil organic carbon decomposition to temperature in temperate grassland

Increasing atmospheric carbon dioxide (CO₂) concentration is both a strong driver of primary productivity and widely believed to be the principal cause of recent increases in global temperature. Soils are the largest store of the world's terrestrial C. Consequently, many investigations have attempted to mechanistically understand how microbial mineralisation of soil organic carbon (SOC) to CO₂ will be affected by projected increases in temperature. Most have attempted this in the absence of plants as the flux of CO₂ from root and rhizomicrobial respiration in intact plant‐soil systems confounds interpretation of measurements. We compared the effect of a small increase in temperature on respiration from soils without recent plant C with the effect on intact grass swards. We found that for 48 weeks, before acclimation occurred, an experimental 3 °C increase in sward temperature gave rise to a 50% increase in below ground respiration (ca. 0.4 kg C m^?2; Q₁₀ = 3.5), whereas mineralisation of older SOC without plants increased with a Q₁₀ of only 1.7 when subject to increases in ambient soil temperature. Subsequent ¹⁴C dating of respired CO₂ indicated that the presence of plants in swards more than doubled the effect of warming on the rate of mineralisation of SOC with an estimated mean C age of ca. 8 years or older relative to incubated soils without recent plant inputs. These results not only illustrate the formidable complexity of mechanisms controlling C fluxes in soils but also suggest that the dual biological and physical effects of CO₂ on primary productivity and global temperature have the potential to synergistically increase the mineralisation of existing soil C.     

Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.     

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases.

After the synthesis of the 5′-carboxymethoxy derivative of enterolactone and 4′-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively.

We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 μmol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography–mass spectrometry (GC–MS). However, the assay results by the present method showed strong correlation with those obtained by GC–MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.     

Pomegranate peel induced biogenic synthesis of silver nanoparticles and their multifaceted potential against intracellular pathogen and cancer

In the field of nano-biotechnology, silver nanoparticles (AgNPs) share a status of high repute owing to their remarkable medicinal values. Biological synthesis of environment-friendly AgNPs using plant extracts has emerged as the beneficial alternative approach to chemical synthesis. In the current study, we have synthesized biogenic silver nanoparticles (PG-AgNPs) using the peel extract of Punica granatum as a reducing and stabilizing agent. The as-synthesized PG-AgNPs were characterized and evaluated for their antibacterial and anticancer potential. UV–Visible spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the formation of biogenic PG-AgNPs. The antibacterial potential was assessed against the biofilm of Listeria monocytogenes. The PG-AgNPs were efficacious against sessile bacteria and their biofilm as well. The as-synthesized nanoparticles at sub-MIC values showed dose-dependent inhibition of biofilm formation. Corroborating results were observed under crystal violet assay, Congo red staining, Confocal microscopy and SEM analysis. The anticancer ability of the nanoparticles was evaluated against MDA-MB-231 metastatic breast cancer cells. As evident from the MTT results, PG-AgNPs significantly reduced the cell viability in a dose-dependent manner. Exposure of MDA-MB-231 cells led to the accumulation of reactive oxygen species (ROS). Morphological changes and DNA fragmentation showed the strong positive effect of PG-AgNPs on the induction of apoptosis. Collectively, the as-synthesized PG-AgNPs evolved with synergistically emerged attributes that were effective against L. monocytogenes and also inhibited its biofilm formation; moreover, the system displayed lower cytotoxic manifestation towards mammalian cells. In addition, the PG-AgNPs embodies intriguing anticancer potential against metastatic breast cancer cells.     

Thioredoxin reductase and cancer cell growth inhibition by organotellurium compounds that could be selectively incorporated into tumor cells

The thioredoxins are small ubiquitous redox proteins with the conserved redox catalytic sequence-Trp-Cys-Gly-Pro-Cys-Lys, where the Cys residues undergo reversible NADPH dependent reduction by selenocysteine containing flavoprotein thioredoxin reductases. Thioredoxin expression is increased in several human primary cancers including lung, colon, cervix, liver, pancreatic, colorectal and squamous cell cancer. The thioredoxin/thioredoxin reductase pathway therefore provides an attractive target for cancer drug development. Organotellurium steroid, lipid, amino acid, nucleic base, and polyamine inhibitors were synthesized on the basis that they might be selectively or differentially incorporated into tumor cells. Some of the newly prepared classes of tellurium-based inhibitors (lipid-like compounds 3b and 3e, amino acid derivative 5b, nucleic base derivative 8b, and polyamine derivatives 14a and 14b) inhibited TrxR/Trx and cancer cell growth in culture with IC(50) values in the low micromolar range.