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Human exposure to environmental contaminants is widespread. Some of these contaminants have the ability to interfere with adipogenesis, being thus considered as obesogens. Recently, obesogens have been singled out as a cause of male infertility. Sertoli cells (SCs) are essential for male fertility and their metabolic performance, especially glucose metabolism, is under a tight endocrine control, being essential for the success of spermatogenesis. Herein, we studied the impact of the model obesogen tributyltin in the metabolic profile of SCs. For that, ex vivo-cultured rat SCs were exposed to increasing doses of tributyltin. SCs proliferation was evaluated by the sulforhodamine B assay and the maturation state of the cells was assessed by the expression of specific markers (inhibin B and the androgen receptor) by quantitative polymerase chain reaction. The metabolic profile of SCs was established by studying metabolites consumption/production by nuclear magnetic resonance spectroscopy and by analyzing the expression of key transporters and enzymes involved in glycolysis by Western blot. The proliferation of SCs was only affected in the cells exposed to the highest dose (1000 nM) of tributyltin. Notably, SCs exposed to 10 nM tributyltin decreased the consumption of glucose and pyruvate, as well as the production of lactate. The decreased lactate production hampers the development of germ cells. Intriguingly, the lowest levels of tributyltin were more prone to modulate the expression of key players of the glycolytic pathway. This is the first study showing that tributyltin reprograms glucose metabolism of SCs under ex vivo conditions, suggesting new targets and mechanisms through which obesogens modulate the metabolism of SCs and thus male (in)fertility.

Metabolic reprogramming has been associated to a plethora of diseases, and there has been increased demand for methodologies able to determine the metabolic alterations that characterize the pathological states and help developing metabolically centered therapies. In this chapter, methodologies for monitoring TCA cycle turnover and its interaction with pyruvate cycling and anaplerotic reactions will be presented. These methodologies are based in the application of stable 13 C isotope "tracers"/substrates and 13 C-NMR isotopomer analysis of metabolic intermediates. These methodologies can be applied at several organizational levels, ranging from isolated organelles and organs to whole organisms/humans. For the sake of simplicity, only very simple and well-defined models will be presented, including isolated heart mitochondria and isolated perfused hearts and livers.

Nuclear magnetic resonance (NMR)-based metabolomics is widely used in the research of metabolic conditions of complex biological systems under various conditions, and its use has been found in the field of male fertility. Here we describe the implementation of total and targeted NMR-based metabolomics in the research on Sertoli cell metabolism. Main principles and techniques of cell medium, cellular extracts, and intact cells are explained, as well as some classical experiments that can give complementary information on the Sertoli cell metabolism.

Although silver nanoparticles (AgNPs) are widely disseminated and show great potential in the biomedical field, there is a recognized need to better understand their action at the metabolic and functional levels. In this work, we have used NMR metabolomics, together with conventional clinical chemistry and histological examination, to characterize multi-organ and systemic metabolic responses to AgNPs intravenously administered to mice at 8 mg/kg body weight (a dose not eliciting overt toxicity). The major target organs of AgNPs accumulation, liver and spleen, showed the greatest metabolic changes, in a clear 2-stage response. In particular, the liver of dosed mice was found to switch from glycogenolysis and lipid storage, at 6 h postinjection, to glycogenesis and lipolysis, at subsequent times up to 48 h. Moreover, metabolites related to antioxidative defense, immunoregulation and detoxification seemed to play a crucial role in avoiding major hepatic damage. The spleen showed several early changes, including depletion of several amino acids, possibly reflecting impairment of hemoglobin recycling, while only a few differences remained at 48 h postinjection. In the heart, the metabolic shift towards TCA cycle intensification and increased ATP production possibly reflected a beneficial adaptation to the presence of AgNPs. On the other hand, the TCA cycle appeared to be down regulated in the lungs of injected mice, which showed signs of inflammation. Thekidneys showed the mildest metabolic response to AgNPs. Overall, this study has shown that NMR metabolomics is a powerful tool to monitor invivo metabolic responses to nanoparticles, revealing unforeseen effects., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Nuclear magnetic resonance (NMR) is a very powerful instrumental technique suited to identify and characterize organic compounds. NMR has been successfully used in the analysis of complex biological and environmental samples; however, these applications are still rather limited. In this work, we describe unsupervised component analysis as a multivariate unsupervised method suited to identify the number of relevant NMR signal contributions and to deconvolve mixed signals into signal individual sources and respective contributions. Using this approach, we were able to advance further in the field of quantification of NMR spectra, and this methodology will help in the characterization of complex biological samples. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Introduction: We recently showed that a single high dose of methamphetamine (METH) induces a persistent frontal cortical monoamine depletion that is accompanied by helpless-like behavior in mice. However, brain metabolic alterations underlying both neurochemical and mood alterations remain unknown., Aims: Herein, we aimed at characterizing frontal cortical metabolic alterations associated with early negative mood behavior triggered by METH. Adult C57BL/6 mice were injected with METH (30 mg/kg, i.p.), and their frontal cortical metabolic status was characterized after probing their mood and anxiety-related phenotypes 3 days postinjection., Results: Methamphetamine induced depressive-like behavior, as indicated by the decreased grooming time in the splash test and by a transient decrease in sucrose preference. At this time, METH did not alter anxiety-like behavior or motor functions. Depolarization-induced glucose uptake was reduced in frontocortical slices from METH-treated mice compared to controls. Consistently, astrocytic glucose transporter (GluT1) density was lower in the METH group. A proton high rotation magic angle spinning (HRMAS) spectroscopic approach revealed that METH induced a significant decrease in N-acetyl aspartate (NAA) and glutamate levels, suggesting that METH decreased neuronal glutamatergic function in frontal cortex., Conclusions: We report, for the first time, that a single METH injection triggers early self-care and hedonic deficits and impairs frontal cortical energetics in mice., (© 2016 John Wiley & Sons Ltd.)

Obesity is a worldwide epidemic, and associated pathologies, including type 2 diabetes and cardiovascular alterations, are increasingly escalating morbidity and mortality. Despite intensive study, no effective simple treatment for these conditions exists. As such, the need for go-to drugs is serious. Bile acids (BAs) present the possibility of reversing these problems, as various in vivo studies and clinical trials have shown significant effects with regard to weight and obesity reduction, insulin sensitivity restoration and cardiovascular improvements. However, the mechanism of action of BA-induced metabolic improvement has yet to be fully established. The currently most accepted model involves non-shivering thermogenesis for energy waste, but this is disputed. As such, we propose to determine whether the BA chenodeoxycholic acid (CDCA) can exert anti-obesogenic effects in vitro, independent of thermogenic brown adipose tissue activation. By exposing differentiated 3 T3-L1 adipocytes to high glucose and CDCA, we demonstrate that this BA has anti-obesity effects in vitro. Nuclear magnetic resonance spectroscopic analysis of metabolic pathways clearly indicates an improvement in metabolic status, as these cells become more oxidative rather than glycolytic, which may be associated with an increase in fatty acid oxidation. Our work demonstrates that CDCA-induced metabolic alterations occur in white and brown adipocytes and are not totally dependent on endocrine/nervous system signaling, as thought until now. Furthermore, future exploration of the mechanisms behind these effects will undoubtedly reveal interesting targets for clinical modulation., (Copyright © 2016 John Wiley & Sons, Ltd.)

The widespread use of silver nanoparticles (AgNPs) is accompanied by a growing concern regarding their potential risks to human health, thus calling for an increased understanding of their biological effects. The aim of this work was to systematically study the extent to which changes in cellular metabolism were dependent on the properties of AgNPs, using NMR metabolomics. Human skin keratinocytes (HaCaT cells) were exposed to citrate-coated AgNPs of 10, 30 or 60 nm diameter and to 30 nm AgNPs coated either with citrate (CIT), polyethylene glycol (PEG) or bovine serum albumin (BSA), to assess the influence of NP size and surface chemistry. Overall, CIT-coated 60 nm and PEG-coated 30 nm AgNPs had the least impact on cell viability and metabolism. The role of ionic silver and reactive oxygen species (ROS)-mediated effects was also studied, in comparison to CIT-coated 30 nm particles. At concentrations causing an equivalent decrease in cell viability, Ag(+ )ions produced a change in the metabolic profile that was remarkably similar to that seen for AgNPs, the main difference being the lesser impact on the Krebs cycle and energy metabolism. Finally, this study newly reported that while down-regulated glycolysis and disruption of energy production were common to AgNPs and H2O2, the impact on some metabolic pathways (GSH synthesis, glutaminolysis and the Krebs cycle) was independent of ROS-mediated mechanisms. In conclusion, this study shows the ability of NMR metabolomics to define subtle biochemical changes induced by AgNPs and demonstrates the potential of this approach for rapid, untargeted screening of pre-clinical toxicity of nanomaterials in general.

Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium ((2)H) enrichment was determined following 6 days in (2)H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day(-1)) were calculated for hepatic TG-glyceryl and FA moieties through (2)H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Type 2 diabetes mellitus is a complex metabolic disease, and cardiovascular disease is a leading complication of diabetes. Epicardial adipose tissue surrounding the heart displays biochemical, thermogenic, and cardioprotective properties. However, the metabolic cross-talk between epicardial fat and the myocardium is largely unknown. This study sought to understand epicardial adipose tissue metabolism from heart failure patients with or without diabetes. We aimed to unravel possible differences in glucose and lipid metabolism between human epicardial and subcutaneous adipocytes and elucidate the potential underlying mechanisms involved in heart failure. Insulin-stimulated [(14)C]glucose uptake and isoproterenol-stimulated lipolysis were measured in isolated epicardial and subcutaneous adipocytes. The expression of genes involved in glucose and lipid metabolism was analyzed by reverse transcription-polymerase chain reaction in adipocytes. In addition, epicardial and subcutaneous fatty acid composition was analyzed by high-resolution proton nuclear magnetic resonance spectroscopy. The difference between basal and insulin conditions in glucose uptake was significantly decreased (P= 0.006) in epicardial compared with subcutaneous adipocytes. Moreover, a significant (P< 0.001) decrease in the isoproterenol-stimulated lipolysis was also observed when the two fat depots were compared, and it was strongly correlated with lipolysis, lipid storage, and inflammation-related gene expression. Moreover, the fatty acid composition of these tissues was significantly altered by diabetes. These results emphasize potential metabolic differences between both fat depots in the presence of heart failure and highlight epicardial fat as a possible therapeutic target in situ in the cardiac microenvironment., (Copyright © 2016 the American Physiological Society.)

Klinefelter syndrome (KS) is the most common genetic cause of human infertility, but the mechanism(s) responsible for its phenotype remain largely unknown. KS is associated with alterations in body composition and with a higher risk of developing metabolic diseases. We therefore hypothesized that KS men seeking fertility treatment possess an altered testicular metabolism profile that may hamper the nutritional support of spermatogenesis. Testicular biopsies from control (46, XY) (n = 6) and KS (47, XXY) (n = 6) individuals were collected and analyzed by proton high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy. The mRNA and protein expression of crucial glycolysis-associated enzymes and transporters were evaluated in parallel by quantitative PCR and Western blot, respectively. Our data revealed altered regulation of glucose transporters (GLUT1 and GLUT3); phosphofructokinase 1, liver isoform (PFKL); and lactate dehydrogenase A (LDHA) expression in the testis of KS patients. Moreover, we detected a severe reduction in lactate and creatine accumulation within testicular tissue from KS men. The aberrant levels of the biomarkers detected in testicular biopsies of KS men may therefore be associated with the infertility phenotypes presented by these men. Mol. Reprod. Dev. 83: 208-216, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia, but in addition has several other biological targets, including the NMDA receptor (NR). Here we describe the inhibitory potencies of 76 pentamidine analogs at 2 binding sites of the NR, the channel binding site labeled with [(3)H]MK-801 and the [(3)H]ifenprodil binding site. Most analogs acted weaker at the ifenprodil than at the channel site. The spermine-sensitivity of NR inhibition by the majority of the compounds was reminiscent of other long-chain dicationic NR blockers. The potency of the parent compound as NR blocker was increased by modifying the heteroatoms in the bridge connecting the 2 benzamidine moieties and also by integrating the bridge into a seven-membered ring. Docking of the 45 most spermine-sensitive bisbenzamidines to a recently described acidic interface between the N-terminal domains of GluN1 and GluN2B mediating polyamine stimulation of the NR revealed the domain contributed by GluN1 as the most relevant target., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30% digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5% (2)H2O over the last six feeding days, and quantifying the incorporation of (2)H into blood glucose and hepatic glycogen by (2)H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P < 0.001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P < 0.001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (sem 1) μmol/kg per h; P < 0.001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

Background: Cancer cells are widely recognized for being able to adapt their metabolism towards converting available nutrients into biomass to increase proliferation rates., Materials and Methods: We will review a series of nuclear magnetic resonance (NMR)-based stable isotope tracer methodologies for probing cancer metabolism., Results: The monitoring of such adaptations is of the utmost importance to unravel cancer metabolism and tumour growth. Several major metabolic targets have been recognized as promising foci and have been addressed by multiple studies in recent years. In this work are presented strategies to quantify glycolysis, pentose phosphate pathway, Krebs cycle turnover and de novo lipogenesis by NMR isotopomer analysis., Conclusions: Being able to adequately define the interplay between metabolic pathways allows the monitoring of their prevalence in tissues and such information is critical for an accurate knowledge of the metabolic distinctive nature of tumours towards devising more efficient antitumorigenic strategies. Discussed methodologies are currently available in the literature, but to date, no single review has compiled all their possible uses, particularly in an interdependent perspective., (© 2015 Stichting European Society for Clinical Investigation Journal Foundation.)

Sources of blood glucose in European seabass (initial weight 218.0±43.0g; mean±S.D., n=18) were quantified by supplementing seawater with deuterated water (5%-(2)H2O) for 72h and analyzing blood glucose (2)H-enrichments by (2)H NMR. Three different nutritional states were studied: continuously fed, 21-day of fast and 21-day fast followed by 3days of refeeding. Plasma glucose levels (mM) were 10.7±6.3 (fed), 4.8±1.2 (fasted), and 9.3±1.4 (refed) (means±S.D., n=6), showing poor glycemic control. For all conditions, (2)H-enrichment of glucose position 5 was equivalent to that of position 2 indicating that blood glucose appearance from endogenous glucose 6-phosphate (G6P) was derived by gluconeogenesis. G6P-derived glucose accounted for 65±7% and 44±10% of blood glucose appearance in fed and refed fish, respectively, with the unlabeled fraction assumed to be derived from dietary carbohydrate (35±7% and 56±10%, respectively). For 21-day fasted fish, blood glucose appearance also had significant contributions from unlabeled glucose (52±16%) despite the unavailability of dietary carbohydrates. To assess the role of hepatic enzymes in glycemic control, activity and mRNA levels of hepatic glucokinase (GK) and glucose 6-phosphatase (G6Pase) were assessed. Both G6Pase activity and expression declined with fasting indicating the absence of a classical counter-regulatory stimulation of hepatic glucose production in response to declining glucose levels. GK activities were basal during fed and fasted conditions, but were strongly stimulated by refeeding. Overall, hepatic G6Pase and GK showed limited capacity in regulating glucose levels between feeding and fasting states., (© 2013.)

The stimulation of hepatic glycogenesis is a ubiquitous response to a glucose challenge and quantifying its contribution to glucose uptake informs its role in restoring euglycemia. Glycogenesis can be quantified with labeled water provided that exchange of glucose-6-phosphate hydrogen 2 (G6P-H2) and body water via glucose-6-phosphate isomerase, and exchange of positions 4, 5 and 6 hydrogens (G6P-H456) via transaldolase, are known. These exchanges were quantified in 24-h fasted rats (Rattus norvegicus; n=6) and 21-day fasted seabass (Dicentrarchus labrax; n=8) by administration of a glucose load (2000mg·kg(-1)) enriched with [U-(2)H7]glucose and by quantifying hepatic glycogen (2)H-enrichments after 2h (rats) and 48h (seabass). Direct pathway contributions of the glucose load to glycogenesis were also estimated. G6P-H2 and body water exchange was 61±1% for rat and 47±3% for seabass. Transaldolase-mediated exchange of G6P-H456 was 5±1% for rat and 10±1% for seabass. Conversion of the glucose load to hepatic glycogen was significant in seabass (249±54mg·kg(-1)) but negligible in rats (12±1mg·kg(-1)). Preload plasma glucose levels were similar for seabass and rats (3.3±0.7 and 4.4±0.1mmol·L(-1), respectively) but post-load plasma glucose was significantly higher in seabass compared to rats (14.6±1.8 versus 5.8±0.3mmol·L(-1), p<0.01). In conclusion, G6P-H2 and body water exchange is incomplete for both species and has to be accounted for in estimating hepatic glycogen synthesis and direct pathway activities with labeled water tracers. Transaldolase-mediated exchange is insignificant. Hepatic direct pathway glycogenesis plays a prominent role in seabass glucose load disposal, but a negligible role in the rat., (© 2013.)

Hepatic glycogen synthesis fluxes from direct and indirect pathways were quantified in seabass by postmortem (2)H NMR analysis of plasma water (PW) and glycogen glucosyl (2)H enrichments from (2)H-enriched seawater. Eighteen fish (28.0 ± 1.7 cm and 218.0 ± 43.0 g) were divided into three groups of 6 and studied over 24 days with transfer to 5% (2)H-seawater after day 21. Over this period, one group was fed daily with fishmeal, a second group was fasted, and a third group was fasted for 21 days followed by 3 days refeeding. Glycogen turnover and sources were determined from the ratio of glucosyl position 5 enrichment to that of plasma water (H5/PW). Glycogen levels of fed fish were significantly higher than fasted (665.4 ± 345.2 μmol.g(-1) liver versus 77.2 ± 59.5 μmol.g(-1) liver, P<0.05) while refed fish had comparable levels to fed (584.6 ± 140.4 μmol.g(-1) liver). Glycogen enrichment of fed fish was undetectable indicating negligible turnover over 3 days. For fasted fish, H5/PW was ~50% indicating that half of the glycogen had turned over via indirect pathway flux. For refed fish, H5/PW was ~100% indicating that the indirect pathway accounted for all net glycogen synthesis. Direct pathway conversion of dietary carbohydrate to glycogen was not detected in any of the groups., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A series of new N,N-dimethylaminopropyl- and 2-imidazolinyl-substituted derivatives of benzo[b]thienyl- and thieno[2,3-b]thienylcarboxanilides and benzo[b]thieno[2,3-c]- and thieno[3',2':4,5]thieno[2,3-c]quinolones were prepared. Quinolones were prepared by the reaction of photochemical dehydrohalogenation of corresponding anilides. Carboxanilides and quinolones were tested for the antiproliferative activity. 2-Imidazolinyl-substituted derivatives showed very prominent activity. By use of the experimentally obtained antitumor measurements, 3D-derived QSAR analysis was performed for the set of compounds. Highly predictive 3D-derived QSAR models were obtained, and molecular properties that have the highest impact on antitumor activity were identified. Carboxanilides 6a-c and quinolones 9a-c and 11a were evaluated for DNA binding propensities and topoisomerases I and II inhibition as part of their mechanism of action assessment. The evaluated differences in the mode of action nicely correlate with the results of the 3D-QSAR analysis. Taken together, the results indicate which modifications of the compounds from the series should further improve their anticancer properties.

Six recently synthesized cyano-substituted heteroaryles, which do not bind to DNA but are highly cytotoxic against the human tumor cell line HeLa, were analyzed for their antitumor mechanisms of action (MOA). They did not interfere with the expression of human papillomavirus oncogenes integrated in the HeLa cell genome, but they did induce strong G1 arrest and result in the activation of caspase-3 and apoptosis. A computational analysis was performed that compared the antiproliferative activities of our compounds in 13 different tumor cell lines with those of compounds listed in the National Cancer Institute database. The results indicate that interference with cytoskeletal function and inhibition of mitosis are the likely antitumor MOA. Furthermore, a second in silico investigation revealed that the tumor cells that are sensitive to the cyano-substituted compounds show differences in their expression of locomotion genes compared with that of insensitive cell lines, thus corroborating the involvement of the cytoskeleton. This MOA was also confirmed experimentally: the cyano-substituted heteroaryles disrupted the actin and the tubulin networks in HeLa cells and inhibited cellular migration. However, further analysis indicated that multiple MOA may exist that depend on the position of the cyano-group; while cyano-substituted naphthiophene reduced the expression of cytoskeletal proteins, cyano-substituted thieno-thiophene-carboxanilide inhibited the formation of cellular reactive oxygen species.

Nuclear magnetic resonance (NMR) spectroscopy is a technique with an increasing importance in the study of metabolic diseases. Its initial important role in the determination of chemical structures (1, 2) has been considerably overcome by its potential for the in vivo study of metabolism (3-5). The main characteristic that makes this technique so attractive is its noninvasiveness. Only nuclei capable of transitioning between energy states, in the presence of an intense and constant magnetic field, are studied. This includes abundant nuclei such as proton ((1)H) and phosphorous ((31)P), as well as stable isotopes such as deuterium ((2)H) and carbon 13 ((13)C). This allows a wide range of applications that vary from the determination of water distribution in tissues (as obtained in a magnetic resonance imaging scan) to the calculation of metabolic fluxes under ex vivo and in vivo conditions without the need to use radioactive tracers or tissue biopsies (as in a magnetic resonance spectroscopy (MRS) scan). In this chapter, some technical aspects of the methodology of an NMR/MRS experiment as well as how it can be used to study mitochondrial bioenergetics are overviewed. Advantages and disadvantages of in vivo MRS versus high-resolution NMR using proton high rotation magic angle spinning (HRMAS) of tissue biopsies and tissue extracts are also discussed.

Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ((2)H(2)O). After transfer to seawater enriched with 4.9% (2)H(2)O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by (2)H NMR and ESI-MS/MS. Plasma water (2)H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72 h. The distribution of (2)H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during (2)H(2)O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h (2)H(2)O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Novel amidino-substituted conformationally restricted derivatives of pentamidine were synthesized and their antiproliferative activity against several human cancer cell lines determined. It was found that introduction of furandicarboxamide core moiety (9, 10) increases antiproliferative activity as well as selectivity against certain tumor cell lines in comparison with amidino-substituted furan-mono-carboxamide (5, 6). Unlike the furan series where iso-propyl substituted amidine (10) exhibits more potent overall antiproliferative activity and selectivity toward certain cell lines, the same was found for unsubstituted amidines in pyridine series. Amongst all tested compounds the compound 10 is the only one that possesses antiproliferative activity against SW 620 cell line (4 μM). Spectroscopic studies of the interactions of prepared diamidines with double-stranded DNA and RNA polynucleotides show that all compounds preferentially bind into the minor groove of DNA, while most of them intercalate into RNA. The structure-dependant biological activity and the lack of DNA/RNA selective binding suggest that the mechanism of action of the here-presented compounds is controlled not only by the interactions with cellular nucleic acids, but also with other more specific protein targets., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Novel cyano- and 2-imidazolinyl-substituted derivatives of pyridylbenzo[b]thiophene-2-carboxamides 4, 5, 10-13 and benzo[b]thieno[2,3-c]naphthyridin-2-ones 6, 7, 14-17 were prepared. All derivatives showed a prominent antiproliferative effect. Extensive DNA binding studies and additional biological evaluations point to various modes/targets of action. The results strongly support intercalation into DNA as a dominant binding mode of fused analogues, which was substantiated using topoisomerase I inhibition assay. Most intriguingly, only minor structural difference between "nonfused" compounds 12 and 13 has strong impact on the interactions with DNA; while 13 binds within the DNA minor groove in the form of dimer, 12 does not form significant interactions with DNA. The assumption that severe mitotic impairment (G2/M phase arrest) induced by 12 could point to tubulin, another important target, was confirmed by its obvious anti-tubulin activity observed in immunofluorescence assay, whereby treated cells showed disruption of microtubule formation comparable to the effect obtained by paclitaxel, a well-known tubulin antagonist chemotherapeutic.

A series of cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and their 'cyclic' derivatives (quinolones) were synthesized. 'Cyclic' compounds displayed a rather strong and differential antiproliferative effect on various cell lines, while the 'acyclic' amidino-substituted compounds were much more active, but showing mostly non-differential cytotoxicity, whereas cyano-substituted compounds (2a,b) produced a strikingly strong effect selectively on HeLa and Hep-2 cell lines. Antiproliferative activity of 'cyclic' derivatives is very likely caused by intercalation into DNA, while their 'acyclic' analogues use other target(s) and/or mechanisms of action.

The title compound, C10H16N3+.Cl-.C2H6O, is an important intermediate in the convergent synthesis of amidine-substituted polycyclic heterocycles, a class of compounds that shows significant anticancer activity. The molecule of (I) is not planar, having a dihedral angle of 25.00 (7) degrees between the aniline and amidine (-C-NH=C=NH2) groups. The protonation of the amidine molecular fragment is accompanied by delocalized C-N bond distances of 1.320 (2) and 1.317 (2) A. The cations and chloride anions are involved in a network of hydrogen bonds, resulting in the formation of infinite chains propagating along the b direction. The chains are further grouped within the ab plane, in such a way that the structure is segregated into layers dominated by hydrophobic interactions involving N-isopropyl residues and layers dominated by N-H...Cl [N...Cl = 3.275 (2)-3.596 (2) A], O-H...Cl [O...Cl = 3.229 (3) A] and N-H...O [N...O = 2.965 (3) A] hydrogen bonds.