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Favipiravir is an antiviral agent effective against several RNA viruses that is converted into an inactive oxidative metabolite (M1), mainly by aldehyde oxidase, in humans. In the present study, the biotransformation of favipiravir into M1 in male and female humans, monkeys, rats, and mice was examined in an in vitro system using liver cytosolic fractions. The kinetics for M1 formation followed the Michaelis-Menten model in all species. The Km , Vmax , and CLint values in humans were 602 µM, 466 pmol/min/mg protein, and 776 nl/min/mg protein in males, respectively, and 713 µM, 404 pmol/min/mg protein, and 567 nl/min/mg protein in females, respectively. Species differences in CLint values were monkeys > humans > mice > rats in both males and females, and the variations for males and females were 120- and 96-fold, respectively. Sex differences in CLint values were males > females in humans and mice, females > males in monkeys and rats, and marked variation (4.3-fold) was noted in mice. This suggests that the roles of aldehyde oxidase in the hepatic metabolism of favipiravir differ extensively depending on the species and sex, and this study will aid in the assessment of the antiviral activities of favipiravir against novel and/or variant viruses.

More detailed investigations on the in vivo redox status are needed to elucidate the mechanisms contributing to damage caused by ionizing radiation. In the present study, the in vivo redox status of mice was examined using in vivo electron spin resonance (ESR) imaging after an intraperitoneal injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) as a probe. ACP is easily hydrolyzed to its hydroxylamine form in the mouse body, and the interconversion between hydroxylamine and the corresponding nitroxyl radical reflects the biological redox status. Liver damage, based on changes in liver weight and plasma aspartate aminotransferase levels, was detected in mice 4 days after X-ray irradiation at 7.5 Gy. ESR imaging showed that the signal intensity of the nitroxyl radical was high at the liver area in both damaged and healthy mice after administration of ACP. Whereas the signal decayed at the liver area for healthy mouse, the decay was negligible in damaged mice. Unlike healthy mouse, signal in the chest for damaged mouse increased with time. The distribution of the sum of hydroxylamine and the nitroxyl radical was similar in damaged and healthy mice. X-ray irradiation slightly lowered the reduction activity of the liver microsomal fraction for the nitroxyl radical. Thiobarbituric acid reactive substances in the liver were higher in damaged mice than in healthy mice; however, no significant differences were noted in reduced glutathione. The present results indicate that the redox status of mice exposed to X-ray irradiation is more oxidative than that in healthy mice.

Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18F-FDG-PET and lactate production, while pO2 was inversely related to lactate production and 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18F-FDG uptake negatively correlated with pO2 in the center of the tumor and positively correlated with pO2 on the periphery. In contrast to pO2 and 18F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. Significance: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis.

The constant increase of plastic waste released into the environment is a global problem which is raising concern to the general population. Although there are many different approaches for recycling plastics, chemical recycling is currently seen as one of the most promising technologies in that it allows plastic waste to fit into a sustainable, circular economy. Herein we investigate the chemical recycling of bisphenol A polycarbonate (BPA-PC) using diols of different chain lengths to yield bisphenol A and innovative carbonate-containing diols. Subsequently, the latter are polymerised into a series of unique value-added aliphatic polycarbonates (APC). The new polymers obtained by this method have shown promising values of ionic conductivity that make them attractive candidates to be implemented as sustainable polymer electrolytes for solid-state batteries. This procedure opens the way for recycling methods to produce unique, innovative materials using plastic waste as an alternative sustainable feedstock.

The odors and emanations released from the human body can provide important information about the health status of individuals and the presence or absence of diseases. Since these components often emanate from the body surface in very small quantities, a simple sampling and sensitive analytical method is required. In this study, we developed a non-invasive analytical method for the measurement of the body odor component 2-nonenal by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry by selective ion monitoring. Using a StableFlex PDMS/DVB fiber, 2-nonenal was efficiently extracted and enriched by fiber exposition at 50 °C for 45 min and was separated within 10 min using a DB−1 capillary column. Body odor sample was easily collected by gauze wiping. The limit of detection of 2-nonenal collected in gauze was 22 pg (S/N = 3), and the linearity was obtained in the range of 1–50 ng with a correlation coefficient of 0.991. The method successfully analyzed 2-nonenal in skin emissions and secretions and was applied to the analysis of body odor changes in various lifestyles, including the use of cosmetics, food intake, cigarette smoking, and stress load.

Drastic sensitivity enhancement of dynamic nuclear polarization is becoming an increasingly critical methodology to monitor real-time metabolic and physiological information in chemistry, biochemistry, and biomedicine. However, the limited number of available hyperpolarized 13C probes, which can effectively interrogate crucial metabolic activities, remains one of the major bottlenecks in this growing field. Here, we demonstrate [1-13C] N-acetyl cysteine (NAC) as a novel probe for hyperpolarized 13C MRI to monitor glutathione redox chemistry, which plays a central part of metabolic chemistry and strongly influences various therapies. NAC forms a disulfide bond in the presence of reduced glutathione, which generates a spectroscopically detectable product that is separated from the main peak by a 1.5 ppm shift. In vivo hyperpolarized MRI in mice revealed that NAC was broadly distributed throughout the body including the brain. Its biochemical transformation in two human pancreatic tumor cells in vitro and as xenografts differed depending on the individual cellular biochemical profile and microenvironment in vivo. Hyperpolarized NAC can be a promising non-invasive biomarker to monitor in vivo redox status and can be potentially translatable to clinical diagnosis.

Tuning the rheological properties of surfactant solutions by charge screening is a convenient formulation tool in cosmetic, household, oil recovery, drag-reduction, and thickening applications. Surfactants self-assemble in water, and upon charge screening and core shielding, they grow into long wormlike micelles (WLMs). These are valuable model systems for soft matter physics, and the most explored formulation is hexadecyl-trimethylammonium bromide (CTAB) and sodium salicylate (NaSal). Replacing NaSal with aromatic salts of altered hydrophobicity results in different penetration of the additive in the CTAB micellar core. This altered penetration depth will determine the anisotropic micellar growth that tailors the viscoelastic response. Sodium 4-methylsalicylate (mNaSal) is a higher hydrophobicity alternative to NaSal, requiring less additive to induce strong changes in the viscoelastic properties. Herein, we provide a comparative study of the mNaSal/CTAB system with the reference NaSal/CTAB over a range of temperatures and salt concentrations. The findings from the well-known NaSal/CTAB pair are transferred to the mNaSal/CTAB system, revealing the origins of the WLM solution’s viscoelastic properties by discerning contributions from charge screening and micellar core shielding upon small differences in hydrophobicity. ISSN:0743-7463 ISSN:1520-5827

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of β-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of β-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular β-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing β-1,2-linked arabinofuranose disaccharide (β-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) β-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the β-Ara2 disaccharide; this pocket is highly conserved among GH121 proteins. The three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all ~270 members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 β-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. β-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the β-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

Peptides are still difficult to synthesize when they contain sterically hindered amino acids, such as α,α-disubstituted amino acids and N-substituted amino acids. In this study, solid-phase syntheses of oligopeptides containing multiple α-aminoisobutyric acid (Aib) residues were performed in high yields by using a nonswellable resin as the solid-support and 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as the condensing reagent.

Water is an essential component of many biochemical reactions. Deuterated water (D2O) has been used to study cell kinetics, protein synthesis, and metabolism. We hypothesized that rapidly proliferating cancer cells would become preferentially labeled with deuterium due to high metabolic activity, thus allowing imaging of biosynthetically labeled metabolites within tumorsin vivo. We initiated systemic D2O labeling in two established tumor xenograft models, HT-29 and MiaPaCa-2 and imaged mice by deuterium magnetic resonance spectroscopic imaging (dMRSI). After 14 days of tumor growth and 7 days ofin vivolabeling, a clear contrast was demonstrated between the xenograft and the contralateral control limb in both models. The origin of the contrast was traced to an aliphatic peak at 1.8 ppm, which was identified byex vivoNMR analysis to originate from cholesterol and cholesterol esters. Cholesterol is important for tumor cell proliferation, signaling, and malignant transformation, while current methods to monitor cholesterol synthesis and accumulation are limited. This deuterated water labeling-imaging approach could complement current cancer imaging techniques, allowing not only imaging of uptake but also synthesis of cholesterol to elucidate effects on tumor cholesterol metabolismin vivo.

The heterocyclic portions of yuzurimine-type alkaloids, such as deoxyyuzurimine and macrodaphnine, were synthesized by using a stereoselective hydroboration-oxidation reaction to install the C20 methyl group, the intramolecular Mitsunobu reaction to construct the E-ring portion, and the intramolecular SN2 reaction to construct the F-ring portion as key steps.

Cationic aliphatic polycarbonates bearing primary ammonium side chains have been developed with relatively high molecular weights and controlled macromolecular architectures. These polycarbonates exhibit reasonable antimicrobial activity against Gram-negative and Gram-positive bacteria. The prepared homopolymers could be effective against Gram-negative bacteria whose growth is usually inhibited by copolymers with hydrophobic comonomer units when quaternary ammonium salts (QAS) are used at the cationic side chains. A methoxyethyl (ME) side chain was explored as a comonomer unit for modulating biological activities, besides conventional hydrophobic side chains including ethyl and benzyl groups. In contrast to the ethyl side chain that increases both antimicrobial and hemolytic activities, the ME side chain serves to enhance the antimicrobial activity, but suppresses the hemolytic activity. This could be attributed to the unique characteristics of an aliphatic polycarbonate bearing a ME side chain: hemocompatibility, cell adhesion property, and selective interactions with proteins. The benefits of blood compatibility of the cationic aliphatic polycarbonates with the use of the primary ammonium side chains have been reported for the first time. The polycarbonate main chain is subjected to hydrolysis, which reduces the inherent cytotoxicity of polycations. This hydrolytic property is specific to these primary ammonium-tagged polycarbonates and could be an advantage over previously reported QAS-tagged antimicrobial polycarbonates.

Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect. Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro. LDH activity was assessed in tumor homogenates. Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro. LDH activity correlated to FX-11 sensitivity. Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137–48. ©2018 AACR.

Euglena gracilis Z is a microorganism classified as a microalga and is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of E. gracilis Z, is reported to affect the immunological system. This study evaluated the symptom-relieving effects of E. gracilis Z and paramylon in rheumatoid arthritis in a collagen-induced arthritis mouse model. The efficacy of both substances was assessed based on clinical arthritis signs, as well as cytokine (interleukin [IL]-17, IL-6, and interferon [IFN]-γ) levels in lymphoid tissues. Additionally, the knee joints were harvested and histopathologically examined. The results showed that both substances reduced the transitional changes in the visual assessment score of arthritis symptoms compared with those in the control group, indicating their symptom-relieving effects on rheumatoid arthritis. Furthermore, E. gracilis Z and paramylon significantly reduced the secretion of the cytokines, IL-17, IL-6, and IFN-γ. The histopathological examination of the control group revealed edema, inflammation, cell hyperplasia, granulation tissue formation, fibrosis, and exudate in the synovial membrane, as well as pannus formation and articular cartilage destruction in the femoral trochlear groove. These changes were suppressed in both treatment groups. Particularly, the E. gracilis Z group showed no edema, inflammation, and fibrosis of the synovial membrane, or pannus formation and destruction of articular cartilage in the femoral trochlear groove. Furthermore, E. gracilis Z and paramylon exhibited symptom-relieving effects on rheumatoid arthritis and suppressed the secretion of cytokines IL-17, IL-6, and IFN-γ. These effects were likely mediated by the regulatory activities of E. gracilis Z and paramylon on Th17 immunity. In addition, the symptom-relieving effects of both substances were comparable, which suggests that paramylon is the active component of Euglena gracilis Z.

AIMS: Evofosfamide (TH-302) is a hypoxia-activated prodrug (HAP) that releases the DNA-damaging bromo-isophosphoramide mustard (Br-IPM) moiety selectively under hypoxic conditions. Since solid tumors are known to have hypoxic regions, HAPs in combination with chemotherapy or radiotherapy (XRT) will be beneficial. We tested the oxygen dependence of release kinetics of Br-IPM using electron paramagnetic resonance (EPR) with spin trapping by monitoring redox cycling of the nitroimidazole moiety of TH-302, and oxygen dependence of TH-302 on in vitro cytotoxicity at different levels of hypoxia was also examined. Two tumor implants (SCCVII and HT29) in mice were studied. RESULTS: TH-302 fragmentation to release Br-IPM was noticed at oxygen levels

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease, and disease activity is associated with serum IFN-α level. Plasmacytoid dendritic cells (pDCs) sense microbial as well as self-nucleic acids by TLRs 7 and 9 and produce a large amount of IFN-α. Here, we show that heat shock protein 90 (Hsp90) associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Inhibition of Hsp90 by various approaches including the use of Hsp90 inhibitor, a geldanamycin derivative, suppressed the Hsp90 association with TLR7/9, which resulted in inhibition of IFN-α production, leading to improvement of SLE symptoms in mice. Notably, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Furthermore, we demonstrated that serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Our data demonstrate that Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases.

Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has β-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum . At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1β, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection.

In this study, the phenylpropanoid composition and antioxidant activity of identified components in fig (Ficus carica L.) leaves were examined. Known polyphenols rutin, isoschaftoside, isoquercetin, and chlorogenic acid were identified. Furthermore, caffeoylmalic acid (CMA) was the most abundant polyphenol and was identified for the first time. CMA exhibited antioxidant activity similar to that of vitamin C or catechin. Psoralen and bergapten were identified as known furanocoumarins, with psoralen being the most abundant. Moreover, psoralic acid glucoside (PAG) was identified for the first time. As a precursor of psoralen, PAG content was equivalent to the psoralen content in moles. Notably, the content of these compounds varied between the five fig varieties, and the furanocoumarin and PAG contents varied more than that of the polyphenols. Further investigations concerning the influence of CMA and PAG on human health are necessary to elucidate functionalities of fig leaves.

Although mutagenic and carcinogenic heterocyclic amines (HCAs) are known to be formed in cooked meat and fish, human HCA exposure and carcinogenic risk have not been elucidated in sufficient detail. In this work, we investigated the formations of HCA–amino acid adducts in a model system by using a liquid chromatography–mass spectrometry to elucidate another source of human HCA exposure. The 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) adduct with glycine was formed easily by heating at 200 °C within 5 min, which is probably based on the dehydration condensation of the amino group of PhIP and carboxyl group of glycine. PhIP and other HCAs such as 2-amino-3-methyl-3H-imidazo[4,5-f]quinolone, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline and 3-amino-1,4-dimethyl-5H-pyrido[3,4-b]indole, also bound with various amino acids by heating. Among these amino acids, proline tends to form adducts with HCAs, but serine, cysteine and lysine hardly bound with HCAs. These results provided a basic understanding of the formation of HCA adducts with amino acids during cooking.

A simple and sensitive method for the determination of ochratoxins A and B in nuts and grain samples was developed using an automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Ochratoxins were separated within 5 min by high-performance liquid chromatography using an Inertsil ODS-3 column with 5mM anmonium acetate/acetonitrile (65/35, v/v) as the mobile phase. Electrospray ionization conditions in the positive ion mode were optimized for mass spectrometric detection of ochratoxins. The pseudo molecular ion [M+H](+) was used to detect ochratoxins with selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample using a Carboxen-1006 PLOT capillary column as an extraction device. The extracted ochratoxins were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME/LC-MS with SIM method, good linearities of the calibration curves (r=0.9993 for ochratoxin A and r=0.9989 for ochratoxin B) were obtained in the concentration range from 0.5 to 20 ng/mL. The detection limits (S/N=3) for ochratoxins A and B were 92 and 89 pg/mL, respectively. The in-tube SPME method showed above 15-19-fold greater sensitivity than the direct injection method (10 μL injection). The within-day and between-day precisions (relative standard deviations) were below 5.1% and 7.7% (n=6), respectively. This method was applied successfully to analysis of nuts and grain samples without interference peaks. The recoveries of ochratoxins spiked into extraction solution from nut samples were above 88%. Ochratoxins were detected at 0.7-8.8 ng/g levels in various nuts and grain samples.

We have developed a simple and sensitive method for the simultaneous determination of the antiviral drug oseltamivir (OS) and its metabolite oseltamivir carboxylate (OSC) in environmental waters by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). OS and OSC were separated within 4 min using an ODS-3 column and 1% acetic acid (aq)/methanol (55/45, v/v) as the mobile phase at a flow rate of 0.2 mL min−1. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection of OS and OSC. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL min−1 using a CP-Pora PLOT amine capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS/MS method, we obtained good linearity of the calibration curve (r ≥ 0.9985) for each compound in the range of 1–500 pg mL−1 using stable isotope-labeled internal standards. The detection limits (S/N = 3) for OS and OSC were 0.078 and 0.16 pg mL−1, respectively. The within-day and between-day precisions (relative standard deviations) were below 7% and 13%, respectively. This method was successfully utilized to analyze OS and OSC in environmental water samples without any other pretreatment or interference peaks, and the recoveries of OS and OSC spiked into river water were each above 91%. OS and OSC were detected at concentrations of several hundred pg mL−1 in these water samples, and their concentrations changed seasonally.

MRI using hyperpolarized (13) C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO(2)) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-(13) C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO(2) after pyruvate injection in hyperpolarized (13) C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies.

Solid-phase oligopeptide synthesis has been well developed and most short oligopeptides can now be easily synthesized. However, when a desired oligopeptide forms a secondary structure or includes less reactive amino acids such as aminoisobutyric acid, its terminal amino groups become less reactive and synthesis of the desired oligopeptides becomes difficult. To expand the number of synthetic peptide sequences, we have developed efficient coupling conditions using 3-nitro-l,2,4-triazol-l-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as a highly reactive condensing reagent on an unswellable solid support. PyNTP demonstrated higher reactivity than conventional condensing reagents and the optical purity of the synthesized oligopeptides was sufficiently high for application to general oligopeptide synthesis.

Purpose: Non-invasive magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (HP) 13C-labeled pyruvate and its metabolite lactate is being used to monitor the metabolic flux in solid tumors. In this study, we evaluate the potential of MRSI of HP [1-13C]-pyruvate and [1-13C]-lactate, in prostate cancer as a predictive biomarker for targeting lactate dehydrogenase. Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured, after intravenous delivery of hyperpolarized [1-13C] pyruvic acid, by MRSI. Steady state metabolomic analysis of xenograft tumors was performed by mass spectrometry and steady state lactate levels were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 µg/mouse/day for 5 days x 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates and oxygen consumption of the prostate cancer cell lines was analyzed in vitro. Protein levels of glycolysis regulators were assessed with immunoblotting. Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with HP [1-13C] MRSI relative to PC3 tumors (21% higher 13C-lactate/pyruvate p Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer xenografts predicted the efficacy of targeting LDH when steady state markers of glycolysis, in vivo and in vitro, did not. Citation Format: Bradley T. Scroggins, Masayuki Matsuo, Ayla O. White, Keita Saito, Jeeva P. Munasinghe, Carole Sourbier, Kazutoshi Yamamoto, Vivian Diaz, James B. Mitchell, Murali C. Krishna, Deborah E. Citrin. Hyperpolarized [1-13C]-pyruvate/lactate magnetic resonance spectroscopic imaging of prostate cancer in vivo predicts response to lactate dehydrogenase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4106.

Recent studies have suggested that TLR9 signaling in early endosomes leads to IFN-α production by plasmacytoid dendritic cells (pDCs), whereas TLR9 signaling in late endosomes induces pDC maturation, IL-6, and TNF-α secretion. In this study, we show that human DNA as well as CpG-oligodeoxynucleotides (ODNs) in complex with heat shock protein 90 (Hsp90) stimulate pDCs to produce large quantities of IFN-α. The Hsp90–CpG-A complexes are targeted into the Rab5+, early endosomal Ag 1+-static early endosome postinternalization by DCs, suggesting that preferential sorting of Hsp90-chaperoned self-DNA/CpG-ODNs to the static endosome is required for signaling through TLR9 for IFN-α production. Interestingly, Hsp90-mediated preferential static early endosomal translocation of CpG-ODNs triggers robust IFN-α production from murine conventional DCs. Thus, extracellular Hsp90 converts inert self-DNA/CpG-ODNs into a potent trigger of IFN-α production via spatiotemporal regulation.

We have developed a simple, rapid, and sensitive method for the determination of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). PFOA and PFOS were separated within 10 min by high-performance liquid chromatography using an Inertsil ODS-3 column and 10 mM ammonium acetate/methanol (35/65, v/v) as a mobile phase at a flow rate of 0.25 mL min(-1). Electrospray ionization conditions in the negative ion mode were optimized for MS detection of PFOA and PFOS. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 microL using a CP-Pora PLOT amine capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS method, good linearity of the calibration curve (r=0.9990 for PFOA, r=0.9982 for PFOS) was obtained in the range of 0.05-5 ng mL(-1) each compound. The detection limits (S/N=3) for PFOA and PFOS were 1.5 and 3.2 pg mL(-1), respectively. The method described here showed about 100-fold higher sensitivity than the direct injection method. The within-day and between-day precisions (relative standard deviations) were below 3.7 and 6.0%, respectively. This method was applied successfully to the analysis of PFOA and PFOS in environmental water samples and to the elution test from a Teflon-coated frying pan without interference peaks. The recoveries of PFOA and PFOS spiked into river samples were above 81%, and PFOA was detected at pg mL(-1) levels in environmental water samples and eluate from the frying pan.

This paper summarizes our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on superoxide radical (O(2) (•-)) scavenging activity followed by characterization of antioxidant properties. Firstly, scavenging activity against O(2) (•-) was extensively screened from ethanol extracts of approximately 1000 kinds of herbs by applying an electron spin resonance (ESR)-spin trapping method, and we chose four edible herbal extracts with prominently potent ability to scavenge O(2) (•-). They are the extracts from Punica granatum (Peel), Syzygium aromaticum (Bud), Mangifera indica (Kernel), and Phyllanthus emblica (Fruit). These extracts were further examined to determine if they also scavenge hydroxyl radical ((•)OH), by applying the ESR spin-trapping method, and if they have heat resistance as a desirable characteristic feature. Experiments with the Fenton reaction and photolysis of H(2)O(2) induced by UV irradiation demonstrated that all four extracts have potent ability to directly scavenge (•)OH. Furthermore, the scavenging activities against O(2) (•-) and (•)OH of the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) proved to be heat-resistant.The results of the review might give useful information when choosing a potent antioxidant as a foodstuff. For instance, the four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, the extracts of the aforementioned three herbs are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance.

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP-protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP-protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H](+) (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat-treated protein by LC-MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP-protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food-borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.

A simple and sensitive method for the determination of patulin in fruit juice and dried fruit samples was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Patulin was separated within 5 min by high-performance liquid chromatography using a Synergi MAX-RP 80A column and water/acetonitrile (80/20, v/v) as the mobile phase. Electrospray ionization conditions in the negative ion mode were optimized for MS detection of patulin. The pseudo-molecular ion [M-H](-) was used to detect patulin in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 microL of sample using a Carboxen 1006 PLOT capillary column as an extraction device. The extracted patulin was readily desorbed from the capillary by passage of the mobile phase, and no carry-over was observed. Using the in-tube SPME LC-MS with SIM method, good linearity of the calibration curve (r=0.9996) was obtained in the concentration range of 0.5-20 ng/mL using (13)C(3)-patulin as an internal standard, and the detection limit (S/N=3) of patulin was 23.5 pg/mL. The in-tube SPME method showed >83-fold higher sensitivity than the direct injection method (10 microL injection volume). The within-day and between-day precision (relative standard deviations) were below 0.8% and 5.0% (n=6), respectively. This method was applied successfully for the analysis of fruit juice and dried fruit samples without interference peaks. The recoveries of patulin spiked into apple juice were >92%, and the relative standard deviations were

It is well known that hydroxyl radicals are generated by ultrasound in water. This study with an electron spin resonance spin-trapping technique showed that hydroxyl radical generation was positively correlated with ultrasound duration and water temperature. The clear fungicidal action against Trichophyton spp. evident by studying cultured cells and the degradation of cytoplasmic and surface structures observed by transmission and scanning electron microscopy suggest that ultrasound in hot water is effective for sterilization of dermatophyte contamination and could be effective for the treatment of tinea infection.

A simple, rapid and sensitive method for the determination of nicotine, cotinine, nornicotine, anabasine, and anatabine in human urine and saliva was developed. These compounds were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Nicotine, cotinine and related alkaloids were separated within 7 min by high performance liquid chromatography (HPLC) using a Synergi 4u POLAR-RP 80A column and 5 mM ammonium formate/methanol (55/45, v/v) as a mobile phase at a flow-rate of 0.8 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of these compounds. The optimum in-tube SPME conditions were 25 draw/eject cycles with a sample size of 40 microL using a CP-Pora PLOT amine capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS method, the calibration curves were linear in the concentration range of 0.5-20 ng/mL of nicotine, cotinine and related compounds in urine and saliva, and the detection limits (S/N=3) were 15-40 pg/mL. The method described here showed 20-46-fold higher sensitivity than the direct injection method (5 microL injection). The within-run and between-day precision (relative standard deviations) were below 4.7% and 11.3% (n=5), respectively. This method was applied successfully to analysis of urine and saliva samples without interference peaks. The recoveries of nicotine, cotinine and related compounds spiked into urine and saliva samples were above 83%, and the relative standard deviations were below 7.1%. This method was used to analyze urinary and salivary levels of these compounds in nicotine intake and smoking.

BACKGROUND: In a previous study with the electron spin resonance (ESR) spin-trapping method on large-scale screening with ethanol extracts of approximately 1000 kinds of herbs, four herbal extracts with prominently potent ability to scavenge superoxide anions were chosen, namely the extracts from Punica granatum (peel), Syzygium aromaticum (bud), Mangifera indica (kernel) and Phyllanthus emblica (fruit)). In the present study, these extracts were further examined to determine if they also scavenge hydroxyl radicals, by applying the ESR spin-trapping method, and if they have heat resistance as a desirable characteristic feature. RESULTS: Experiments with the Fenton reaction and UV irradiation of hydrogen peroxide, both of which generate hydroxyl radicals, showed that all four extracts had potent ability to directly scavenge hydroxyl radicals. Each extract exerted more potent hydroxyl radical-scavenging activity than mannitol and salicylic acid. Furthermore, the scavenging activities against superoxide anions and hydroxyl radicals of the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) proved to be heat-resistant. CONCLUSION: The four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, three of them are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance. Copyright © 2008 Society of Chemical Industry

To search for edible herbal extracts with potent antioxidant activity, we conducted a large scale screening based on the superoxide scavenging activity. That is, scavenging activity against superoxide anions were extensively screened from ethanol extracts of approximately 1,000 kinds of herbs by applying an electron spin resonance (ESR)-spin trapping method. Among them we chose four edible herbal extracts with prominently potent ability to reduce the signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH, a spin adduct formed by DMPO and superoxide anion. They are the extracts from Punica granatum (Peel), Syzygium aromaticum (Bud), Mangifera indica (Kernel), and Phyllanthus emblica (Fruit), and are allowed to be used as foodstuffs according to the Japanese legal regulation. The ESR-spin trapping method coupled with steady state kinetic analysis showed that all of the four extracts directly scavenge superoxide anions, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. Furthermore, polyphenol determination indicates that the activity is at least in part attributable to polyphenols. These results with such large scale screening might give useful information when choosing a potent antioxidant as a foodstuff.

Purpose: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized 13C-MRI enabled detecting metabolic changes in tumors using a tracer [1-13C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized 13C-MRI. Experimental Design: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-13C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and 13C-MR signals were acquired using a 4.7 T scanner. Results: [1-13C]pyruvate and [1-13C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-13C]pyruvate administration. The [1-13C]lactate to [1-13C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on 13C-MRI. Conclusions: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized 13C-MRI could be useful for the evaluation of early response to radiotherapy. Clin Cancer Res; 21(22); 5073–81. ©2015 AACR. See related commentary by Lai et al., p. 4996

The electron spin resonance (ESR) spin-trapping technique coupled with iron–dithiocarbamate complexes is one of the most specific methods for nitric oxide (NO) detection. In this study, we applied this method for the evaluation of the substrate and the inhibitors of NO synthase (NOS). A three-line ESR signal was detected from the mixture of inducible NOS (iNOS), l -arginine (Arg), nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin, dithiothreitol, and Fe2+-N-(dithiocarboxy) sarcosine (DTCS–Fe), and the signal intensity increased time-dependently. The signal was not observed by excluding either Arg or NADPH, and it was decreased by the addition of hemoglobin, which is an NO scavenger, and NG-monomethyl- l -arginine ( l -NMMA), NG-nitro- l -arginine ( l -NAME), and aminoguanidine (AG), which are NOS inhibitors, depending on the concentration. In comparison with l -NAME and AG, l -NMMA strongly inhibited iNOS activity. By using this method, the Km value of Arg and the Ki value of l -NMMA for iNOS were determined to be 12.6 and 6.1 μM, respectively. These values are consistent with the reported values measured by the oxyhemoglobin and citrulline assays. These results suggest that the ESR spin-trapping technique coupled with the iron–dithiocarbamate complex can be applied for the evaluation of substrates and inhibitors of NOS, and it would be a powerful tool due to its simplicity and high specificity to NO.

The roles of Phe-120 and Glu-222 in the oxidation of chiral substrates bunitrolol (BTL) and bufuralol (BF) by CYP2D6 are discussed. Wild-type CYP2D6 (CYP2D6-WT) oxidized BTL to 4-hydroxybunitrolol (4-OH-BTL) with substrate enantioselectivity of (R)-(+)-BTL > (S)-(-)-BTL. The same enzyme converted BF into 1''-hydroxybufuralol with substrate enantioselectivity of (R)-BF >> (S)-BF and metabolite diastereoselectivity of (1''R)-OH < (1''S)-OH. The substitution of Phe-120 by alanine markedly increased the apparent K(m) and V(max) values for enantiomeric BTL 4-hydroxylation by CYP2D6. In contrast, the same substitution caused an increase only in V(max) values of (S)-BF 1''-hydroxylation without changing apparent K(m) values, while kinetic parameters (K(m) and V(max) values) for (R)-BF 1''-hydroxylation remained unchanged. Furthermore, the substitution of Glu-222 as well as Glu-216 by alanine remarkably decreased both the apparent K(m) and V(max) values without changing substrate enantioselectivity or metabolite diastereoselectivity. A computer-assisted simulation study using energy minimization and molecular dynamics techniques indicated that the hydrophobic interaction of an aromatic moiety of the substrate with Phe-120 and the ionic interaction of a basic nitrogen atom of the substrate with Glu-222 in combination with Glu-216 play important roles in the binding of BF and BTL by CYP2D6 and the orientation of these substrates in the active-site cavity. This modeling yielded a convincing explanation for the reversal of substrate enantioselectivity in BTL 4-hydroxylation between CYP2D6-WT and CYP2D6-V374M having methionine in place of Val-374, which supports the validity of this modeling.

1-Acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) is a unique probe for in vivo measurements of reactive oxygen species (ROS), because it is hydrolyzed by esterase to a hydroxylamine form (CP-H), which is oxidized to an electron spin resonance-detectable nitroxyl radical (CP) by a reaction with superoxide anion radical, etc. Although a knowledge of pharmacokinetics is essential for the use of ACP in vivo, such information is limited. We investigated the pharmacokinetics of ACP in mice by examining the time course of the tissue distribution of ACP, CP-H, and CP after intravenous or intraperitoneal injection of ACP. Esterase activity for ACP in tissue homogenates was also measured. The concentration of ACP decreased in all tissues obeying a one-compartment model. ACP was hydrolyzed to CP-H in the liver and kidney predominantly, and the first-pass effect of liver on the hydrolysis of ACP was very large. A homogeneous biodistribution of CP-H was obtained 10 min after the injection of ACP regardless of the injection route, and concentrations remained stable over at least 20 min. Because of these pharmacokinetic properties, ACP should be suitable for the imaging of ROS in animals.

Photoimmunotherapy (PIT) using the near-infrared-absorbing photosensitizing phthalocyanine dye, IRDye 700DX (IR-700), conjugated with a tumor-targeting antibody such as panitumumab (Pan) has shown efficacy in in vitro studies and several preclinical models in mice with promise for clinical translation. PIT results in rapid necrotic cell death in vitro and tumor shrinkage in vivo. Photochemical studies with the Pan-IR-700 conjugate showed that this agent can support generation of singlet oxygen and also generate reactive oxygen species after exposure to near-infrared (NIR) light. Moreover, in vitro studies using A431 cells, singlet oxygen scavengers abrogated the efficacy of PIT with Pan-IR-700, while oxygen depletion to undetectable levels in the exposure chamber almost completely inhibited the cellular cytotoxicity of PIT. Survival of tumor bearing mice was prolonged in PIT-treated animals but mice whose tumors were made transiently hypoxic prior to PIT had no benefit from the treatment. The results from this study support a central role for molecular oxygen-derived species in cell death caused by PIT.

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2

Excess reactive oxygen species (ROS) generated from ionizing radiation (IR) or endogenous sources like cellular respiration and inflammation produce cytotoxic effects that can lead to carcinogenesis. Resveratrol (RSV), a polyphenol with antioxidant and anticarcinogenic capabilities, has shown promise as a potential radiation modifier. The present study focuses on examining the effects of RSV or RSV metabolites as a radiation modifier in normal tissue. RSV or a RSV metabolite, piceatannol (PIC) did not protect human lung fibroblasts (1522) from the radiation-induced cell killing. Likewise, neither RSV nor PIC afforded protection against lethal total body IR in C3H mice. Additional research has shown protection in cells against hydrogen peroxide when treated with RSV. Therefore, clonogenic survival was measured in 1522 cells with RSV and RSV metabolites. Only the RSV derivative, piceatannol (PIC), showed protection against hydrogen peroxide mediated cytotoxicity; whereas, RSV enhanced hydrogen peroxide sensitivity at a 50 µM concentration; the remaining metabolites evaluated had little to no effect on survival. PIC also showed enhancement to peroxide exposure at a higher concentration (150 µM). A potential mechanism for RSV-induced sensitivity to peroxides could be its ability to block 1522 cells in the S-phase, which is most sensitive to hydrogen peroxide treatment. In addition, both RSV and PIC can be oxidized to phenoxyl radicals and quinones, which may exert cytotoxic effects. These cytotoxic effects were abolished when HBED, a metal chelator, was added. Taken together RSV and many of its metabolic derivatives are not effective as chemical radioprotectors and should not be considered for clinical use.

Perfluorooctane sulfonate (PFOS) is a chemically stable compound extensively used as oil and water repellent, surface active agents in our daily life. Accumulative research evidence gradually appears the toxicity of PFOS against mammals, but the whole figure remains to be elucidated. The present study was conducted to know the effects of PFOS on human hepatic drug metabolizing-type cytochrome P450 (CYP) isoenzymes such as CYP1A2 (7-ethoxyresorufin as a substrate), CYP2A6 (coumarin), CYP2B6 (7-ethoxy-4-trifluoromethylcoumarin), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (bufuralol), CYP2E1 (chlorzoxazone) and CYP3A4 (testosterone) in human livers employing their typical substrates. Although all of the oxidation reactions tested were more or less inhibited by PFOS, diclofenac 4'-hydroxylation mediated mainly by CYP2C9 was most strongly inhibited (K(i) value of 40 nM), followed by paclitaxel 6α-hydroxylation mediated mainly by CYP2C8 (K(i) value of 4 μM). The substrate oxidation reactions catalyzed by CYP2A6, CYP2B6, CYP2C19 and CYP3A4 were moderately (K(i) values of 35 to 45 μM), and those by CYP1A2, CYP2D6 and CYP2E1 were weakly inhibited by PFOS (K(i) values of 190-300 μM). The inhibition by PFOS for coumarin 7-hydroxylation mainly catalyzed by human liver microsomal CYP2A6 as well as by the recombinant enzyme was found to be enhanced by the preincubation of PFOS with human liver microsomes and NADPH as compared to the case without preincubation. The inhibition of the human liver microsomal cumarin 7-hydroxylation was PFOS concentration-dependent, and exhibited pseudo-first-order kinetics with respect to preincubation time, yielding K(inact) and K(I) values of 0.06 min(-1) and 23 μM, respectively. These results suggest that the metabolism of medicines which are substrates for CYP2C9 may be altered by PFOS in human bodies, and that PFOS is a mechanism-based inhibitor of CYP2A6.

Previous studies have suggested that increased levels of endocannabinoids in various cardiovascular disorders (e.g., various forms of shock, cardiomyopathies, atherosclerosis) through the activation of CB(1) cannabinoid receptors may promote cardiovascular dysfunction and tissue injury. We have investigated the role of the main endocannabinoid anandamide-metabolizing enzyme (fatty acid amide hydrolase; FAAH) in myocardial injury induced by an important chemotherapeutic drug, doxorubicin (DOX; known for its cardiotoxicity mediated by increased reactive oxygen and nitrogen species generation), using well-established acute and chronic cardiomyopathy models in mice. The DOX-induced myocardial oxidative/nitrative stress (increased 4-hydroxynonenal, protein carbonyl, and nitrotyrosine levels and decreased glutathione content) correlated with multiple cell death markers, which were enhanced in FAAH knockout mice exhibiting significantly increased DOX-induced mortality and cardiac dysfunction compared to their wild type. The effects of DOX in FAAH knockouts were attenuated by CB(1) receptor antagonists. Furthermore, anandamide induced enhanced cell death in human cardiomyocytes pretreated with FAAH inhibitor and enhanced sensitivity to ROS generation in inflammatory cells of FAAH knockouts. These results suggest that in pathological conditions associated with acute oxidative/nitrative stress FAAH plays a key role in controlling the tissue injury that is, at least in part, mediated by the activation of CB(1) receptors by endocannabinoids.

A simple and sensitive automated method, consisting of in-tube solid-phase microextraction (SPME) coupled with high-performance liquid chromatography-fluorescence detection (HPLC-FLD), was developed for the determination of 15 polycyclic aromatic hydrocarbons (PAHs) in food samples. PAHs were separated within 15 min by HPLC using a Zorbax Eclipse PAH column with a water/acetonitrile gradient elution program as the mobile phase. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 microL of sample using a CP-Sil 19CB capillary column as an extraction device. Low- and high-molecular weight PAHs were extracted effectively onto the capillary coating from 5% and 30% methanol solutions, respectively. The extracted PAHs were readily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME HPLC-FLD method, good linearity of the calibration curve (r>0.9972) was obtained in the concentration range of 0.05-2.0 ng/mL, and the detection limits (S/N=3) of PAHs were 0.32-4.63 pg/mL. The in-tube SPME method showed 18-47 fold higher sensitivity than the direct injection method. The intra-day and inter-day precision (relative standard deviations) for a 1 ng/mL PAH mixture were below 5.1% and 7.6% (n=5), respectively. This method was applied successfully to the analysis of tea products and dried food samples without interference peaks, and the recoveries of PAHs spiked into the tea samples were >70%. Low-molecular weight PAHs such as naphthalene and pyrene were detected in many foods, and carcinogenic benzo[a]pyrene, at relatively high concentrations, was also detected in some black tea samples. This method was also utilized to assess the release of PAHs from tea leaves into the liquor.

This study compared the superoxide detection abilities of four spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(diphenylphosphinoyl)-5-methyl-1pyrroline N-oxide (DPPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in living cells. Electron spin resonance (ESR) signals of the superoxide adducts were observed when spin traps were added to a suspension of human oral polymorphonuclear leukocytes (OPMNs) stimulated by phorbol 12-myristate 13-acetate. The ESR signal of the CYPMPO-superoxide adduct (CYPMPO-OOH) increased for 24 min after the initiation of the reaction, whereas the signals from DMPO-OOH and DPPMPO-OOH peaked at 6 and 10 min, respectively. The maximum concentrations of DMPO-OOH, DPPMPO-OOH and CYPMPO-OOH in OPMNs were 1.9, 6.0 and 10.7 microM, respectively. Furthermore, CYPMPO could more efficiently trap superoxide in blood PMNs compared with DEPMPO. From these results, it was concluded that CYPMPO performs better than DMPO, DPPMPO and DEPMPO for superoxide measurements in living cell systems because it has lower cytotoxicity and its superoxide adduct has a longer lifetime.

A simple and sensitive method for the determination of musty odorants, 2-methylisoborneol (MIB) and geosmin (GSM), in environmental water was developed by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry. MIB and GSM were separated within 10 min using a DB-1 capillary column and detected in the selective ion monitoring mode. HS-SPME using a polydimethylsiloxane/divinylbenzene fiber provided effective sample enrichment, and was carried out by fiber exposition at 70 °C for 30 min. Using this method, the calibration curves of MIB and GSM were linear in the range of 0–500 pg/mL, with a correlation coefficient above 0.9977 (n = 24). The detection limits (S/N = 3) of MIB and GSM were 0.9 and 0.6 pg/mL, respectively. This method was successfully applied to the analysis of environmental water samples without interference peaks.

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3′-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3′-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB ≫ (S)-HB and metabolite diastereoselectivity of 3′(R) < 3′(S) in 3′-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3′-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB < (S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3′-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3′(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19. Chirality, 2007. © 2007 Wiley-Liss, Inc.

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one-third of the medicines in current clinical use and exhibits genetic polymorphism with interindividual differences in metabolic activity. To precisely investigate the effect of CYP2D6*10B and CYP2D6*36 frequently found in Oriental populations on mexiletine metabolism in vitro, CYP2D6 proteins of wild-type (CYP2D6.1) and variants (CYP2D6.10 and CYP2D6.36) were heterologously expressed in yeast cells and their mexiletine p - and 2-methyl hydroxylation activities were determined. Both variant CYP2D6 enzymes showed a drastic reduction of CYP2D6 holo- and apoproteins compared with those of CYP2D6.1. Mexiletine p - and 2-methyl hydroxylation activities on the basis of the microsomal protein level at the single substrate concentration (100 μM) of variant CYP2D6s were less than 6% for CYP2D6.10 and 1% for CYP2D6.36 of those of CYP2D6.1. Kinetic analysis for mexiletine hydroxylation revealed that the affinity toward mexiletine of CYP2D6.10 and CYP2D6.36 was reduced by amino acid substitutions. The V max and V max / K m values of CYP2D6.10 on the basis of the microsomal protein level were reduced to less than 10% of those of CYP2D6.1, whereas the values on the basis of functional CYP2D6 level were comparable to those of CYP2D6.1. Although it was impossible to estimate the kinetic parameters for the mexiletine hydroxylation of CYP2D6.36, the metabolic ability toward mexiletine was considered to be poorer not only than that of CYP2D6.1 but also than that of CYP2D6.10. The same tendency was also observed in kinetic analysis for bufuralol 1″-hydroxylation as a representative CYP2D6 probe. These findings suggest that CYP2D6*36 has a more drastic impact on mexiletine metabolism than CYP2D6*10 .

In vitro quantitative studies of the oxidative metabolism of (5-methoxy-N,N-diisopropyltryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) metabolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics. Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-deisopropylase activities. The apparent Km value of CYP2D6 was close to that for 5-MeO-DIPT O-demethylation, and the Km values of other CYP enzymes were similar to those of the low-Km (CYP2C19), intermediate-Km (CYP1A2, CYP2C8 and CYP3A4) and high-Km phases (CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition studies, quinidine (1 microM), an inhibitor of CYP2D6, almost completely inhibited human liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10 microM. Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9 inhibitor and ketoconazole, a CYP3A4 inihibitor (5 microM each) suppressed about 60%, 45%, 15% and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50 microM substrate. In contrast, omeprazole (10 microM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by human liver microsomes, whereas at the same concentration the inhibitor suppressed the reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-MeO-DIPT N-deisopropylase enzymes in the human liver.

The exposure of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) to hydroxyl radical ((.)OH) led to the appearance of a new triplet ESR signal along with a reduction in the intensity of the TEMPOL signal. The observed spectrum was in good agreement with the spectrum simulated with 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPOL, and the addition of sodium borohydride to the product solution led to the disappearance of the new signal. These results indicate the formation of TEMPON during the reaction. A comparison of the concentration of TEMPON formed with that of nitroxyl compounds that lost the electron spin resonance (ESR) signal showed that the rate of TEMPON formation is less than one-tenth that of the paramagnetism loss caused by (.)OH in the pH range 6.5-8.5. These results indicate that TEMPOL has two reaction sites with (.)OH; the nitroxyl group and the 4-position of the piperidine ring. The reaction at the 4-position should contribute to the (.)OH scavenging activity of TEMPOL together with that at the nitroxyl group, although the rate of the former reaction is relatively low.