Mayu Onozato

Polycyclic Aromatic Hydrocarbons in Sediments and Bivalves on the Pacific Coast of Japan: Influence of Tsunami and Fire

Surface sediments and at least one edible bivalve species (Ruditapes philippinarum, Mytilus galloprovincialis, and Crassostrea gigas) were collected from each of seven intertidal sites in Japan in 2013. The sites had experienced varying levels of tsunami and fire disturbance following the major earthquake of 2011. Eight polycyclic aromatic hydrocarbons (PAHs) were identified and analyzed by gas chromatography–mass spectrometry. Total sediment PAH concentration (CT), the sum of the average concentrations of the eight PAHs, was 21–1447 μg kg-1-dry. Relative to the average level of one type of PAH in sediments collected around Japan in 2002 (benzo[a]pyrene = 21 μg kg-1-dry), five of the seven sites showed concentrations significantly lower than this average in 2013. The CTs for the three bivalves (134–450 μg kg-1-dry) were within the range of the previous reports (2.2–5335 μg kg-1-dry). The data suggest that the natural disaster did not increase PAH concentrations or affect the distribution within sediment or bivalves in Tohoku district. Although PAH concentrations at the sites pose no risk to human health, the findings highlight that the observed PAH levels derive from pre- rather than post-quake processes.

ANALYSIS OF MUTAGENICITY OF NITROBENZANTHRONES BY MOLECULAR ORBITAL CALCULATIONS

3-Nitrobenzanthrone (3-NBA) detected in diesel exhaust was found to be a very strong mutagen. 9-NBA, however, is less potent than 3-NBA and 11-NBA is devoid of mutagenicity. To explain the difference in mutagenicity among the three isomers theoretically, we proposed the metabolic activation pathway of NBAs and performed molecular orbital calculations. In the pathway, NBAs are transformed to nitrosobenzanthrone, N-hydroxy-3-aminobenzanthrone (N-OH-ABA), N-acetoxy-3-aminobenzanthrone (N-AcO-ABA), and finally ultimate carcinogens to form NBA-DNA adducts. We calculated the heat of formation, HOMO and LUMO energy for parent molecules and their activated intermediates. The activation energy (Ea) was also calculated for the binding reaction of the ultimate carcinogens with the DNA bases. A correlation was found between the mutagenicity of NBAs and the LUMO energy of parent molecules and the intermediates, nitrosobenzanthrone, N-OH-ABA, and N-AcO-ABA. The Ea values were not correlated with mutagenicity. This indicates that the production rate and stability of ultimate carcinogens are more important in determining the mutagenicity than the reaction rate of their binding to DNA.

A high-performance liquid chromatography assay with a triazole-bonded column for evaluation of d-amino acid oxidase activity

Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia.

DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) IN SEDIMENTS AND BENTHOS COLLECTED ON THE COAST OF CHIBA

Eight polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene were analyzed in sediment and benthos collected on the coast of Chiba Prefecture in Japan by gas chromatography-mass spectrometry (GC/MS). The total concentration of the PAHs ranged from 8 to 18 μ g/kg-dry in the sediments and from 36 to 59 μ g/kg-wet in the benthos, Mactra quadrangularis, Scoletoma nipponica, and Arenicola basiliensis. The PAH composition in the sediments and the benthos was similar at four sampling points and dominated by phenanthrene (Phe), fluoranthene (Flu), and pyrene (Pyr). The ratios of abundance of Flu to that of Pyr, Flu/Pyr, and of Phe/anthracene suggest that the PAHs in the benthos are derived from the combustion of fossil fuels. The ratio, R, of the PAH concentration in the benthos to that in the sediment is 2.0 for Scoletoma nipponica and 1.8 for Arenicola basiliensis in average, indicating that the bioaccumulation is not significant in the benthos. However, the R value for perylene in Scoletoma nipponica was 3.7 times as large as the average.

Enantiomeric Separation of Monosubstituted Tryptophan Derivatives and Metabolites by HPLC with a Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phase and Its Application to the Evaluation of the Optical Purity of Synthesized 6-Chloro-l-Tryptophan

6-Chlorotryptophan possesses unique bioactivity and can be used as a precursor for several bioactive compounds in medicinal chemistry. It was enantioselectively synthesized by condensing 6-chloroindole with racemic N-acetylserine, followed by enzymatic hydrolysis with L-aminoacylase (EC 3.5.1.14). The optical purity was examined by conducting high-performance liquid chromatography with a Cinchona alkaloid-based zwitterionic chiral stationary phase (CSP) [CHIRALPAK® ZWIX(+)], which bears a chiral trans-2-aminocyclohexanesulfonic acid moiety tagged at C-9 of the Cinchona alkaloid. The zwitterionic CSP enabled efficient enantiomeric separations of monosubstituted tryptophan derivatives 1-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-methoxytryptophan, and 6-chlorotryptophan with a methanol/H2O (98/2) mobile phase containing formic acid (FA) and diethylamine (DEA) additives. The mobile phase contains 25–75 mM FA and 20–50 mM DEA, enabling good separation of the enantiomers of each tryptophan derivative (α > 1.25). Thus, the optical purity of the synthesized 6-chloro-L-tryptophan was easily determined (greater than 99.0%) using HPLC with the zwitterionic CSP.

ANALYSIS OF THE METABOLIC TRANSFORMATION OF MUTAGENIC NITROBENZOBENZANTHRONES BY MOLECULAR ORBITAL CALCULATIONS

The mutagenic activity of three nitrobenzobenzanthrones (NBBAs) and two dinitrobenzobenzanthrones (DNBBAs) was found to depend largely on the position and the number of the nitro groups as well as that of nitrobenzanthrones (NBAs). Two NBBAs exhibit the activity approximately two and a half times as large as that of 3-NBA, whereas the other less than one-hundredth. The activity of the DNBBAs is smaller than that of structurally corresponding NBBAs. To elucidate these differences in the mutagenicity, we postulated a metabolic activation pathway of NBAs and NBBAs and calculated physicochemical properties of the parent molecules, metabolites, and ultimate mutagens by the semi-empirical molecular orbital method. Correlation of the results with the mutagenic activity revealed that important factors determining the activity are the LUMO energies of the parent molecules and nitroso-metabolites and the HOMO energies of the hydroxylamino metabolites, which almost the same as those obtained in the case of NBAs and DNBAs.

CONFORMATIONAL ANALYSIS OF 2-(DIPHENYLPHOSPHANYL)-N, N-DIMETHYL-1-BENZ AMIDE AND 2-(DIPHENYLPHOSPHANYL)-PHENYL-PYRROLIDIN-1-YL-METHANONE BY NMR SPECTROSCOPY

We have synthesized 2-(Diphenylphosphanyl)-N, N-dimethyl-1-benzamide (1) and 2-(Diphenylphosphanyl)- phenyl-pyrrolidin-1-yl-methanone (2), and examined their conformations on the basis of NMR spectral data. Conformational analysis of the compounds is useful in deducing the structure in which they are active as a catalyst. In the present NMR measurements, 1H-X fg-JHMBC (field gradient J-Resolved Hetero-nuclear Multiple-Bond Correlation) spectroscopy was implemented as a tool for the determination of hetero-nuclear three bond, phosphorus and protons and carbon-protons coupling constants. By fitting a sine curve to the experimental data by the method of 3D J-resolved HMBC NMR measurements, accurate n J HX coupling constants were obtained. From the coupling constants, the corresponding dihedral angles, H3-C2-C3-P, H12-C12-C11-P, H16-C16-C11-P, H18-C18-C17-P, and H22-C22-C17-P, of compounds (1) and (2) were determined. The optimized structures of the compounds were obtained by molecular orbital calculations in which the dihedral angles experimentally determined were used.

ANALYSIS OF THE METABOLIC TRANSFORMATION OF HETEROCYCLIC AMINES BY MOLECULAR ORBITAL CALCULATIONS

Heterocyclic amines (HCAs), contained in charred parts of broiled fish and meat, show a variety of mutagenic activities. To investigate the structure-activity correlation of HCAs, molecular orbital calculations were performed for 19 HCAs. We focused our attention on the binding reaction of the nitrenium ion of HCAs to bases of DNA and calculated the activation energy for the reaction. The activation energies for guanine were found to be the smallest of those for the four bases. This explains the experimental result that HCAs react preferentially with guanine in DNA. Correlations, however, were poor between the activation energy and the mutagenicity of HCAs, which indicates that the production rate and stability of nitrenium ions are more important in the mutagenicity of HCAs than the reaction rate of the ions.

Alteration in plasma and striatal levels of d-serine after d-serine administration with or without nicergoline: An in vivo microdialysis study

Aims d-Serine (d-Ser), a co-agonist of N-methyl-d-aspartate receptor (NMDAR), is effective for treating schizophrenia. The present study investigated changes in plasma and striatal d-Ser levels in Sprague-Dawley (SD) rats after intraperitoneal d-Ser administration alone or together with nicergoline (Nic), a commercial cerebral ameliorating drug, using in vivo microdialysis (MD) to explore the function of Nic. Main methods Phosphate-buffered saline (PBS) or Nic (0, 1.0, or 3.0 mg/kg) followed by d-Ser (5.0, 10.0, 20.0, and 50.0 mg/kg for PBS or 20.0 mg/kg for Nic) was administered intraperitoneally to male SD rats, and the profiles of d-Ser levels in plasma and striatal MD samples were examined by high-performance liquid chromatography (HPLC) with fluorescence detection. The area under the curve (AUC) for the MD and plasma samples was also calculated and statistically compared among groups. Key findings AUC values of d-Ser increased in a d-Ser dose-dependent manner in plasma samples, while a proportional increase in the AUC values of striatal MD samples was only observed in d-Ser doses up to 20 mg/kg. The Nic co-administered group showed a significant increase in the AUC of plasma d-Ser in a Nic dose-dependent manner, but the AUC in striatal d-Ser significantly decreased with increasing Nic doses suggesting that Nic may prevent excess d-Ser from penetrating the central nervous system (CNS). Significance Nic may prevent an excessive distribution of exogenous d-Ser, such as that from a dietary origin, into the CNS by suppressing excitatory neurotransmission through NMDAR.

Effect of risperidone on plasma d-serine concentration in rats post-administered with d-serine.

AIMS Risperidone (Ris) is a second-generation antipsychotic (SGA) used to treat patients with schizophrenia. Additional interventions that increase plasma d-serine (d-Ser) levels could provide improved amelioration of the negative symptoms of schizophrenia. In the present study, we studied whether Ris pretreatment altered the concentration of plasma d-Ser administered intraperitoneally. In addition, the effects of Ris and its main metabolite, 9-hydroxyrisperidone (9-OHRis), on rat d-amino acid oxidase (DAO) activity were examined in vitro. MATERIALS AND METHODS Ris (0, 0.5, 1.0, or 3.0mg/kg), followed by d-Ser (20mg/kg), were administered intraperitoneally (i.p.) to male Sprague-Dawley rats, and the time-courses of plasma d-Ser, Ris, and 9-OHRis concentrations were examined. Inhibition of DAO activity in rat cerebellar and kidney preparations by Ris and 9-OHRis were measured spectrophotometrically. KEY FINDINGS Significant increases in plasma d-Ser levels were observed in rats treated with both Ris and d-Ser. This effect occurred in a Ris dose-dependent manner, and the areas under the plasma d-Ser concentration-time curves were similar in rats treated with Ris (1.0mg/kg) and with a commercial DAO inhibitor, 3-methylpyrazole-5-carboxylic acid (1.0mg/kg). Rat plasma analyses showed that 9-OHRis was rapidly produced from Ris; however, high concentrations of Ris and 9-OHRis produced weak DAO inhibition in vitro, suggesting that some other pharmacological effect of Ris and/or 9-OHRis might contribute to its effects on plasma d-Ser levels. SIGNIFICANCE The combined administration of Ris and d-Ser may increase plasma d-Ser levels, suggesting that this approach could reduce the dose of d-Ser required for these patients.