Yukiko Hanasaki

The correlation between active oxygens scavenging and antioxidative effects of flavonoids.

The abilities of 15 flavonoids as a scavenger of active oxygens (hydroxyl radical and superoxide anion) were studied. Hydroxyl radical (.OH) was generated by the Fenton system, and assayed by the determination of methanesulfonic acid (MSA) formed from the reaction of dimethyl sulfoxide (DMSO) with .OH. (+)-Catechin, (-)-epicatechin, 7,8-dihydroxy flavone, and rutin showed the .OH scavenging effect 100-300 times superior to that of mannitol, a typical .OH scavenger. The other flavonoids showed no .OH scavenging effect at their concentrations up to 50 microM. Baicalein, quercetin, morin, and myricetin unexpectedly increased the .OH production in the Fenton system. The flavonoids tested now, except monohydroxy flavones, were more or less inhibitive to the superoxide anion (O2) generation in the hypoxanthine-xanthine oxidase system. A great part of this inhibitory effect was likely owing to suppression of xanthine oxidase activity by the flavonoids. The flavonoids, which scavenged .OH or O2-, were necessarily antioxidants to the peroxidation of methyl linoleate. However, there was a type of flavonoid such as morin, which have neither .OH nor O2- scavenging effect, but was a strong antioxidant.

High-performance liquid chromatographic determination of methanesulphinic acid as a method for the determination of hydroxyl radicals

For the determination of hydroxyl radicals, dimethyl sulphoxide was used as a molecular probe and the methanesulphinic acid produced was determined by high-performance liquid chromatography of its Fast Yellow GC salt derivative. The results for hydroxyl radicals formed using the Fenton and hypoxanthine-xanthine oxidase systems agreed well with the theoretical values. Interferences from phenols, aromatic amines and amino acids, which give coloured substances by reaction with the diazonium salt, could be avoided. The recovery of methanesulphinic acid added to liver homogenates and incubated for 1 h at 37 degrees C was 70.2 +/- 2.1%. The detection limit for methanesulphinic acid in a sample solution was ca. 8 ng/ml.

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.

Determination method of singlet oxygen in the atmosphere by use of α-terpinene

Abstract A chemical determination method of singlet oxygen in the atmosphere was established. The method employs a specific reaction of α -terpinene with singlet oxygen to produce the single product, ascaridole. Amberlite XAD-2 coated with α -terpinene was packed into a glass tube shielded from light and sample air was passed through the tube. Ascaridole formed was extracted with hexane from Amberlite XAD-2 and was determined by gas chromatography. The amount of singlet oxygen was calculated from that of ascaridole. Ascaridole was not formed by oxidation of α -terpinene with ozone, hydrogen peroxide and hydroxyl radical, and ascaridole formed by singlet oxygen was stable against these oxidants. The method was applied to actual polluted air and diurnal variations in the singlet oxygen were observed.

Determination of the potent mutagen 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-franone (MX) in water by gas chromatography with electron-capture detection

Abstract A highly sensitive method for the determination of 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-franone (MX) in water by gas chromatography with electron-capture detection is described. MX was derivatized with 2,2,3,3,3-pentafluoropropanol and the product was easily decomposed by UV irradiation. The disappearance of the peak on the chromatogram after the irradiation was used for the identification of MX; the amount of MX was calculated from the decrease in peak height. The method was applied to the analysis of five samples of chlorinated domestic sewage. MX (141.6-2.2 ng/1) was detected in all samples with a detection limit of 0.8 ng/1. The recovery of MX was more than 97.5% at the level of 20.0 ng/1 added.

Diurnal changes of singlet oxygen like oxidants concentration in polluted ambient air

The long-term measurement of singlet oxygen like oxydants (SOLO) in polluted ambient air by use of the chemical determination method with α-terpinene (Ter) as a substrate was carried out. α-Terpinene endoperoxide, ascaridole (Asc), was produced by the reaction of SOLO with Ter in a similar manner to singlet oxygens (1O2). Asc formed was analyzed by gas chromatography-mass fragmentgraphy with selected ion monitoring mode.The concentration of SOLO in sample air was calculated from the amount of Asc determined, and expressed as O2 in active state. The concentration of 4 – 21 ppb of SOLO was detected in the daytime, and it reached to the maximum at around noon. SOLO were also detected in the nighttime and its concentration reached to the maximum at around midnight. The pattern of diurnal change of SOLO concentration was apparently different from that of photochemical oxidants including ozone, but identical with that of NOx. SOLO are not conventional photochemical oxidants such as ozone, but must be α-terpinene endoperoxide producible oxydants having similar reactivity to 1O2 and probably including 1O2 themselves.

Removal of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in water by oxidative, reductive, thermal or photochemical treatments

Abstract Removal of a highly potent Ames mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) with decrease of its mutagenic potency from an aqueous solution by oxidative (ozonation and addition of H2O2 with or without ferrous ion), reductive (addition of Na2SO3, Na2S2O4 and ascorbic acid), thermal (heating in a boiling water bath) and photochemical (UV irradiation) treatments were investigated. More than 97% of removal of MX (initial amount of 1 mM) and its mutagenicy was achieved by the treatments with O 3 Fe 2+ and H 2 O 2 Fe 2+ . Hydroxyl radical was an important reactant in these treatments, since the removal of MX was strongly inhibited by the addition of typical hydroxyl radical scavengers, mannitol and L-tryptophan methyl ester. The removal of 1 mole of MX required 2 moles of hydroxyl radical accompanied by the release of 2 moles of chloride ion. In the thermal and photochemical treatments, 59% and 76% of MX and the same portions of its mutagenic potency were removed during 60 min, respectively. The release of 2 moles of chloride ion from 1 mole of MX were also observed in these treatments, but hydroxyl radical did not participitate in the decomposition. In the reductive treatments, 68% of MX was removed by the addition of Na2SO3 during 60 min. However, the decrease of mutagenic potency was 48%. It was strongly suggested that the lack of regularity between the amount of removed MX and the decrease of its mutagenic potency is due to weak addition of SO32− to MX rather than reduction of MX.