Shunjiro Ogawa

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.

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[a]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.

Mutagenicity modulating effect of quercetin on aromatic amines and acetamides

The effect of quercetin on the mutagenicity of 32 kinds of aromatic amines and their acetamides were investigated using Salmonella typhimurium TA98 with a mammalian metabolic activation system (S9 mix). Quercetin enhanced the mutagenicity of the tricyclic aromatic amines (aminofluorene, aminoanthracene and aminophenanthrene) and their acetamides by 1.2-5.9-fold. Whereas, quercetin depressed the mutagenicity of aniline derivatives, biphenyl derivatives, and bi- and tetra-cyclic amino derivatives. The modulation of mutagenicity of Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2 (heterocyclic amines) by quercetin were liable to be affected by the content of S9 in the S9 mix. It seems that quercetin does not have the same effect as norharman, because quercetin did not enhance the mutagenicity of aniline. It is suggested that the modulation of the mutagenicity of aromatic amines and acetamides is caused by the modulation of the balance between the mutagenic activation and inactivation in the metabolism of these amines and acetamides in the presence of quercetin. In this modulation, quercetin may participate through its effects on the promotion of N-hydroxylation and the inhibition of arylhydroxylation and transacylation. The presence of tricyclic aromatic rings of amines and acetamides is a structural requirement for the mutagenicity enhancement by quercetin.

The effect of quercetin, a mutagenicity-enhancing agent, on the metabolism of 2-acetylaminofluorene with mammalian metabolic activation systems

The effect of quercetin as the comutagen on 2-acetylaminofluorene (AAF) was investigated. AAF was metabolized with mammalian metabolic systems (S9 mix) in the presence or absence of quercetin in vitro, and its metabolites were determined by high-performance liquid chromatography. In the presence of quercetin, the total metabolic rate of AAF decreased compared with that in the absence of quercetin, whereas the formation of N-hydroxy-AAF (N-OH-AAF) and 2-aminofluorene (AF) increased. Since the main metabolic pathway of AAF is aryl-hydroxylation, it is suggested that the decrease of total metabolic rate of AAF is due to the inhibition of aryl-hydroxylation by quercetin. From these results, it seems probable that the comutagenic effect of quercetin on AAF is due to the inhibition of aryl-hydroxylation (the detoxifying pathway) and the promotion of N-hydroxylation and deacetylation (the activating pathway) in the AAF metabolism with S9 mix.

Formation of non-volatile potent mutagens in domestic sewage by chlorination

Abstract XAD extracts obtained from chlorinated domestic sewage samples were evaluated for mutagenicity by Ames test. The sewage before chlorination did not show obvious mutagenicity in S. typhymurium TA 100. The sewage after chlorination exhibited dose-related mutagenic activity ( 5000 ∼ 30000 rev./ 1 ) in S. typhimurium TA 100 without S9 mix. The activity was enhanced 5 ∼ 50 fold over that of non-chlorinated sewage. Formation of both volatile and non-volatile mutagens by the chlorination of sewage was confirmed by a taped plate assay and Ames assay of the XAD extracts. It was estimated that the mutagens consisted of several substances from results of fractionation of the XAD extracts.

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.

Mutagenicity-enhancing effect of quercetin on the active metabolites of 2-acetylaminofluorene with mammalian metabolic activation systems

The effects of quercetin on the mutagenicity of 2-acetylaminofluorene (AAF) and its 3 active metabolites, N-hydroxy-AAF (N-OH-AAF), aminofluorene (AF) and N-acetoxy-AAF(N-OAc-AAF) were investigated. The mutagenicity assays were carried out with Salmonella typhimurium TA98, and S9, microsomes and cytosol were used as metabolic activation systems. In the presence of S9, quercetin enhanced the mutagenicity of AAF, N-OH-AAF, AF and N-OAc-AAF by 6.9-, 4.3-, 3.6- and 3.9-fold, respectively. Quercetin enhanced the mutagenicity of these substrates with microsomes, whereas it depressed the mutagenicity of these substrates with cytosol. From these results, it seemed probable that quercetin promotes the N-hydroxylation and deacetylation in the microsomes, whereas it inhibits the deacetylation in the cytosol. It was shown that in the metabolism of AAF and its metabolites, quercetin modulates the balance between the mutagenicity activation and inactivation processes, which is catalysed by the enzymes in the microsomes and cytosol, and causes enhancement of the mutagenicity of AAF.

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.