Tetsuta Kato

DNA strand-breaking activity and mutagenicity of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), a Maillard reaction product of glucose and glycine

Aqueous solution of glucose and glycine was heated under reflux for 4 h and extracted with ethyl acetate. Reversed phase HPLC of the extract revealed a new DNA strand-breaking substance, which was purified by repeated HPLC and identified as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP). DDMP induced DNA strand breaking in a dose- and time-dependent manner. It was active to break DNA strands at pH 7.4 and 9.4. Its pyranone skeleton was destroyed at the pH values. DNA strand breaking by DDMP was inhibited by superoxide dismutase, catalase, scavengers for hydroxyl radical, spin trapping agents and metal chelators, and the breaking was enhanced by Fe(III) ion. A mixture of DDMP and a spin trap DMPO gave electron spin resonance signals of a spin adduct DMPO-OH, indicating generation of hydroxyl radical. DDMP was found to be mutagenic to Salmonella typhimurium TA100 without metabolic activation. These results show DDMP generated active oxygen species to cause DNA strand breaking and mutagenesis.

Formation of yellow, orange, and red pigments in the reaction of alk-2-enals with 2-thiobarbituric acid

The reaction of four to eight carbon straight-chain alk-2-enals with 2-thiobarbituric acid (TBA) produced yellow 455-nm-, orange 495-nm-, and red 532-nm-absorbing pigments depending upon the reaction conditions. The 1:1 reaction of the aldehydes with TBA in 15% acetic acid at 100 degrees C produced the yellow pigment at 0.25 h and the red at 6 h. The reaction of the aldehydes with TBA in excess at 100 degrees C produced the yellow at 0.25 h, the orange at 2-6 h, and the red at 0.25-6 h. The formation of these pigments required molecular oxygen. These pigments could be separated from each other on HPLC. The red pigment formed from the aldehydes could not be distinguished from the red 1:2 malonaldehyde-TBA adduct by absorption spectrum and HPLC. The red color yield was the highest in the 1:1 reaction and retarded in the reaction with TBA in excess. The red color due to these aldehydes may contribute in part to the color formed in the general TBA test of lipid oxidation. The 1:1 reaction initially produced colorless 1:1 adducts X, which were subsequently converted into the yellow and red pigments under aerobic conditions. The reaction of the aldehydes with TBA in excess might initially produce X and then another colorless 1:2 adducts Y; the latter being converted into yellow, orange, and red pigments under aerobic conditions.

DAMAGE OF AMINO ACIDS AND PROTEINS INDUCED BY NITROGEN DIOXIDE, A FREE RADICAL TOXIN, IN AIR

Damage of amino acids and proteins induced by nitrogen dioxide, a free radical toxin in polluted air, was investigated. When nitrogen dioxide (30-90 ppm) in air was exposed to a solution of an amino acid at pH 7.5 for several hours, tryptophan and tyrosine were damaged. Degradation of tryptophan was accompanied by formation of a nitroindole derivative. Decrease of tyrosine was accompanied by formation of 3-nitrotyrosine and fluorescent dityrosine. When nitrogen dioxide was exposed to a solution of bovine serum albumin, human gamma-globulin and bovine eye lens alpha-crystallin, the proteins were crosslinked by nondisulfide bonds. Tryptophan and tyrosine residues in the proteins were extensively decreased, and significant amounts of 3-nitrotyrosine and fluorescent dityrosine were formed. The modification of the proteins with nitrogen dioxide in air may have toxicological significance. Because fluorescent dityrosine is detected in a wide variety of natural proteins, nitrogen dioxide may play a role in its occurrence in natural proteins.

Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides

Formation of fluorescence by the reaction of various amino acids with lipid hydroperoxides,i.e., linoleic acid 13-monohydroperoxide, methyl linoleate 13-monohydroperoxide and phosphatidylcholine hydroperoxide, in the presence of methemoglobin was investigated. Two types of fluorescence were produced: fluorescent dityrosine (3,3′-dityrosine) from tyrosine, and unidentified fluorophores with α- and ε-amino groups of various amino acids. While the former was stable after treatment with borohydride, the latter fluorophores were readily destroyed. The rate of dityrosine formation was rapid, and the yield of dityrosine was dependent on the concentrations of tyrosine and the lipid hydroperoxides. Butylated hydroxytoluene and tocopherol inhibited the formation of dityrosine, but did not affect the formation of fluorophores on the amino groups. Dityrosine appears to be formed by radical reaction of the lipid hydroperoxides, while the other fluorophores seem to be created by nonradical mechanisms.

Formation of red pigment by a two-step 2-thiobarbituric acid reaction of alka-2,4-dienals. Potential products of lipid oxidation

Reaction of 2,4-hexadienal, 2,4-nonadienal and 2,4-decadienal with 2-thiobarbituric acid (TBA) in aqueous acetic acid produced a 532-nm absorbing red pigment. While the 1∶1 reaction of the aldehyde and TBA produced little pigment, reaction of the aldehyde with an excess amount of TBA produced significant amounts. Instant heating of the reaction mixture did not produce the pigment. However, initial reaction at 5°C and subsequent heating to 100°C produced the pigment efficiently (two-step reaction). Pigment formation required water and dissolved oxygen. The yield of the pigment from the alka-2,4-dienals was 1/10–1/20 of that from malonaldehyde. In the first step of the reaction at 5°C, the 1∶1 adducts of the aldehydes at the5-position of TBA and several other uniden-tified adducts were formed. In the second step, these adducts were converted at 100°C, in the presence of water and oxygen, into the red pigment. The structure of the red pigment from 2,4-hexadienal was elucidated to be the 1∶2 adduct of malonaldehyde and TBA. 2-Hexenal andt-butylhydroperoxide showed marked synergistic effects on the pigment formation from the alka-2,4-dienals. Red pigment formation due to the alka-2,4-dienals may be enhanced by the presence of other aldehydes and hydro-peroxides.

Identification of hydroxyhydroquinone in coffee as a generator of reactive oxygen species that break DNA single strands

A component in instant coffee that caused DNA single strand breaks was isolated by successive ethyl acetate:ethanol extraction, silica gel column chromatography and high performance liquid chromatography using a reversed phase column. The active component was identified as hydroxyhydroquinone (HHQ). Incubation of supercoiled pBR 322 DNA with HHQ at 0.1 mM in phosphate buffer (pH 7.4) at 37 degreesC for 1 h caused single strand breaks, and reactive oxygen species, hydrogen peroxide and hydroxyl radical, were involved in DNA breaking by HHQ. Genotoxic effects of HHQ including DNA breaking activity through generation of reactive oxygen species have been well-demonstrated because the component is considered to be an important genotoxic intermediate metabolite of benzene. Occurrence of HHQ in coffee must have an important significance to consider genotoxicity of coffee.

Determination of malonaldehyde in oxidized lipids by the Hantzsch fluorometric method

Malonaldehyde is a secondary product formed during lipid oxidation. We developed a sensitive and reliable Hantzsch fluorometric method for determination of malonaldehyde in oxidized lipids. The principle of the method is based on the formation of highly fluorescent 1,4-dimethyl-1,4-dihydropyridine-3,5-dicarbaldehyde MI by reaction of malonaldehyde, methylamine, and acetaldehyde under neutral conditions. Compound MI formed could be estimated by high-performance liquid chromatography. Free malonaldehyde, that liberated under neutral conditions (labile forms) and that liberated by acid pretreatment (acid labile forms), could be determined by use of the calibration curves of MI versus malonaldehyde sodium salt. Oxidized methyl linoleate with a peroxide value of 1600 neq/mg contained 0.95 (free and labile) and 1.3 nmol (acid labile) malonaldehyde/mg, oxidized sardine oil with a peroxide value of 640 neq/mg contained 1.1 (free and labile) and 3.0 nmol (acid labile) malonaldehyde/mg, and the lipid fraction of oxidized rat liver microsomes contained less than 0.2 (free and labile) and 0.8 nmol (acid labile) malonaldehyde/mg. The malonaldehyde contents were much lower than those obtained by traditional 2-thiobarbituric acid test. It appears likely that the malonaldehyde contents, both free and labile, and acid labile forms, in oxidized lipids are too low to be taken into account.

DNA strand breaking by the carbon-centered radical generated from 4-(hydroxymethyl) benzenediazonium salt, a carcinogen in mushroom Agaricus bisporus

4-(Hydroxymethyl)benzenediazonium salt (HMBD), a carcinogen in mushroom Agaricus bisporus, was found to generate a carbon-centered radical, 4-(hydroxymethyl)phenyl radical, during incubation at pH 7.4 and 37 degrees C, when estimated by Electron Spin Resonance (ESR) spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), N-tert-butylphenyl-alpha-nitrone (PBN) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS). Formation of a substantial amount of benzyl alcohol during incubation of HMBD in the presence of a hydrogen donor, ethanol, supported the generation of the carbon-centered radical. When plasmid supercoiled DNA was incubated with HMBD at pH 7.4 and 37 degrees C for 30 min, the supercoiled DNA was converted into a nicked circular relaxed form and subsequently into a linear form. Sequence analysis indicated that the compound cleaved the plasmid DNA strand non-specifically. The intracellular double stranded DNA of Escherichia coli was fragmented by the compound, which may be responsible for its cytotoxicity. The compound induced mouse micronucleated peripheral reticulocytes. The compound was active in breaking DNA strands in the absence of molecular oxygen and in the presence of superoxide dismutase and catalase, indicating that no oxygen-derived radicals participated in the breaking. DNA breaking was inhibited by hydrogen donors butyl hydroxyanisole and ethanol, thiol compounds L-cysteine and 2-mercaptoethanol, and spin-trapping agents DMPO and PBN, indicating the direct contribution of the carbon-centered radical to the breaking.

DNA strand break by 2,5-dimethyl-4-hydroxy-3(2 H)-furanone, a fragnant compound in various foodstuffs

2,5-Dimethyl-4-hydroxy-3(2 H)-furanone (DMHF), produced by Maillard reaction of sugar/amino acid and found in various foodstuffs, showed mutagenicity to Salmonella typhimurium TA100 strain with and without S9 mix, and induced micronucleated mouse peripheral reticulocytes. DNA strand breaking activity of the compound at pH 7.4 increased with the increasing dose of the compound and with the increasing incubation time. The breaking activity was inhibited in the presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin trapping agents, thiol compounds and metal chelators, and also by removal of dissolved oxygen from the incubation mixture. Addition of Fe(III) ion to the incubation mixture enhanced the breaking activity. Incubation of DMHF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave electron spin resonance signals characteristic to DMPO-OH adduct, indicating generation of hydroxyl radical. It was found that DMHF generated hydroxyl radical with an aid of a trace amount of metal ions, and induced DNA strand breaking. Mutagenicity and induction of micronucleated reticulocytes by DMHF may be caused as a result of DNA modification via hydroxyl radical.

DNA strand breakage by hydroxyphenyl radicals generated from mutagenic diazoquinone compounds.

The mutagenic diazoquinone compounds p-diazoquinone (p-DQ), o-diazoquinone (o-DQ) and 3-diazo-N-nitrosobamethan (D-BM) cleaved the phosphodiester bond of lambda DNA, phi X174 RFI DNA and M13mp8ss DNA. p-DQ also cleaved the phosphodiester bond of bis(p-nitrophenyl)phosphate. The breakage of the phosphodiester bond was inhibited by the antioxidant butyl hydroxyanisole (BHA), ethanol, the spin trapping agent DMPO, cysteine and 2-mercaptoethanol. While incubation of p-DQ and o-DQ alone gave p-hydroquinone and catechol, respectively, incubation of these compounds in the presence of BHA and ethanol gave phenol in large yields. Incubation of p-DQ and o-DQ with the spin trapping agents DMPO and PBN gave spin adducts assignable as p- and o-hydroxyphenyl adducts, respectively. The breakage of the phosphodiester bond of DNA by the diazoquinone compounds is suggested to be due to the hydroxyphenyl radicals generated during incubation.