Kiyomi Kikugawa

Evidence that reactive oxygen species do not mediate NF‐κB activation

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)‐κB activation. This hypothesis is mainly based on the findings that N‐acetyl‐L‐cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF‐κB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF‐κB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)‐induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IκB–ubiquitin ligase activity in the cell‐free system where extracellular stimuli‐regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF‐κB signaling, but instead lower the magnitude of its activation.

Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.

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.

Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins

Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and malonaldehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5-dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Schiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanals, alk-2-enals and alka-2,4-dienals can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.

Thiobarbituric acid-reactive substances from peroxidized lipids.

The thiobarbituric acid (TBA) reaction was performed on linoleic acid 13-monohydroperoxide, autoxidized fatty esters, edible fats and oils, rat liver microsomal lipids, and on human erythrocyte ghost lipids in order to determine which substances from peroxidized lipids are TBA-reactive. The reaction was carried out in 2% acetic acid containing butylated hydroxytoluene using two different reaction modes: a one-step mode which involves heating at 100°C, and a two-step mode which involves first treatment at 5°C and subsequent heating at 100°C. Yields of the red 1∶2 malonaldehyde/TBA adduct, as estimated by absorbance, fluorescence intensity and high-performance liquid chromatography, were much higher than the malonaldehyde content as determined by direct chemical analysis. Yields of red pigment obtained by the two-step mode were slightly higher than those obtained by the one-step mode. Pigment yields were dramatically increased by addition oft-butyl hydroperoxide. Red pigment formation from alkenals and alkadienals was similarly enhanced by the two-step mode or by addition oft-butyl hydroperoxide, whereas pigment formation from malonaldehyde was not. It appears likely that a component of the total red pigment formed from the peroxidized lipids was due to aldehyde species other than malonaldehyde.

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.

Potential thiobarbituric acid-reactive substances in peroxidized lipids

This paper describes what components produce a red pigment in the thiobarbituric acid (TBA) test of peroxidized lipids. Alk-2-enals, alka-2,4-dienals and hydroperoxide functions are potential candidates for the red pigment in the TBA test. The TBA test can be regarded as an excellent method to reflect the combined effect of these components and thus the degree of lipid oxidation.

Thiobarbituric acid reaction of aldehydes and oxidized lipids in glacial acetic acid

Thiobarbituric acid (TBA) reaction of several aldehydes and oxidized lipids in glacial acetic acid was performed. All the samples were freely soluble in the solvent used. Saturated aldehydes produced a stable yellow pigment with an absorption maximum at 455 nm, a red pigment derived from malonaldehyde at 532 nm, and an orange pigment due to dienals at 495 nm. The absorbance maximum was 7–9 per μmol for saturated aldehydes, 27.5 per μmol for malonaldehyde and about 2 per μmol for dienals. Autoxidation of unoxidized lipids increased progressively in glacial acetic acid. When the TBA test was performed under nitrogen, autoxidation of unoxidized lipids was inhibited completely. While saturated aldehydes produced no yellow pigment under nitrogen, oxidized lipids produced a considerable amount of stable yellow pigment. The value for absorbance at 455 nm as a function of autoxidation time paralleled those of peroxide values. The absorbance of most oxidized lipids at 455 nm was higher than at 532 nm. Yellow pigment formation in the TBA test under nitrogen could not be ascribed to free saturated aldehydes but rather to unspecified closely related substances. The stable yellow pigment was found to be an excellent indicator of lipid oxidation.

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.