加藤 陽二

The protective activity of rosmarinic acid from Perilla frutescens on liver injury induced by LPS in D-GalN-sensitized mice was examined. We also investigated the effects of antitumor necrosis factor-a antibody (anti-TNF), superoxide dismutase (SOD), and aminoguanidine (AG) on this model in order to elucidate the mechanism of rosmarinic acid protection. Perilla extract (PE) and rosmarinic acid (RA) treatments significantly reduced the elevation of plasma asparatate aminotransferase levels, as well as anti-TNF and SOD treatment, compared with controls, but this reduction was not seen in the AG group. These results were confirmed by histological examination using hematoxylin-eosin and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Increases in tumor necrosis factor-alpha (TNF-alpha) mRNA expression in liver and in plasma TNF-alphalevels, which were observed in control mice, were not significantly reduced by PE or RA. PE and RA treatments also did not significantly diminish iNOS mRNA expression or plasma nitrate/nitrite levels. Nitrotyrosine and N-epsilon-(hexanonyl)lysine (HEL) production, the residue of oxidative stress, was observed in livers from controls, but not in those mice pretreated with PE or RA. These results suggest that the liver protection of RA is due to the scavenging or reducing activities-superoxide or peroxynitirite rather than to inhibition of TNF-alpha production. (C) 2002 Elsevier Science Inc.

&nbsp;&nbsp;New biomarkers for oxidative damage, were used to identify whether hyperglycemia caused oxidative stress in diabetic Akita mice. At 13 weeks of age, the tissues of these mice were obtained, and the levels of N^&epsilon;-(hexanonyl)lysine (HEL) and dityrosine (DT) were measured, these being related to lipid peroxide-derived protein covalent modification and protein cross-linking. The levels of HEL and DT in the kidneys of Akita mice were significantly increased compared with the control mice without any accumulation of thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified protein. Immunopositive staining was clearly observed in the kidneys of the Akita mice when using the anti-HEL antibody or anti-DT antibody. These results suggest that hyperglycemia in Akita mice induced oxidative stress and increased these markers in the kidneys.<br>

We have shown that the orally administered cyanidin 3-O-β-D-glucoside (C3G) attenuates the hepatic ischemia-reperfusion (I/R) injury, which was used as a model for oxidative stress through a decrease in neutrophil chemoattractant production in rats. The rats were subjected to hepatic I/R at 30 min after the administration of C3G (0.9 mmol/kg body weight) or vehicle. I/R treatment resulted in the elevation of oxidative stress marker [liver thiobarbituric acid-reactive substance, N^ε-(hexanonyl) lysine and dity-rosine] levels in the liver and of the serum activities of marker enzymes for liver injury. The administration of C3G significantly suppressed these elevations, which had been caused by hepatic I/R. Liver myeloperoxidase activity, a useful marker for neutrophil infiltration into tissues, and the plasma and liver concentration of cytokine-induced neutrophil chemoattractant-1(CINC-1), which has a potent chemotactic activity, were markedly elevated in the control group after hepatic I/R. However, these elevations were significantly suppressed in the C3G group. C3G and its metabolites in the plasma and liver were detected in the C3G group after hepatic I/R. These results suggest that the absorbed C3G and/or its metabolites can act as antioxidants in the blood and liver and scavenge the reactive oxygen species, and brought on a decrease in neutrophil infiltration into the liver through the suppression of CINC-1 production and the tissue damage caused by neutrophils after I/R is attenuated.

Endogenous lipid peroxidation products react with DNA and form exocyclic DNA adducts. The purpose of this study was to investigate the in vivo formation of 7-(2-oxo-heptyl)-substituted 1,N-2-etheno-2'-deoxyguanosine adduct (Oxo-heptyl-epsilondG), one of the major products from the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with dG. The monoclonal antibody specific to Oxo-heptyl-epsilondG was prepared using a chemically synthesized conjugate of Oxo-heptyl-epsilondG and carrier protein as immunogen. The characterization showed that the obtained antibody (mAb6A3) is specific to the Oxo-heptyl-epsilondG moiety. Using the novel antibody, the presence of the Oxoheptyl-epsilondG adduct in vivo was immunohistochemically revealed in the liver of rats fed a choline-deficient, L-amino acid-defined diet, an endogenous carcinogenesis model, for 3 days. In addition, the Oxo-heptyl-epsilondG formation was confirmed in DNAs treated with peroxidized linoleic acid, arachidonic acid and gamma-linolenic acid, respectively, suggesting that the hydroperoxides of omega-6 polyunsaturated fatty acids could be the potential sources of Oxo-heptyl-epsilondG formation in vivo. Collectively, the results in this study suggest the first evidence that the formation of Oxo-heptyl-epsilondG, the omega-6 lipid hydroperoxide-mediated DNA adduct, may be a potential biomarker for the early phase of carcinogenesis.

Dityrosine formation leads to the cross-linking of proteins intra- or intermolecularly. The formation of dityrosine in lens proteins oxidized by metal-catalyzed oxidation (MCO) systems was estimated by chemical and immunochemical methods. Among the four MCO systems examined (H2O2/Cu, H2O2/Fe-ethylenediaminetetraacetic acid (Fe-EDTA), ascorbate/Cu, ascorbate/Fe-EDTA), the treatment with H2O2/Cu preferentially caused dityrosine formation in the lens proteins. The success of oxidative protein modification with all the MCO systems was confirmed by carbonyl formation estimated using 2,4-dinitrophenylhydrazine. The loss of tyrosine by the MCO systems was partly due to the formation of protein-bound 3,4-dihydroxyphenylalanine. The formation of dityrosine specific to H2O2/Cu was confirmed by using poly-(Glu, Ala, Tyr) and N-acetyl-tyrosine as a substrate. The dissolved oxygen concentration in the H2O2/Cu system hardly affected the amount of dityrosine formation, suggesting that dityrosine generation by the H2O2/Cu system is independent of oxygen concentration. Moreover, the combination of copper ion with H2O2 is the most effective system for dityrosine formation among various metal ions examined. The addition of reducing agents, glutathione or ascorbic acid, into the H2O2/Cu system suppressed the generation of the dityrosine moiety, suggesting effective quench of tyrosyl radicals by the reducing agents. (C) 2001 Elsevier Science Inc.

Dityrosine is one of the specific biomarkers for protein oxidation. We prepared an antibody specific for protein dityrosine using a dimer of 3-p(hydroxyphenyl)propionic acid (di-HP) as a hapten. Three clones (A8, G6, and 1C3) were obtained, and the antibody from the A8 clone reacted with the di-HP-conjugated protein but not with a dityrosine conjugate. The others (G6 and 1C3 clones) recognized both the di-HP and dityrosine conjugates. The antibodies reacted with peptidyl dityrosine, derived from Thr-Tyr-Ser, rather than the free dityrosine. The reactivity of the latter two antibodies with lens proteins oxidized by incubation with H2O2/Cu was in good accordance with the formation of the dityrosine-like fluorescence. Using the obtained monoclonal antibody, the immunopositive staining of atherosclerotic lesions in Apo E-deficient mice was confirmed by an immunohistochemical technique. (C) 2000 Academic Press.

The monoclonal antibody to N-epsilon-(hexanonyl) lysine (HEL), a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, has been prepared and characterized. The obtained antibody specifically recognized the HEL moiety, Using the monoclonal antibody, we evaluated the protective effects of feeding eriocitrin, which is one of flavonoids in lemon fruit, on oxidative modification induced by exercise in rats. The supplementation of eriocitrin significantly suppressed the increase in HEL in the skeletal muscle by exercise. The result suggests that the determination of HEL may be a good method for evaluation of the protective effect of beneficial food factors against oxidative stress. (C) 2000 Academic Press.

こんにゃくは、水酸化カルシウム溶液に浸されたアルカリ性を呈する食品であるが、腸管出血性大腸菌O157との関わりを検討した報告はない。そこで、本研究では、こんにゃく液中におけるO157の生存状況と、こんにゃく液の抗菌効果(静菌作用あるいは殺菌作用)について検討した。こんにゃく液はいずれもpH12前後であり、この高いpH域では、O157は反応させるだけで検出限界までに激減することがわかった。初発菌数が多い場合でも1日の反応(保存)でほとんどが死滅した。また、この抗菌作用は大腸菌の株による違いや温度差には影響されなかった。一方、食中毒菌であるサルモネラ菌、腸炎ビブリオ菌および黄色ブドウ球菌についても同様に比較検討したところ、こんにゃくの液の抗菌効果は、O157と比べると同等かあるいはやや弱いようであった。しかし、これらの食中毒菌もこんにゃく液中では時間とともに減少し、2〜3日後ではほとんどが死滅することが判明した。さらに、市販こんにゃく液中の細菌の存在有無を検討したが、調べた限り、開封直後のこんにゃく液中には菌は検出されなかった。以上の結果から、こんにゃく液は衛生学的に安全性の高いことが証明され、静菌作用(菌の増殖抑制・阻害作用)というよりは、殺菌作用(生菌数の低減化作用)をもつことが明らかとなった。こんにゃくのような高アルカリ性呈示食品は、少数の生菌数の摂取でも発症するような腸管出血性大腸菌O157などの感染症防止に対しても、大きな役割を果たしているものと考えられる。

The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employing N-benzoyl-glycyl-L-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-N-epsilon-(hexanonyl)lysine. To prove the formation of N-epsilon-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.

Lipofuscin is a yellowish brown fluorescent pigment which is sequestered within cytoplasmic granules during aging. To examine the contribution of protein oxidation to lipofuscin accumulation, we performed immunohistochemical detection of dityrosine, which is considered one of the specific markers for protein oxidation, in lipofuscin in the aged human brain using an antibody specific to dityrosine. By characterization using competitive enzyme-linked immunosorbent assay, the specificity of the antibody to dityrosine was confirmed, None of the other tyrosine-related compounds such as L-tyrosine, 3-nitrotyrosine, 3-chlorotyrosine, or 3,4-dihydroxyphenylalanine cross-reacted with the antibody. The anti-dityrosine antibody reacted with lipofuscin granules in the pyramidal neurons of the aged human brain, The results suggest that protein oxidation by free radicals and/or peroxidases may play an important role in lipofuscin accumulation. (C) 1998 Federation of European Biochemical Societies.

The reaction of lipid hydroperoxide with protein was investigated using an antibody, which was prepared using 15-hydroperoxyeicosatetraenoic acid (15-HPETE)-modified keyhole limpet hemocyanin as an immunogen. The obtained antibody recognized not only 15-HPETE-modified bovine serum albumin (BSA) but also 13-hydroperoxyoctadecadienoic acid (13-HPODE)-modified BSA. Glutaroyl-BSA adduct, which was prepared by the reaction of glutaric anhydride with protein, was also recognized by the antibody. The results revealed that the carboxyl terminus of lipid moiety in adducts was required for an appearance of the antigenicity. The cross-reactivity of phosphatidylcholine hydroperoxide-modified BSA (PCAOOH-BSA) with the antibody was examined. The antibody could not recognize the intact PCAOOH-BSA, whereas alkaline-treated modified BSA revealed the antigenicity. Furthermore, stearic acid at the 1 position in the phospholipid was liberated from the PCAOOH-BSA following treatment with 0.25 N NaOH. The result showed that the phospholipid moiety could be covalently bound to the protein molecule. The formation of esterified fatty acid-protein adduct during oxidation was confirmed using low-density lipoprotein (LDL). During oxidation of LDL by copper ion or 2,2'-azo-bis(2-amidinopropane)dihydrochloride, the formation of antigenic materials was observed in a time-or dose-dependent fashion, The antigenicity was significantly enhanced by the alkaline treatment on the oxidized LDL, suggesting that considerable amounts of oxidized esterified fatty acids can covalently react with apoprotein B-100 in oxidatively modified LDL. (C) 1998 Academic Press.

To investigate the prevention of protein modification by food components, effects of natural antioxidants on in vitro modification of collagen by peroxynitrite were examined using a polyclonal antibody specific to 3-nitrotyrosine, To perform the assay with many samples at a time, the inhibitory effects of antioxidants were evaluated using an enzyme-linked immunosorbent assay. Polyphenols such as caffeic acid, curcumin, and flavonoids showed strong inhibitory effects on the formation of 3-nitrotyrosine in peroxynitrite-modified collagen. The inhibitory effects of caffeic acid and its related compounds on nitration of Tyr were then further investigated. Both caffeic acid, ferulic acid, and p-coumaric acid inhibited the modification, whereas cinnamic acid did not. By the treatment of p-coumaric acid with peroxynitrite, nitrated p-coumaric acid was detected in the reaction mixture using a liquid chromatograph-mass spectrometer (LC-MS). These results suggest that some phenolic antioxidants may prevent tissue injury by oxidants including peroxynitrite in vivo.

Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal antibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 11E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODE-KLH) as immunogen. The antibody recognized 13-HPODE-modified bovine serum albumin (BSA), but not aldehyde-modified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13-HPODE and 13S- hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOTRE(alpha)). The oxidized alpha-linolenic acid- and linoleate-protein adducts were recognized by the antibody. Oxidized phospholipid-protein adducts were scarcely recognized by the antibody. However, when ester bonds of phospholipids containing linoleic acid were hydrolyzed by alkaline treatment, the cross-reactivities appeared. The result suggests that a phospholipid hydroperoxide can react with a protein directly or indirectly, and a carboxyl terminal (COOH) of the lipid in an adduct was needed as an epitope. Oxidized LDL (ox-LDL) was prepared by the incubation of LDL with copper ion or 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), and the formation of lipid hydroperoxide-modified apolipoprotein was confirmed using the antibody. A slight immunoreactivity was observed in ox-LDL without alkaline treatment. When the ox-LDL was treated with alkali to hydrolyze the ester bonds of the lipid, enhanced antigenicity appeared with time-dependency. The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.

The aim of this study was to clarify the mechanism of loss of Trp residues in proteins exposed to peroxynitrite. The Trp residues in bovine serum albumin and collagen IV were decreased by peroxynitrite treatment. To identify the degradation products of the Trp residue by peroxynitrite, tert-butoxycarbonyl-L-tryptophan (Boc-Trp) was used as a model of the Trp residue in proteins, and the products formed from peroxynitrite-treated Boc-Trp were then isolated. Boc-Trp decreased with an increase in peroxynitrite concentration. N-Formylkynurenine, oxindole, and hydropyrroloindole were identified as major products. The formation of these products may account for the losses of Trp residues in proteins by peroxynitrite. (C) 1997 Academic Press.

The objective of this study was to detect carboxymethyllysine (CML) moieties in biological materials using immunological methods. We prepared a polyclonal antibody specific to the carboxymethylated protein. The antibody recognizes the CML moiety in protein. Protein-bound carboxymethylcysteine (CMS) is scarcely recognized by the antibody. By use of the antibody, the formation of carboxymethylated apo B in copper-oxidized low density lipoprotein (ox-LDL) was immunochemically confirmed. To clarify the source compounds of carboxymethylation, various aldehyde-modified BSAs were prepared, and their reactivities with the antibody were investigated. Among the aldehyde-modified BSAs used, the glyoxal-BSA adduct showed a strong antigenicity. As Ear as we know, this is the first report of the detection of the CML moiety in ox-LDL using immunological methods. (C) 1996 Academic Press, Inc.

Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.

Riboflavin-sensitized photodynamic modification of collagen led to significant formation of cross-linked molecules. Sodium azide or 1,4-diazabicyclo(2,2,2)octane, which are known to be singlet oxygen quenchers, and catalase could not inhibit the modification. Surprisingly, the collagen modification was accelerated in the presence of superoxide dismutase. The aggregation was accompanied by the loss of tyrosine and histidine residues in the collagen. An inhibitory effect of dissolved oxygen on the modification of collagen was observed. Similarly, the loss of tyrosine residues in the irradiated collagen was inhibited in the presence of dissolved oxygen. Dityrosine formation was also observed with the loss of tyrosine. These results indicate that photodynamic modification of tyrosine probably contributes to the riboflavin-sensitized cross-linking of collagen through the formation of dityrosine.

Oxidative degradation of collagen and the model peptides by Cu(II)/H2O2 has been studied. The depolymerization of collagen was predominantly observed by use of gel filtration chromatography. Polyproline was used as a model for collagen, and the oxidative modification was examined by amino acid analysis. Glutamic acid and gamma-aminobutyric acid were identified in the hydrolysates of oxidized polyproline. The formation of glutamic acid was reduced by treatment with NaBH4. The model peptide, (Pro-Pro-Gly)10, was also degraded by Cu(II)/H2O2, and a new N-terminal glycine was generated in proportion to the reaction time. Hydroxyl radical scavengers show only partial inhibition of the degradation of (Pro-Pro-Gly)10. In order to estimate the fragmentation mechanism, we used N-tert-butoxycarbonyl (Boc)-L-prolylglycine as a model for collagen and (Pro-Pro-Gly)10. The degradation products were isolated and characterized. Then N-tert-Boc-2-pyrrolidone, which provides gamma-aminobutyric acid by acid hydrolysis, was identified. The formation of a 2-pyrrolidone compound from oxidized BOC-L-prolylglycine is direct evidence for the scission of the peptide bond. The time-dependent formation of N-tert-Boc-2-pyrrolidone and liberation of glycine from N-tert-Boc-L-prolylglycine exposed to Cu(II)/H2O2 was observed. These results suggest that the cleavage of the peptide bond (Pro-Gly) was caused by oxidation of the proline residue, which led to the formation of the 2-pyrrolidone compound. We confirmed that proline oxidation leads to the fragmentation of proteins, accompanied by the formation of a 2-pyrrolidone structure.

The effects of ultraviolet irradiation on collagen and its model peptides were studied. Degradation of collagen was predominant in the system using gel filtration chromatography. The fragmentation was presumably due to oxidation of proline, since collagen is a proline-rich protein and proline residues on collagen markedly decreased with irradiation. To clarify the fragmentation mechanism, poly(L-proline) and (Pro-Pro-Gly)10 as models of a collagen molecule were used and their oxidation was investigated. Glutamic acid, gamma-aminobutyric acid (GABA), and ammonia from the hydrolysates of the irradiated prolyl peptides were identified by amino acid analysis. It was presumed that GABA was generated from a 2-pyrrolidone structure by acid hydrolysis. To confirm this prediction, N-tert-butoxycarbonyl (Boc)-L-proline and N-tert-Boc-L-prolylglycine were exposed to ultraviolet light, and the irradiation products were isolated and characterized. Then, N-tert-Boc-2-pyrrolidone was identified from both UV-irradiated N-tert-Boc-L-proline and N-tert-Boc-L-prolylglycine. We proposed that the formation of the 2-pyrrolidone compound must contribute to the fragmentation of prolyl peptide on the basis of its structural property.

Oxidative degradation of collagen by metal-catalyzed free radical generating systems (Cu(II)/ascorbate, Fe(II)/ascorbate, Cu(II)/H2O2, and Fe(II)/H2O2) has been studied. SDS-PAGE revealed that the reaction of collagen with these systems gave rise to depolymerization of protein. The amino acid analysis of the oxidized collagens indicated significant loss of proline, which was accompanied by the formation of hydroxyproline, aspartic acid, and glutamic acid. In addition, among these free radical generating systems, we found that collagen was most sensitive to oxidation by Cu(II)/H2O2. This preferential reactivity of Cu(II)/H2O2 was also confirmed by using other prolyl polypeptides such as polyproline and poly(Pro-Gly-Pro). We confirmed that the acid hydrolysis of the oxidized prolyl polypeptide generated considerable amounts of gamma-aminobutyric acid other than hydroxyproline and glutamic acid.