Sanae Muraoka

Lipid peroxidation induced by indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals.

Some of the side-effects of using indomethacin (IM) involve damage to the gastric mucosa and liver mitochondria. On the other hand, neutrophils infiltrate inflammatory sites to damage the tissues through the generation of reactive oxygen species by myeloperoxidase. The stomach and intestine have large amounts of peroxidase. These findings suggest that peroxidases are involved in tissue damage induced by IM. To clarify the basis for the tissue damage induced by IM in the presence of horseradish peroxidase (HRP) and H2O2 (HRP-H2O2), lipid peroxidation was investigated. When IM was incubated with liver microsomes in the presence of HRP-H2O2 and ADP-Fe3+, lipid peroxidation was time-dependent. Catalase and desferrioxamine almost completely inhibited lipid peroxidation, indicating that H2O2 and iron are necessary for lipid peroxidation. Of interest, superoxide dismutase strongly inhibited lipid peroxidation, and it also inhibited the formation of bathophenanthroline-Fe2+, indicating that reduction of the ferric ion was due to superoxide (O2-). ESR signals of IM radicals were detected during the interaction of IM with HRP-H2O2. However, the IM radical by itself did not reduce the ferric ion. These results suggest that O2- may be generated during the interaction of IM radicals with H2O2. Ferryl species, which are formed during the reduction of iron by O2-, probably are involved in lipid peroxidation.

DNA damage induced by catechol derivatives.

We investigated the effect of catechol derivatives, including dopa, dopamine, adrenaline and noradrenaline, on DNA damage and the mechanisms of DNA strand breakage and formation of 8-hydroxyguanine (8HOG). The catechol derivatives caused strand breakage of plasmid DNA in the presence of ADP-Fe(3+). The DNA damage was prevented by catalase, mannitol and dimethylsulfoxide, suggesting hydroxyl radical (HO..)-like species are involved in the strand breakage of DNA. Iron chelators, such as desferrioxamine and bathophenanthroline, and reduced glutathione also inhibited the DNA damage. Deoxyribose, a molecule that is used to detect HO,, was not degraded by dopa in the presence of ADP-Fe(3+). By adding EDTA, however, dopa induced the marked deoxyribose degradation in the presence of ADP-Fe(3+), indicating that EDTA may extract iron from ADP-Fe(3+) to catalyze HO. formation by dopa. Thus, EDTA was a good catalyst for HO.-generation, whereas it did not promote the strand breakage of DNA. However, calf thymus DNA base damage, which was detected as 8-HOG formation, was caused by dopa in the presence of EDTA-Fe(3+), but not in the presence of ADP-Fe(3+). The 8HOG formation was also inhibited by catalase and HO. scavengers, indicating that HO&z.rad; was involved in the base damage. These results suggest that DNA strand breakage is due to ferryl species rather than HO., and that 8HOG formation is due to HO. rather than ferryl species.

Inactivation of creatine kinase during the interaction of mefenamic acid with horseradish peroxidase and hydrogen peroxide: participation by the mefenamic acid radical.

Creatine kinase (CK) was used as a marker molecule to examine the side effects of damage to tissues by mefenamic acid, an effective drug to treat rheumatic and arthritic diseases, with horseradish peroxidase and hydrogen peroxide (HRP-H(2)O(2)). Mefenamic acid inactivated CK during its interaction with HRP-H(2)O(2). Also, diphenylamine and flufenamic acid caused a loss of CK activity, indicating the imino group, not substituent groups, in the phenyl rings have a crucial role in CK inactivation. Rapid change in mefenamic acid spectra was detected, suggesting that mefenamic acid is efficiently oxidized by HRP-H(2)O(2). Peroxidases oxidize xenobiotics to free radicals by a one-electron transfer. However, direct detection of mefenamic acid radicals by electron spin resonance (ESR) was unsuccessful. Reduced glutathione and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) in the reaction mixture containing mefenamic acid with HRP-H(2)O(2) produced ESR signals consistent with a DMPO-glutathionyl radical adduct. These results suggest that inactivation of CK is probably caused through formation of mefenamic acid radicals. Sulfhydryl groups and tryptophan residues of CK were diminished by mefenamic acid with HRP-H(2)O(2). Other SH enzymes, including alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were very sensitive to mefenamic acid with HRP-H(2)O(2). Inactivation of SH enzymes may explain some deleterious actions of mefenamic acid.

Inactivation of creatine kinase induced by quercetin with horseradish peroxidase and hydrogen peroxide. pro-oxidative and anti-oxidative actions of quercetin.

Pro-oxidative and anti-oxidative actions of quercetin were examined through inactivation of CK and inhibition of lipid peroxidation. Quercetin induced inactivation of creatine kinase (CK) during the interaction with horseradish peroxidase and hydrogen peroxide (HRP-H(2)O(2)). CK activity in heart homogenate was also reduced by quercetin with HRP-H(2)O(2). Flavonoids that have a catechol structure in the B ring, such as taxifolin, catechin and luteolin, also induced CK inactivation. These flavonoids strongly inhibited NADPH and ADP-Fe(3+)-dependent microsomal lipid peroxidation. These results suggest a close relationship between pro-oxidative and anti-oxidative actions of quercetin. Electron spin resonance (ESR) signals of the quercetin radical was emitted during the interaction of quercetin with HRP-H(2)O(2) in the presence of Zn(2+) as a stabilizer. Adding CK diminished the ESR signals of quercetin radicals, suggesting CK efficiently scavenged quercetin radicals. Sulfhydryl groups and tryptophan residues in CK decreased during the interaction of quercetin with HRP-H(2)O(2). The kinetic parameters of K(m) and V(max) for ADP and creatine phosphate changed rapidly, suggesting that the inactivation of CK was induced through conformational change of the enzyme. Glyceraldehyde-3-phosphate dehydrogenase had a higher sensitivity to quercetin with HRP-H(2)O(2) than CK. Quercetin radicals may mediate between pro-oxidative and anti-oxidative action.

Inactivation of creatine kinase by Adriamycin® during interaction with horseradish peroxidase

Oxidative damage of creatine kinase (CK) induced by Adriamycin((R)) (ADM) with peroxidase was investigated using horseradish peroxidase (HRP). ADM oxidatively inactivated CK during its interaction with HRP in the presence of H(2)O(2) (HRP-H(2)O(2)). The red color of ADM was lost during oxidation by HRP-H(2)O(2). Adding catalase stopped the color change of ADM induced by HRP-H(2)O(2), indicating that ADM was oxidized by HRP complex I or II. CK was inactivated readily, even when it was added to the reaction mixture containing colorless ADM. Some sulfhydryl groups of CK, which have an important role in its enzyme activity, were very sensitive to ADM activated by HRP-H(2)O(2), suggesting that inactivation of CK is due to oxidation of SH groups at the active center. Presumably, oxidative ADM quinone is involved dominantly in the inactivation of CK. Among the anthracycline drugs tested in this study, only ADM and epirubicin caused inactivation of CK and alcohol dehydrogenase and loss of the red color during oxidation by HRP-H(2)O(2).

Inactivation of creatine kinase induced by dopa and dopamine in the presence of ferrylmyoglobin

We investigated the effect of dopa and dopamine on creatine kinase (CK) activity in the presence of ferrylmyoglobin (ferrylMb). CK was sharply inhibited by dopa and dopamine in the presence of ferrylMb. Dopa and dopamine markedly promoted the reduction of ferrylMb to metmyoglobin (metMb). The semiquinone from dopa and dopamine may be involved in CK inactivation. During inactivation of the enzyme, both kinetic parameters Vmax and Km changed. In addition, reduced glutathione restored the activity of CK at an early stage. These results suggest that inactivation of CK is dominantly due to oxidation of sulfhydryl (SH) groups of the enzyme. Other catechols, such as adrenaline and noradrenaline, little inactivated CK activity, whereas they promoted the reduction of ferrylMb to metMb. Other SH enzymes, including alcohol dehydrogenase (ADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were inactivated to a lesser extent by dopa and dopamine in the presence of ferrylMb. Adrenaline and noradrenaline did not significantly prevent the inactivation of ADH and very slightly inhibited GAPDH. These results suggest that dopa and dopamine act as prooxidants to inactivate SH enzymes in the presence of ferrylMb.

Oxidative damage to bovine serum albumin induced by hydroxyl radical generating systems of xanthine oxidase + EDTA−Fe3+ and ascorbate + EDTA−Fe3+

Oxidative damage to bovine serum albumin (BSA) was induced by hydroxyl radical (HO.) generating systems of xanthine oxidase (XO) + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. Formation of bityrosine and loss of tryptophan were observed in the ascorbate + EDTA-Fe3+ system and carbonyl formation was induced by both systems. Mannitol and ethanol very strongly inhibited the carbonyl and/or bityrosine formation, indicating that the oxidative damage to BSA was due to HO(.). The sulfhydryl (SH) groups of BSA were very sensitive to the XO + EDTA-Fe3+ but not to the ascorbate + EDTA-Fe3+ system. Catalase but not hydroxyl radical scavengers or superoxide dismutase strongly inhibited the loss of SH groups, indicating that H2O2 is involved in their oxidation. Fragmentation of BSA was observed during exposure to the XO + EDTA-Fe3+ and ascorbate + EDTA-Fe3+ systems and the products presented a broad band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Little formation of amine groups was observed in these systems, indicating that little peptide bond cleavage occurred. BSA exposed to the ascorbate + EDTA-Fe3+ system was more readily degraded by trypsin than that exposed to the XO + EDTA-Fe3+ system. Elastase degraded BSA exposed to the ascorbate + EDTA-Fe3+ system but not to the XO + EDTA-Fe3+ system.

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide.

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.

Lipid peroxidation induced by phenylbutazone radicals

Lipid peroxidation was investigated to evaluate the deleterious effect on tissues by phenylbutazone (PB). PB induced lipid peroxidation of microsomes in the presence of horseradish peroxidase and hydrogen peroxide (HRP-H2O2). The lipid peroxidation was completely inhibited by catalase but not by superoxide dismutase. Mannitol and dimethylsulfoxide had no effect. These results indicated no paticipation of superoxide and hydroxyl radical in the lipid peroxidation. Reduced glutathione (GSH) efficiently inhibited the lipid peroxidation. PB radicals emitted electron spin resonance (ESR) signals during the reaction of PB with HRP-H2O2. Microsomes and arachidonic acid strongly diminished the ESR signals, indicating that PB radicals directly react with unsaturated lipids of microsomes to cause thiobarbituric acid reactive substances. GSH sharply diminished the ESR signals of PB radicals, suggesting that GSH scavenges PB radicals to inhibit lipid peroxidation. Also, 2-methyl-2-nitrosopropan strongly inhibited lipid peroxidation. R-Phycoerythrin, a peroxyl radical detector substance, was decomposed by PB with HRP-H2O2. These results suggest that lipid peroxidation of microsomes is induced by PB radicals or peroxyl radicals, or both.

Phenylbutazone Radicals Inactivate Creatine Kinase

Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by phenylbutazone (PB), an effective drug to treat rheumatic and arthritic diseases, with horseradish peroxidase and hydrogen peroxide (HRP-H2O2). PB inactivated CK during its interaction with HRP-H2O2, and inactivated CK in rat heart homogenate. PB carbon-centered radicals were formed during the interaction of PB with HRP-H2O2. The CK efficiently reduced electron spin responance signals of the PB carbon-centered radicals. The spin trap agent 2-methyl-2-nitrosopropane strongly prevented CK inactivation. These results show that CK was inactivated through interaction with PB carbon-centered radicals. Sulfhydryl groups and tryptophan residues in CK were lost during the interaction of PB with HRP-H2O2, suggesting that cysteine and tryptophan residues are oxidized by PB carbon-centered radicals. Other enzymes, including alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, but not lactate dehydrogenase, were also inactivated. Sulfhydryl enzymes seem to be sensitive to attack by PB carbon-centered radicals. Inhibition of SH enzymes may explain some of the deleterious effects induced by PB.