Yoshito Kumagai

Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage.

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 micrograms) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide.

Apparent inhibition of superoxide dismutase activity in vitro by diesel exhaust particles

The inhibitory effects of diesel exhaust particles (DEP) on superoxide dismutase (SOD) activity were examined in vitro because intratracheal administration of DEP to mice resulted in a suppression of the pulmonary enzyme activity (Sagai et al., Free Radic. Biol. Med. 14:37-47; 1993). Superoxide production, based on the reduction of cytochrome c, was suppressed considerably by the soluble fraction of mouse lung and by purified SOD from bovine erythrocytes, but the suppression was drastically diminished in the presence of methanol-extractable compounds of DEP. Inhibition of SOD by diethyldithiocarbamate was irreversible, but that by 1,2-naphthoquinone (1,2-NQ) and the methanol extract of DEP was removed by dialysis. Inhibition of superoxide mediated cytochrome c reduction by Tiron, a scavenging agent for superoxide, was blocked by the methanol extract and 1,2-NQ in a concentration-dependent manner. In contrast, addition of a large amount of SOD to the reaction mixture resulted in an almost complete disappearance of inhibitory action of not only 1,2-NQ but also the methanol extract. The existence of carbonyl compounds in the DEP was confirmed by thin-layer chromatography (TLC) with 2,4-dinitrophenylhydrazine reagent. Electron spin resonance (ESR) spectra of an incubation mixture of oxidized 1,2-dihydroxynaphthalene in the absence and presence of cytochrome c indicated a reaction between the semiquinone radical of 1,2-NQ and cytochrome c. These results indicate that the apparent reduction in SOD activity by DEP is due to the chemical reaction of superoxide with components like quinones, which reduce levels of superoxide.

Plasma protein-bound sulfhydryl group oxidation in humans following a full marathon race

Physical exercise can induce oxidative stress in humans. We studied the influence of aerobic exercise on the status of plasma protein-bound sulfhydryl groups in seven moderately-trained male college students who participated in a full marathon race. The plasma protein-bound sulfhydryl group values were significantly declined immediately after the race (-22%, p < 0.01), and 24 h (-12%, p < 0.01) and 48 h (-13%, p < 0.01) after the race, as compared to the baseline value. The plasma concentrations of thiobarbituric acid reactive substances were unchanged following the race, while the activities of plasma creatine kinase were significantly increased, indicating skeletal muscle damage. These results suggested that prolonged exercise may cause the oxidation of plasma proteins.

Direct Detection of Mercury-Bound Metalloproteins (metallothionein and Cu, Zn-Superoxide Dismutase) Using a Combination of Gel Electrophoresis and One Dimensional Synchrotron Radiation X-Ray Fluorescence Analysis

Abstract Metallothionein-II (MT-II) and Cu, Zn-superoxide dismutase (Cu, Zn-SOD) interacted with mercury were detected by a new method utilizing isoelectric focusing-agarose or -polyacrylamide gel electrophoresis (IEF-AGE or IEF-PAGE) and nondestructive one-dimensional synchrotron radiation X-ray fluorescence (SR-XRF) analysis. When MT-II reacted with mercuric chloride, an obvious change of isoelectric point (pI = 3.7 - 4.7) for the intact form to alkaline pI (9.4) was observed. This marked migration of MT-II by the metal was blocked by addition of glutathione, suggesting that sulfhydryl functions participate in the pI variation. In contrast, interaction of Cu, Zn-SOD with mercury did not cause any changes of its pI although the metal bound tightly to Cu, Zn-SOD after electrophoresis; however, the enzyme activity was drastically suppressed. These observations indicate that combination of electrophoresis with SR-XRF analysis is an useful technique for detecting structural or functional alteration of protei...