Masaru Shinyashiki

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.

Electrophilic and redox properties of diesel exhaust particles

The adverse health effects of air pollutants have been associated with their redox and electrophilic properties. Although the specific chemical species involved in these effects are not known, the characterization of their general physical and chemical properties is important to our understanding of the mechanisms by which they cause health problems. This manuscript describes results of a study examining the partition properties of these activities in aqueous and organic media. The water and dichloromethane (DCM) solubility of redox active and electrophilic constituents of seven diesel exhaust particle (DEP) samples were determined with assays developed earlier in this laboratory. The constituents exhibiting redox activity, which included both metals and nonmetal species, were associated with the particles in the aqueous suspensions. Portions of the redox active compounds were also DCM-soluble. In contrast, the electrophilic constituents included both water-soluble and DCM-soluble species. The role of quinones or quinone-like compounds in redox and electrophilic activities of the DCM-soluble constituents was assessed by reductive acetylation, a procedure that inactivates quinones. The results from this experiment indicated that most of the activities in the organic extract were associated with quinone-like substances. The partition properties of the reactive species are important in exposure assessment since the toxicokinetics of particles and solutes are quite distinct.

REDOX AND ELECTROPHILIC PROPERTIES OF VAPOR- AND PARTICLE-PHASE COMPONENTS OF AMBIENT AEROSOLS

Particulate matter (PM) has been the primary focus of studies aiming to understand the relationship between the chemical properties of ambient aerosols and adverse health effects. Size and chemical composition of PM have been linked to their oxidative capacity which has been postulated to promote or exacerbate pulmonary and cardiovascular diseases. But in the last few years, new studies have suggested that volatile and semi-volatile components may also contribute to many adverse health effects. The objectives of this study were: (i) assess for the first time the redox and electrophilic potential of vapor-phase components of ambient aerosols and (ii) evaluate the relative contributions of particle- and vapor-fractions to the hazard of a given aerosol. To achieve these objectives vapor- and particle-phase samples collected in Riverside (CA) were subjected to three chemical assays to determine their redox and electrophilic capacities. The results indicate that redox active components are mainly associated with the particle-phase, while electrophilic compounds are found primarily in the vapor-phase. Vapor-phase organic extracts were also capable of inducing the stress responding protein, heme-oxygenase-1 (HO-1), in RAW264.7 murine macrophages. These results demonstrate the importance of volatile components in the overall oxidative and electrophilic capacity of aerosols, and point out the need for inclusion of vapors in future health and risk assessment studies.

Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury

Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg-1 day-1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1. 6-1.9-fold, 5-8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000xg supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11-43 microM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.

Nitroxyl-mediated disruption of thiol proteins: inhibition of the yeast transcription factor Ace1.

Among the biologically and pharmacologically relevant nitrogen oxides, nitroxyl (HNO) remains one of the most poorly studied and least understood. Several previous reports indicate that thiols may be a primary target for the biological actions of HNO. However, the intimate details of the chemical interaction of HNO with biological thiols remain unestablished. Due to their ability to grow under a variety of conditions, the yeast Saccharomyces cerevisiae represents a unique and useful model system for examining the chemistry of HNO with thiol proteins in a whole-cell preparation. Herein, we have examined the effect of HNO on the thiol-containing, metal-responsive, yeast transcription factor Ace1 under a variety of cellular conditions as a means of delineating the chemistry of HNO interactions with this representative thiol protein. Using a reporter gene system, we find that HNO efficiently inhibits copper-dependent Ace1 activity. Moreover, this inhibition appears to be a result of a direct interaction between Ace1 thiols and HNO and not a result of any chemistry associated with HNO-derived species. Thus, this report indicates that thiol proteins can be a primary target of HNO biochemistry and that HNO-mediated thiol modification is likely due to a direct reaction of HNO.

Selective inhibition of the mouse brain Mn-SOD by methylmercury.

Changes in mRNA levels, protein contents and enzyme activities for brain Cu,Zn- and Mn-SOD by methylmercury chloride (MMC) administration, were examined, over a period of 12 days in ICR male mice. After subcutaneous administration of MMC (10 mg/kg) to mice, brain mercury content reached a maximum at 2 days and remained at that level for at least 5 days. MMC exposure resulted in a time-dependent decrease in the Mn-SOD activity: the enzyme activity at 5 days after exposure to MMC was about 60% of control level whereas this exposure was without effect on the Cu,Zn-SOD activity, indicating differential sensitivity of SOD isozymes to the metal. However, levels of mRNA and protein synthesis for Mn-SOD were unaffected by MMC administration. The direct effect of MMC on the both SOD activities were further examined with purified enzyme preparations. After each SOD isozyme (10 U) was incubated with 0.2 mM MMC for 24 h at pH 7.8, the enzyme activities for Cu,Zn- and Mn-SOD were 90% and 37% of control, respectively. Incubations at a ratio of SOD to MMC (1 : 600) for 24 h resulted in a substantial decrease in the enzyme activity of the Mn form; this isozyme-selective inactivation was noted at alkaline pH. A combination of isoelectric focusing-agarose gel electrophoresis (IEF-AGE) and synchrotron radiation X-ray fluorescence (SR-XRF) analysis revealed that Mn-SOD rather than Cu,Zn-SOD underwent modification. Furthermore, a decrease in native form of Mn-SOD protein after MMC exposure was confirmed by gel filtration chromatography. These results indicate that Mn-SOD, but not Cu,Zn-SOD, is susceptible to modification by MMC and the resulting alteration in structure appears to cause a loss of enzyme activities.

Post-transcriptional elevation of mouse brain Mn-SOD protein by mercuric chloride

Alterations in gene expression, protein content and enzyme activity of brain Mn-SOD following mercuric chloride (HgCl2) exposure were examined in ICR male mice. Subcutaneous administration of HgCl2 (1 mg Hg/kg) resulted in a significant increase (4-fold) in the brain Mn-SOD content at 6 h after injection while the total mercury concentration was about 0.11 microg/g of brain. The enhancement of Mn-SOD protein caused by HgCl2 was completely abolished by pretreatment with dexamethasone (3 mg/kg) 1 h prior to HgCl2 administration, suggesting involvement of inflammation in inorganic mercury-induced increase in the antioxidant enzyme. This increase in level of Mn-SOD content coincided with a substantial rise in the enzyme activity; however, Northern blot analysis revealed that the induction of protein level was not due to that of its gene expression. The results of the present study indicate that mouse brain Mn-SOD appears to undergo post-translational modification by the environmental toxic metal, and induction of the antioxidant enzyme could be of an initial response to the metal-induced oxidative stress.

An efficient method for purification of cuprozinc superoxide dismutase from bovine erythrocytes.

Cuprozinc superoxide dismutase (Cu,Zn-SOD) was isolated from bovine erythrocytes by pH-controlled ammonium sulfate-methanol extraction (ASME extraction). Adjustment of the pH of a suspension of the lysed red cells in the presence of ammonium sulfate (90% saturation) to pH 5.0, followed by partition with an equal amount of methanol, resulted in isolation of the enzyme with specific, activity of greater than 2000 units/mg of protein. Further purification using DEAE-cellulose column chromatography gave a highly purified Cu,Zn-SOD showing a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure about 14 mg of pure Cu,Zn-SOD with a specific activity of 4728 units/mg of protein can be recovered from one liter of bovine blood. The enzyme was characterized and the results obtained were in agreement with earlier reports. This procedure appears, therefore, to be a convenient method for isolating the enzyme.

Alterations in Superoxide Dismutase Isozymes by Methylmercury

A study of alterations in two mammalian superoxide dismutases (SODs), Cu,Zn-SOD and Mn-SOD, caused by methylmercury has been reviewed. Mechanisms for the isozyme-selective decrease in MnSOD activity by the organometallic compound observed in vivo have been extensively examined by experiments with purified enzyme preparations.

Mercury dynamics in hair of rats exposed to methylmercury by synchrotron radiation X-ray fluorescence imaging

Two-dimensional distribution of mercury (Hg) in hair samples of rats exposed to methylmercury (MeHg) was analyzed by synchrotron radiation X-ray fluorescence (SR-XRF) imaging. Experiments with endogenous- and exogenous-model for MeHg exposure revealed that the metal level was obviously higher in the hair cortex after the former exposure whereas a dominant site that Hg distributed after the latter exposure was the cuticle. The method also provided us the Hg profile along the hair length with a single hair obtained by the endogenous model. Thus application of SR-XRF analysis to hair sample would facilitate biological monitoring to not only distinct Hg exposure but also determine its dynamics with only the specimen.