Makihiko Masuda

Electrooxidation of methanol on platinum bonded to the solid polymer electrolyte, Nafion

The electrooxidation of methanol was enhanced on PtSn-SPE, PtRu-SPE and PtIr-SPE in sulfuric acid solution, when compared with the activity of Pt-SPE, which has already been shown to have a higher activity than a Pt electrode. SPE is an abbreviation for Nafion, a solid polymer electrolyte. It is suggested that this dual enhancement of the oxidation rate for PtSn-SPE and PtRu-SPE catalysts is due to the modification of the oxidation state of Pt by Sn and Ru and to the presence of H2O and CH3OH, both modified by the SPE matrix. This modification appears to weaken their hydrogen bonds in solution. Both Pt and Ir have catalytic properties for methanol oxidation, but a PtIr-SPE catalyst showed a more enhanced catalytic activity than either of them. This will be discussed in terms of Ir, oxidized at relatively low positive potentials, assisting the redox process of Pt0/Pt2+ or Pt2+/Pt4+ in the SPE matrix, where CH3OH and H2O are present in modified forms. For comparison, IrPd-SPE was also used as an electrode and showed a higher activity than Ir alone, although Pd did not have any activity toward methanol oxidation in sulfuric acid solution. Irrespective of the kind of Pt-SPEs, the Tafel slope was approximately 120 mV; the CH3OH concentration dependence was of the order of 0.2–0.6. The pH dependence was nearly 0.5 against NHE. The activation energy of the Pt-SPEs for the reaction ranged between 20 and 33 kJ mol−1.

Mechanism of enhanced lipid peroxidation in the liver of Long-Evans cinnamon (LEC) rats

Abstract The Long-Evans Cinnamon (LEC) rat is a mutant strain of rats that accumulate copper (Cu) in the liver in much the same way as individuals who suffer from Wilsons disease (WD) and has been suggested as a model for this disease. Lipid peroxidation (LPO) is considered to be involved in the toxic action of Cu in the livers of LEC rats. We investigated the mechanism of LPO in the livers of LEC rats showing apparent signs of hepatitis. Several-fold higher LPO levels were observed in post-mitochondrial supernatant (S-9) fraction of livers from hepatitic LEC rats than in those from Wistar rats. To mimic living cells, we introduced NADPH-generating system (NADPH-gs) into the S-9 incubation system. Thus was ensured a constant supply of NADPH to vital enzymes that may be directly or indirectly involved in the generation and/or elimination of reactive oxygen species (ROSs), such as glutathione reductase (GSSG-R), which require NADPH for their reactions. The levels of LPO in liver S-9 from hepatitic LEC rats were further increased by incubating liver S-9 at 37 °C in the presence of NADPH-gs. This increase was inhibited by EDTA, butylated hydroxytoluene (BHT), and catalase (CAT), suggesting that some metal, most likely the accumulated Cu, and ROSs derived from hydrogen peroxide (H2O2) are involved in the increased levels of LPO in the livers of hepatitic LEC rats. The requirement of NADPH-gs for enhanced LPO in the livers of hepatitic LEC rats indicates the consumption of NADPH during reactions leading to LPO. It is known that H2O2, and consequently hydroxyl radical are generated during Cu–catalyzed glutathione (GSH) oxidation. The cyclic regeneration of GSH from GSSG by NADPH-dependent GSSG-R in the presence of NADPH-gs may cause sustained generation of hydroxyl radical in the presence of excess free Cu. The generation of H2O2 in S-9 fraction of livers from hepatitic LEC rats was observed to be significantly higher than that in S-9 fraction of livers from non-hepatitic LEC rats and Wistar rats. Moreover, in addition to the reported decrease in glutathione peroxidase (GPX) activity, we found that CAT activity was markedly decreased in LEC rats with hepatitis. The increased generation of H2O2 with reduced activities of GPX and CAT may result in cellular accumulation of H2O2 in the liver of hepatitic LEC rats. Taken altogether, it is suggested that the accumulated H2O2 undergoes the Fenton-type reaction with also accumulated free Cu, thus generating hydroxyl radical in the livers of hepatitic LEC rats and increasing LPO levels in these animals.

Bilayer model for zero order desorption

Abstract A bilayer model is presented to interpret the zero order desorption of adsorbates on solid surfaces. The adsorbate is separated into two phases coexisting in equilibrium, the dense phase and the dilute phase. The dense phase exists in the first layer on the substrate surface while the dilute phase, pictured as a two-dimensional ideal gas, covers not only the substrate surface unoccupied by the dense phase but also the dense phase itself. The desorption is assumed to occur unimolecularly in the dilute phase only. It is shown in transition state theory that the desorption rate per unit area is equal everywhere on the surface even though the adsorbate in the dilute phase may have different potential energies and different vibrational characteristics in the two layers, i.e., that the desorption obeys zero order kinetics. Activation energies are connected to thermodynamic functions of the adsorption phase and related to each other.

P450 in wild animals as a biomarker of environmental impact

The impact of environmental pollution on selected animals was tested by monitoring the hepatic content of cytochromes P450 and their enzyme activities or by calculating TEQ values from the concentration of pollutants in the body. Fish-eating Stellars Sea Eagles, Haliaeetus pelagicus, found dead in the northern part of Hokkaido island accumulated high levels of PCBs and DDT and metabolites. The TEQ values calculated from the PCB concentration in the eagles were high enough to cause a significant toxic effect in other birds living in the same environment. Some of these birds were also contaminated with high concentrations of lead. Spotted seals, Phoca largha, captured along the coast-line of Hokkaido accumulated PCBs in their fat at about 100 million times the concentrations in the surface sea water. The levels of expressions of hepatic microsomal CYP 1A1and related enzyme activities in these seals showed good correlation to the levels of PCBs accumulated in the fat. The fresh water crabs, Eriocheir japonicus, were captured from three different rivers with various degrees of pollution. The P450 content and the related enzyme activities showed good correlation to TEQ values obtained from the concentrations of PCBs and PCDDs in the crabs from the rivers. The wild rodents, Clethrionomys rufocanus, were captured from urban, agricultural, and forest areas in Hokkaido. Those from the forest area had the lowest CYP content and related enzyme activities, comparable to those in laboratory-raised animals. Those from the urban areas, presumably contaminated with PAHs from fuel combustion, showed increased CYP 1A1 content and related enzyme activities. Those from the agricultural areas showed increased levels of CYP 1A1, 2B, 2E1. Rats treated with some of the agrochemicals used in the area resulted in a similar pattern of induction. It is concluded that P450 can be a useful biomarker for assessing the environmental impact of chemical pollutants on wild animals.

Limit cycle in electrochemical oscillation—potential oscillation during anodic oxidation of H2

Abstract A limit cycle was obtained by simulating kinetic equations describing the potential oscillation due to coupling of the anodic oxidation of H2 with Ag deposition and dissolution. Attractive interaction between Ag adatoms on Pt test electrodes was found to be essential for producing the limit cycle.

Strain differences in CYP3A-mediated C-8 hydroxylation (1,3,7-trimethyluric acid formation) of caffeine in Wistar and dark Agouti rats : Rapid metabolism of caffeine in debrisoquine poor metabolizer model rats

We observed significant strain differences [Dark Agouti (DA) > Wistar] in 1,3,7-trimethyluric acid formation (C-8 hydroxylation) during caffeine metabolism, though not in N-demethylations, in adult male DA and Wistar rats. In contrast, adult female and immature male rats of both DA and Wistar strains did not show significant differences in activity levels of C-8 hydroxylation. Kinetic studies using liver microsomes revealed that adult male DA rats have a larger Vmax for C-8 hydroxylation than do Wistar rats. Troleandomycin (TAO), known as a cytochrome P450 (CYP) 3A inhibitor, and an anti-rat CYP3A2 polyclonal antibody effectively reduced C-8 hydroxylation by rat liver microsomes in a concentration-dependent manner, suggesting that C-8 hydroxylation in rats is mediated largely by an isoform(s) of the CYP3A subfamily. Troleandomycin and the antibody did not inhibit the N-demethylations of caffeine by rat liver microsomes. Treatment of rats with CYP3A inducers caused a marked increase in C-8 hydroxylase activity. These results indicate that the rat CYP3A subfamily is capable of catalyzing C-8 hydroxylation of caffeine as is the case for human CYP3A4. The results of western blotting analysis using anti CYP3A antiserum showed that the staining intensity of the protein band in DA rat liver microsomes was higher than that in Wistar rat liver microsomes. We concluded that marked sex-dependent strain differences in C-8 hydroxylation of caffeine between Wistar and DA rats are due to the differences in the levels of expression of CYP3A in these strains of rats.

Mechanism for mouse strain differences in the protective effect of Sudan III against the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene

The effect of Sudan III-pretreatment on the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene (DMBA) was investigated using C57BL/6 (B6) and DBA/2 (D2) mice. A significant increase in the frequency of micronucleated reticulocytes was observed in both strains of mice treated with DMBA. The increase was significantly reduced in B6 but not D2 mice by Sudan III-pretreatment. However, enhancement of metabolic activation was found in the Ames assay in the hepatic post-mitochondrial supernatant fraction (S9) from Sudan III-treated animals. It was greater with S9 from B6 than S9 from the D2 group. When the assay was performed in the presence of glutathione, this enhancement was significantly reduced. Sudan III induced some drug metabolizing enzymes, mainly CYP1A and glutathione S-transferase was also induced. The induction of CYP1A was more effective in B6 than D2 mice. These results support our hypothesis that the simultaneous induction of Phase I and II drug metabolizing enzymes is the mechanism for the chemoprevention by Sudan III and suggest that strong induction of CYP1A might be essential for a protective effect.

Potential oscillation during anodic oxidation of hydrogen at a platinum electrode—II. Kinetic analysis

A kinetic model was constructed representing the coupling of the anodic oxidation of H2 with Ag deposition and dissolution. The calculated potential oscillation curve reproduced well the observed one. Furthermore, the model was compatible with the following experimental results reported in the preceding paper. (1) The potential oscillation is observed in a certain range of the applied current, I, and a plot of log I against the maximum potential, φMAX, in oscillation gives a straight line. (2) On the other hand, in the region where I is lower than a threshold value, potential does not oscillate and a plot of log I vs the steady potential, φ, is found to be linear. (3) A smaller bubbling rate of H2 results in a longer period, τ, and a higher φMAX.

Anti-peptide antibodies to the P4502D subfamily in rat, dog and man

1. In order to obtain specific antibodies of the P4502D subfamily, we generated two anti-peptide antibodies against synthetic peptides, DPAQPPRD (peptide A) and DPTQPPRH (peptide B). The sequence of peptide A occurs in rat P4502D2, P4502D4 and human P4502D6, whereas the sequence of peptide B occurs in the dog P4502D subfamily. These sequences are closely related to an epitope of liver/kidney microsomal autoimmune hepatitis. 2. In immunoblotting studies, the anti-peptide antibody against peptide A recognized a 49-KDa protein in microsomes derived from human lymphoblasts expressing P4502D6 and rat liver. It showed no crossreactivity with microsomes from dog liver. In contrast, the anti-peptide antibody against peptide B recognized a 49-KDa protein only in microsomes of dog liver. These indicate that each anti-peptide antibody has the specificity for the respective sequences of the members of P4502D subfamily, with the species investigated herein. 3. In immunoinhibition studies, the anti-peptide antibodies against peptide B inhibited bunitrolol 4-hydroxylation and propranolol 4,5-hydroxylation, which are mediated by the dog P4502D subfamily. These data suggest that the anti-peptide antibodies against peptide B bind to the native and denatured forms of the P4502D subfamily. 4. The present study has demonstrated that the anti-peptide antibodies against this region are useful for studying the members of the P4502D subfamily.

Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21.

1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the N-deethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS-21, and the cyclohexane-4-cis- and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.