Yashige Kotake

Stabilities of hydroxyl radical spin adducts of PBN-type spin traps

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.

Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)‐spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane‐associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.

Inhibition of NF-κB, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl-N-tert-butylnitrone (PBN)

Previously, the spin trapping agent phenyl-N-tert-butylnitrone (PBN) has been shown to decrease the level of nitric oxide synthase mRNA in vivo. This inhibition is suggested to be an underlying mechanism for PBNs wide variety of pharmacological actions in animal models. However, the determination of PBNs cellular pharmacological activities has not been carried out, but is necessary for the understanding of the effects in vivo. Since the known pharmacological effects of PBN are primarily anti-inflammatory in nature, in this study we determined the inhibitory activities of PBN against two inflammatory factors: inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX2). We show here that PBN decreases steady state COX2 mRNA level and COX2 catalytic activity in macrophage cell culture at supra-pharmacological concentrations. While PBN decreases iNOS mRNA, it does not inhibit iNOS catalytic activity, which is consistent with previous in vivo studies. We also studied nuclear factor kappaB (NF-kappaB), a transcription factor that can rapidly activate the expression of genes involved in inflammatory, immune and acute phase responses. The binding of NF-kappaB to iNOS gene has been shown to be critical for iNOS gene expression, and the promoter region of COX2 gene contains NF-kappaB consensus sequence. We show that PBN inhibits lipopolysaccharide-mediated increase of NF-kappaB DNA binding activity with a lower concentration than that for the non-steroidal anti-inflammatory drug (NSAID), salicylate. Furthermore, we show that PBN inhibits COX2 catalytic activity, suggesting that PBN has an NSAID-like function.

Synthesis and characterization of a practically better DEPMPO-type spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO)

5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), a new cyclic DEPMPO-type nitrone was evaluated for spin-trapping capabilities toward hydroxyl and superoxide radicals. CYPMPO is colorless crystalline and freely soluble in water. Both the solid and diluted aqueous solution did not develop electron spin resonance (ESR) signal for at least 1 month at ambient conditions. CYPMPO can spin-trap superoxide and hydroxyl radicals in both chemical and biological systems, and the ESR spectra are readily assignable. Half life for the superoxide adduct of CYPMPO produced in UV-illuminated hydrogen peroxide solution was approximately 15 min, and in biological systems such as hypoxanthine (HX)/xanthine oxidase (XOD) the half-life of the superoxide adduct was approximately 50 min. In UV-illuminated hydrogen peroxide solution, there was no conversion from the superoxide adduct to the hydroxyl adduct. Although overall spin-trapping capabilities of CYPMPO are similar to DEPMPO, its high melting point, low hygroscopic property, and the long shelf-life would be highly advantageous for the practical use.

Continuous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex

Nitric oxide (NO) generation in murine macrophages was determined in real time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl D-glucamine dithiocarbamate was utilized as the spin trap. This spin trapping compound reacts with NO in solution to form a specific room-temperature stable, mononitrosyl complex which is readily detected and identified by EPR spectroscopy. Mouse peritoneal macrophages were placed in an EPR sample-cell and activated by lipopolysaccharide and gamma-interferon at 37 degrees C, followed by an additional incubation in oxygenated medium without these activation agents. After various incubation periods, spin trap solution was infused to replace the medium in the sample-cell, and the time-evolution of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes of the increase in the EPR intensity with time. In comparison to the NO (or NO2-) generation rate obtained under similar experimental conditions using the Griess reaction assay, the spin trapping method was found to be more sensitive, with a lowest limit of the detection of 3 pmol/min. In addition, by using the spin trapping method, NO generation from the same cells could be measured consecutively during various stages of activation, because infusion of the spin trap solution did not affect the viability of macrophages.

An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals.

A new method is proposed for the evaluation of oxygen radical absorbance capacity (ORAC). The current fluorescence-based ORAC assay (ORAC-FL) is an indirect method that monitors the antioxidants ability to protect the fluorescent probe from free radical-mediated damage, and an azo-radical initiator, AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), has been used as a thermal free radical source. The new ORAC assay employs a short in situ photolysis of AAPH to generate free radicals. The electron paramagnetic resonance (EPR) spin trapping method was employed to identify and quantify AAPH radicals. In the presence of antioxidant, the level of AAPH radicals was decreased, and ORAC-EPR values were calculated following a simple kinetic formulation. Alkyl-oxy radical was identified as the sole decomposition product from AAPH; therefore, we concluded that ORAC-FL is the assay equivalent to alkyl-oxy radical scavenging capacity measurement. ORAC-EPR results for several antioxidants and human serum indicated that the overall tendency is in agreement with ORAC-FL, but absolute values showed significant discrepancies. ORAC-EPR is a rapid and simple method that is especially suitable for thermally labile biological specimens because the sample heating is not required for free radical production.

Optimal time and dosage of phenyl N-tert-butyl nitrone (PBN) for the inhibition of nitric oxide synthase induction in mice.

We have previously reported that phenyl N-tert-butyl nitrone (PBN) inhibits the induction of inducible nitric oxide synthase (iNOS) and, thus, prevents the overproduction of nitric oxide (NO), resulting in the reduction of endotoxin-mediated death in mice. In this study, to examine the effect of PBN in detail, we investigated the dose- and administration-timing dependence of PBN on endotoxin-induced NO generation in mice. NO generation was monitored in the mouse liver after administration of lipopolysaccharide (LPS) by the in vivo NO-spin trapping method using the iron complex of N-methyl-D-glucamine dithiocarbamate (MGD) as a spin trap, followed by ex vivo EPR measurement of the liver tissue. PBN was effective in reducing liver NO generation monitored 6 h after endotoxin injection when it was administered shortly before or after LPS injection. The maximum inhibition of liver NO was obtained when PBN was administered 30 min before LPS injection. ID50 for the inhibition was estimated to be approximately 200 mg/kg when the LPS dose of 50 mg/kg was used. Expression of mRNA for iNOS in the liver as estimated by reverse transcription polymerase chain reaction was decreased when PBN was given 30 min before LPS injection, indicating that the reduction of expression of iNOS protein by PBN, which has been shown previously, is at least in part caused by a decrease in mRNA expression.

Region‐selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain

Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl‐tert‐butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hafter endotoxin injection. Animals were killed 7 days post‐injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open‐channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (two‐ to threefold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (> 50%) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl‐tert‐butyl nitrone treatment resulted in a partial inhibition (approx. 25% decrease) of the lipopolysaccharide‐induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide‐induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimers disease.

LINE-1 Hypomethylation in a Choline-Deficiency-Induced Liver Cancer in Rats: Dependence on Feeding Period

Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.

Reactive oxygen species in choline deficiency induced carcinogenesis and nitrone inhibition

Reactive oxygen species and free radical processes have been considered important in cancer development for many years. Much research demonstrates that the choline-deficiency induced hepatocarcinogenesis model prominently involves reactive oxygen species. We present a summary of results obtained in our original studies of this model over the last 4 years. We have shown that α-phenyl-tert-butyl nitrone (PBN) and some of its hydroxylated derivatives (the 4- and 3-hydroxylated compounds) prevent hepatocarcinogenesis in this model. Mechanistic studies have demonstrated that isolated mitochondria from the livers of rats fed the choline-deficiency defined amino acid diet produce significantly much more H2O2 per NADH reducing equivalents oxidized. Based on these observations, we postulate that H2O2 is a primary carcinogenic factor in this model. Based on studies of the action of PBN on isolated mitochondria, we postulate that the inhibiting action of PBN involves suppression of H2O2 production of mitochondria and generally decreasing the oxidative stress within the preneoplastic lesions. The net effect of the activity of the nitrone compounds appears to be due to their ability to shift the apoptosis/neoplastic tendency balance toward apoptosis of the cells within the preneoplastic lesions. This is considered to be the primary reason the size of the preneoplastic lesions are significantly decreased and why the nitrones are potent anti-carcinogenic agents in this model.