Kieko Saito

Generation of Nitric Oxide from Spin-trapping Agents under Oxidative Conditions

Nitric oxide (NO) generation from the spin-trapping agents, phenyl-tert-butylnitrone (PBN), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), under UV irradiation in the presence of dissolved oxygen and by oxidation with the Fenton reagent was examined by using ESR spin-trapping and spectrophotometric methods. A triplet signal at g=2.041 was observed after the ferrous complex of dithiocarbamate [Fe(MGD)2] had been added to a solution of these trapping agents treated with UV irradiation and the Fenton reagent, showing that NO was trapped with Fe(MGD)2. The concentration of nitrite induced from NO was determined via the Griess reaction to increase with the time of the treatment. It is speculated by reference to the ESR signal observed at the position around g=2.006 that the C=N double bond might have been cleaved by oxidation, resulting in the formation of a nitroso compound, and that NO was then generated by the fission of the C-N bond of the nitroso compound. NO generated in this way activated guanylate cyclase, from which it can be expected that a spin-trapping agent acts as an NO generator in vivo as well as a free radical scavenger.

Protective Effect of Spin Trap Agent, N - tert -butyl-α-phenylnitrone on Hyperoxia-induced Oxidative Stress and Its Potential As a Nitric Oxide Donor

We have previously suggested that the spin trap agent, N - tert -butyl- f -phenylnitrone (PBN) can function not only as an antioxidant but also as a nitric oxide (NO) donor. To characterize the pharmacological activities of PBN against oxidative damage, we examined the effect of PBN on NO generation under hyperoxic conditions. The formation of NO in mice exposed to 95% oxygen was determined using a NOx analyzer and electron spin resonance (ESR). Levels of NOx, an oxidative product of NO, increased in the blood of mice under these conditions. However, the increase was returned to a normal level by the NOS (nitric oxide synthase) inhibitor, L-NMMA, indicating that the NO was formed via a biosynthetic pathway. In addition, ESR spectra of the liver and brain of control and experimental mice that were measured using Fe(DETC) 2 as an NO trap reagent showed strong ESR signals from NO complexes in the livers of mice exposed to 95% oxygen. When examining the effect of PBN in mice, PBN reduced the NOx formation in the blood under the same hyperoxic conditions. In addition, the ESR intensity of the NO complex was weaker in the PBN-treated mice than in the non-treated mice, showing that PBN possess anti-inflammatory properties. However, under a normal atmosphere, NOx and ESR analyses showed that NO levels increased in PBN-treated mice but not in control mice. These findings suggested that PBN functions as an NO donor under specific physiological conditions. PBN appears to protect against hyperoxia-induced NO toxicity by anti-inflammatory action rather than by serving as an NO donor.

Effect of Radical Scavenger N-Tert-Butyl-α-Phenylnitrone on Stroke in a Rat Model Using a Telemetric System

We used malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) as a stroke model to explore the effects of the radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN) on stroke. PBN was administrated in drinking water to M-SHRSP. Circadian rhythms in heart rate, blood pressure, and locomotive activity in M-SHRSP were monitored with a telemetric system, in addition to measurement of water intake and body weight. Stroke-onset was assessed by changes in neurological symptoms, water intake, and body weight. Circadian rhythms were basically the same between PBN-treated and control rats several days after stroke onset. Significant differences were seen in blood pressure, relative weight of brain and water intake, heart rate, and locomotive activity between two groups. As a result, no significant difference in age of stroke onset was seen between PBN-treated and control rats, but PBN-treated rats displayed prolonged mean life spans. PBN might be effective in prolonging life span.

ESR Imaging on a Solid-tumor-bearing Mouse Using Spin-labeled Dextran

Imaging of a tumor with ESR was tried using two different types of spin probes, a low molecular weight spin probe, CPROXYL, and a polymer spin probe, TEMPO-DX. Spin probes were administered to a mouse bearing a solid tumor that was a transplanted Ehrlich’s ascites carcinoma in the back, using two methods, conventional intraperitoneal injection and continuous intravenous injection with a micro-feeder. First, the accumulation of the probe was examined by X-band ESR. CPROXYL, which was administered to a mouse intraperitoneally, was exclusively retained in urine, showing that it was rapidly excreted into the bladder, while TEMPO-DX was absorbed from the peritoneal cavity with difficulty to the vessel. Using continuous intravenous injection, CPROXYL was also rapidly excreted, but it was confirmed that TEMPO-DX concentrated in tumor tissue because it has a long half-life in vivo. In addition, measurement of ESR imaging was done to measure the distribution of spin probes with continuous intravenous injection. The strongest spot of CPROXYL was observed on ESR images, showing the accumulation at the bladder, while the spot of TEMPO-DX was observed in the solid tumor of the back of the mouse. These results suggest that TEMPO-DX could stay much longer than a low molecular weight spin probe in vivo and concentrate at the tumor. TEMPO-DX may be useful for developing specific ESR imaging agents for tumor.

ESR Characterization of a Novel Spin-trapping Agent, 15N-Labeled N-tert-Butyl-α-phenylnitrone, as a Nitric Oxide Donor

We previously found that one of the pharmacological effects of N-tert-butyl-α-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15N to identify its corresponding 15NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15N-PBN, the starting material, ammonium-15N chloride, was converted to 2-amino-15N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-15N, and finally reacted with benzaldehyde to give 15N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)2, as a trapping agent to measure the NO levels of 15N-PBN or 14N-PBN in vitro, the peak intensity of 15NO[Fe(MGD)2] was over 50% stronger than that of 14NO[Fe(MGD)2], and that 15NO and 14NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15NO derived from 15N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15NO from PBN and 14NO generated from cells. These results suggested that 15N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.

Nitric Oxide and Effect of a Radical Scavenger N-tert-butyl-α-phenylnitrone on Stroke in a Rat Model

To characterize the role of nitric oxide (NO) in stroke, NO was measured using an in vivo microdialysis technique and electron spin resonance spectrometry in malignant stroke-prone spontaneously hypertensive rats (M-SHRSP), stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The brain dialysate NO level was higher in SHRSP than in WKY. NO was not detected in M-SHRSP hippocampus microdialysate after stroke except after the administration of N-tert-butyl-α-phenylnitrone (PBN). In addition, very little NO was generated in M-SHRSP brain tissue with hemorrhage. These data demonstrate an association between NO and stroke in M-SHRSP. Further, PBN administration results in maintenance of NO levels after stroke in M-SHRSP.

Nitric oxide formation during light-induced decomposition of phenyl N-tert-butylnitrone

Phenyl N-tert-butylnitrone (PBN) is a spin trap commonly employed in free radical research. PBN has been shown to have adverse and beneficial effects on various biological systems. We report here evidence that photolysis (or even ambient light) decomposes PBN to nitric oxide in aqueous solutions. Non-heme and heme proteins have been employed to form nitrosyl complexes, which were detected using EPR spectroscopy. Concomitantly, nitrite formation was detected after light-induced decomposition of PBN. In addition, we found that tert-nitrosobutane and decomposed PBN caused an activation of guanylate cyclase. We propose a mechanism where PBN is decomposed by light to tert-nitrosobutane. The latter compound is, in turn, decomposed to nitric oxide. This study suggests the possibility that PBN or PBN radical adducts may be sources of nitric oxide in biological environments. When using PBN as a spin trap in biological samples, not only is the trapping of reactive free radicals operative, but nitric oxide produced from PBN decomposition may play an important role in altering biological functions.

Roots of hydroponically grown tea (Camellia sinensis) plants as a source of a unique amino acid, theanine.

The beneficial effects of green tea are well documented. However, most research has reported the effects of green tea brewed solely from leaves or leaf extracts. We focused on tea roots and developed a hydroponic system to explore the effect on roots that biosynthesize one of the rarest functional amino acids, theanine. The level of theanine in tea roots was much higher than in leaves, which was analyzed using HPLC. Moreover, a higher level of theanine was detected in white rootlets than in lignified roots. Thus, tea roots cultured hydroponically in a controlled environment might be considered a natural drug containing theanine, which could lead to synergistic effects with other ingredients of the root. This novel medicinal material from the roots demonstrates a significant medical function for tea that extends beyond its leaves. Original Research Article American Journal of Experimental Agriculture, 4(2): 125-129, 2014 126