Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used as a free radical scavenging drug for the treatment of acute ischemic stroke in Japan since 2001. Edaravone is given to patients intravenously; therefore, it is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because it is present as edaravone anion, which is capable of transferring an electron to free radicals including oxygen, and becomes edaravone radical. We observed the formation of hydrogen peroxide and edaravone trimer when aqueous edaravone solution was kept at 60°C for 4 weeks. We proposed the mechanism of edaravone trimer formation from edaravone radicals. Lowering the pH and deoxygenation can effectively increase the stability of aqueous edaravone solution, since the former reduces edaravone anion concentration and the latter inhibits edaravone radical formation. Addition of sodium bisulfite partially stabilized aqueous edaravone solutions and partially inhibited the formation of edaravone trimer. Formation of bisulfite adduct was suggested by 13C NMR and HPLC studies. Therefore, the stabilizing effect of sodium bisulfite is ascribed to the formation of a bisulfite adduct of edaravone and, consequently, reduction in the concentration of edaravone anion.
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has garnered attention since its approval for amyotrophic lateral sclerosis in Japan (2015) and the United States (2017). Edaravone is administered intravenously, and as such, is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because they present as edaravone anions, which become edaravone radicals when the anion donates an electron to free radicals including oxygen. In this study, glutathione (GSH) stabilized an aqueous edaravone solution during storage at 60°C for 4 weeks, and prevented the formation of potentially carcinogenic phenylhydrazine, while cysteine did not. One possible explanation is that GSH undergoes intermolecular hydrogen bonding with edaravone anions, while cysteine does not, as it favors intramolecular hydrogen boding. The combination of GSH and sodium bisulfite (NaHSO3) stabilized aqueous edaravone at room temperature for more than 1 year even under aerobic conditions. However, the U.S. Food and Drug Administration cautioned that NaHSO3 may cause allergic reactions. Therefore, we developed a stable edaravone aqueous solution without using NaHSO3, namely a combination of GSH with deoxygenation, which resulted in better stabilization of aqueous edaravone than the combination of GSH and NaHSO3.
Uric acid quenches singlet oxygen physically or reacts with it, but the oxidation product has not been previously characterized. The present study determined that the product is parabanic acid, which was confirmed by LC/TOFMS analysis. Parabanic acid was stable at acidic pH (<5.0), but hydrolyzed to oxaluric acid at neutral or alkaline pH. The total yields of parabanic acid and oxaluric acid based on consumed uric acid were ~100% in clean singlet oxygen production systems such as UVA irradiation of Rose Bengal and thermal decomposition of 3-(1,4-dihydro-1,4-epidioxy-4-methyl-1-naphthyl)propionic acid. However, the ratio of the amount of uric acid consumed to the total amount of singlet oxygen generated was less than 1/180, indicating that most of the singlet oxygen was physically quenched. The total yields of parabanic acid and oxaluric acid were high in the uric acid oxidation systems with hydrogen peroxide plus hypochlorite or peroxynitrite. They became less than a few percent in peroxyl radical-, hypochlorite- or peroxynitrite-induced oxidation of uric acid. These results suggest that parabanic acid could be an in vivo probe of singlet oxygen formation because of the wide distribution of uric acid in human tissues and extracellular spaces. In fact, sunlight exposure significantly increased human skin levels of parabanic acid.
Congenital diaphragmatic hernia (CDH) is a life-threatening neonatal disease that leads to lung hypoplasia and pulmonary hypertension. We recently found that maternal prenatal administration of Saireito (TJ-114) ameliorates fetal CDH in a nitrofen-induced rat model. Here, we studied the role of iron and oxidative stress in neonates of this model and in lung fibroblasts IMR90-SV in association with nitrofen and Saireito. We observed increased immunostaining of 8-hydroxy-2'-deoxyguanosine in the lungs of neonates with CDH, which was ameliorated by maternal Saireito intake. Pulmonary transferrin receptor expression was significantly decreased in both CDH and CDH after Saireito in comparison to normal controls, indicating functional lung immaturity, whereas catalytic Fe(II) and pulmonary DMT1/ferroportin expression remained constant among the three groups. Saireito revealed a dose-dependent scavenging capacity with electron spin resonance spin trapping in vitro against hydroxyl radicals but not against superoxide. Finally, nitrofen revealed dose-dependent cytotoxicity to IMR90-SV cells, accompanied by an increase in oxidative stress, as seen by 5(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and catalytic Fe(II). Saireito ameliorated all of these in IMR90-SV cells. In conclusion, catalytic Fe(II)-dependent oxidative stress by nitrofen may be the pathogenic cause of CDH, and the antioxidative activity of Saireito is at least partially responsible for improving nitrofen-induced CDH.
Iron is an essential nutrient for life and is involved in many important processes such as oxygen transport and DNA synthesis. However, excess amounts of iron can cause carcinogenesis by producing reactive oxygen species. Thus, the cellular transport of iron must be tightly regulated. In the human body, iron may be present as heme or non-heme iron. The mechanisms governing the cellular transport of these forms have not been clearly elucidated. We previously reported that the expression of an important heme transporter, heme carrier protein 1 was regulated by cancer-specific reactive oxygen species derived from mitochondria. In this study, we have asked if mitochondrial reactive oxygen species may also be related with non-heme iron transport. In order to address this question, we have investigated the relationship between mitochondrial reactive oxygen species and accumulation of cellular non-heme iron in a rat gastric normal, cancer and manganese superoxide dismutase-overexpressing cancer cell line, in which reactive oxygen species from mitochondria are specifically scavenged. We have also analyzed the expression of divalent metal transporter 1 and ferroprotin, involved in the incorporation and excretion of non-heme iron, respectively, as well as a hypoxia-related transcription factor HIF-1α, to elucidate the molecular mechanism of non-heme iron accumulation.
Monascus purpureus is a red dye derived from yeast rice and has been used as color additives for food in East Asia. Monascus purpureus consists of several bioactive components. Some of these components work as a radical scavenger, thus monascus purpureus would also eliminate reactive oxygen species. Cancer cells maintain the high level of reactive oxygen species than normal cell and are death by imbalance in pro-oxidant/antioxidant homeostasis. In this study, we investigated whether monascus purpureus induced cancer specific cell death by scavenging reactive oxygen species. Compared to normal cell, monascus purpureus had cancer specific cytotoxicity. Monascus purpureus and lovastatin, its component, scavenged free radicals caused by a xanthine/xanthine oxidase system, thus Monascus purpureus is likely to scavenge reactive oxygen species by a synergistic effect between lovastatin and other components. Monascus purpureus also decreased reactive oxygen species derived from mitochondria in cancer cells, and cellular apoptosis was induced via activation of caspase-9. Induction of apoptosis by reduction of reactive oxygen species generation decreased acid ceramidase, and this mechanism could be involved with increasing ceramide accumulation in cells.
As the beneficial effects of the Mediterranean diet on human health are well established, the phenolic compounds in olive oil have been gaining interest. Oleuropein, a major phenolic compound in olives, is known to reduce the blood glucose levels in alloxan-induced diabetic rats and rabbits, however, its effect on type 2 diabetes caused by obesity is not clear. The purpose of this study is clarifying the effect of oleuropein on the glucose tolerance in skeletal muscle under the condition of lipotoxicity caused by type 2 diabetes. Oleuropein enhanced glucose uptake in C2C12 cells without insulin. Translocation of glucose transporter 4 (GLUT4) into the cell membrane was promoted by activation of adenosine monophosphate-activated protein kinase (AMPK) but not protein kinase B (Akt). Physiological concentration of oleuropein (10 µM) was sufficient to express beneficial effects on C2C12 cells. Oleuropein prevented palmitic acid-induced myocellular insulin resistance. Furthermore, in gastrocnemius muscles of mice fed a high fat diet, oleuropein also induced the GLUT4 localization into cell membrane. These results suggest the possibility of oleuropein to be effective for type 2 diabetes by reducing insulin resistance in skeletal muscles.
Acetaminophen is a commonly used analgesic. However, an overdose of acetaminophen causes severe hepatotoxicity via depletion of hepatic glutathione. Here, we investigated the protective effects of sake lees hydrolysate against acetaminophen-induced hepatotoxicity in mice. Sake lees hydrolysate was administered orally to ICR mice for seven days. Six hours after acetaminophen treatment, the mice were sacrificed, and blood and liver samples were collected for analysis. Treatment with acetaminophen markedly increased the levels of serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase. Pretreatment with sake lees hydrolysate significantly prevented the increases in the serum levels of these enzymes and inhibited acetaminophen-mediated glutathione depletion. In addition, histopathological evaluation of the livers also revealed that sake lees hydrolysate prevented acetaminophen-induced centrilobular necrosis. The expression of γ-glutamylcysteine synthetase (γ-GCS), hemeoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the liver were decreased after acetaminophen treatment, whereas pretreatment with sake lees hydrolysate led to an increased expression of all three proteins. Furthermore, sake lees hydrolysate induced the expression of these proteins in HepG2. These results suggested that sake lees hydrolysate could induces HO-1 and γ-GCS expression via activation of the Nrf2 antioxidant pathway, and protects against acetaminophen-induced hepatotoxicity in mice.
Epidemiological and animal studies have demonstrated that ingestion of green tea enhances healthy life. However, caffeine in green tea can interfere with sleep. In this report, we examined the effect of green tea with lowered caffeine, low-caffeine green tea, on stress and sleep of the elderly. The participants (n = 10, mean age 89.3 ± 4.2 years) drank five cups/day of standard green tea for 1 week. Subsequently, they drank five cups/day of low-caffeine green tea for 2 weeks. Salivary α-amylase activity (sAA) was measured as a stress marker. Sleep stages were measured using a portable electroencephalography (n = 7, 6 female and 1 male). The level of sAA in the morning (sAAm) was significantly lower when the participants drank low-caffeine green tea than standard green tea. While the levels of sAAm were different among individuals, lower sAAm correlated with a higher quality of sleep. In those participants whose sAAm was lowered by the ingestion of low-caffeine green tea, some sleep parameters improved. Daily ingestion of low-caffeine green tea may be a beneficial tool for improving the quality of sleep of the elderly via the suppression of stress, although further research is required to fortify this hypothesis.
We assessed whether gut microbial functional profiles predicted from 16S rRNA metagenomics differed in Japanese type 2 diabetic patients. A total of 22 Japanese subjects were recruited from our outpatient clinic in an observational study. Fecal samples were obtained from 12 control and 10 type 2 diabetic subjects. 16S rRNA metagenomic data were generated and functional profiles predicted using “Phylogenetic Investigation of Communities by Reconstruction of Unobserved States” software. We measured the parameters of glucose metabolism, gut bacterial taxonomy and functional profile, and examined the associations in a cross-sectional manner. Eleven of 288 “Kyoto Encyclopedia of Genes and Genomes” pathways were significantly enriched in diabetic patients compared with control subjects (p<0.05, q<0.1). The relative abundance of almost all pathways, including the Insulin signaling pathway and Glycolysis/Gluconeogenesis, showed strong, positive correlations with hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG) levels. Bacterial taxonomic analysis showed that genus Blautia significantly differed between groups and had negative correlations with HbA1c and FPG levels. Our findings suggest a novel pathophysiological relationship between gut microbial communities and diabetes, further highlighting the significance and utility of combining prediction of functional profiles with ordinal bacterial taxonomic analysis (UMIN Clinical Trails Registry number: UMIN000026592).
Transarterial chemoembolization and hepatic arterial infusion chemotherapy are recommended for the treatment in patients with intermediate stage of hepatocellular carcinoma. Impaired liver function was sometime observed in patients with hepatocellular carcinoma after transarterial chemoembolization or hepatic arterial infusion chemotherapy. However, what kinds of factors deeply influence in impaired liver function are not clear. A retrospective study was performed to evaluate the risk factors of impaired liver function in cisplatin-naïve patients treated with these therapies using cisplatin. Prior to and 2 months after these therapies, we analyzed the liver function by Child-Pugh score in these patients. For assessing the severity of chemotherapy-induced nausea and vomiting, we utilized the Common Terminology Criteria for Adverse Events ver. 4.0. In hepatocellular carcinoma patients received these therapies using cisplatin, the cancer stage and treatment without neurokinin-1 (NK1) antagonist were found to be independent risk factors of the impaired liver function. The treatment with NK1 antagonist was effective in reducing chemotherapy-induced nausea and vomiting and patients treated with NK1 antagonist kept their liver functions after cisplatin-used these therapies. The treatment with NK1 antagonist was effective in chemotherapy-induced nausea and vomiting and prevented the impaired liver function associated with cisplatin-used these therapies in hepatocellular carcinoma patients.
Endothelial dysfunction is the early predictive factor for the development of atherosclerosis and future cardiovascular diseases in adulthood. The prevalence of endothelial dysfunction in children and early adolescents is increasing worldwide. Peripheral arterial tonometry is a noninvasive technique for assessing peripheral microvascular function and is used as a validated marker of endothelial function. We assessed anthropometric parameters, blood pressure, arterial stiffness, and peripheral endothelial function in 157 Japanese early adolescents (75 boys and 82 girls). We measured peripheral endothelial function by using peripheral arterial tonometry to determine the reactive hyperemia index, and assessed the association of reactive hyperemia index with parameters of anthropometry and arterial stiffness. The mean reactive hyperemia index of all subjects was 1.85 ± 0.6, and there was no difference of reactive hyperemia index according to sex. Reactive hyperemia index was significantly associated with systolic and diastolic blood pressures, and had no correlation with anthropometric parameters and arterial stiffness markers. The reactive hyperemia index values among Japanese early adolescents were similar to those reported in previous studies on children and early adolescents. This noninvasive technique may be useful for the assessment of microvascular endothelial function among children and early adolescents.