Kunie Nakamura

Cell death by reactive oxygen species generated from water-soluble cationic metalloporphyrins as superoxide dismutase mimics

We investigated the effect on cell death of reactive oxygen species induced by water-soluble cationic metalloporphyrins with superoxide dismutase (SOD) activity. The SOD activity of 5,10,15,20-tetrakis(4-N-methylpyridyl)]porphine (MPy(4)P) containing Fe, Mn or Cu was measured using a cytochrome c assay by the xanthine/xanthine oxidase system and stopped-flow kinetic analysis. Cell viability of four cell lines treated with metalloporphyrins, mitomycin c (MMC), or cisplatin was estimated by a trypan blue exclusion assay. FeMPy(4)P with a high SOD activity showed a significant cytotoxicity compared with MMC and cisplatin, while CuMPy(4)P without SOD activity exhibited no cytotoxicity. However, MnMPy(4)P showing an SOD activity as high as that of FeMPy(4)P did not indicate cytotoxicity. These findings suggest that FeMPy(4)P as SOD mimic converts intracellular O2(*-) to H(2)O(2) and that it rapidly reacts with H(2)O(2) to form *OH, causing DNA damage and inducing cell death. On the other hand, MnMPy(4)P did not participate in the Fenton reaction, so that DNA damage in the cells treated with MnMPy(4)P was not observed. In addition, the cytotoxicity by the metalloporphyrin was inversely correlated with the SOD activity of the cells and the selective damage at cellular and DNA levels was confirmed. We believe that for an anticancer drug with antioxidant ability O(2)(*-) is useful as a target molecule to induce selective cell death between cancer and normal cells and that metalloporphyrins showing SOD activity and Fenton-like reaction are a new class of anticancer agents.

Effect of coenzyme Q10 on superoxide production in rats with reperfusion injuries.

We examined the effect of coenzyme Q10 (Co Q10) on superoxide radical (O2-) production in a model of rat reperfusion injury. The chemiluminescence method using a derivative of luciferin was used to quantify O2- production by erythrocytes in the reperfused limb after a period of ischaemia. A total of 20 limbs from Lewis rats were preserved at 4 degrees C in Euro-Collins solution for 72 hours, and were grafted orthotopically to syngeneic rats by a microsurgical technique. In the treated group (n = 10), Co Q10 (10 mg/kg) was injected intraperitoneally into the recipients one hour before reperfusion. In the control group (n = 10), the same dose of solvent was given. To measure the extent of oxidative stress, heparinised blood from the treated and control recipients was collected before, and at 15, 30, and 60 minutes after reperfusion for the measurement of chemiluminescence. O2- production in the Co Q10-treated group was significantly lower than in the control group (p < 0.05). Although these findings suggest that Co Q10 scavenged O2- that was produced in the replanted limbs as a result of ischaemia-reperfusion injury, we should consider other possible mechanisms by which this agent may protect against ischaemia-induced reperfusion injury.

Prevention and reversible solubilization of advanced glycation and products (AGE) by organic germanium compounds as derivatives of amino acids.

SummaryThe amino-carbonyl reaction (The Maillard reaction) of bovine lens crystallin, serum albumin or skin collagen with glucose was investigated to find effective means to prevent the formation of Advanced Glycation End Products (AGE) and induce the reversible solubilization of polymerized glycated proteins. The organic germanium compounds (Ge-132, 373, 385), derivatives of amino acids containing germanium as the linker of framework, were combined by the box titration method to determine the dose that would be most effective, compared with Aminoguanidine-HCl (AMG),α-tocopherol (VE), and pirenoxine (Catalin-K, CK). Although AMG suppressed the formation of AGE, effective concentrations were higher than 20 mM. Ge-385, when administered by itself at a low dose, induced the reversible solubilization of AGE made from crystallin, and albumin. The addition of any two reagents such as AMG, VE, CK and Ge-132 or 385 together to proteins lessened the effective range, and the peaks of smaller molecules in the profiles of HPLC and PAGE were quite remarkable. Examination was made of the effects of Ge-132 on the eyes of SAM mice, which show senescence accelerated cataracts at a relatively young age. The prevention of cataract-genesis and induction of reversible transparency of turbid lenses became evident following the administration of Ge-132 to the eyes 4 times a day. The mode of action of organic germanium compounds was demonstrated quite capable of disconnecting the sugar-parts from AGE by decarbonylation, resulting in the formation of glucosone and amino residues, and further leading subsequently to fewer AGE.

Nitric oxide metabolite in pregnant women before and after delivery

Background. During pregnancy, systemic vascular resistance as well as vascular sensitivity to vasoconstrictive agents decreases in pregnant women. Methods and materials. The vascular resistance of the fetus is also maintained in the presence of low blood pressure. We believe that the main factors in this phenomenon are nitric oxide (NO), along with prostaglandin. NO is an unstable compound with a short half‐life; it is easily converted to nitrite (NO2) and nitrate (NO3). Since NO cannot be precisely quantified, we measured the NO2 concentration in maternal blood just before and after delivery and compared it with the values of non‐pregnant women. Results. NO2. concentrations in the 13 women who received cesarean deliveries under epidural anesthesia were not significantly changed by the anesthesia. Before anesthesia the NO2 concentration was 216 ± 115 pmol/mg protein, and after the induction of anesthesia, but before surgery, it was 218 ± 112 pmol/mg protein. The NO2 concentration then fell significantly after the surgery to 174 ± 75 pmol/mg protein (P < 0.05). In addition, after vaginal delivery, in 17 other patients we observed marked decreases in NO2 concentration, falling from 160 ± 82 to 125 ± 61 pmol/mg protein (p < 0.05). These values were significantly higher than those of nonpregnant women (3.4 ± 2.1 μM. 33 ± 22 pmol/mg protein) (p < 0.000I) Conclusion. These results suggest that NO contributes to the low vascular resistance observed in the mother and fetus during pregnancy.

Effect of SOD-Mimetic Fe-Chlorine e6-Na on the Level of Brain Lipid Peroxide of Rat Fetal Brains Exposed to Reactive Oxygen Species Leading to Intrauterine Growth Retardation

The influence of oxidative stress in rat brain and liver on intrauterine growth retardation (IUGR) in rat fetuses was examined. Twenty pregnant Wistar rats were used. On the 15th day of pregnancy, uterine artery and vein were ligated bilaterally using a modified Wigglesworth method. On the 21st day of pregnancy, the fetuses were delivered by hysterotomy. Fetal blood was collected by cardiac puncture. Fetal brain and liver were removed for the analysis of lipid peroxide. Sham surgical operations were performed in the control rats. Within the ligated group, a superoxide dismutase mimicking substance, Fe-chlorine e6-Na (FeCNa), was injected intraperitoneally once a day from day 15 of gestation to day 20. Fetal blood, brain, and liver were stored at –70°C until analysis. Control rats received an equivalent volume of saline. In growth-retarded fetuses, both superoxide released from erythrocytes and brain lipid peroxide showed significantly higher levels, but not superoxide dismutase in erythrocytes and liver lipid peroxide. These changes were alleviated by injection of superoxide dismutase-mimicking substance, FeCNa. Rat fetuses with intrauterine growth retardation suffered from a significant oxidative stress in utero. The increase in reactive oxygen species was alleviated by an injection of FeCNa.

Autogenous production of hydroxyl radicals from thiopental

It is generally believed that barbiturates can protect neural tissues from the damage induced by cerebral hypoxia. One of the mechanisms for protecting neurons is through the inhibition of lipid peroxidation (LPO). We therefore examined LPO in rat brain, liver and kidney by measuring the accumulation of thiobarbituric acid‐reactive substances (TBAR) after thiopental administration under 21% O2. We also designed an in vitro study to gain insight into free radical generation leading to the formation of LPO from thiopental by electron spin resonance (ESR). An accumulation of TBAR in the rat liver was observed after the administration of a large dose of thiopental (70 mg/kg intraperitoneally). However, no change in LPO in the brain and kidney was observed. In the in vitro study, thiopental could scavenge superoxide (O2‐) radicals, while it spontaneously generated hydroxyl radicals (·OH) in solution. We conclude that thiopental can scavenge O2‐, while producing ·OH, subsequently resulting in membrane lipid peroxidation under physiologic O2 conditions. This formation of ·OH may damage cell membrane lipids.

REGULATION OF THE MAILLARD REACTION BY ORGANIC GERMANIUM COMPOUNDS. MOLECULAR MECHANISM ON THE PREVENTION AND REVERSIBLE SOLUBILIZATION OF ADVANCED GLYCATION ENDPRODUCTS (AGE)

Kunie Nakamura*,Toshihiko Osawa, Shunro Kawakishi, and Norihiro Kakimoto 1 Molecular Biology Laboratory, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa, 228 Japan 2 Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464 Japan, 3 Asai Germanium Research Institute, 1-6-4 Izumi-Honcho, Komae, Tokyo, 201 Japan

Suppression Of Ages Formation In Spontaneous Diabetic OLETF Rat By Organic Germanium Compound [Poly-trans-(2-carboxyethyl) Germasesquioxane] (Ge-132)

The model rat for the type 2 diabetes mellitus (NIDDM) in human were observed for 72 weeks after birth, administering an organic germanium compound [Bis(2-Carboxyethyl) germasesquioxane, Ge-132] perorally 100 mg/kg/day since 24 weeks old. Clinical examinations were followed throughout the observation period. Blood and urinary glucose in positive control OLETF rats tended to be higher than those treated with Ge-132. At the end of the 72nd week, animals were sacriificed to examine the pathological changes, specifically in pancreas, kidney and brain. Anti-AGE antibody stained proximal and distal tubles and basement membrane of glomerules in kidney, and accumulated AGE masses in cortex, hippocampus and cerebellum of OLETF rats brain. Amyloid stains by basic congo red on kidney and brain revealed that the deposits of amyloid in kidney mesangium and in cortex, hippocampus and cerebellum were observable in OLETF rats. Ge-132 suppressed the deposition of amyloid tangles in kidneys and brains. Anti rat complement...

Original Article: Nitric oxide metabolite in pregnant women before and after delivery

Background. During pregnancy, systemic vascular resistance as well as vascular sensitivity to vasoconstrictive agents decreases in pregnant women. Methods and materials. The vascular resistance of the fetus is also maintained in the presence of low blood pressure. We believe that the main factors in this phenomenon are nitric oxide (NO), along with prostaglandin. NO is an unstable compound with a short half-life; it is easily converted to nitrite (NO2) and nitrate (NO3). Since NO cannot be precisely quantified, we measured the NO2 concentration in maternal blood just before and after delivery and compared it with the values of non-pregnant women. Results. NO2. concentrations in the 13 women who received cesarean deliveries under epidural anesthesia were not significantly changed by the anesthesia. Before anesthesia the NO2 concentration was 216 ± 115 pmol/mg protein, and after the induction of anesthesia, but before surgery, it was 218 ± 112 pmol/mg protein. The NO2 concentration then fell significantly a...