Abstract
Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca2+ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca2+ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.
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Borg LA, Eide SJ, Anderson A and Hellerstrom C (1979) Effect in vitro of alloxan on the glucose metabolism of mouse pancreatic β-cells. Biochem J 182: 797-802.
Eisenbarth GS (1986) Type I diabetes mellitus: A chronic autoimmune disease. N Eng J Med 314: 1360-1368.
Gorus FK, Malaisse WJ and Pipelees DG (1982) Selective uptake of alloxan by pancreatic β-cells. Biochem J 208: 513-515.
Halliwell B and Gutteridge JMC (1990) Role of free radicals and catalytic metal ions in human disease: An overview. Methods in Enzymol 186: 1-85.
Hanaoka K (2001) Antioxidant effects of reduced water produced by electrolysis of sodium chloride. J Appl Electrochem 31: 1307-1313.
Janciauskiene S and Ahren B (1998) Different sensitivity to the cytotoxic action of IAPP fibrils in two insulin-producing cell lines, HIT-T15 and RINm5F cells. Biochem Biophys Res Commun 251: 888-893.
Kim H, Rho H, Park B, Park J, Kim J and Kim UH (1994) Role of Ca2+ in alloxan-induced pancreatic β-cell damage. Biochim Biophys Acta 1227: 87-91.
Krowlewski A, Warram J, Rand L and Kahn C (1987) Epidemiologic approach to the etiology of type I diabetes mellitus and its complications. N Eng J Med 317: 1390-1398.
LeBel CP, Ishiropoulos H and Bondy SC (1992) Evolution of the probe 2′,7′-dichlorofluorescein as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5: 227-231.
Ludin A (1978) Determination of creatine kinase isoenzyme in human serum by an immunological method using purified firefly luciferase. Methods Enzymol 57: 56-65.
Malaisse WJ and Lea MA (1982) Alloxan toxicity to the pancreatic β-cell. A new hypothesis. Biochem Pharmacol 31: 3527-3534.
Masumoto N, Tasaka K, Miyake A and Tanizawa O (1990) Superoxide anion increases intracellular free calcium in human myometrial cells. J Biol Chem 265: 22533-22536.
Mossman T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.
Oda M, Kusumoto K, Teruya K, Hara T, Maki T, Kabayama Y, Katakura Y, Otsubo K, Morisawa S, Hayashi H, Ishii Y and Shirahata S (1999) Electrolyzed and natural reduced water exhibit insulin-like activity on glucose uptake into muscle cells and adipocytes. In: A Bernard et al. (eds), Animal Cell Technology: Products from Cells, Cells as Products (pp. 425-427) Kluwer Academic Publishers, The Netherlands.
Okamoto H (1985) Molecular basis of experimental diabetes: Degeneration, oncogenesis and regeneration of pancreatic β-cells of islet of Langerhans. BioEssay 2: 15-21.
Orrenius S, McConkey D J, Bellomo G and Nicotera P (1989) Role of Ca2+ in toxic cell killing. Trends Pharmacol Sci 10: 281-285.
Park B, Rho H, Park J, Cho C, Kim J, Chung H and Kim H (1995) Protective mechanism of glucose against alloxan-induced pancreatic β-cells damage. Biochem Biophys Res Commun 210: 1-6.
Rho H, Lee J, Kim H, Park B and Park J (2000) Protective mechanism of glucose against alloxan-induced β-cell damage: Pivotal role of ATP. Exp Mol Med 32: 12-17.
Sakurai K, Katoh M, Someno K and Fujimoto Y (2001) Apoptosis and mitochondrial damage in INS-1 cells treated with alloxan. Biol Pharm Bull 24: 876-882.
Shirahata S (2000) Regulation of functions of animal cells by reduced water and its medical application. Nippon Nogei Kagaku Kaishi 74: 994-998.
Shirahata S (2002) Reduced water for prevention of diseases. In: Animal Cell Technology: Basic and Applied Aspects, Vol. 12 (pp. 25-30) Kluwer Academic Publishers, The Netherlands.
Shirahata S, Kabayama S, Nakano M, Miura T, Kusumoto K, Gotoh M, Hayashi H, Otsubo K, Morisawa S and Katakura Y (1997) Electrolyzed-reduced water scavenge active oxygen species and protects DNA from oxidative damage. Biochem Biphys Res Commun 234: 269-274.
Shirahata S, Nishimura T, Kabayama S, Aki D, Teruya K, Otsubo K, Morisawa S, Ishii Y, Gadek Z and Katakura Y (2001) Anti-oxidative water improves diabetes. In: E Linder-Olsson et al. (eds) Animal Cell Technology: From Target to Market (pp. 574-577) Kluwer Academic Publishers, The Netherlands.
Suresh Y and Das UN (2001) Protective action of arachidonic acid against alloxan-induced cytotoxicity and diabetes mellitus. Prostaglandins, Leukotrienes and Essential Fatty Acids 64: 37-52.
Szkudelski T (2001) The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Rev 50: 537-546.
Takasu N, Asawa T, Komiya I, Nagasawa Y and Yamada T (1991) Alloxan-induced DNA strand breaks in pancreatic islets: Evidence for H2O2 as an intermediate. J Biol Chem 266: 2112-2114.
Tashiro H (1999) Clinical examination of alkaline ion water. Abstract book of Symposium 'Electrolyzed functional water in therapy' in 25th Meeting of Japanese Medical Society, pp. 6-7.
Tomita T, Lacy PE, Natschinsky FM and McDaniel ML (1994) Effect of alloxan on insulin secretion in isolated rat islets perfused in vitro. Diabetes 23: 517-524.
Wogensen L, Lee MS and Sarvetnick N (1994) Production of interleukin 10 by islet cells accelerates immune-mediated destruction of beta cells in nonobese diabetic mice. J Exp Med 179: 1379-1384.
Yamamoto H, Uchigata Y and Okamoto H (1981) Streptozotocin and alloxan induce DNA strand breaks and poly(ADP-ribose) synthetase in pancreatic islets. Nature 294: 284-286.
Zhang H, Ollinger K and Brunk U (1995) Insulinoma cells in culture show pronounced sensitivity to alloxan-induced oxidadive stress. Diabetologia 38: 635-641.
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Li, Y., Nishimura, T., Teruya, K. et al. Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species. Cytotechnology 40, 139–149 (2002). https://doi.org/10.1023/A:1023936421448
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DOI: https://doi.org/10.1023/A:1023936421448