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Vol. 234, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS bated with the mixture of AsA and Cu(II), the amount TABLE 1 of super-coil DNA gradually decreased (FIG. 4). How- Effect of Reduced Water, SOD, Catalase, and Various ever, reduced water significantly inhibited the singlethe Cu(II)-Catalyzed Oxidation of AsA Radical Scavengers on Single-Strand Breakage of DNA by strand breakage by the mixture of AsA and Cu(II). As shown in Table 1, reduced water inhibited the DNA breaking reaction in a dose-dependent manner. Catalase also inhibited the breaking reaction but SOD did Reduced water 3.7 IC50SO units Addition Concn Specificity Inhibin, % 19 7.5 IC50SO units 38 15 IC50SO units 49 SOD 150 U/ml O 2 2 Catalase 0.8 U/ml •0 H2O2 6 4 U/ml H2O2 36 20 U/ml H2O2 90 AET a 4 1 10 05 gereral 44 KI 1 1 10 02 •OH 18 5 1 10 02 •OH 53 NaN3 1 1 10 02 1O2 14 5 1 10 02 M 1O2 43 aAET Å 2-(aminoethyl)isothiuroium. not, indicating that H2O2 participated in the breaking reaction, but O •0 2 did not. Both radical scavengers specific to • OH or 1 O2 inhibited the breaking reaction. Theseresultsindicatedthatreducedwatercanprevent DNA damage caused by active oxygen species such as H2O2, • OH, and 1 O2 produced by the Cu(II)-catalyzed oxidationof AsA.Significantsynergistic effectbetween reduced water and the radical scavengers used here was not observed (data not shown). It is noteworthy that reduced water can prevent DNA damage even in thepresenceofmetallicionswhichcatalyzetheautooxidation of most established antioxidants and reverse their protective effect against active oxygen species. However, the water must have a higher reducing ability for this purpose than scavenging O •0 2 . Althoughaerobicorganismshaveevolvedbyacquisition of the ability to utilize oxygen, oxygen is principally a toxic substance. Recently biological activation ofhydrogenbyhydrogenaseswasreported(13).Hydrogenases, which are among in the oldest enzymes (3.8 billion years old), can reversibly split molecular hydrogen to produce active atomic hydrogen. Active atomic FIG.4. Inhibitoryeffectofreducedwateronsingle-strandbreakage of DNA caused by oxygen radicals produced by the Cu(II)-cata- hydrogen may have participated in the redox regulalyzed oxidation of AsA. (A) Electrophoresis of plasmid DNA treated tion of cellular functions. Water can permeate everywith the mixture of AsA and Cu(II) in the presence of 4.1 IC50SO where in the body and penetrates every membrane in- units of reduced water (RP, 0659 mV; pH 10.5; IC50SO(18%)) or NaOH solution (pH 10.5). SC, super-coil DNA. OC, open-circular cluding the blood-brain barrier. To neutralize the toxic DNA. Lane 1, marker (l DNA-HindIII); lane 2, control DNA (2 action of active oxygen species, electrolyzed-reduced hours);lane3,DNA/Cu(II)(2hours);lane4,DNA/AsA(2hours). water may be an ideal and very powerful antioxidant. Lanes 5, 7, 9, 11 and 13 show the DNA breaking reaction by the Furtherintensiveinvestigationontheeffectofreduced mixture of AsA and Cu(II) in the presence of NaOH solution for 0, water on cell biology, immunology and oncology should 15, 30, 60 and 120 minutes, respectively. Lanes 6, 8, 10, 12 and 14 be promoted. show the DNA breaking reaction by the mixture of AsA and Cu(II) in the presence of reduced water for 0, 15, 30, 60 and 120 minutes, respectively. (B) Decrease of the relative amount (%) of super-coil DNA duringthe incubation ofplasmid DNA withthe mixtureof AsA ACKNOWLEDGMENTS and Cu(II) in the presence of reduced water () or NaOH solution (l).Averagevaluesoftwoindependentexperiments(nÅ2,each)were We are grateful to Dr. David Barnes, Oregon State University, shown. The vertical bars show the SD errors. and Mr. Mark Selby for their critical reading of the manuscript. 273
Vol. 234, No. 1, 1997 REFERENCES BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 7. Tanaka, T. (1969) Chemistry of Free Radical, Iwanami Shoten, Tokyo. 1. Sohal, R.S., and Weindruch, R. (1996) Science 273, 59–63. 8. Langmuir, I. (1927) Ind. Eng. Chem. 19, 667–674. 9. Sasamori, R., Okaue, Y., Isobe, T., and Matsuda, Y. (1994) Science 265, 1691–1693. 2. Feig, D.I., Reid, T.M., and Loeb, L.A. (1994) Cancer Res. 54, 1890s–1894s. 10. Shinohara, K., So, M., Nonaka, M., Nishiyama, K., Murakami, 3. Hayashi, H. (1995) Explore 6, 28–31. H., and Omura, H. (1983) J. Nutr. Sci. Vitaminol. 29, 481–488. 4. Nakano,M.,Sugioka,K.,Ushijima,Y.,andGoto,T.(1986)Anal. 11. Chiou, S.-H. (1983) J. Biochem. 94, 1259–1267. Biochem. 159, 363–369. 12. Scarpa, M., Stevanato, R., Viglino, P., and Rigo, A. (1983) J. 5. Gadgil, K., and Gonzalez, R.D. (1975) J. Catal. 40, 190–196. Biol. Chem. 258, 6695–6697. 13. Happe, R.P., Roseboom, W., Plerik, A.J., Albracht, S.P.J., and 6. Austin, L.G. (1961) Ind. Eng. Chem. 53, 659–661. Bagley, K.A. (1997) Nature 385, 126. 274
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CLINICAL STUDIES OF ALKALINE WATER
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a fluid replacement solution is rec
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to suppress single-strand breakage
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Inactivation of Escherichia coli (O
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PMID: 12857518 [PubMed - indexed fo
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"Ionized alkaline antioxidant water
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"Mr. Yamada, the head of the Police
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Alkaline Water and Obesity Prof. Ha
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Since the introduction of alkaline
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studied safety and usefulness of al
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serum climb up and resume to normal
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intestinal fermentation is more inh
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decalcification has not happened ye
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