Slight Deuterium Enrichment in Water Acts as an Antioxidant: Is Deuterium a Cell Growth Regulator?

Abstract

Small admixtures in water, e.g. of metal ions, often act as cell growth regulators. Here we report that enrichment of deuterium content in water, normally found at 8 mm concentration, two-three folds increases cell proliferation and lowers the oxidative stress level as well. Acting as an anti-oxidant, deuterium-enriched water prevents the toxic effect of such oxidative agents as hydrogen peroxide and auranofin. This action is opposite to that of deuterium depletion that is known to suppress cell growth and induce oxidative stress in mitochondria. We thus hypothesize that deuterium may be a natural cell growth regulator that controls mitochondrial oxidation-reduction balance. Because growth acceleration is reduced approximately by half by addition to water a minute amount (0.15%) of 18O isotope, at least part of the deuterium effect on cell growth can be explained by the isotopic resonance phenomenon. A slight (≈2-fold) enrichment of deuterium in water accelerates human cell growth. Quantitative MS based proteomics determined changes in protein abundances and redox states and found that deuterium-enriched water acts mainly through decreasing ROS production in mitochondria. This action is opposite to that of deuterium depletion that suppresses cell growth by inducing oxidative stress. Thus deuterium may be a natural cell growth regulator that controls mitochondrial oxidation-reduction balance. The role of isotopic resonance in this effect was validated by further experiments on bacteria. Small admixtures in water, e.g. of metal ions, often act as cell growth regulators. Here we report that enrichment of deuterium content in water, normally found at 8 mm concentration, two-three folds increases cell proliferation and lowers the oxidative stress level as well. Acting as an anti-oxidant, deuterium-enriched water prevents the toxic effect of such oxidative agents as hydrogen peroxide and auranofin. This action is opposite to that of deuterium depletion that is known to suppress cell growth and induce oxidative stress in mitochondria. We thus hypothesize that deuterium may be a natural cell growth regulator that controls mitochondrial oxidation-reduction balance. Because growth acceleration is reduced approximately by half by addition to water a minute amount (0.15%) of 18O isotope, at least part of the deuterium effect on cell growth can be explained by the isotopic resonance phenomenon. A slight (≈2-fold) enrichment of deuterium in water accelerates human cell growth. Quantitative MS based proteomics determined changes in protein abundances and redox states and found that deuterium-enriched water acts mainly through decreasing ROS production in mitochondria. This action is opposite to that of deuterium depletion that suppresses cell growth by inducing oxidative stress. Thus deuterium may be a natural cell growth regulator that controls mitochondrial oxidation-reduction balance. The role of isotopic resonance in this effect was validated by further experiments on bacteria. Deuterium (D) is the natural heavy stable isotope of hydrogen. The relative D content in natural water is on average 0.015% (150 ppm), which is equivalent to 8 mm concentration. Deuterium fractionation occurs during vapor–liquid–ice (snow) phase transitions and during sorption and filtration in natural processes, and as a result deuterium content in water varies in terrestrial conditions within the range between 79 ppm and 195 ppm (1Ferronskii V. Polyakov V. Isotopy of the Earth's Hydrosphere. Springer, Netherlands2012Crossref Google Scholar). By many physico-chemical manipulations, including distillation, diffusion, and chemical reactions, the concentration of deuterium in water can be made arbitrary high or low. Because the discovery of deuterium by Urey et al. in 1931 (2Szent-Györgyi A. The living state and cancer.Proc. Natl. Acad. Sci. U S A. 1977; 74: 2844-2847Crossref PubMed Scopus (70) Google Scholar, 3Gat J.R. Gonfiantini R. Stable isotope hydrology.Deuterium and oxygen-18 in the water cycle. Wiley, Hoboken1981Google Scholar), the biological effects of deuterium enrichment have been examined in a host of experimental situations (4Enright J. Heavy water slows biological timing processes.Z Vergl. Physiol. 1971; 72: 1-16Crossref Scopus (44) Google Scholar, 5Harvey E.N. Biological effects of heavy water.Biol. Bull. 1934; 66: 91-96Crossref Google Scholar, 6Mosin, O., and Ignatov, I., (2012) Isotope effects of deuterium in bacterial and microalgae cells at growth on heavy water (D2O).Google Scholar). Although bacteria can endure up to 90-100% D (v/v) in deuterium enriched water (DEW), very high deuterium concentrations turned out to be incompatible with highly organized life, as they slow down cellular metabolism and cause mitotic inhibition of the prophase (4Enright J. Heavy water slows biological timing processes.Z Vergl. Physiol. 1971; 72: 1-16Crossref Scopus (44) Google Scholar). Plant cells can develop normally in up to 75% DEW, whereas animal cells – not more than 30% DEW (7Mosin O. Shvets V. Skladnev D. Ignatov I. Studying of microbic synthesis of deuterium labelled l-phenylalanine by methylotrophic bacterium brevibacterium methylicum on media with differeny content of heavy water.Russ. J. Biopharmaceut. 2012; 4: 11-22Google Scholar). That high concentration of deuterium has a strong biological effect is not unexpected given its factor of two mass difference with hydrogen atom. More surprising was the finding made in 1930s that relatively small deviations from normal deuterium content, such as 4-fold deuterium enrichment, have a marked effect on various organisms (8Barnes T.C. A possible physiological effect of the heavy isotope of h in water.J. Am. Chem. Soc. 1933; 55: 4332-4333Crossref Scopus (8) Google Scholar, 9Barnes T.C. Larson E.J. Further experiments on the physiological effect of heavy water and of ice water.J. Am. Chem. Soc. 1933; 55: 5059-5060Crossref Scopus (5) Google Scholar, 10Barnes T.C. The effect of heavy water of low concentration on euglena.Science. 1934; 79 (370): 370Crossref PubMed Scopus (6) Google Scholar, 11Lockemann G. Leunig H. Über den Einfluß des schweren Wassers”.Ber. dtsch. Chem. Ges. A/B. 1934; 67: 1299-1302Crossref Google Scholar, 12Barnes T.C. Larson E.J. The influence of heavy water of low concentration on Spirogyra, Planaria and on enzyme action.Protoplasma. 1935; 22: 431-443Crossref Scopus (6) Google Scholar, 13Lobyshev V.I. Tverdislov V.A. Vogel J. Iakovenko L.V. Activation of Na,K-ATPase by small concentrations of D2O, inhibition by high concentrations.Biofizika. 1978; 23: 390-391PubMed Google Scholar, 14Lobyshev V.I. Fogel I. Iakovenko L.V. Rezaeva M.N. Tverdislov V.A. D2O as a modifier of ionic specificity of Na, K-ATPase.Biofizika. 1982; 27: 595-603PubMed Google Scholar). The growth rate and morphology of Spirogyra, flatworms, and Euglena in 0.06% D (600 ppm DEW) water have been found significantly affected (8Barnes T.C. A possible physiological effect of the heavy isotope of h in water.J. Am. Chem. Soc. 1933; 55: 4332-4333Crossref Scopus (8) Google Scholar, 9Barnes T.C. Larson E.J. Further experiments on the physiological effect of heavy water and of ice water.J. Am. Chem. Soc. 1933; 55: 5059-5060Crossref Scopus (5) Google Scholar, 10Barnes T.C. The effect of heavy water of low concentration on euglena.Science. 1934; 79 (370): 370Crossref PubMed Scopus (6) Google Scholar, 12Barnes T.C. Larson E.J. The influence of heavy water of low concentration on Spirogyra, Planaria and on enzyme action.Protoplasma. 1935; 22: 431-443Crossref Scopus (6) Google Scholar). On the other hand, in the same studies fermentation reaction slowed down by ≈15%. Lockemann and Leunig studied the effect ≤0.54% DEW upon E. coli and Pseudomonas proliferation. They observed that concentrations as low as 0.04% D enhanced growth (11Lockemann G. Leunig H. Über den Einfluß des schweren Wassers”.Ber. dtsch. Chem. Ges. A/B. 1934; 67: 1299-1302Crossref Google Scholar). In another study, Aspergillus grew 10% faster in 0.05% D water (15Curry J. Pratt R. Trelease S.F. Does Dilute Heavy Water Influence Biological Processes?.Science. 1935; 81: 275-277Crossref PubMed Scopus (5) Google Scholar). Second world war interrupted these early studies, and atomic bomb project classified the deuterium-related research. But starting from 1960s, open research was resumed, and in 1970s Lobyshev et al. discovered that the Na, K-ATPase activity increases by up to 50% at 0.04-0.05% D, i.e. at a 2-3-fold enrichment (13Lobyshev V.I. Tverdislov V.A. Vogel J. Iakovenko L.V. Activation of Na,K-ATPase by small concentrations of D2O, inhibition by high concentrations.Biofizika. 1978; 23: 390-391PubMed Google Scholar, 14Lobyshev V.I. Fogel I. Iakovenko L.V. Rezaeva M.N. Tverdislov V.A. D2O as a modifier of ionic specificity of Na, K-ATPase.Biofizika. 1982; 27: 595-603PubMed Google Scholar, 16Lobyshev V.I. Tverdislov V.A. Vogel J. Iakovenko L.V. Activation of Na,K-ATPase by small concentrations of D2O, inhibition by high concentrations.Biofizika. 1978; 23: 390-391PubMed Google Scholar). In early 1990s, Somlyai et al. confirmed that increasing deuterium concentration to ≈600 ppm enhances the growth rate of mammalian cells (fibroblasts) (17Somlyai G. Jancsó G. Jákli G. Vass K. Barna B. Lakics V. Gaál T. Naturally occurring deuterium is essential for the normal growth rate of cells.FEBS Lett. 1993; 317: 1-4Crossref PubMed Scopus (75) Google Scholar). However, neither of the previous studies has suggested a credible mechanism for growth acceleration by DEW. On the other side of the enrichment scale, it has been found that deuterium depleted water (DDW) with deuterium content of 20-130 ppm suppresses cell growth, inhibiting cancer cell proliferation and tumor growth (18Somlyai G. Laskay G. Berkényi T. Galbács Z. Galbács G. Kiss S. Jákli G. Jancsó G. The biological effects of deuterium-depleted water, a possible new tool in cancer therapy.Zeitschrift fur Onkologie. 1999; 30: 91-94Google Scholar, 19Krempels K. Somlyai I. Somlyai G. A retrospective evaluation of the effects of deuterium depleted water consumption on 4 patients with brain metastases from lung cancer.Integr. Cancer Ther. 2008; 7: 172-181Crossref PubMed Scopus (32) Google Scholar, 20Somlyai G. Molnár M. Laskay G. Szabó M. Berkényi T. Guller I. Kovács A. Biological significance of naturally occurring deuterium: the antitumor effect of deuterium depletion.Orv. Hetil. 2010; 151: 1455-1460Crossref PubMed Google Scholar). Despite dozens of research reports, this the mechanism of growth has been we have studied the effect of ppm in human lung cells with chemical proteomics and found that mitochondrial redox which to oxidative stress M. 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PubMed Scopus Google Scholar), the for cell of the DEW effect is a we the DEW concentration to ppm and the range The cells proliferation was enhanced by with in the of between ppm and ppm D. a of this ppm DEW was for further a in cell proliferation was with deuterium at a for growth, ppm was In for of and in cells with and DEW was to the difference between the two conditions The and of the in cells in ppm DEW to and classified the in and the metabolism and found as the enriched biological in cells as an of they reduced and H. of Scholar). of and and Because is the of mitochondrial ROS production E.J. J. of is the of mitochondrial production in in early 2012; PubMed Scopus Google Scholar), this result that DEW may act through of cellular redox to M. A. effect of deuterium depleted water redox to oxidative PubMed Scopus Google Scholar), in the opposite be that the from in previous M. 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The effect of on ROS production is of in the media and redox proteomics on cells to the with the effect of and acceleration effect of DEW cells growth was the concentration of D was and in ppm DEW Thus these deuterium concentrations for as and DEW In and in in was to the difference The and in cells in ppm DEW to and classified with of the in cells in ppm DEW at and activity was of the enriched proteomics was in cells The average level in cells in ppm DEW was than in and significantly than in ppm This result was further confirmed by the which significantly ROS in cells in DEW with and with The with in redox or and of the reduced enriched in which was In the to That to hypothesize that DEW may accelerates cell growth by This was validated by finding significantly of cellular in and cells in DEW with and and of cells is affected by cellular such as and and Because in cellular these highly with often growth an in the through role as enzyme The of PubMed Scopus Google Scholar). in cell of A. J. R. H. of cell proliferation by PubMed Scopus Google or human A. J. R. R. The of human cell proliferation by and its by Physiol. 1977; PubMed Scopus Google was found to be by concentrations to of cells in the On the other hand, proliferation of these cell at of decreasing at concentration is an regulator of cellular growth and and the of in normal and 1978; PubMed Scopus Google Scholar, a regulator of growth and PubMed Scopus Google Scholar). with is by a concentration that with concentrations to faster growth concentration is finding is that deuterium cell growth within the concentration range between ppm and ppm that D concentration cell whereas D concentration to faster growth. a cell growth deuterium acts by the in cellular mitochondrial redox The mechanism is to be for action we have an M. A. effect of deuterium depleted water redox to oxidative PubMed Scopus Google Scholar). But on a more the of cell growth by deuterium may be a of the more such as the isotopic resonance G. B. H. Z. Somlyai I. and the effect of deuterium J. Scholar). The isotopic resonance to the of at of isotopic which to faster of The for the isotopic resonance is the that to as as that the as that to a in the of of In isotopic resonance is not is e.g. average isotopic mass with the The isotopic resonance for normal isotopic of the and and of D is found in the range between and ppm D S. A. resonance at ppm deuterium of by 8 PubMed Scopus Google Scholar), in with the growth of cells observed in resonance often enhances the reaction that ppm D water by 30% the rate of by S. A. resonance at ppm deuterium of by 8 PubMed Scopus Google Scholar). The isotopic resonance the from isotopic the effect or of the reaction or In this with growth by isotopic resonance is at least for the observed growth of bacteria in the with concentration of D was was discovered that ppm suppresses bacterial growth, whereas ppm DEW enhances it with at two 18O and The of 18O concentration was by to growth media a small of water with 10% 18O or the same of isotopic resonance be at deuterium concentration The of two experiments in the isotopic the growth of DEW more than with conditions at the same deuterium concentration. normal deuterium concentration, at which the isotopic resonance conditions more the same of 18O concentration effect on cell growth. at least part of the DEW growth acceleration effect is of the isotopic resonance phenomenon. Here we confirmed the acceleration effect of DEW on cell proliferation and discovered on the it acts mainly through of mitochondrial redox states in the opposite than on level DEW acts as an On the protein DEW the of in metabolism and with many in mitochondria. The that the concentration of D is than the normal of the oxidative stress down cell proliferation. However, D concentration than cell growth is through inhibition of ROS The growth acceleration effect to in a range of deuterium concentrations between and with ppm the of that or this is the for growth acceleration by the isotopic resonance S. A. resonance at ppm deuterium of by 8 PubMed Scopus Google Scholar, R. K. A. H. in mass 2010; Scholar), which is by previous (13Lobyshev V.I. Tverdislov V.A. Vogel J. Iakovenko L.V. Activation of Na,K-ATPase by small concentrations of D2O, inhibition by high concentrations.Biofizika. 1978; 23: 390-391PubMed Google Scholar, 14Lobyshev V.I. Fogel I. Iakovenko L.V. Rezaeva M.N. Tverdislov V.A. D2O as a modifier of ionic specificity of Na, K-ATPase.Biofizika. 1982; 27: 595-603PubMed Google Scholar). Because the growth acceleration effect at ppm is the resonance conditions at least part of the effect be of the isotopic et al. have not confirmed the effect of deuterium content in water on the of that this effect is of water than in of the cells Lobyshev V.I. The activity of in deuterium depleted Scopus Google Scholar).

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