https://orcid.org/0000-0001-6812-3048
Figures
Abstract
Numerous enzymatic reactions involve hydrolysis, making water indispensable for sustaining life. Some water includes hydrogen isotopes, deuterium or tritium, with larger atomic weights. Heavy water consisting of deuterium is toxic to living organisms and induces cell death; however, the extent and underlying mechanisms of this toxicity remained elusive. Herein, we demonstrate that 100% heavy water triggers a remarkably heightened apoptotic response in human cells, compared to exposure to high-dose ionizing radiation. This pronounced effect of heavy water on cellular function may stem from the quantum-level mechanisms of kinetic isotope effects inherent to water isotopes, leading to a deceleration in enzymatic hydrolysis reactions. Notably, dilution of heavy water by approximately ten-fold with ordinary light water abolishes its isotope effect on enzymatic hydrolysis reactions, concomitant with the disappearance of DNA repair inhibition and cell death induction in human cells. These findings reveal that high concentrations of water isotopes containing heavier hydrogen have extreme cell death-inducing toxicity, yet this toxicity disappears upon dilution, thereby offering crucial insights into environmental considerations.
Citation: Yasuda T, Nakajima NI, Yanaka T, Gotoh T, Tajima K (2025) Heavy water toxicity via isotope effects: Stronger than high-dose radiation, neutralized by light water. PLOS Water 4(1): e0000292. https://doi.org/10.1371/journal.pwat.0000292
Editor: Gaurav Saxena, Mandsaur University, INDIA
Received: July 30, 2024; Accepted: January 7, 2025; Published: January 29, 2025
Copyright: © 2025 Yasuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are available in the manuscript and its Supporting Information.
Funding: This research was supported by JSPS KAKENHI, Grant Number 20K12177 to T. Yasuda, Grant Number 20K08071 to K.T. and T. Yasuda, and Grant Number 22K08414 to T.G. and T. Yasuda. The funders had no role in the study design, data collection, interpretation, or decision to submit the work for publication.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Various harmful factors in our environment induce cell death. For example, ionizing radiation, such as X-rays and γ rays, induces cell death by causing DNA breaks in cells [1,2]. In response to a DNA double strand break (DSB), the ATM protein kinase is activated and initiates the DNA damage response signaling pathway by phosphorylating various proteins, including the histone variant H2AX in the vicinity of the DSB site and the Chk2 kinase protein [3]. The phosphorylated Chk2 kinase activates a cell cycle checkpoint mechanism that temporarily arrests the cell cycle until the damaged DNA is repaired by various DNA repair proteins that are accumulated at the DSB sites [4,5].
Irreparable DNA damage activates apoptosis, eliminating damaged cells [6]. For example, tissue damage in the hematopoietic and gastrointestinal tracts caused by high-dose radiation exposure will induce apoptotic cell death [7,8]. The transcription factor p53 is involved in inducing the expression of both cell cycle checkpoint-related genes and apoptosis-inducing genes. The p53 functions are regulated by internal phosphorylation sites, and phosphorylation at serine 46 (S46) is required for the expression of apoptosis-related genes [9]. Upon Ser46 phosphorylation, apoptosis-inducing genes such as Bax, Bid, and Puma are expressed by p53-mediated activation. These apoptosis-inducing factors increase mitochondrial membrane permeability, resulting in the release of cytochrome c from mitochondria into the cytoplasm. Cytochrome c binds to Apaf-1 to form a complex named the apoptosome, which cleaves and activates pre-processed caspase-9. The activated caspase-9 induces the sequential cleavage-mediated activations of downstream caspase-7/caspase-3 to execute apoptosis. Another apoptosis-inducing pathway involves the cleavage-mediated activation of caspase-8, which is induced by signaling from the binding of DNA damage-induced TNFα to its cell surface receptors [10].
Water is involved in myriad reactions in living cells and is therefore essential for life on earth [11-13]. In contrast to ordinary water, heavy water, in which the hydrogen in the water molecule is replaced by its isotope, deuterium, induces cell death [14-19]. Unlike the radioactive isotope tritium, deuterium is a stable isotope, so the cytotoxicity of heavy water is not caused by radiation. We have shown that the heavy water-induced cytotoxicity is related to quantum-level effects in enzyme-mediated hydrolysis reactions [20]. Many intracellular enzymatic reactions involve hydrolysis, in which water molecules directly participate in the chemical reaction [21-25]. In several types of enzyme-mediated hydrolysis reactions, the reaction rate was decreased when the water in the reaction solution was replaced with heavy water [20]. This phenomenon is called the kinetic isotope effect [26,27]. One reason for the kinetic isotope effect is that the probability of quantum tunneling is lower for deuterium with atomic weight 2 than for hydrogen with atomic weight 1 in the chemical reactions of hydrogen through quantum tunneling [28]. Another reason is that the vibrational potential energy of the chemical bonds involved in a chemical reaction depends on the mass difference between hydrogen and deuterium [28]. Both reasons are related to quantum-level mechanisms [28].
For in vitro enzymatic chemical reactions, such as deacetylation reactions using purified proteins, the kinetic isotope effect of heavy water reduced the enzymatic reaction rate by at most 1/2. However, cells exposed to heavy water are affected by unexpectedly strong isotope effects [20]. For example, the DNA homologous recombinational repair associated with the in vitro enzymatic reactions was almost completely inhibited with heavy water in human cells [20]. We speculate that the reason could be due to the synergistic kinetic isotope effects of a very large number of hydrolysis reactions in the cells [20]. In this study, we clarified the magnitude of the effects of heavy water on human cells in comparison with exposure to high-dose radiation, which induces apoptotic cell death.
Materials and methods
Reagents
The following reagents were used: BSA (B9001, New England Biolabs, Ipswich, MA, USA), I-SceI (R0694, New England Biolabs), H2O (06442-95, Nacalai Tesque, Inc., Kyoto, Japan), D2O (D214H, Nacalai Tesque, Inc.), and Hydroxyurea (4987481379657, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan).
Cell culture
U937 cells were cultured in RMPI 1640 medium (11875, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin (PS). For experiments comparing the isotope effects due to H2O or D2O in the cells, the medium was prepared by dissolving RMPI 1640 powder (31800, Thermo Fisher Scientific) in H2O (06442-95, Nacalai Tesque, Inc.) or D2O (D214H, Nacalai Tesque, Inc.).
HeLa pDR-GFP cells, obtained from Dr. M. Jasin [29], were cultured in minimum essential medium (MEM) (1030700, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% PS. For experiments comparing the isotope effects due to H2O or D2O in these cells, the MEM solutions were prepared by dissolving MEM powder (61100, Thermo Fisher Scientific) in H2O (06442-95, Nacalai Tesque, Inc.) or D2O (D214H, Nacalai Tesque, Inc.).
Irradiation
Irradiation of cells with γ rays was performed as described [30]. Irradiation of cells with X-rays was performed as described [31].
Immunoblotting
Immunoblotting analyses were performed essentially as described previously [30]. Proteins were separated by 12% SDS-PAGE and subsequently transferred onto Immobilon-P membranes (Merck Millipore, Whitehouse Station, NJ, USA). The target proteins were detected by using the Western-Light PlusTM Chemiluminescent Detection system (Tropix, Bedford, MA, USA) with an LAS-4000 mini luminoimaging analyzer (Fujifilm, Tokyo, Japan) or with Biomax XAR film (Carestream, Rochester, NY, USA). The antibodies used for immunoblotting analyses are listed (S1 Table).
Microscopy
The antibodies used for immunofluorescence microscopic analyses are listed (S1 Table). The immunofluorescence microscopic analysis of the HeLa pDR-GFP cells irradiated with γ rays or treated with hydroxyurea (HU) was performed with anti-53BP1, anti-phospho-BRCA1 at Ser1254, and anti-γH2AX antibodies, using an IX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with an ORCA-R2 cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) and x100 lens (UPLSAPO100XO, Olympus), as described previously [30]. For the quantitative colocalization analysis, the total number of 53BP1 or phospho-BRCA1 foci and the number of their foci colocalized with γH2AX were counted in each cell. The percentage of colocalized foci was calculated by dividing the number of colocalized foci by the total number of foci.
The immunofluorescence microscopic analysis of the U937 cells irradiated with X-rays was performed with an anti-γH2AX antibody, with an all-in-one fluorescence microscope (BZ-X700, Keyence, Osaka, Japan) equipped with a x100 lens. Cell staining with 5-ethynyl-2′-deoxyuridine (EdU) was performed with a Click-iT™ EdU Cell Proliferation Kit for Imaging (Thermo Fisher Scientific), as described previously [31]. The total area of γH2AX foci in each cell was analyzed with the BZ-X700 microscope.
In vitro I-SceI-mediated DNA cleavage assay
The in vitro I-SceI-mediated DNA cleavage assays were performed as described [20]. The pGP2 NotI-linearized control plasmid (New England Biolabs), which contains a single I-SceI site, was used as the substrate. The substrate DNA (50 ng) was incubated with the I-SceI enzyme (0.1 units/μl) in 10 μl of CutSmart buffer [50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9], containing the indicated concentrations of H2O (06442-95, Nacalai Tesque, Inc.) and D2O (D214H, Nacalai Tesque, Inc.), at 15°C for 20 min. After the reaction, the samples were analyzed by 0.8% agarose gel electrophoresis with ethidium bromide staining. The gel images were captured using a BioSpectrum Imaging System (UVP, Upland, CA, USA) and an LAS-4000 mini imaging analyzer (Fujifilm). Quantification was performed using the Multi Gauge software (Fujifilm).
Cell survival assays
To monitor the cell viability by a WST-1 assay, U937 cells were seeded in 96-well flat-bottom plates (4,000 cells/well) and cultured in medium containing the indicated concentration of D2O (D214H, Nacalai Tesque, Inc.). After 2 days, the cell viability was examined with the ROCHE Cell Proliferation Reagent (WST-1) (05015944001, Merck & Co., Inc.), according to the manufacturer’s instructions, using an ARVO X5 microplate reader (Perkin Elmer).
I-SceI-based reporter assay for HR repair
For the HR repair assay, Hela pDR-GFP cells were seeded in 12-well plates (5x105 cells/well) and cultured in standard cell culture medium. At 24 h, the culture medium was changed to fresh medium containing 0, 10, 20, 50 or 100% D2O, made with H2O (06442-95, Nacalai Tesque, Inc.) and D2O (D214H, Nacalai Tesque, Inc.), and the cells were transfected with 2.5 μg of the I-SceI expression plasmid, pCMV-NLS-I-SceI [29]. The cells were cultured at 31, 33, 35, 37, or 39˚C. At 48 h after the plasmid-transfection, the cells were harvested by trypsinization and analyzed with an SA3800 Spectral Cell Analyzer (SONY Biotechnology, San Jose, CA, USA).
Statistical analysis
The KaleidaGraph software (version 5.0), was used for statistical analyses. Statistical analyses for multiple comparisons were performed using a one-way ANOVA, as described [32]. Statistical analyses between the data of two groups were performed using an unpaired Student´s t-test, as described [32].
Results
Caspase activation is extremely higher with D2O than with exposure to high-dose radiation
Since U937 human monocyte cells are prone to apoptosis, cleavage-mediated activation of caspase proteins can be easily detected. Therefore, to examine the effect of D2O on cellular caspase activation, we chose U937 cells in this study. At first, U937 cells were cultured in regular cell culture medium containing H2O, and then the cells were cultured in cell culture media prepared with either H2O or D2O immediately after irradiation (Fig 1). In cells cultured in the medium prepared with H2O, caspase-3 cleavage was detected 4 hours after 24 Gy of γ-ray irradiation. In contrast, cells cultured in the medium prepared with D2O after irradiation showed remarkably enhanced γ-irradiation-induced cleavage of caspase-3. Surprisingly, caspase-3 cleavage occurred even without irradiation in D2O-treated cells. Moreover, the extent of the caspase-3 cleavage induced by D2O treatment was much stronger than that by the high dose of γ-irradiation (Fig 1).
U937 cells cultured in standard media containing H2O were seeded in 3.5 cm plastic dishes (2x105 cells/dish) and cultured in fresh medium prepared with H2O or D2O immediately before γ-ray irradiation. After irradiation with the indicated dose of γ-rays, the cells were cultured for the indicated times. The cell extracts were prepared with U937 cells cultured in standard media without irradiation and U937 cells cultured in fresh medium with or without irradiation, and were subjected to immunoblotting analyses with the indicated antibodies.
Next, we examined the cleavage of the other caspases, caspase-7 and caspase-8. Caspase-7 cleavage was induced more strongly in cells cultured for 2 hours in the medium prepared with D2O, compared to those cultured for 6 hours after 24 Gy γ-ray irradiation (Fig 2A). In addition, cleavage-mediated caspase-8 activation did not occur in cells after 6 hours of 24 Gy γ-ray irradiation, but did occur in cells exposed to D2O for 2 hours (Fig 2B). These results indicate that cleavage-mediated activations of caspase proteins are induced more extensively in cells by exposure to D2O than to high doses of ionizing radiation.
(A-E) U937 cells cultured in standard media containing H2O were seeded in 3.5 cm plastic dishes (2x105 cells/dish) and cultured in fresh medium prepared with H2O or D2O. U937 cells cultured in fresh medium containing H2O were irradiated with 24 Gy of γ-rays and cultured for 6h after irradiation. U937 cells cultured in fresh medium containing D2O were cultured for 2h. The cell extracts of U937 cells without treatment (-) and U937 cells treated with D2O or γ-ray irradiation were subjected to immunoblotting analyses with the indicated antibodies.
High activation of cellular signaling for apoptosis and checkpoint with D2O
In the pathway upstream of apoptosis induction by the series of caspase cleavages, the p53 protein is phosphorylated at S46 [9]. Induction of p53 phosphorylation at S46 was detected in cells exposed to D2O for 2 hours. Meanwhile, in cells exposed to 24 Gy of γ-rays for 6 hours, p53 phosphorylation at S46 was not induced and the level was comparable to that in non-irradiated cells (Fig 2C).
We next examined the effect of D2O on the activation of the cell cycle checkpoint mechanism. Activation of a DNA damage checkpoint protein, Chk2, by phosphorylation was detected in cells exposed to D2O for 2 hours. In contrast, in cells exposed to 24 Gy of γ-rays for 6 hours, Chk2 phosphorylation was not induced and was comparable to that in non-irradiated cells (Fig 2D).
DSBs are induced with D2O but less than with ionizing irradiation
The activation of Chk2 by phosphorylation is induced by the generation of DSBs. Therefore, we investigated whether DSBs are induced in cells exposed to D2O. H2AX phosphorylated at S139, γH2AX, is an indicator of DSBs. An immunoblotting analysis showed that the induction of γH2AX was detected in U937 cells exposed to D2O for 2 hours (Fig 2E).
Based on the results of the immunoblotting experiments examining the activations of caspases and the phosphorylations of p53 and Chk2, we expected that more DSBs would be induced in cells exposed to D2O than in irradiated cells. We performed immunofluorescence microscopy experiments with γH2AX staining in U937 (Fig 3) and Hela-DR-GFP cells (S1 Fig). Small amounts of γH2AX-positive cells are generally detected even in non-irradiated cells [33,34]. Among the irradiated cells, all were stained with γH2AX. However, contrary to our speculation, only some γH2AX-positive cells were detected among those exposed to D2O, and the percentage of γH2AX-positive cells was significantly lower than that of the irradiated cells (Fig 3, and S1 Fig). Since 5-ethynyl-2’-deoxyuridine (EdU) is incorporated into DNA during DNA synthesis, the cells in S phase of the cell cycle can be distinguished with EdU staining [31]. The γH2AX positivity of cells exposed to D2O was compared between EdU-positive and negative cells, and the number of EdU-positive cells among the D2O-exposed cells was slightly higher than the EdU-negative cells (Fig 3). This tendency was also observed in cells cultured in H2O. DSBs in non-irradiated cells are thought to be associated with DNA replication stressors, which cause DSBs by inhibiting DNA synthesis. Thus, these results suggest that D2O-exposure might enhance DNA replication stress in cells.
(A) U937 cells were cultured with 5-ethynyl-2′-deoxyuridine (EdU) for 30 min before irradiation. Immediately before irradiation, the culture medium was changed to fresh medium prepared with H2O or D2O containing EdU. Two hours after exposure to irradiation or heavy water, cells were subjected to immunofluorescent staining with an anti-γH2AX (green) antibody, DAPI (blue), and EdU (red). (B) The total area of γH2AX foci in each cell was analyzed in each indicated condition. The graph shows the mean values with dots representing each data value.
DNA replication stress-induced accumulation of DSB repair proteins at DSB sites is inhibited with D2O-treatment
We have recently reported that DSB repair mechanisms mediated by homologous recombination (HR), single-strand annealing (SSA), and nonhomologous end joining (NHEJ) are severely inhibited in cells exposed to D2O [20]. However, it was not clear whether D2O affects the localization of DSB repair enzymes to the DSB sites induced by DNA replication stress. Hydroxyurea (HU) is used to induce DNA replication stress and works by depleting the intracellular dNTP pool required for DNA synthesis [35,36]. 53BP1 and BRCA1 are DSB repair proteins that localize well to DSB sites. 53BP1 promotes nonhomologous end joining (NHEJ) repair, a major pathway of DSB repair, while BRCA1 is phosphorylated after DNA damage and required for the progression of homologous recombination (HR) repair, another major pathway of DSB repair [37,38]. Therefore, we investigated the effect of exposing cells to heavy water on the localizations of 53BP1 and phosphorylated BRCA1 to DSB sites elicited by HU-induced DNA replication stress. In cells cultured in medium containing normal H2O, 53BP1 and phosphorylated BRCA1 localized very well to γH2AX, a marker of DSB sites generated by exposure of cells to HU. In contrast, HU-induced DNA replication stress strongly inhibited the localization of 53BP1 and phosphorylated BRCA1 to DSB sites in cells exposed to D2O (Fig 4). This result is consistent with our experimental finding that DSB repair is severely impaired in cells exposed to D2O.
(A-D) HeLa pDR-GFP cells were cultured in fresh media containing H2O or D2O for 1 h before HU-treatment. In (A) and (B), without HU or at 2 h after addition of HU (2 mM), cells were subjected to immunofluorescent staining with an anti-53BP1 (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). In (C) and (D), without HU or at 6 h after addition of HU (2 mM), cells were subjected to immunofluorescent staining with an anti-phospho-BRCA1 at Ser1254 (green) antibody, an anti-γH2AX (red) antibody, and DAPI (blue). In (B) and (D), the percentages of 53BP1 (B) or phospho-BRCA1 (D) foci colocalized with γH2AX were calculated by dividing the number of 53BP1 (B) or phospho-BRCA1 (D) foci colocalized with γH2AX by the total number of 53BP1 (B) or phospho-BRCA1 (D) foci in each cell, respectively.
Concentration-dependent kinetic isotope and cellular effects of heavy water
In chemical reactions involving hydrogen transfer, replacing hydrogen of mass 1 with deuterium of mass 2 or tritium of mass 3 causes a kinetic isotope effect, in which the rate of a chemical reaction decreases as the mass of the hydrogen isotope increases [20,26,27,39-42] (S2 Fig). In the case of hydrolysis reactions, the vibrational potential energy required to break the bond between the hydrogen and oxygen in the water molecule is larger for O-H, O-D, and O-T, in that order, because the E0 energy of the ground state is different [26-28] (S2A Fig). Meanwhile, if the potential barrier cannot be overcome to allow a chemical reaction to proceed, then small quantum-level reactants such as protons can pass through the potential barrier by the quantum tunneling effect. The probability of the quantum tunneling effect decreases with increasing mass in the order of H, D, and T [28] (S2B Fig). For these two reasons, a kinetic isotope effect occurs, in which the reaction rate differs depending on the mass of the reacting hydrogen isotope [26-28] (S2C Fig).
With respect to the above mechanism, we next examined the extent to which the effect of the kinetic isotope effects due to heavy water, which reduces the reaction rate of the enzymatic hydrolysis reaction, is attenuated by dilution with normal water. For this purpose, we used the kinetic isotope effects on the hydrolytic DNA cleavage by the I-SceI nuclease as an experimental model system [20] (Fig 5A). We performed experiments under the conditions where a significant effect on the reaction rate appears between the reaction solution with 100% D2O and the control solution with 100% H2O. As a result, dilution of D2O with H2O to less than 10% eliminated the significant differences in reaction rates between the diluted and control samples (Fig 5B).
(A) Schematic representation of the kinetic isotope effect on I-SceI-mediated DNA cleavage in the presence of H2O or D2O. (B) In vitro I-SceI-mediated DNA cleavage assays were performed at 15˚C for 20 min in the presence of the indicated concentrations of H2O and D2O, as described in the Materials and Methods. The relative band intensities of full-length DNA are shown in the graph. The mean values and standard errors of the mean from 4 independent experiments were plotted, with dots representing each data value. Statistically significant differences were examined between each sample in lanes 3 through 9 and lane 2 (*P < 0.05, ***P < 0.001 and N.S., not significant by a one-way ANOVA).
Next, the effect of the heavy water concentration on cell survival was examined. After 2 days of exposure to heavy water, U937 cell viability decreased in a heavy water concentration-dependent manner, and was almost 0% at 100% D2O. Cell viability was about 40% at 50% D2O and approximately 80% at 5–20% D2O concentrations when the concentration of D2O was diluted with H2O (Fig 6). In addition, dilution with H2O also dramatically suppressed the cleavage-mediated activations of caspase-3 and caspase-7 induced by exposure to heavy water. At a 50% D2O concentration, the cleavage-mediated activations of caspase-3 and caspase-7 were efficiently suppressed, while at a 5% D2O concentration, the cleavages of caspase-3 and caspase-7 were suppressed to almost the same level as that at a 0% D2O concentration (Fig 7).
U937 cells were cultured in media containing various concentrations of D2O for 2 days. Cell survival was examined by a WST-1 assay, as described in the Materials and Methods. The graph shows the mean values and standard errors of the mean from 4 samples, with linear curve fitting.
(A and B) U937 cells cultured in standard media containing H2O were seeded in 3.5 cm plastic dishes (2 × 105 cells/dish) and cultured in fresh medium containing various concentrations of D2O for the indicated times. The cell extracts were subjected to immunoblotting analyses with the indicated antibodies.
Finally, we examined the effect of the heavy water concentration on DSB repair, which is critical for cell survival. DNA homologous recombination (HR) is one of the DSB damage repair mechanisms that mediates a recombination reaction between homologous DNA sequences [5] (Fig 8A). We used a human cell line bearing a GFP-based reporter gene cassette, which allows us to specifically analyze the efficiency of HR repair [29,30] (Fig 8B). The frequency of GFP-expressing cells can be used to determine the efficiency of HR repair of a specific DSB site induced by the I-SceI nuclease. Exposure to 100% heavy water almost completely inhibited HR repair in the temperature range of 31°C–39°C, where cells could grow, in addition to the normal culture temperature of 37°C (Fig 8C). In contrast, when D2O was diluted with H2O, the heavy water-mediated HR inhibition was reduced in all temperature ranges, with the 10% D2O concentration reaching a level similar to that of the 0% D2O concentration.
(A) Schematic of the homologous recombination (HR) repair of an I-SceI-induced DSB site. (B) Schematic of the HR repair reporter assay. HR repair at I-SceI-induced specific DSB sites produces GFP-positive cells. (C) I-SceI-based reporter assays for HR were performed in HeLa pDR-GFP cells cultured in medium containing the indicated concentration of D2O at the indicated temperature, as described in the Materials and Methods. The graph shows the mean values and standard errors of the mean from 4 samples, with dots representing each data value.
Discussion
This study shows for the first time that a high concentration of heavy water is more toxic to human cells in terms of inducing apoptotic cell death than a high-dose radiation exposure of 24 Gy (S3 Fig). This radiation dose is larger than the estimated radiation dose received by those who died from high-dose exposure in the Tokai-mura JCO criticality accident in Japan [43]. Heavy water is known to be toxic to human cells, but it is surprising that the toxicity is so much greater than anticipated. Deuterium in heavy water is not a radioactive isotope, unlike tritium, another isotope of hydrogen. Thus, heavy water induces cell death by a different mechanism than radiation, which primarily targets DNA for cell death induction.
DSBs were also induced in cells exposed to heavy water, but only in some cells and primarily those in S-phase. Inhibition of DNA synthesis in S-phase is known to result in DSBs. DSBs are induced even under normal growth conditions, mainly in S-phase cells, suggesting that DSBs are due to defective DNA synthesis. Based on our findings, we propose the following mechanism of apoptosis induction by heavy water (Fig 9). Exposure to heavy water might cause cells to have more impaired DNA synthesis than under normal growth conditions, which in turn might cause the induction of DSBs (Fig 9B). The major difference is that DSB repair by HR is almost completely inhibited when cells are exposed to D2O, in comparison to H2O. In cells grown in H2O, even if radiation exposure produces more DSBs than D2O exposure, the DSBs are repaired by DNA repair mechanisms. In contrast, in cells cultured in D2O, even if the DSB induction is less than that with radiation exposure, the complete inhibition of DSB repair with D2O induces cell death more robustly (Fig 9B).
(A) Since the proton transfer reaction is affected by kinetic isotope effects, the 1H transfer reaction is faster than the 2D transfer reaction. Hydrolytic enzymatic reactions, which involve proton transfer, are affected by the kinetic isotope effects of water isotopes. In intracellular reactions, a series of hydrolytic enzyme reactions would experience synergistic effects on reaction rates. Thus, if there are 10 reaction steps subjected to synergistic effects that reduce the reaction rate by 1/2, then the final reaction would be almost completely impeded due to these effects. (B) Based on our experimental results, we propose a model of apoptosis induction by heavy water. DNA replication involves hydrolytic enzymes that are subjected to the isotope effects of heavy water. Thus, heavy water would induce DNA replication stress, inhibiting DNA synthesis and causing DSB formation. Heavy water inhibits DSB repair mechanisms (HR, SSA, and NHEJ), leaving DSB sites unrepaired [20]. CHK2 phosphorylation by DNA damage signaling activates a checkpoint, arresting the cell cycle. Phosphorylation of p53 at serine 46 induces apoptosis-related gene transcription, activating caspase proteins and triggering apoptotic cell death.
Heavy water’s strong cytotoxicity likely stems from unique quantum effects (S3 Fig), unlike mechanisms seen with radiation or poisons [20]. Our studies have shown that quantum tunneling effects are involved in the enzymatic hydrolysis reactions of protein deacetylation, protein cleavage, and DNA cleavage, and that the kinetic isotope effects associated with the quantum effects reduce the enzymatic chemical reaction rates when D2O is used instead of H2O in the hydrolysis reaction buffer (Fig 9A) [20]. These enzymatic chemical reactions are part of a very wide range of intracellular chemical reactions involved in various biological phenomena [21-23]. Quantum effects may be similarly involved in the ATP hydrolysis by various enzymes that produces the energy of life in cells [25]. Unlike radiation and poisons, which primarily target specific molecules within cells for induction of cell death [44-46], the kinetic isotope effects by heavy water target chemical reactions by a wide range of molecules within cells. Even if the rate-decreasing effect on individual chemical reactions is at most one-half, it can be assumed that for successive chemical reactions in cells, the synergistic effect would completely halt the reaction pathway (Fig 9A). If all possible reaction pathways in cells are blocked by the kinetic isotope effect of heavy water through such a mechanism, then very strong cytotoxicity will be generated in the cells. Our analyses of the isotope effect of heavy water on intracellular histone acetylation levels showed that hydrolysis-independent acetylation reactions are not affected by the isotope effect, but deacetylation reactions are, and thereby the balance between acetylation and deacetylation reactions is upset, resulting in an increase in histone acetylation levels (S4A Fig) [20]. As a result, the regulation of gene expression via histone acetylation was significantly altered [20]. As shown in this example, hydrolysis-independent reactions in cells are not subject to the isotope effects caused by heavy water, and it might be thought that heavy water alters the balance of various reaction equilibria in cells, resulting in a major distortion that generates intracellular stress (S4B Fig). This mechanism also might be one of the causes of the cytotoxicity induced by heavy water.
Unlike human cells, E. coli can grow in the presence of a high concentration of heavy water, although its growth rate is decreased [47,48]. A temperature change of about 1 degree Celsius relative to the normal temperature has a significant effect on the physical condition of human cells [49], while E. coli can grow at temperatures ranging from 25 to 40 degrees Celsius [50,51]. Since temperature changes affect the rates of chemical reactions, such as enzyme reactions, it is thought that E. coli is resistant to changing rates of intracellular chemical reactions, while human cells may be more vulnerable to such changes. Based on this sensitivity to chemical reaction rate changes, we speculate that heavy water, which affects chemical reaction rates, may have a greater effect on humans than on E. coli.
Tritium, which is of concern for its environmental effects [52-54], has a greater kinetic isotope effect relative to hydrogen than deuterium [26–28,39-42]. Therefore, high concentrations of tritiated water may be more cytotoxic than deuterated water, via the kinetic isotope effect. However, the strong cytotoxicity is an effect seen in undiluted, highly concentrated heavy water, and diluted heavy water is no longer cytotoxic (Figs 6-8, and S3 Fig). This is thought to be due to the fact that the dilution almost eliminates the kinetic isotope effect on chemical reaction rates (Fig 5). Heavy water, which originally contains ~0.028% of the hydrogen on earth [55], has no effect on life at all because it is present in such small concentrations. As in the case of heavy water, when tritiated water is diluted with H2O, its cytotoxicity is expected to disappear. In conclusion, heavy water exerts a greater toxicity on human cells than one might imagine, but only at high concentrations without dilution, due to kinetic isotope effects associated with quantum effects (S3 Fig).
Supporting information
S1 Table. List of antibodies used in this study.
Each antibody used in this study is listed along with its catalog number, specific target protein, antibody dilution ratio used for western blotting (WB) or immunofluorescence microscopic analysis (IF), and source.
https://doi.org/10.1371/journal.pwat.0000292.s001
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S1 Fig. Comparison of γH2AX positive cells induced by treatment of Hela-DR-GFP cells with D2O or γ-ray irradiation.
HeLa pDR-GFP cells were cultured in medium prepared with H2O or D2O. At the indicated time after exposure to irradiation or heavy water, the cells were subjected to immunofluorescent staining with an anti-γH2AX (red) antibody and DAPI (blue). The cells cultured in medium with H2O without irradiation were also subjected to immunofluorescent staining as a negative control.
https://doi.org/10.1371/journal.pwat.0000292.s002
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S2 Fig. Schematic representations of mechanisms of kinetic isotope effects.
(A) Differences in zero-point vibrational energies among hydrogen (H), deuterium (D), and tritium (T) to explain the kinetic isotope effects [27-29]. (B) Differences in probabilities of quantum tunneling through a potential barrier among H, D, and T to explain the kinetic isotope effects [27-29]. (C) Due to the kinetic isotope effects, the rates of chemical reactions involving H decrease when hydrogen is replaced by the heavier isotopes D or T.
https://doi.org/10.1371/journal.pwat.0000292.s003
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S3 Fig. Graphical summary of the results.
100% heavy water triggers an apoptotic response far higher than high-dose radiation. Heavy water toxicity to human cells disappears upon light water dilution. Heavy water toxicity may stem from the kinetic isotope effects inherent to hydrogen isotopes.
https://doi.org/10.1371/journal.pwat.0000292.s004
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S4 Fig. Possible mechanism of cell death by disruption of intracellular equilibrium via heavy water isotope effects.
(A) Intracellular histone acetylation levels are controlled by the balance between acetylation and deacetylation. Heavy water’s isotope effect slows deacetylation, without affecting acetylation. Consequently, histone acetylation increases, enhancing gene expression, including apoptosis-inducing genes. (B) If the intracellular equilibrium depends on both hydrolysis-dependent and -independent reactions, and only hydrolysis-dependent reactions are affected by heavy water’s isotope effects, then the heavy water may disrupt the equilibrium, altering product levels. This disruption could cause intracellular stress, leading to cell death.
https://doi.org/10.1371/journal.pwat.0000292.s005
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S1 Raw images. The original blot and gel images contained in the manuscript’s main figures.
The cropped areas used in the manuscript’s main figures are surrounded by red lines.
https://doi.org/10.1371/journal.pwat.0000292.s006
(PDF)
Acknowledgment
We thank Dr. M. Jasin (Memorial Sloan-Kettering Cancer Center, NYC, USA) for the DR-GFP assay materials.
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