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Heavy Water Affects Vital Parameters of Human Melanoma Cells in vitro

Purpose Although regular water is composed of two hydrogens and one oxygen, referred to as H2O, a small amount of water on this planet contains alternative forms of elements with different molecular weights because of the addition of neutrons. The present study was dedicated to studying the effect of heavy water (D2O), in which the two hydrogens become substituted by deuterium, on the cell physiology of different human cells with particular focus on malignant melanoma cells. Methods Cells were cultured in regular medium in which the content of H2O was gradually substituted by D2O or deuterium-depleted water (DDW). Following this, the changes of basic cellular parameters, such as morphology, migration, proliferation, cell cycle, apoptosis and microtubule integrity were examined. Results It was found that raising the D2O content above the standard levels led to a concentration-dependent decrease in proliferation. Lowering the D2O levels below this level had no effect. Likewise, elevated D2O levels hampered migration. Moreover, cell-cycle analysis showed an increase of sub-G1 cells. Corroboratively, markers for apoptosis were induced (histone-associated DNA fragments, Bax, and PARP). In regard to microtubule integrity, only very high levels of D2O (75%) caused partial filament condensation. Conclusion D2O, although chemically identical with H2O, shows proapoptotic and antiproliferative effects on melanoma cells. These findings give a closer look of this interesting compound.


Introduction
Although water is omnipresent in all physiological processes, it is mostly considered as an unavoidable medium for enzymatic reactions and not as a promising therapeutic target. In the following, we introduce an idea of how to interfere with cellular water and how to use this for therapeutic purposes.
Regular water is a molecule composed of two hydrogens and one oxygen referred to as H 2 O. In addition to this, a small amount is composed of stable isotopes, which are alternative forms of elements with different atomic weights because of the addition of neutrons. For hydrogen ( 1 H) which has a relative abundance of 99.985%, there exists the heavier isotope 2 H, known as deuterium, with an abundance of 0.015%. For regular oxygen ( 16 O) the relative abundance is 99.759%, the two stable isotopes, 17 O and 18 O, have an abundance of 0.037% and 0.204%, respectively. 1 In the following, we focus on the role of deuterium, which can substitute one or both hydrogens in the water molecule (HDO, D 2 O). The content of deuterium in surface water from different regions varies slightly from 0.0130% in arctic surface water to 0.0162% in the river Nile in Egypt. 2,3 Consequently, a small amount of deuterium-containing water is present in living matter. Although isotopes are chemically identical, they differ in their physical properties. Of note, it was observed that changes in the natural content of deuterization exert physiological responses. Some organisms such as some algae, bacteria, yeasts and molds can be propagated in media consisting of almost 100% D 2 O, however with lower growth rates and phenotypic alterations. In contrast, higher plants and animals do not survive a full exchange of hydrogen by deuterium. 4 Rats given a regular diet with 50% D 2 O in tap water showed severe generalized symptoms after 10 days. At this time the amount of D 2 O in the body water was 15%. At exchange rates higher than 30% rats become comatose. 5 Besides rats, a body water deuterization of 30-40% was shown to be lethal also in mice and dogs. 6 However, several studies revealed that a moderate body deuterization had little or no adverse effects and was well tolerated by mice and rats. 7 In humans, the amount of total body water can change inter-individually with age, body weight, obesity and sex. 8 As a rough estimate, an amount of 50 to 60% of the total body weight can be assumed. 9 Humans supplied with 140-250g D 2 O, corresponding to 0.5% of total body water, show only temporary symptoms such as vertigo, maybe due to perturbations in the vestibular fluid by heavy water. 6 In a more recent paper using D 2 O as a tracer to study muscle metabolism, humans were given boluses of D 2 O to achieve an amount of 0.4 to 0.8% within the body water. 10 No side effects were reported.
Because of its growth inhibitory effects, D 2 O seems to be an interesting compound in tumor therapy. In this paper, we focus on melanoma, which is still a therapeutically challenging condition despite the progress in treatment emerging in the recent years. In an in vitro attempt, we evaluated the susceptibility of melanoma cells on different amounts of D 2 O in the culture medium.

Ethics Statement
This study was conducted according to the Declaration of Helsinki Principles and in agreement with the Local Ethic Commission of the faculty of Medicine of the Johann Wolfgang Goethe University (Frankfurt am Main, Germany). The Local Ethic Commission waived the need for consent.

Cell Culture and Application of D 2 O
The malignant melanoma cell lines A375, SK-Mel-28 and SK-Mel-30 (all from ATCC) and normal human skin fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose, Gibco, Karlsruhe, Germany) with 5% fetal calf serum (FCS, Gibco, Karlsruhe Germany) and 1% penicillin-streptomycin solution (Biochrom KG, Berlin, Germany) and 1% Asp (L-ascorbic acid 2-phosphate, Sigma-Aldrich, Taufkirchen, Germany). Normal human melanocytes isolated from foreskins were propagated in DermaLife M medium (CellSystem, Troisdorf, Germany). All cells were held at 37°C in a 5% CO 2 atmosphere. The medium was renewed twice a week. In order to study the effect of heavy water, powdered DMEM medium was reconstituted using deuterium dioxide (D 2 O, 99.9%) or deuterium-depleted water (DDW, D 2 O ≤ 1ppm), both from Sigma-Aldrich (Taufkirchen, Germany), to the indicated concentrations.

Cell Proliferation Assay
Cells were seeded in 96-microwell plates at a density of 1.0×10 4 cells/per well. After 24 h the media were exchanged to the indicated D 2 O concentrations. For the last 16 hrs, cells were pulsed with 5-bromo-2ʹ-deoxyuridine (BrdU). Subsequently, the incorporation rate of BrdU was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Roche, Mannheim, Germany). Briefly, cells were fixed and immune complexes were formed using peroxidase-coupled BrdU-antibodies. A colorimetric reaction with tetramethylbenzidine (TMB) as a substrate gave rise to a reaction product measured at 450 nm in a scanning multiwell spectrophotometer (ELISA-Reader ASYS Expert 96, Deelux Labortechnik, Gödenstorf, Germany).

Immunocytochemistry
Coverslips were placed into 12-well plates and cells were seeded at a density of 6×10 4 cells per well in 1.5mL DMEM high glucose for 24 hrs at 37°C and 5% CO 2 . Cells were treated with different D 2 O concentrations for 24 hrs at 37°C and 5% CO 2. For displaying microtubules, cells were fixed with 4% paraformaldehyde (PFA) for 10 mins at room temperature. After removal of PFA, cells were washed 3 times with cold PBS for 5 mins each and then permeabilized with 0.1% Triton-X-100 in PBS (10 mins, RT). Unspecific antibody-binding sites were blocked with 1% BSA in 0.1% Tween 20 in PBS for 30 mins at RT. Microtubule antibody anti-human alpha-tubulin (DM1A, Cell Signaling, Frankfurt, Germany) was diluted 1:1000 in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA) and applied to the cells over night at 4°C. After three washes with PBS, cells were incubated with alexa 546-coupled goat anti-mouse (life technologies, Darmstadt Germany, 1:100 in PBS) for 60 mins at RT. Nuclei were stained with DAPI for 1 min. Indirect immunofluorescence was examined with the Axioskop (Zeiss, Jena, Germany).

Morphologic Analysis and Migration Assay
Cells were seeded at a cell density of 3×10 5 cells/mL in 12-well plates. Consecutively, the medium was replaced with the indicated D 2 O concentrations. Live cell microscopy of the scratch closure was performed every 2 hrs for up to 24 hrs with an incubator microscope unit (IncuCyte ZOOM, EssenBioScience, Hertfordshire, UK). In order to study the migration of cells standardized scratches were applied to confluent cultures by using pipettor tips. The closure of the cell-free space was analyzed using the ImageJ software. 11

Histone-Associated DNA Fragments
Apoptosis was quantified on the basis of cytoplasmic histone-associated DNA fragments using the Cell Death Detection ELISA (Roche, Mannheim, Germany) according to the manufacturer's manual. In brief, cells were cultured in microwell plates (2×10 4 cells per 0.33cm 2 ) and treated with the indicated D 2 O concentrations. After 24 h cytosolic fraction (200 g supernatant) was used as antigen source in a sandwich enzyme-linked immunosorbent assay with primary anti-histone antibody coated to a microtiter plate and secondary anti-DNA antibody coupled to peroxidase. Optical density was measured at 530nm in an ELISA reader (ELISA-Reader ASYS Expert 96, Deelux Labortechnik, Gödenstorf, Germany).

Western Blot Analysis
Protein extracts of cells treated with D 2 O for 48 hrs were separated by SDS-polyacrylamide gels and electroblotted as described. 12 Membranes were incubated with the following primary antibodies: Bcl-2, Bcl-XL, Bax, AIF, cleaved PARP, surviving (all purchased from Cell Signaling, Frankfurt, Germany) and actin (Heidelberg, Santa Cruz). Primary antibody application was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit IgG, Amersham, Uppsala, Sweden). Blots were developed using an enhanced chemiluminescence detection system (ECL) (Amersham) according to the manufacturer's instructions.
Annexin-V-Fluorescein Isothiocyanate/ Propidium Iodide (AV-FITC/PI) Double-Staining Assay 9×10 4 cells per were seeded in a 12-well plate for 24 hrs at 37°C and 5% CO 2 and treated with D 2 O as indicated for 24 hrs. Early apoptosis and late apoptosis were detected by using TACS Annexin V-FITC Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer´s protocol. Briefly, all cells were collected by centrifugation at 300g for 5 mins at RT and pellet was washed once with 500µL cold PBS and the centrifugation step was repeated. 100µL Annexin V/propidium iodide incubation reagent was applied to the cell pellet for 15 mins in the dark at RT and 400µL 1× binding buffer was applied. Analysis of Annexin V/propidium iodide was assessed by a BD FACScan Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) within 1 hr.

Fluorescence-Activated Cell Sorting Analysis
Cells were incubated with D 2 O as indicated for 24 hrs. Nocodazole (1µg/mL, applied for 2 hrs) was used as positive control to induce G2/M arrest. After fixation in ice-cold 70% ethanol cells were incubated in PBS containing 40g/mL RNase A for 30 mins at 37°C and resuspended in PBS containing 50g/mL propidium iodide. Analysis of cell cycle was assessed by a FACScan Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as described. 13

Statistical Analysis
All data are presented as mean values ± standard deviation. Statistical significance of the data was calculated by Mann-Whitney-Rank Sum test (BIAS, Frankfurt, Germany). Each set of data was related to the referring control or as indicated in the legends.

Results
In order to study the effect of D 2 O on melanoma cells, a set of culture media were tested which differ in their D 2 O content. Medium with a content of 0.014% D 2 O represents control condition.
In Figure 1  Consecutively, the effect of D 2 O on DNA synthesis was tested in different melanoma cells (Figure 2). Lowering the amount of D 2 O shows no significant effect on DNA synthesis as measured by incorporation of 5-bromo-2´-deoxyuridine (BrdU) in the DNA in A375 cells (Figure 2A), SK-Mel-28 ( Figure 2B) and SK-Mel-30 ( Figure 2C). In contrast, increased levels of D2O above the normal range offer inhibition of DNA synthesis. In A375 cells already 5% D 2 O showed a significant decrease in DNA synthesis. Cultivation in 100% D 2 O decreased the level to 18% (EC50: 41.3% D 2 O, calculated according to 14 ). In SK-Mel -28 75% D 2 O showed a significant inhibition; at 100% D 2 O the DNA-synthesis was inhibited to 74% (EC50: 44.6% D 2 O). In SK-Mel-30 cells a slight but significant reduction was present at 75% D 2 O; at 100% D 2 O DNA-synthesis was reduced to 25% (EC50: 79.9% D 2 O). In order to capture D 2 O effects on non-malignant cells normal human melanocytes and fibroblasts were tested ( Figure S1). It was found that D 2 O also offers anti-proliferative effects in fibroblasts ( Figure S1A; EC50: 45.6% D 2 O). Interestingly, in melanocytes D 2 O shows no anti-proliferative effect ( Figure S1B; EC50: 171.5% D 2 O).
From the above-described experiments, it became evident that D 2 O inhibits DNA-synthesis and also leads to dramatic morphological changes. These findings indicate that D 2 O triggers processes in the context of cell death. Therefore, A375 melanoma cells, which show the clearest effect in response to D 2 O, were tested on apoptosis ( Figure 3). It was found that D 2 O containing medium induced cytoplasmic histone-associated DNA fragments ( Figure 3A) which indicates advanced apoptosis. 15 In 40% D 2 O the amount of cytoplasmic histone-associated DNA fragments was increased to 344%, in 50% D 2 O to 355% and in 100%    ( Figure 3D). In order to learn more about the molecular processes involved in D 2 O-stimulated apoptosis Western blot analysis was performed using a panel of pro-and antiapoptotic markers ( Figure 3E). Compared to control conditions with 0.014% D 2 O cells cultured with 40 and 50% D 2 O feature higher levels anti-apoptotic Bcl-2 and Bcl-XL but also lower levels of survivin. Moreover, pro-apoptotic Bax and cleaved PARP were elevated. The level of caspaseindependent AIF was unchanged. In sum, these results speak for a complex regulation of pro-apoptotic and also counterregulating effectors. After giving proof for the induction of apoptosis it was investigated whether increased levels of D 2 O have also impact on the cell cycle ( Figure 4). Analysis of cell cycle distribution was investigated by FACS using propidium iodide staining to determine the cellular DNA content ( Figure 4A-D). Summarized results given in Figure 4E show no significant alterations in the amount of cells in the S-and G2/M-phase by D 2 O (40% and 50%) compared to control conditions (0.014% D 2 O). However, the amount of G1/G0 cells decreased with a concomitant increase of sub-G1 cells. The latter finding is in concert with the above described pro-apoptotic action of D 2 O.
In order to study a possible effect of D 2 O on tumor dissemination, a migration assay was performed ( Figure 5). After application of standardized scratches closure of the cell-free space was monitored by live cell microscopy for 24 hrs ( Figure 5A). Quantitative measurements of gap closure demonstrated an inhibition to circa 20% by D 2 O (40% and 50%) compared to control conditions (0.014% D 2 O) ( Figure 5B).
Several studies describe an effect of D 2 O on microtubule stability in cell-free systems. Therefore, the effect of D 2 O on the microtubule filament was investigated in in vitro cultured melanoma cells. Indirect immunofluorescence against alpha-tubulin displayed a finely woven maze under control conditions (0.014% D 2 O) ( Figure 6A). Treatment with 50% D 2 O for 24 hrs had no visible impact on the microtubule organization ( Figure 6B). An increase to 75% D 2 O lead to a partially blurred picture indicating microtubule disintegration with small anti-tubulin reactive aggregates ( Figure 6C).

Discussion
In the present study, we investigated the effect of D 2 O on basal physiological parameters in melanoma cells. Increasing the D 2 O concentration above the normal range causes a massive decrease of cells at 50% D 2 O already after 24 hrs as detected by live microscopy; a total replacement of regular H 2 O by D 2 O leads to complete cell disruption. The growth inhibitory effect of D 2 O was further substantiated by showing a concentration-dependent decrease of DNA synthesis. This suits well to the anti-tumor effect of D 2 O which was detected in a colon and SCC xenograft tumor model in mice. Here the drinking water was replaced by 30% D 2 O, which reduced the tumor growth in a range from 22% to 65% compared to the controls after approximately 30 days. The D 2 O concentration in the serum was 16%. 16 Also, a protective effect of D 2 O on tumor growth was described. Mice which were supplied with 30% D 2 O drinking water for 7 days and then inoculated with pancreatic cancer cells developed smaller tumors. 17 However, this effect was only transient.
Interestingly, also a decrease of the D 2 O concentration below the normal range using DDW was shown to have anti-tumor effects. [18][19][20] It is thought that DDW diminishes the amount of deuterium of sugar-phosphates in the DNA backbone stabilizing the hydrogen bond network and therefore protecting against aneuploidy and strand breaks. 21 In our model using melanoma cells, we found no effects of DDW in regard to cell morphology and DNA synthesis. Therefore, we focused on the following only on effects caused by elevated D 2 O levels. In order to characterize growth inhibition by D 2 O cell cycle distribution and apoptosis were analyzed. In the former case, no specific cell cycle arrest was found. Others found cell accumulation in the G2/M phase in a model using other malignant cells 22,23 speaking for a cell-specific mode of action. Confirmatory, an upregulation of apoptosis was found in some cell lines and in others not. 23 However, in our model using A375 cells a significant increase of cytoplasmic histone-associated DNA fragments, which is typical for advanced apoptosis, was found. Also, Annexin V, an early apoptosis marker, was increased by D 2 O. However, besides the upregulation of pro-apoptotic markers (Bax, cleaved PARP) and the downregulation of an anti-apoptotic marker (Survivin) also some anti-apoptotic markers became slightly upregulated (Bcl-2, Bcl-XL) which speaks for a complex regulation of pro-and anti-apoptotic cascades which in sum shifted the equilibrium towards apoptosis.
In order to specify the anti-tumor effect of D 2 O cell migration, which plays an important role in tumor dissemination, was regarded. Utilizing a scratching assay shows a significantly reduced cell migration. This finding is in concert with others who found a suppression of cell locomotion by D 2 O in mouse fibroblasts. 24 Cell shape and cell migration are controlled by filaments of the cytoskeleton.
Besides the microfilament and the intermediate filament also microtubules contribute to polarized cell migration. 25 Microtubules are of particular interest in this context as it was found that D 2 O enhances tubulin polymerization in a cell-free system. 26 In cultured cells, it was found that D 2 O promotes the assembly of microtubules from tubulin and stabilized them. 27 Moreover, also stabilization of the spindle apparatus was found in the eggs of the sea urchin resulting in a mitosis halt. 27,28 In general, D 2 O seems to stabilize microtubule and make them more resistant towards exogenous noxa reversing effects of compounds interacting with microtubule stability (demecolcine, depolymerization; taxol, microtubule stabilization). 29,30 The sensitivity of microtubules against D 2 O might explain the overall resistance of prokaryotes. Our results with melanoma cells also display a well-ordered microtubule network at 50% D 2 O, which may be more rigid than under control conditions explaining the decreased migratory activity. However, a further increase in the D 2 O concentration up to 75% caused a partial disruption of the fine woven microtubule filament. The manifestation of anti-tubulin reactive aggregates speaks for a general toxic effect.
In sum, our results demonstrate that a D 2 O, a chemically identical isotope of H 2 O exerts massive effects on pivotal cell parameters such as growth and apoptosis. However, the sensitivity towards D 2 O differed markedly between the different melanoma cells tested. Surprisingly, D 2 O shows, in contrast to the melanoma cells tested, no anti-proliferative effect in normal human melanocytes. If D 2 O is in general more effective in malignant cells should be investigated in future studies. However, experiments with fibroblasts speak against a tumorspecific action of D 2 O. Hence, possible adverse health effects have to be carefully evaluated before considering a clinical application. It is an interesting issue to identify cellular determinants that correspond to the sensitivity against D 2 O. First indications suggest that Aquaporin-11 is relevant for the transmission of the effect. 31 In sum, in tumors with a high sensitivity towards D 2 O an exchange of normal water by D 2 O may be therapeutic option particularly in combination with other measures.

Conclusion
Commonly most active substances are developed on the basis of chemical structures. Here we show that a compound, chemically identical with regular water, but different on the physical level, has an impact on melanoma cell physiology. These findings suggest a closer look at stable isotypes. The replacement of regular atoms in a lead compound by one or more stable isotypes may lead to new and more effective molecules. Although the mechanism of the D 2 O effect is not clear, it could be speculated that the deuterium ion interferes with the Akt hydrogen network. 32 B A C Figure 6 Indirect immunofluorescence against microtubules in D 2 O-treated A375 melanoma cells. Cells were held (A) under control conditions (0.014% D 2 O) or treated with (B) 50% or (C) 75% D 2 O for 24 hrs. After fixation, cells were incubated with an anti-human alpha-tubulin antibody followed by incubation with an alexa 546-coupled goat anti-mouse antibody (for details please see "Materials and Methods"). The images show representative sections. Scale bar = 20μM.