Hydrogen has been shown in several clinical trials to be completely safe without adverse events and there are no warnings in the literature of its toxicity or adverse effects during long-term exposure. Molecular hydrogen has proven useful and convenient as a novel antioxidant and modifier of gene expression in many conditions where oxidative stress and changes in gene expression result in cellular damage. Our intracellular biomarker studies have shown that a hydrogenized water drink formula containing 2.6 ppm dissolved hydrogen was able to penetrate cellular membranes and function as an antioxidant in human liver cells (HePG2) utilizing the Cellular Antioxidant Assay (CAA). This assay uses the protection of a fluorescent probe as a marker for cellular damage by reactive oxygen species (ROS), such as peroxyl radical, and compares this to the known antioxidant standard, Quercetin. Using this system oxidative damage was reduced in a dose-dependent manner. One ml of hydrogenized water was found to possess antioxidant capacity equivalent to 0.05 μmole of quercetin. When examined in a human colon cell line (Caco-2 cells), hydrogenized water demonstrated a dose- and time-dependent permeability inhibition of an intracellular fluorescent glucose derivative (2-NBDG), indicating decreased glucose uptake. In another study, the impact of hydrogenized water on Akt phosphorylation (Ser473), a biomarker for insulin signaling, was monitored in human skeletal muscle cells. The hydrogenized water treatment markedly elevated the level of phosphorylation of Akt (Ser473) in a dose-dependent manner. The anti-aging effects of hydrogenized water were examined utilizing SIRT1 expression as a biomarker of aging in human umbilical cells (HUVECs). Hydrogenized water increased dose-dependent SIRT1 gene expression. Hydrogenized water also increased telomerase activity (an anti-aging biomarker in HUVEC cells) up to 148% when cells were treated with media containing 25% hydrogenized water formula. Increased telomerase activity caused by hydrogenized water may be able to protect telomeres from degradation, suggesting the possible use of hydrogenized water in therapeutic interventions for age-related diseases. These studies show that commercial hydrogenized water improved the levels or activities of a few intracellular biomarkers specific for antioxidant activity, glucose uptake, insulin signaling and SIRT 1 and telomerase activities. Industrial Relevance: The molecular hydrogen used in this study indicates that certain commercial sources of hydrogenized water can provide similar antioxidant and gene expression modifications seen in other sources of molecular hydrogen. The biomarkers evaluated here lend well to hydrogenized water’s biological activity relating to health conditions and aging.
Hydrogen is the smallest and most abundant element, and virtually non-toxic. For some time H2 was not considered a bioactive molecule, but a recent study found that inhaled hydrogen gas protects the brain against ischemia-reperfusion injury and stroke, and this was likely due to an antioxidant mechanism that reduces free radical damage .
Hydrogen can be delivered by gas inhalation, injection of hydrogen-rich saline solutions or by drinking hydrogen-rich water (hydrogenized water). Using these methods of administration H2 has been shown to have various preventive and therapeutic effects useful in emergency and critical care medicine . In animal model studies hydrogen gas has been shown to ameliorate intracerebral hemorrhage  and reduce hyperoxic lung injury in vivo . Hydrogen gas has also been useful in diagnostic medicine. For example, a hydrogen breath test has been used in clinical practice to detect abnormal bacterial overgrowth in the small intestine or to diagnose the malabsorption of lactose or fructose .
It was reported that molecular hydrogen could improve the damage caused by cerebral ischemia-reperfusion injury and selectively reduce strong cytotoxic oxygen radicals, including hydroxyl radical (•OH) and peroxynitrite (ONOO−) [2,5]. Molecular hydrogen has also proven to be useful in tissue injury and aging [1,2,5,7,8,9,10,11]. In this process, free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated as by-products of oxidative metabolism. Since H2 can rapidly diffuse across cell membranes, it is an effective ·OH radical scavenger in cultured cells. ROS/RNS can induce cumulative oxidative damage to cellular macromolecules, eventually resulting in cellular dysfunction, cell death and in some cases, leading to or assisting in the development of various diseases.
It has also been demonstrated that H2 rapidly diffuses across cell membranes and is an effective ·OH radical scavenger in cultured cells and this has been related to its ability to rapidly diffuse across cell membranes. Since then, numerous studies have focused on understanding the potential therapeutic value of hydrogen and hydrogen-rich water in various disease models [12,13].
A comprehensive review the literature on the effects of molecular hydrogen (H2) on animals and human subjects and patients with a variety of diagnoses, such as metabolic, rheumatic, cardiovascular, neurodegenerative disease, infections, radiation damage, exercise as well as effects on aging has recently been published. .
Here we investigated the effects of hydrogenized water on several intracellular biomarkers related to human health, metabolism and longevity, such as those specific for antioxidant activity weight management/diabetic functional improvement insulin receptor signaling and anti-aging indicators [17,18].
2. Materials and Methods
Hydrogenized water identified as “IZUMIO Water” was acquired from Naturally Plus Co. Ltd. Roppongi, Minato-ku, Tokyo, Japan. At the time of initial packaging, the infused hydrogen within the test water was analyzed by both manometric and diaphragm polarographic electrode measurements at final concentration of 2.6 ppm and pH 7.0. The human Caco-2 cell line and mouse C2C12 myoblast cell line were obtained from American Type Culture Collection (ATCC, Manassas, USA). HG- DMEM (Dulbecco’s modified Eagle’s medium with high glucose) was from GIBCOTM (Grand Island, NY, USA). Fetal bovine serum (FBS) and horse serum were purchased from Hyclone (Pittsburg, PA, USA). Insulin, fatty acid-free bovine serum albumin (BSA) was obtained from (Sigma, St. Louis, MO, USA). The sources of other materials are indicated in individual method sections.
2.1. Cellular Antioxidant Assay
The Cellular Antioxidant Analysis (CAA) analyzes the capacity of hydrogenized water to protect a fluorescent probe (as a marker) from damage by reactive oxygen species (ROS) in intracellular environments. The assay was carried out as previously described . First, the probe was incubated with representative human liver cells (HePG2 cells obtained from ATCC, Manassas, VA, USA). Once inside cells the loss of probe fluorescence functions as an indication of the extent of damage the probe experiences from the oxygen radicals. Various concentrations of hydrogenized water were incubated with the cells to allow its natural absorption. Then an oxygen radical inducer, (AAPH) ,( was introduced into cellular environment, which triggers the release of peroxyl radicals.
Without excess antioxidants present outside or inside the cells, oxygen radicals permeate through cellular membranes and oxidize the marker probe, and when in excess the oxygen radicals damage the cells. In contrast, when excess antioxidant material is present inside cells, damage does not occur. The cellular antioxidant effect of the hydrogenized water can be measured by assessing the preservation effect of hydrogenized water absorbed inside of cells. quercetin, a known bio-effective antioxidant, is used as the positive control. The cellular antioxidant effect of the test material is compared with that of quercetin, and the comparison result is then expressed as μmole quercetin equivalency (QE) per gram (or milliliter) of a tested material.
2.2. Glucose uptake in Caco-2 human Cells
This study investigates the effect of hydrogenized water on the permeability of glucose across a Caco-2 cell monolayer. In this study, Caco-2 human cells were grown at 37°C in 5% CO2 and at 95% relative humidity using Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 0.05% penicillin/streptomycin/amphotericin. Cells were removed at 80–90% confluence using a 0.25% trypsin/0.20% ethylene diamine teraacetic acid (EDTA) solution and replated. Media were changed approximately every 48 h. For the transport experiments Caco-2 cells (passages 26–40) were seeded at 6.5×104 cells/cm2 into polycarbonate 12-well Transwell® plates (Corning Costar Corporation, Cambridge, MA, USA) (0.4μm mean pore size) and used 21–28 days after seeding. 2-NBDG (2-(N- (7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose), a fluorescently-labeled deoxy glucose analog, was used as a probe for the detection of glucose movement across Caco-2 cell monolayers. Six concentrations of hydrogenized water (0, 3.125, 6.25, 12.5, 25 or 50% v/v hydrogenized water in ionic and osmotic balanced DMEM), along with 200 μg/ml 2-NBDG were added to the apical side of confluent monolayers of Caco-2 cells, and the monolayer cultures were then incubated for 30 min and 1 hr., respectively. At each time point, the receiver (basolateral) sides of the cell media were removed for analysis. The degree of glucose permeation across the cell monolayer was monitored using changes in the fluorescent intensity of 2-NBDG . Statistical assessments were performed by t-test analyses.
2.3. Cellular Insulin Receptor Signaling Assessment Using Akt phosphorylation
The effects of hydrogen water on insulin signaling in human skeletal muscle cells was studied using Akt phosphorylation as the cellular biomarker. C2C12 murine myoblasts (ATCC, , CRL-1772) were cultivated in DMEM (Lonza, ) containing 4.5 g/L glucose, 2 mM glutamine and 10% FCS (Thermo Scientific). Cells were plated and grown to approximately 80–90% confluence. The cells were serum starved (DMEM 0.5% FCS) overnight, then treated with hydrogenized water at 0, 3.125, 6.25, 12.5, 25 or 50% v/v hydrogenized water in ionic and osmotic balanced DMEM for 60 min. Cells were also treated with concentrations of 30 nM insulin (Sigma, Aldrich, St. Louis, MO, USA) for 15 min as a control. After the hydrogenized water treatment, cells were then washed in PBS and lysed in lysis buffer (Thermo Fisher Scientific, Fremont, CA, USA). After protein measurement, SDS loading buffer (3% (w/v) SDS, 3% (v/v) β-mercaptoethanol, 10 mM EDTA (pH 8), 20% (v/v) glycerol, and 0.05% (w/v) bromphenol blue) was added, and the samples were boiled for 5 min at 95 °C. Proteins were separated by SDS- polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose filter. The membranes were blocked for minimum of 1 h in Net-G [150 mM NaCl, 5 mM EDTA (pH 8), 50 mM Tris–HCl (pH 7.5), 0.05% (v/v) Triton X-100, 0.25% (w/v) gelatin] and incubated with primary antibody overnight at 4 °C. Proteins were detected by horseradish peroxidase-conjugated secondary antibody using an ECL reagent as described by the manufacturer (Thermo Scientific, Fremont, CA, USA).
2.4. Cellular Aging Assessment
The impact of hydrogenized water on the cellular aging process was carried out via cellular assessment of two aging biomarkers: SIRT1 and telomerase activity, respectively.
2.4.1. SIRT1 as Biomarker for Aging
Cellular aging via SIRT1 assessment measures the ability to prevent or augment cellular SIRT1 production. In this assay, human umbilical vein endothelial cells (HUVECs) were treated with 0, 3.125, 6.25, 12.5, 25 or 50% v/v hydrogenized water in the buffer and in duplicate and incubated continued for up to 24 hr. One group of cells was treated with 100 μM of Resveratrol (Sigma Aldrich, St. Louis, MO, USA) for 24 hr as the control. Subsequently, the cells were lysed, and SIRT1 expression levels were analyzed via Western blot.
2.4.2. Telomerase Activity as an Aging Biomarker
Cellular aging via telomerase activity assessment investigates the effect of hydrogenized water on aging via changes in telomerase activities in human umbilical vein endothelial cells (HUVECs). Here, HUVECs were treated with 0, 3.125, 6.25, 12.5, 25 or 50% v/v hydrogenized water in the buffer and in duplicate for 24 hr. One cell sample was treated with 10 nM raloxifene (Sigma, Aldrich, St. Louis, MO, USA) for 24 hr as a positive control. Telomerase activity was determined using the TRAPeze® XL Telomerase Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.
3. Results and Discussion
3.1. Cellular Antioxidant Assay
CAA analyzes the capacity of a material to protect a fluorescent probe (the biomarker) from damage by reactive oxygen species (ROS) in the intracellular environment. In this assay, peroxyl is used as the free radical (ROS), and human liver cells are used as the cellular model . Quercetin is used as the standard, and the results are expressed as μmole quercetin equivalency (QE) per gram (or milliliter) of the tested material. Our analysis determined that the CAA value of the hydrogenized water was 0.05 μmole QE per mL, which indicates that one ml of hydrogenized water possesses antioxidant capacity equivalent to 0.05 μmole of quercetin. This analysis indicates that commercial hydrogenized water can permeate cellular membranes and function as an in vivo antioxidant in human cells. An example of CAA value of a fruit rich in antioxidants, e.g., cranberries, has CAA value of ~ 12.5 μmole QE per gram of fresh fruit . Compared with cranberries, the CAA value of hydrogenized water is relatively low. However, the usual daily intake of water is much higher than fruit alone. Another point that must be taken into consideration is that the active evaporation of hydrogen from hydrogenized water can significantly impact the actual amount of hydrogen that can permeate into the cells and function as a cellular antioxidant. In human bodies, such hydrogen evaporation may be significantly reduced or the effect of evaporation may be different.
3.2. Impact of Hydrogenized Water on Glucose Uptake Using the Caco-2 Membrane Model
Differentiated polarized human Caco-2 colon epithelial cells express a wide range of transporter proteins on their cell membranes that mimic human intestinal epithelia . The Caco-2 absorption study is considered to be the reference standard in pharmaceutical and nutraceutical industries for in vitro prediction of in vivo human intestinal absorption and bioavailability of orally administered substances. In the drug discovery field, the Caco-2 cell permeability assay is also used to predict drug-drug interactions (DDIs) of orally administered drugs…