Female C57BL/6CrSlc mice (7 weeks old, 15–20 g) for the nephrotoxicity studies, male ddY mice (4 weeks old, 18–20 g) for the tumor studies, and male SD rats (7 weeks old, 210–230 g) for the measurement of hydrogen concentration in blood were purchased from Nippon SLC (Hamamatsu, Shizuoka, Japan). Mice were fed ad libitum and housed in a temperature-controlled room (22–24°C) under a 12-h light/dark cycle. The care and treatment of experimental animals were in accordance with institutional guidelines. This study was approved by the Animal Care and Use Committee of Nippon Medical School.

S-180 sarcoma (CFW sarcoma 180, mouse) and L-1210 (lymphocytic leukemia, mouse) cell lines were obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). S-180 cells were maintained in MEME medium supplemented with 10% fetal calf serum, 1% NEAA and penicillin/streptomycin. L-1210 cells were maintained in RPMI1640 medium supplemented with 10% fetal calf serum and penicillin/streptomycin.

Cisplatin (25 mg/50 mL) was purchased from Yakult Honsha Co., Ltd. (Tokyo, Japan). All other chemicals and reagents were of analytical grade.

C57BL/6 mice were divided randomly into five groups. Group I (CTL) received physiological saline (0.9% NaCl) by intraperitoneal injection. Groups II–V received a single dose of CDDP (17 mg/kg) by intraperitoneal injection. Groups II [HG (+)] and III [HG (−)] inhaled air with or without hydrogen, respectively. Groups IV [HW (+)] and V [HW (−)] were allowed to freely drink water with or without hydrogen, respectively. Lee et al. [16] described renal injury was clearly seen with a dose of 20 mg/kg cisplatin at 72 h after the cisplatin treatment in C57BL/6 mice. However the lethality caused by a dose of 20 mg/kg cisplatin reached 67% in our preliminary experiment (n = 10; data not shown). To obtain almost 50% lethal dose of cisplatin, we used a dose of 17 mg/kg cisplatin in this experiment,

Mice were housed in a standard cage with food and water available ad libitum and the cage was placed into a semi-closed box (55 × 35 × 30 cm; length × width × height), into which 1% H₂ in air was introduced at a rate of 10 L/min throughout the experiments. The box was placed in a temperature-controlled room (22–24°C) under a 12-h light/dark cycle. In the control group, air was administered at the same rate for the same time period. During each experiment, the concentration of hydrogen in the box was monitored using a gas analyzer (TGA-2000, Teramecs Co., Kyoto, Japan).

Molecular hydrogen (H₂) was dissolved in water under high pressure (0.4 MPa) to a supersaturated level using hydrogen water-producing apparatus (ver. 2) produced by Blue Mercury Inc. (Tokyo, Japan). The saturated hydrogen water was stored in an aluminum bag. Hydrogen water was freshly prepared every week, which ensured that a concentration of more than 0.6 mM was maintained. We confirmed the hydrogen content with a hydrogen electrode (ABLE). Each day, hydrogen water from the aluminum bag was placed into a closed glass vessel (70 mL) equipped with an outlet line containing two ball bearings, which kept the water from being degassed. This vessel ensured that the hydrogen concentration was more than 0.4 mM after 1 day. Hydrogen water degassed by gentle stirring was used for control animals; the complete removal of hydrogen gas was confirmed with a hydrogen electrode.

Three days after cisplatin injection, animals were killed under anesthesia, blood was collected from the heart, and the kidneys were obtained. The left kidney was used for measurement of the level of malondialdehyde (MDA) and the right kidney was used for H&E and TUNEL staining. Serum levels of creatinine and BUN were measured using a Creatinine Testwako kit and a Urea N B Testwako kit (Wako Pure Chemical Industries Ltd., Osaka, Japan), respectively. MDA levels in the kidney were determined using a BIOXYTHCH MDA-586 Assay kit (OxisResearch, Oregon, USA) as described previously [17].

Rat received hydrogen water orally by stomach gavage at 15 mL/kg. Three minutes after administration, the rat was killed under anesthesia and blood was collected from the heart. Hydrogen concentration in blood was measured as described previously [14]. In brief, 5 mL of blood was kept in a closed aluminum bag with 25 mL air to transfer the hydrogen from blood to the air. The amount of hydrogen in the air was measured by gas chromatography.

The kidney was fixed with 4% paraformaldehyde in PBS. The tissues were dehydrated, embedded in paraffin, sectioned at 5-μm thickness, and stained by hematoxylin and eosin (H&E) for histopathological analysis. The degree of injury was scored according to the following scale: 0 no pathological findings, 1 mild, 2 moderate, 3 severe. Apoptosis was detected by DNA strand breaks using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) according to the procedure of the manufacturer (Chemicon International).

S-180 (1 × 10⁴ mL⁻¹) or L-1210 (5 × 10⁴ mL⁻¹) cells were seeded in 24-well plates. The cells were treated with various concentrations of cisplatin or PBS and cultured in medium with or without 0.6 mM hydrogen. After 72-h incubation, dead cells were assessed with 0.2% trypan blue staining [18] and scored viable cells. Under serum-free conditions, S-180 cells (2 × 10⁴ mL⁻¹) were seeded in 24-well plates and trypan blue assay was performed after 120-h incubation with cisplatin. We repeated independent experiments using 3 wells for each concentration.

Cell culture in medium with or without hydrogen was performed as described previously [14]. In brief, we dissolved hydrogen into medium by bubbling hydrogen gas (75% H₂, 20% O₂ and 5% CO₂). We used medium bubbled with control gas (75% N₂, 20% O₂ and 5% CO₂) as a control. The cells were maintained at 37°C in a humidified box filled with gas with or without hydrogen gas.

S-180 cells (3 × 10⁶ cells/mouse) were subcutaneously inoculated into the back of ddY mice. One week later, the tumors had grown to 70–130 mm³, and the mice were randomly divided into three groups. The first group received physiological saline and the second and third groups received three consecutive daily injections of cisplatin (5 mg/kg). The second and third groups were given water with or without hydrogen throughout the experiment, as described above. Tumor volume was measured with LaTheta LCT-100, X-ray CT for experimental animals (Aloka Co., Ltd., Tokyo, Japan) after the administration of Omnipaque 300, a contrast medium (Daiichi Sankyo Co., Ltd., Tokyo, Japan).

We performed statistical analysis using StatView software (SAS Institute) by applying an unpaired two-tailed Student’s t test and ANOVA followed by Fisher’s exact test as described previously [14].

To investigate the effect of hydrogen gas on cisplatin-induced toxicity, mice were intraperitoneally injected with a single dose of cisplatin (17 mg/kg) and housed in a box filled with 1% H₂ in air, as described in “Materials and methods”. We monitored their survival rate daily (Fig. 1a). In the control air group, mice started to die on Day 2 and only 60% of mice survived to Day 6. In contrast, all mice survived to Day 5 and 80% of mice survived to Day 9 in the hydrogen gas group. No mice died after Day 9 in all groups. Body-weight loss in the control group on Day 3 was 9.7%, whereas inhalation of hydrogen gas significantly suppressed body-weight loss to only 3.5% on Day 3 (Fig. 1b).

Fig. 1 Hydrogen gas (HG) reduces mortality, body-weight loss and nephrotoxicity induced by cisplatin (CDDP). Mice were injected intraperitoneally with a single dose of cisplatin (17 mg/kg) (Day 0). Hydrogen gas was administered by inhalation (1% H₂ in air) throughout the experiments (from Day 2 to Day 10). HG (+) and HG (−) were mice that inhaled air with or without hydrogen, respectively. a Survival rate was monitored daily (n = 10). b Body weight of each mouse was measured on Day 3 (n = 12). c Serum creatinine and d BUN levels were measured on Day 3 (n = 5). Data are the means ± SEM. Difference in body-weight loss was significant (*P < 0.05) by Student’s t test. Differences in creatinine and BUN levels were significant (*P < 0.05) by one-way ANOVA

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Next we measured the levels of serum creatinine and blood urea nitrogen (BUN) to assess the functional effect of hydrogen on cisplatin-induced renal dysfunction (Fig. 1c, d). Cisplatin increased the levels of serum creatinine and BUN by two- and fourfold, respectively, at 72 h after administration with cisplatin as compared with the non-treatment group. Inhalation of hydrogen gas decreased the levels of serum creatinine (9.6 ± 1.5 (SEM) vs. 5.7 ± 1.0 (SEM) mg/L) and BUN (863 ± 170 (SEM) vs. 477 ± 135 (SEM) mg/L) as compared with the control group with cisplatin and without hydrogen.

Hydrogen gas may be inconvenient for daily intake; thus, we examined whether hydrogen can be administered as hydrogen water (water containing hydrogen) instead of hydrogen gas. Molecular hydrogen is dissolved in water at the saturated level of 0.8 mM [14]. Blood of several milliliters is necessary to measure the hydrogen concentrations in blood. Because it is difficult to obtain a sufficient volume of blood from mice, we used rats for the measurement of hydrogen concentration in the blood. We placed hydrogen water at 3.5 mL/230 g (15 mL/kg) in the stomach of a rat via a catheter in the fed and fasted state, and measured the concentration of hydrogen in blood after 3 min as described [14]. The concentration of hydrogen increased 3.7-fold and 7.6-fold in the fed and fasted state, respectively (Fig. 2a, b), suggesting that orally administered hydrogen can be incorporated into the body.

Fig. 2 Hydrogen is detected in blood after oral administration of hydrogen water and reduced oxidative stress in the kidney. a Rats (approximately 230 g) were administered 3.5 mL of hydrogen water (0.8 mM H₂ in water) into the stomach via a catheter. After 3 min, hydrogen concentration in blood was quantified using gas chromatography, as described in “Materials and methods”. Representative profiles of gas chromatography for detecting molecular hydrogen are shown. b Hydrogen concentration in blood was quantified in fasted and fed state as described in a (n = 5 for fasted group and n = 3 for fed group). Data are the means ± SD. Differences in hydrogen concentration were significant (**P < 0.01, ***P < 0.001) by Student’s t test. c Mice were injected intraperitoneally with a single dose of cisplatin (17 mg/kg) (Day 0). Hydrogen water (0.8 mM H₂ in water) was available ad libitum throughout the experiments (from Day 2 to Day 3). HW (+) and HW (−) were mice given water with or without hydrogen, respectively. MDA was measured on Day 3 (n = 15). Data are the means ± SEM. Differences in the MDA level were significant (*P < 0.05) by one-way ANOVA

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Next hydrogen water was given to mice ad libitum as described in “Materials and methods”. We measured the consumed volume of hydrogen water and degassed control water in mice. Water intake was nearly the same (194 ± 12 (SD) vs. 188 ± 15 (SD) mL/(kg day)) between groups drinking hydrogen water and degassed control water. In addition, a 24-h water intake ad libitum (194 mL/kg) was almost 13-fold higher compared with a single water intake given by a catheter as mentioned above (15 mL/kg); thus we used the method in which hydrogen water was available ad libitum throughout the whole period.

Cisplatin stimulates the generation of ROS such as hydroxyl radicals and renal lipid peroxidation [19]. We examined the effect of hydrogen on oxidative stress in the kidney as judged by the level of malondialdehyde (MDA), an oxidative stress marker derived from lipid peroxides [20]. Mice were given hydrogen water freely throughout the experiment. Three days after cisplatin administration, the MDA level in the kidney fell to nearly the normal level in mice drinking hydrogen water (Fig. 2c), indicating that daily consumption of hydrogen water suppresses oxidative stress.

We tested the possibility that hydrogen impairs anti-tumor activity of cisplatin using cultured cells. Hydrogen and oxygen concentrations were maintained in culture medium as described [14], where pH is not influenced by hydrogen. S-180 sarcoma and L-1210 leukemia cells were exposed to various concentrations of cisplatin to induce cell death and continued to culture in medium with or without 0.6 mM hydrogen (Fig. 5a–c). Cell death was assessed using trypan blue staining [18]. Hydrogen did not suppress cell death induced by cisplatin in vitro (Fig. 5a–c).

Fig. 5 Hydrogen does not impair cytotoxicity by cisplatin. a–c Hydrogen does not influence cytotoxicity of cisplatin against tumor cell lines in vitro. Relative cell number of (a, b) sarcoma 180 and (c) L-1210 cells were examined under (a, c) serum-containing or (b) serum-free medium. Cells were cultured in medium with or without 0.6 mM H₂ and treated with various concentrations of cisplatin for 72 (a, c) or 120 h (b). Cell number was measured by counting viable cells as described in “Materials and methods”. Data show relative cell number at 72 (a, c) or 120 h (b) against the starting cell number. Data are the means ± SD. d, e Hydrogen does not inhibit anti-tumor activity of cisplatin in vivo. Sarcoma 180 cells were subcutaneously transplanted into ddY mice in the back. After their tumor volumes reached almost 100 mm³ (Day 0), mice received three consecutive daily injections of cisplatin (5 mg/kg). Hydrogen water (0.8 mM H₂ in water) was available ad libitum throughout the experiments (from Day 2 to Day 7). On Days 0, 3, and 7, their tumor sizes were evaluated with a CT scan. d Representative images of CT scanning on Day 7 are shown. Tumor areas are indicated with white dot lines and arrows. e Tumor volumes were calculated by serial CT scan images, as described in “Materials and methods”. Data are the means ± SEM. CTL were mice that received saline instead of cisplatin (n = 4). HW (+) and HW (−) were mice given water with or without hydrogen, respectively (n = 4 for each group)

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We next evaluated the effects of hydrogen on anti-tumor activity of cisplatin using tumor-bearing mice in vivo [21]. As the sublethal dose of cisplatin described above is not applicable for actual clinical uses, we examined anti-tumor activity of a safe dose of cisplatin using a transplantation model. To obtain an optimal dose and times, cisplatin was injected with different doses (5, 10, or 15 mg/kg) and times (once, twice or three times) (n = 6 in each experiment). Treatment of three consecutive daily injections of cisplatin (5 mg/kg) inhibited tumor growth and caused only a little weight loss. Higher doses of cisplatin (10 or 15 mg/kg, single injection) caused apparent weight loss (10–30%). Therefore, the regimen (5 mg/kg, three times) was used in this study. We transplanted S-180 sarcoma cells into ddY mice and monitored the tumor mass with a CT scan. When tumor-bearing mice received an injection of physiological saline instead of cisplatin, the tumor tissue increased in mass by twofold on Day 7 (Fig. 5d, e). Administration of three consecutive daily injections of cisplatin (5 mg/kg) inhibited tumor growth. Notably, cisplatin inhibited tumor growth in the group consuming hydrogen water ad libitum to the same level as in the group without hydrogen water. We measured levels of serum creatinine and BUN as described above (Fig. 1c, d) to assess nephrotoxicity. Giving hydrogen water freely decreased serum creatinine (6.4 ± 0.7 (SEM) vs. 4.1 ± 0.4 (SEM) mg/L) and BUN levels (302 ± 47 (SEM) vs. 217 ± 25 (SEM) mg/L) compared with cisplatin alone. These results clearly indicated that hydrogen does not interfere with the chemotherapeutic activity of cisplatin and attenuate cisplatin-induced nephrotoxicity.