Keywords: molecular hydrogen, radiation-induced damage, medical application, radioprotective agent, non-DNA target, intracellular response, oxidation, inflammation, apoptosis, gene expression
This paper outlines the paper：《Molecular Hydrogen as a Potential Clinically Applicable Radioprotective Agent》
Hirano SI, Ichikawa Y, Sato B, Yamamoto H, Takefuji Y, Satoh F.
Radioprotective Effects of H2 in Animal Models
As for the radioprotective effects of H2 in animal models, protective effects on cognitive function, the immune system, lungs, heart, digestive organs, hematopoietic organs, testis, skin, and cartilage disorders have been reported An inhibitory effect on thymic lymphoma caused by radiation has also been reported . The following is a summary of the literature that reports specific examples of the protective effects of H2 against different radiation disorders.
1. Protective Effects on Cognitive Impairment
Liu et al. investigated the effect of H2-rich water (0.8–0.9 ppm) on radiation-induced cognitive dysfunction . Rats were continuously administered with H2-rich water for 30 days before and after whole-brain irradiation using electron beams. Spatial recordings of the rats using the Morris water maze showed significant improvements in cognitive function in the H2-rich water group compared to the control group. In the H2-rich water group, the levels of superoxide dismutase (SOD), glutathione (GSH), and brain-derived neurotrophic factor (BDNF) in the brain were significantly higher, and the levels of MDA and 8-OHdG were significantly lower. In addition, mRNA and protein levels of BDNF and brain-derived neurotrophic factor receptor (TrkB) were also significantly higher in the H2-rich water group . As such, they reported that the protective effect of H2 against radiation-induced cognitive dysfunction involves an antioxidant response, anti-inflammatory response, and protection of neonatal neurons by regulating the BDNF-TrkB signaling pathway.
2. Protective Effects on the Immune System
Radiation often causes the depletion of immune cells in tissues and blood, which leads to immunosuppression. Qian et al. reported the radioprotective effect of H2 on cultured human lymphocyte (AHH-1) cells . Pretreatment with H2-rich PBS (1.2 ppm) prior to irradiation significantly reduced the levels of MDA and 8-OHdG in AHH-1 cells compared to untreated controls . Furthermore, Qian et al. also reported the radioprotective effect of H2 in AHH1 cells in another paper. They showed that pretreatment with H2-rich saline (0.6 ppm) increased the viability of AHH-1 cells and inhibited apoptosis compared to non-treated cells .
Yang et al. also investigated the radioprotective effects of H2 on radiated AHH-1 cells, mouse thymocytes, and spleen cells . Pretreatment with H2-rich medium (1.2 ppm) significantly reduced •OH in cultured AHH-1 cells compared to cells with no pretreatment. In addition, intraperitoneal administration of H2-rich saline (1.2 ppm) alleviated the apoptosis of thymocytes and splenocytes in mice, and inhibited the activation of caspase-3 compared to saline alone in mouse experiments. Moreover, H2-rich saline significantly ameliorated the depletion of white blood cells (WBCs) and platelets (PLT) in the peripheral blood of mice Zhao et al. reported the results of an experiment in which H2 protected against radiation-induced immune dysfunction . They found that when H2-rich saline (1.2 ppm) was administered intraperitoneally prior to irradiation, H2 increased the spleen index calculated from mouse body weight and spleen weight and suppressed histopathological spleen damage. H2 also decreased ROS levels in spleen tissue, suppressed spleen apoptosis, and down-regulated pre-apoptotic proteins. In addition, H2 ameliorated radiation-induced T cell imbalance and regulated CD4+ T cell localization, Th-type cytokine, and pro-inflammatory cytokine levels. Based on these results, Zhao et al. reported that H2 has a radioprotective effect by scavenging ROS .
3. Protective Effects against Lung Injury
Terasaki et al. reported the protective effect of H2 against radiation-induced lung injury . They irradiated A549 cells, a cell line of human lung epithelial cells, to induce radiation injury, and examined the effects of H2-rich PBS and H2-rich medium (both 1.2 ppm). In addition, mice were irradiated, and H2 gas (3%) inhalation or oral intake of H2-rich water (0.8 ppm) was used to reduce lung injury. H2 improved the survival rate of A549 cells, suppressed ROS production, and improved oxidative stress and apoptosis markers . In the in vivo experiments using mice, H2 similarly attenuated oxidative stress and apoptotic markers, which were measured as acute lung injuries. H2 also alleviated chest computed tomography (CT), Ashcroft score (an index of lung fibrosis), and type III collagen deposition, which were each measured as indicators of chronic lung injury. They reported that H2 inhibited not only acute lung injury, but also chronic lung injury (lung fibrosis) . A549 cells are tumor lines of lung adenocarcinoma; therefore, the fact that the survival rate of radiation-induced damaged cells was improved by H2 treatment may indicate that the antitumor effects of radiation may be compromised. However, because the rates of improvement in cell death by H2 is small compared to those by H2 for other oxidative stress markers, the attenuation of the antitumor effects may have little effect . Indeed, in clinical trials on cancer patients receiving radiotherapy, Kang et al., as well as our own study, demonstrated that the antitumor effects by H2 were not compromised .
4. Protective Effects on Myocardial Injury
Qian et al. investigated the effects of pretreatment with H2-rich water (1.2 ppm) on radiation-induced myocardial damage in mice . H2-rich water improved the survival rate and histopathological damage of the myocardium in mice compared to control groups. In addition, H2-rich water reduced myocardial MDA and 8-OHdG levels. Moreover, H2-rich water increased myocardial SOD and GSH levels compared to controls and alleviated myocardial cell DNA damage, as measured by the comet assay. Qian et al. reported that H2 has a cardioprotective effect against radiation-induced damage .
MicroRNAs (miRNAs) constitute a large class of post-transcriptional regulators of gene expression, and it is estimated that miRNAs regulate up to 30% of human protein-coding genes. They are implicated in many pathological processes, including radiation damage. Kura et al. investigated the involvement of miRNA-1, -15b, and -21 in the protective effects of H2-rich water (1.2 ppm) on rat myocardium damaged by radiation . Radiation increased MDA and tumor necrosis factor (TNF-α) levels in myocardium, but H2 decreased these levels. miRNA-1, which is involved in myocardial hypertrophy, was decreased by irradiation, but H2 mitigated this decrease. miRNA-15b, which is involved in anti-fibrotic, anti-hypertrophic, and antioxidant effects, was decreased by radiation, but H2 reversed this effect. Furthermore, miRNA-21, which is involved in cardiac fibrosis, was increased by radiation, but H2 also reduced this increase. Based on these results, Kura et al. reported that the cardioprotective effects of H2 against radiation involves the regulation of miRNA-1, -15b, and -21.
5. Protective Effects against Gastrointestinal Disorders
Qian et al. reported the radioprotective effects of H2 on cultured intestinal cells. Pretreatment of human intestinal crypt cells (HIEC) with H2-rich PBS (1.2 ppm) prior to irradiation significantly inhibited apoptosis and increased the cell viability of HIEC cells compared to those with no pretreatment. They also examined the radioprotective effects of H2 in mice via the intraperitoneal administration of H2-rich saline before irradiation . The results showed that, compared to the control group, the H2 group significantly alleviated the histopathological damage in the intestinal tract of mice, increased the levels of SOD and GSH in plasma, and significantly decreased the levels of MDA and 8-OHdG.
Xiao et al. reported the alleviating effects of H2-rich water (1.6 ppm) on gastrointestinal toxicity in a model produced by irradiating mice . H2-water was administered orally by gavage before and after radiation. The results showed that H2-water significantly improved the survival rate and body weight of mice compared to the control, and furthermore improved the function of the intestine as seen from the gene expression of the intestinal epithelium. In a microarray analysis of the small intestine, H2-water down-regulated myeloid differentiation factor 88 (MyD88) expression. Furthermore, in high-throughput screening, H2-water improved the balance of intestinal bacteria impaired by radiation . Xiao et al. reported that H2-water reduces radiation-induced gastrointestinal toxicity through the action of MyD88 on intestinal bacteria.
Qiu et al. investigated the radioprotective effects of H2 in animal experiments using mice and in cellular experiments using the intestinal crypt epithelial cell (IEC-6) line . H2-rich saline (1.2 ppm) improved mouse survival and intestinal mucosal damage and function, as well as oxidative stress and inflammatory response. In vitro experiments using IEC-6 cells showed that H2 improved survival and inhibited ROS production. H2 inhibited mitochondrial depolarization, cytochrome c release, and the activities of caspase-3, caspase-9, and polymerase (PARP). In addition, H2 recovered from the decreased expression of B cell lymphoma-extra-large (Bcl-xl) and B cell lymphoma-2 (Bcl-2), proteins that suppress apoptosis, and suppressed the increased expression of BCL2-associated X protein (Bax), a protein that promotes apoptosis. They suggested that the protective effects of H2 against radiation damage may involve the blockage of the mitochondrial apoptotic pathway.
6. Protective Effects against Hematopoietic Cell Injury
Zhang et al. reported the mitigating effects of H2-rich water on radiation-induced hematopoietic stem cell injury . Mice were irradiated and orally administrated with H2-rich water (1.6 ppm) before and after the radiation. The results showed that H2 mitigated the injury of blood stem cells. In H2-treated c-kit+ cells, the mean fluorescence intensity of phosphorylated H2AX (γ-H2AX) and the percentage of 8-oxoguanine-positive cells were significantly decreased, suggesting that H2 alleviates radiation-induced DNA damage and oxidative DNA damage . Furthermore, proteins related to the cell cycle, apoptosis, and oxidative stress were significantly ameliorated by H2 in irradiated mouse c-kit+ cells .
7. Protective Effects on Sperm Dysfunction
Chuai et al. reported the protective effects of H2 against radiation-induced impairment of spermatogenesis and hematopoiesis . Intraperitoneal administration of H2-rich saline (1.2 ppm) prior to the radiation of mice significantly improved testicular sperm count and impaired spermatogenesis by histopathological analysis. In addition, the impairment of hematopoietic function by endogenous hematopoietic spleen colony formation (endoCFU), bone marrow nucleated cells (BMNC), and WBC in peripheral blood were significantly improved by pretreatment with H2-rich saline. They reported that H2-rich saline partially protected the spermatogenesis and hematopoietic functions of irradiated mice .
Chuai et al. also showed that in a cell-free system, •OH produced by the Fenton reaction and the radiolysis of water was reduced by H2 . Furthermore, they found that the intraperitoneal administration of H2-rich saline (1.2 ppm) to mice prior to radiation significantly suppressed the reaction between •OH and intracellular macromolecules, indicating that radiation causes lipid peroxidation, protein carbonyls, and oxidative DNA damage . In addition, Chuai et al. demonstrated the radioprotective effect of H2 on male germ cells from morphological changes in testicular tissue, apoptosis analysis and sperm quality test .
Jiang et al. reported the protective effects of H2-rich saline and amifostine (WR2721) against radiation-induced testicular damage in rats. H2-rich saline (1.6 ppm) or WR2721 (200 mg/kg) was administered intraperitoneally before radiation. The results showed that testis weight, testis dimensions, sperm count, and sperm motility were all decreased by radiation in the control group, but significant improvement on these decreases was observed in the H2-rich saline and WR2721 groups. In addition, the control group showed a decrease in apoptotic index and SOD activity and an increase in MDA level, while H2-rich saline and WR2721 groups showed significant improvements in these parameters. Moreover, the H2-rich saline and WR2721 groups showed that the recovery of serum testosterone levels decreased with radiation .
8. Protective Effects against Skin Damage
The occurrence of dermatitis is a frequent side effect of radiotherapy in head and neck cancer patients. Therefore, Mei et al. examined the radioprotective effects of H2 (1.2 ppm) and its mechanism under local, single, and fractionated radiation conditions using human keratinocyte HaCaT cells and rats . In experiments using HaCaT cells, the effects of H2 medium on cell viability, apoptosis, and biochemical assays were measured. The results showed that H2 significantly reduced the severity of dermatitis, accelerated tissue recovery, and inhibited weight loss in rats. H2 also showed protective effects when irradiated in three different increments. Moreover, H2 protected cells from radiation injury by improving the survival rate of HaCaT cells, inhibiting apoptosis, increasing SOD and GSH activities, and decreasing MDA levels . Based on these results, they showed that H2 is useful in acute radiation-induced dermatitis.
Watanabe et al. examined the effects of prior inhalation of H2 gas (1.3%) on a radiation-induced skin injury model . Inhalation of H2 significantly reduced the severity of radiation dermatitis and accelerated the repair of wounds with impaired healing. The percentage and staining levels of apoptotic keratinocytes in irradiated skin were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 8-OHdG staining. These results were significantly lower in the H2-inhaled rats than in the non-H2-inhaled rats. In addition, the H2 inhalation group significantly reduced the delay in recovery of full-thickness skin wounds made at the site of X-ray irradiation . These results suggest that prior inhalation of H2 may mitigate radiation-induced skin damage.
Zhou et al. investigated the effects of oral intake of H2-rich water (1.0 and 2.0 ppm) on a rat model with radiation-induced skin damage . The H2 group significantly shortened healing times and increased healing rates of damaged skin, decreased the MDA and IL-6 levels, and increased the SOD activity and epidermal growth factor (EGF) content compared to the control group . These results suggest that H2 promotes wound healing in radiation-induced skin lesions through its antioxidant and anti-inflammatory effects.
9. Protective Effects against Cartilage Damage
Although radiotherapy is a useful treatment for head and neck cancers, unexpected cartilage necrosis of the jaw often occurs as a radiation injury. Chen et al. investigated the protective effects of H2-rich saline (1.2 ppm) on a rat model with chondrocyte necrosis of the jaw induced by radiation in in vitro and in vivo experiments. Treatment of bone marrow-derived mesenchymal stem cells (BMSCs) with H2 prior to irradiation significantly increased cell viability and differentiation potential and decreased ROS production compared to untreated controls. Rats in the control group showed an accumulation of myofibroblasts in and around the fibrotic medulla, but the accumulation was reduced in the H2 pre-treated group . Chen et al. reported that the use of H2 against osteonecrosis of the jaw cartilage could be an important preventive and therapeutic strategy.
10. Inhibitory Effects on Carcinogenesis (Thymic Lymphoma)
Although radiation is a well-known carcinogen, the pathogenesis of radiation-induced thymic lymphoma is not well understood. Zhao et al. examined the protective effects of H2 on radiation-induced thymic lymphoma in mice . The control group was irradiated for four weeks, and the H2 group was given H2-rich saline (1.2 ppm) intraperitoneally 5 min before each irradiation. As a result, the survival rate of mice, the incidence of lymphoma, the production of ROS in peripheral blood mononuclear cells (PBMC), and the levels of SOD, GSH, and MDA in plasma were all improved in the H2 group compared to the control group . Zhao et al. reported that H2 protects against radiation-induced thymic lymphoma.
In this review, we reported the radioprotective effects of H2 in cellular and animal models on cognitive function, immune system, lung, heart, digestive organs, hematopoietic organs, testis, skin, and cartilage damages . We also reported the inhibitory effect of H2 on thymic lymphoma induced by radiation . As for radioprotective effects in clinical trials, improvement of QOL with H2 in patients with liver cancer receiving radiotherapy was reported by Kang et al. . We also recently reported that the inhalation of H2 gas reduced bone marrow damage in end-stage cancer patients receiving IMRT . The mechanism of the radioprotective effects of H2 is thought to be related to the scavenging of free radicals such as •OH, but it is also suggested to be indirectly related to anti-inflammatory and anti-apoptotic characteristics, as well as the regulation of gene expression of intracellular signaling. H2 has been widely applied clinically in areas other than radiation damage and has been reported to be a substance with excellent efficacy and safety. Therefore, H2 may have potential clinical applications as a radioprotective agent, and could be used therapeutically against radiation damage in the future.