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Molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked acute lung injury through a reduction in oxidative stress and inflammation

Laboratory Investigation (2019) | Download Citation


Although inhibition of epidermal growth factor receptor (EGFR)-mediated cell signaling by the EGFR tyrosine kinase inhibitor gefitinib is highly effective against advanced non-small cell lung cancer, this drug might promote severe acute interstitial pneumonia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive anti-oxidant. Here, we show that treatment with H2 effectively protects the lungs of mice from severe damage caused by oral administration of gefitinib after intraperitoneal injection of naphthalene, the toxicity of which is related to oxidative stress. Drinking H2−rich water ad libitum mitigated naphthalene/gefitinib-induced weight loss and significantly improved survival, which was associated with a decrease in lung inflammation and inflammatory cytokines in the bronchoalveolar lavage fluid. Naphthalene decreased glutathione in the lung, increased malondialdehyde in the plasma, and increased 4-hydroxy-2-nonenal production in airway cells, all of which were mitigated by H2-rich water, indicating that the H2-rich water reverses cellular damage to the bronchial wall caused by oxidative stress. Finally, treatment with H2 did not interfere with the anti-tumor effects of gefitinib on a lung cancer cell line in vitro or on tumor-bearing mice in vivo. These results indicate that H2-rich water has the potential to improve quality of life during gefitinib therapy by mitigating lung injury without impairing anti-tumor activity.

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  1. 1.

    Sim EH, Yang IA, Wood-Baker R, et al. Gefitinib for advanced non-small cell lung cancer. Cochrane Database Syst Rev. 2018;1:CD006847.

  2. 2.

    Nakagawa K, Kudoh S, Ohe Y, et al. Postmarketing surveillance study of erlotinib in Japanese patients with non-small-cell lung cancer (NSCLC): an interim analysis of 3488 patients (POLARSTAR). J Thorac Oncol. 2012;7:1296–303.

  3. 3.

    Cohen MH, Johnson JR, Chattopadhyay S, et al. Approval summary: erlotinib maintenance therapy of advanced/metastatic non-small cell lung cancer (NSCLC). Oncologist. 2010;15:1344–51.

  4. 4.

    Kudoh S, Kato H, Nishiwaki Y, et al. Interstitial lung disease in Japanese patients with lung cancer: a cohort and nested case-control study. Am J Respir Crit Care Med. 2008;177:1348–57.

  5. 5.

    Harada C, Kawaguchi T, Ogata-Suetsugu S, et al. EGFR tyrosine kinase inhibition worsens acute lung injury in mice with repairing airway epithelium. Am J Respir Crit Care Med. 2011;183:743–51.

  6. 6.

    Stohs SJ, Ohia S, Bagchi D. Naphthalene toxicity and antioxidant nutrients. Toxicology. 2002;180:97–105.

  7. 7.

    Phimister AJ, Nagasawa HT, Buckpitt AR, et al. Prevention of naphthalene-induced pulmonary toxicity by glutathione prodrugs: roles for glutathione depletion in adduct formation and cell injury. J Biochem Mol Toxicol. 2005;19:42–51.

  8. 8.

    Van Winkle LS, Isaac JM, Plopper CG. Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse. Am J Pathol. 1997;151:443–59.

  9. 9.

    Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med. 2007;13:688–94.

  10. 10.

    Oláh O, Tóth-Szűki V, Temesvári P, et al. Delayed neurovascular dysfunction is alleviated by hydrogen in asphyxiated newborn pigs. Neonatology. 2013;104:79–86.

  11. 11.

    Fukuda K, Asoh S, Ishikawa M, et al. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress. Biochem Biophys Res Commun. 2007;361:670–4.

  12. 12.

    Hayashida K, Sano M, Kamimura N, et al. H2 gas improves functional outcome after cardiac arrest to an extent comparable to therapeutic hypothermia in a rat model. J Am Heart Assoc. 2012;1:e003459.

  13. 13.

    Oharazawa H, Igarashi T, Yokota T, et al. Protection of the retina by rapid diffusion of hydrogen: administration of hydrogen-loaded eye drops in retinal ischemia-reperfusion injury. Invest Ophthalmol Vis Sci. 2010;51:487–92.

  14. 14.

    Ohta S. Molecular hydrogen as a novel antioxidant: overview of the advantages of hydrogen for medical applications. Methods Enzymol. 2015;555:289–317.

  15. 15.

    Slezák J, Kura B, Frimmel K, et al. Preventive and therapeutic application of molecular hydrogen in situations with excessive production of free radicals. Physiol Res. 2016;65(Suppl 1):S11–28.

  16. 16.

    Terasaki Y, Ohsawa I, Terasaki M, et al. Hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress. Am J Physiol Lung Cell Mol Physiol. 2011;301:L415–426.

  17. 17.

    Nakashima-Kamimura N, Mori T, Ohsawa I, et al. Molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice. Cancer Chemother Pharmacol. 2009;64:753–61.

  18. 18.

    Itoh T, Hamada N, Terazawa R, et al. Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages. Biochem Biophys Res Commun. 2011;411:143–9.

  19. 19.

    Koyama Y, Taura K, Hatano E, et al. Effects of oral intake of hydrogen water on liver fibrogenesis in mice. Hepatol Res. 2014;44:663–77.

  20. 20.

    Iketani M, Ohshiro J, Urushibara T, et al. Preadministration of hydrogen-rich water protects against lipopolysaccharide-induced sepsis and attenuates liver injury. Shock. 2017;48:85–93.

  21. 21.

    Kajiyama S, Hasegawa G, Asano M, et al. Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance. Nutr Res. 2008;28:137–43.

  22. 22.

    Nakao A, Toyoda Y, Sharma P, et al. Effectiveness of hydrogen rich water on antioxidant status of subjects with potential metabolic syndrome-an open label pilot study. J Clin Biochem Nutr. 2010;46:140–9.

  23. 23.

    Aoki K, Nakao A, Adachi T, et al. Pilot study: Effects of drinking hydrogen-rich water on muscle fatigue caused by acute exercise in elite athletes. Med Gas Res. 2012;2:12.

  24. 24.

    Yoritaka A, Takanashi M, Hirayama M, et al. Pilot study of H2 therapy in Parkinson’s disease: a randomized double-blind placebo-controlled trial. Mov Disord. 2013;28:836–9.

  25. 25.

    Nishimaki K, Asada T, Ohsawa I, et al. Effects of molecular hydrogen assessed by an animal model and a randomized clinical study on mild cognitive impairment. Curr Alzheimer Res. 2018;15:482–92.

  26. 26.

    Kawai S, Takagi Y, Kaneko S, et al. Effect of three types of mixed anesthetic agents alternate to ketamine in mice. Exp Anim. 2011;60:481–7.

  27. 27.

    Wu Q, Zhang J, Wan Y, et al. Hydrogen water alleviates lung injury induced by one-lung ventilation. J Surg Res. 2015;199:664–70.

  28. 28.

    Okuma T, Terasaki Y, Sakashita N, et al. MCP-1/CCR2 signalling pathway regulates hyperoxia-induced acute lung injury via nitric oxide production. Int J Exp Pathol. 2006;87:475–83.

  29. 29.

    Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988;41:467–70.

  30. 30.

    Shimamura T, Lowell AM, Engelman JA, et al. Epidermal growth factor receptors harboring kinase domain mutations associate with the heat shock protein 90 chaperone and are destabilized following exposure to geldanamycins. Cancer Res. 2005;65:6401–8.

  31. 31.

    Murakami Y, Ito M, Ohsawa I. Molecular hydrogen protects against oxidative stress-induced SH-SY5Y neuroblastoma cell death through the process of mitohormesis. PLoS One. 2017;12:e0176992.

  32. 32.

    Tozan A, Sehirli O, Omurtag GZ, et al. Ginkgo biloba extract reduces naphthalene-induced oxidative damage in mice. Phytother Res. 2007;21:72–77.

  33. 33.

    Buckpitt A, Chang AM, Weir A, et al. Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats, and hamsters. Mol Pharmacol. 1995;47:74–81.

  34. 34.

    Shultz MA, Choudary PV, Buckpitt AR. Role of murine cytochrome P-450 2F2 in metabolic activation of naphthalene and metabolism of other xenobiotics. J Pharmacol Exp Ther. 1999;290:281–8.

  35. 35.

    Wong AP, Keating A, Waddell TK. Airway regeneration: the role of the Clara cell secretory protein and the cells that express it. Cytotherapy. 2009;11:676–87.

  36. 36.

    Reynolds SD, Giangreco A, Hong KU, et al. Airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inflammation and alveolar dysfunction. Am J Physiol Lung Cell Mol Physiol. 2004;287:L1256–1265.

  37. 37.

    Nagata K, Nakashima-Kamimura N, Mikami T, et al. Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice. Neuropsychopharmacology. 2009;34:501–8.

  38. 38.

    Shimouchi A, Nose K, Shirai M, et al. Estimation of molecular hydrogen consumption in the human whole body after the ingestion of hydrogen-rich water. Adv Exp Med Biol. 2012;737:245–50.

  39. 39.

    Ohsawa I, Nishimaki K, Yamagata K, et al. Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice. Biochem Biophys Res Commun. 2008;377:1195–8.

  40. 40.

    Cardinal JS, Zhan J, Wang Y, et al. Oral hydrogen water prevents chronic allograft nephropathy in rats. Kidney Int. 2010;77:101–9.

  41. 41.

    Suzuki Y, Sato T, Sugimoto M, et al. Hydrogen-rich pure water prevents cigarette smoke-induced pulmonary emphysema in SMP30 knockout mice. Biochem Biophys Res Commun. 2017;492:74–81.

  42. 42.

    Ito M, Hirayama M, Yamai K, et al. Drinking hydrogen water and intermittent hydrogen gas exposure, but not lactulose or continuous hydrogen gas exposure, prevent 6-hydorxydopamine-induced Parkinson’s disease in rats. Med Gas Res. 2012;2:15.

  43. 43.

    Petty WJ, Dragnev KH, Memoli VA, et al. Epidermal growth factor receptor tyrosine kinase inhibition represses cyclin D1 in aerodigestive tract cancers. Clin Cancer Res. 2004;10:7547–54.

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We thank Naoki Maruyama for his continuous encouragement and support. This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (Grant numbers 16K16616 and 16H03267).

Author information


  1. Department of Analytic Human Pathology, Nippon Medical School, Tokyo, Japan

    • Yasuhiro Terasaki
    • , Mika Terasaki
    • , Naomi Kuwahara
    • , Yusuke Kajimoto
    •  & Akira Shimizu
  2. Biological Process of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

    • Tetsuya Suzuki
    • , Kozue Tonaki
    • , Jumi Ohsiro
    • , Masumi Iketani
    • , Mayumi Takahashi
    •  & Ikuroh Ohsawa
  3. Department of Life Sciences, Faculty of Life Science, Toyo University, Gunma, Japan

    • Tetsuya Suzuki
    • , Jumi Ohsiro
    •  & Hideo Kawaguchi
  4. Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan

    • Kozue Tonaki
    •  & Seisuke Hattori
  5. Department of Physiology, Toho University School of Medicine, Tokyo, Japan

    • Makoto Hamanoue


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The authors declare that they have no conflict of interest.

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Correspondence to Ikuroh Ohsawa.

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