Archives of Toxicology

, Volume 90, Issue 3, pp 617–632 | Cite as

Polyhexamethylene guanidine phosphate aerosol particles induce pulmonary inflammatory and fibrotic responses

  • Ha Ryong Kim
  • Kyuhong Lee
  • Chang We Park
  • Jeong Ah Song
  • Da Young Shin
  • Yong Joo Park
  • Kyu Hyuck ChungEmail author
Organ Toxicity and Mechanisms


Polyhexamethylene guanidine (PHMG) phosphate was used as a disinfectant for the prevention of microorganism growth in humidifiers, without recognizing that a change of exposure route might cause significant health effects. Epidemiological studies reported that the use of humidifier disinfectant containing PHMG-phosphate can provoke pulmonary fibrosis. However, the pulmonary toxicity of PHMG-phosphate aerosol particles is unknown yet. This study aimed to elucidate the toxicological relationship between PHMG-phosphate aerosol particles and pulmonary fibrosis. An in vivo nose-only exposure system and an in vitro air–liquid interface (ALI) co-culture model were applied to confirm whether PHMG-phosphate induces inflammatory and fibrotic responses in the respiratory tract. Seven-week-old male Sprague–Dawley rats were exposed to PHMG-phosphate aerosol particles for 3 weeks and recovered for 3 weeks in a nose-only exposure chamber. In addition, three human lung cells (Calu-3, differentiated THP-1 and HMC-1 cells) were cultured at ALI condition for 12 days and were treated with PHMG-phosphate at set concentrations and times. The reactive oxygen species (ROS) generation, airway barrier injuries and inflammatory and fibrotic responses were evaluated in vivo and in vitro. The rats exposed to PHMG-phosphate aerosol particles in nanometer size showed pulmonary inflammation and fibrosis including inflammatory cytokines and fibronectin mRNA increase, as well as histopathological changes. In addition, PHMG-phosphate triggered the ROS generation, airway barrier injuries and inflammatory responses in a bronchial ALI co-culture model. Those results demonstrated that PHMG-phosphate aerosol particles cause pulmonary inflammatory and fibrotic responses. All features of fibrogenesis by PHMG-phosphate aerosol particles closely resembled the pathology of fibrosis that was reported in epidemiological studies. Finally, we expected that PHMG-phosphate infiltrated into the lungs in the form of aerosol particles would induce an airway barrier injury via ROS, release fibrotic inflammatory cytokines, and trigger a wound-healing response, leading to pulmonary fibrosis. A simultaneous state of tissue destruction and inflammation caused by PHMG-phosphate had whipped up a “perfect storm” in the respiratory tract.


Polyhexamethylene guanidine phosphate Aerosol particles Humidifier disinfectant Pulmonary fibrosis 



This work was partly supported by the Korea Environmental Industry and Technology Institute (Grant Number: 2012001370006).


  1. Aleshina EY, Yudanova TN, Skokova IF (2001) Production and properties of polyvinyl alcohol spinning solutions containing protease C and polyhexamethylene guanidine. Fibre Chem 33:421–423Google Scholar
  2. Alexander DJ, Collins CJ, Coombs DW, Gilkison IS, Hardy CJ, Healey G, Karantabias G, Johnson N, Karlsson A, Kilgour JD, McDonald P (2008) Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals. Inhal Toxicol 20:1179–1189CrossRefPubMedGoogle Scholar
  3. Alfaro-Moreno E, Nawrot TS, Vanaudenaerde BM, Hoylaerts MF, Vanoirbeek JA, Nemery B, Hoet PH (2008) Co-cultures of multiple cell types mimic pulmonary cell communication in response to urban PM10. Eur Respir J 32:1184–1194CrossRefPubMedGoogle Scholar
  4. Bonner JC (2008) Respiratory toxicity. In: Smart RC, Hodgson E (eds) Molecular and biochemical toxicology, 4th edn. Wiley, New York, pp 639–670CrossRefGoogle Scholar
  5. Bonner JC (2010) Mesenchymal cell survival in airway and interstitial pulmonary fibrosis. Fibrogenesis Tissue Repair 3:15PubMedCentralCrossRefPubMedGoogle Scholar
  6. Brandenberger C, Rothen-Rutishauser B, Mühlfeld C, Schmid O, Ferron GA, Maier KL, Gehr P, Lenz AG (2010) Effects and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial airway model. Toxicol Appl Pharmacol 242:56–65CrossRefPubMedGoogle Scholar
  7. Chapman HA (2011) Epithelial-mesenchymal interactions in pulmonary fibrosis. Annu Rev Physiol 73:413–435CrossRefPubMedGoogle Scholar
  8. Diabaté S, Mülhopt S, Paur HR, Krug HF (2008) The response of a co-culture lung model to fine and ultrafine particles of incinerator fly ash at the air-liquid interface. Altern Lab Anim 36:285–298PubMedGoogle Scholar
  9. Duncan MR, Berman B (1991) Stimulation of collagen and glycosaminoglycan production in cultured human adult dermal fibroblasts by recombinant human interleukin 6. J Invest Dermatol 97:686–692CrossRefPubMedGoogle Scholar
  10. Fukuda Y, Ishizaki M, Kudoh S, Kitaichi M, Yamanaka N (1998) Localization of matrix metalloproteinases-1,-2, and-9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases. Lab Invest 78:687–698PubMedGoogle Scholar
  11. Gilbert P, Moore LE (2005) Cationic antiseptics: diversity of action under a common epithet. J Appl Microbiol 99:703–715CrossRefPubMedGoogle Scholar
  12. Giri SN (2003) The combined treatment with taurine and niacin blocks the bleomycin-induced activation of nuclear factor-kappaB and lung fibrosis. Adv Exp Med Biol 526:381–394CrossRefPubMedGoogle Scholar
  13. Gorguner M, Aslan S, Inandi T, Cakir Z (2004) Reactive airways dysfunction syndrome in housewives due to a bleach-hydrochloric acid mixture. Inhal Toxicol 16:87–91CrossRefPubMedGoogle Scholar
  14. Grainger CI, Greenwell LL, Lockley DJ, Martin GP, Forbes B (2006) Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier. Pharm Res 23:1482–1490CrossRefPubMedGoogle Scholar
  15. Hansen K, Mossman BT (1987) Generation of superoxide (O2-.) from alveolar macrophages exposed to asbestiform and nonfibrous particles. Cancer Res 47:1681–1686PubMedGoogle Scholar
  16. Hay J, Shahzeidi S, Laurent G (1991) Mechanisms of bleomycin-induced lung damage. Arch Toxicol 65:81–94CrossRefPubMedGoogle Scholar
  17. Hayashi T, Stetler-Stevenson WG, Fleming MV, Fishback N, Koss MN, Liotta LA, Ferrans VJ, Travis WD (1996) Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. Am J Pathol 149:1241–1256PubMedCentralPubMedGoogle Scholar
  18. Heinzer R, Ribordy V, Kuzoe B, Lazor R, Fitting JW (2004) Recurrence of acute respiratory failure following use of waterproofing sprays. Thorax 59:541–542PubMedCentralPubMedGoogle Scholar
  19. Herzog F, Clift MJ, Piccapietra F, Behra R, Schmid O, Petri-Fink A, Rothen-Rutishauser B (2013) Exposure of silver-nanoparticles and silver-ions to lung cells in vitro at the air-liquid interface. Part Fibre Toxicol 10:11. doi: 10.1186/1743-8977-10-11 PubMedCentralCrossRefPubMedGoogle Scholar
  20. Hong SB, Kim HJ, Huh JW, Do KH, Jang SJ, Song JS, Choi SJ, Heo Y, Kim YB, Lim CM, Chae EJ, Lee H, Jung M, Lee K, Lee MS, Koh Y (2014) A cluster of lung injury associated with home humidifier use: clinical, radiological and pathological description of a new syndrome. Thorax. doi: 10.1136/thoraxjnl-2013-204135
  21. Ishii H, Hayashi S, Hogg JC, Fujii T, Goto Y, Sakamoto N, Mukae H, Vincent R, van Eeden SF (2005) Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment. Resp Res 6:87CrossRefGoogle Scholar
  22. Kacmarek RM, Stoller JK, Heuer AH (2013) Egan’s fundamentals of respiratory care, 10th edn. Mosby, St. LouisGoogle Scholar
  23. Keane MP, Arenberg DA, Lynch JP 3rd, Whyte RI, Iannettoni MD, Burdick MD, Wilke CA, Morris SB, Glass MC, DiGiovine B, Kunkel SL, Strieter RM (1997) The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 159:1437–1443PubMedGoogle Scholar
  24. Kim JY, Kim HH, Cho KH (2013) Acute cardiovascular toxicity of sterilizers, PHMG, and PGH: severe inflammation in human cells and heart failure in zebrafish. Cardiovasc Toxicol 13:148–160CrossRefPubMedGoogle Scholar
  25. Kim HJ, Lee MS, Hong SB, Huh JW, Do KH, Jang SJ, Lim CM, Chae EJ, Lee H, Jung M, Park YJ, Park JH, Kwon GY, Gwack J, Youn SK, Kwon JW, Yang BG, Jun BY, Kim Y, Cheong HK, Chun BC, Kim H, Lee K, Koh Y (2014a) A cluster of lung injury cases associated with home humidifier use: an epidemiological investigation. Thorax. doi:  10.1136/thoraxjnl-2013-204132
  26. Kim KW, Ahn K, Yang HJ, Lee S, Park JD, Kim WK, Kim JT, Kim HH, Rha YH, Park YM, Sohn MH, Oh JW, Lee HR, Lim DH, Choung JT, Han MY, Lee E, Kim HY, Seo JH, Kim BJ, Cho YA, Do KH, Kim SA, Jang SJ, Lee MS, Kim HJ, Kwon GY, Park JH, Gwack J, Youn SK, Kwon JW, Jun BY, Pyun BY, Hong SJ (2014b) Humidifier disinfectant-associated children’s interstitial lung disease. Am J Respir Crit Care Med 189:48–56PubMedGoogle Scholar
  27. Kinnula VL, Fattman CL, Tan RJ, Oury TD (2005) Oxidative stress in pulmonary fibrosis: a possible role for redox modulatory therapy. Am J Respir Crit Care Med 172:417–422PubMedCentralCrossRefPubMedGoogle Scholar
  28. Klein SG, Serchi T, Hoffmann L, Blömeke B, Gutleb AC (2013) An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung. Part Fibre Toxicol 10:31. doi: 10.1186/1743-8977-10-31 PubMedCentralCrossRefPubMedGoogle Scholar
  29. Kolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J (2001) Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest 107:1529–1536PubMedCentralCrossRefPubMedGoogle Scholar
  30. Korea Centers for Disease Control and Prevention (2011) Interim report of epidemiological investigation on lung injury with unknown cause in Korea. Public Health Weekly Report KCDC 4:817–832 (In Korean). Accessed 26 July 2012Google Scholar
  31. Lemjabbar H, Gosset P, Lechapt-Zalcman E, Franco-Montoya ML, Wallaert B, Harf A, Lafuma C (1999) Overexpression of alveolar macrophage gelatinase B (MMP-9) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment. Am J Respir Cell Mol Biol 20:903–913CrossRefPubMedGoogle Scholar
  32. Limper AH, Broekelmann TJ, Colby TV, Malizia G, McDonald JA (1991) Analysis of local mRNA expression for extracellular matrix proteins and growth factors using in situ hybridization in fibroproliferative lung disorders. Chest 99(Suppl 3):55S–56SCrossRefPubMedGoogle Scholar
  33. Matsui H, Verghese MW, Kesimer M, Schwab UE, Randell SH, Sheehan JK, Grubb BR, Boucher RC (2005) Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces. J Immunol 175:1090–1099CrossRefPubMedGoogle Scholar
  34. Oggionni T, Morbini P, Inghilleri S, Palladini G, Tozzi R, Vitulo P, Fenoglio C (2009) Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis. Eur J Histochem 50:317–326Google Scholar
  35. Oulé MK, Quinn K, Dickman M, Bernier AM, Rondeau S, De Moissac D, Boisvert A, Diop L (2012) Akwaton, polyhexamethylene-guanidine hydrochloride-based sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections. J Med Microbiol 61:1421–1427CrossRefPubMedGoogle Scholar
  36. Pardo A, Selman M (2006) Matrix metalloproteases in aberrant fibrotic tissue remodeling. Proc Am Thorac Soc 3:383–388CrossRefPubMedGoogle Scholar
  37. Piguet PF, Collart MA, Grau GE, Kapanci Y, Vassalli P (1989) Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J Exp Med 170:655–663CrossRefPubMedGoogle Scholar
  38. Proud D (2008) The pulmonary epithelium in health and disease. Wiley, New YorkCrossRefGoogle Scholar
  39. Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J 22:659–661CrossRefPubMedGoogle Scholar
  40. Rodes C, Smith T, Crouse R, Ramachandran G (1990) Measurements of the size distribution of aerosols produced by ultrasonic humidification. Aerosol Sci Tech 13:220–229CrossRefGoogle Scholar
  41. Schröder JM, Sticherling M, Henneicke HH, Preissner WC, Christophers E (1990) IL-1 alpha or tumor necrosis factor-alpha stimulate release of three NAP-1/IL-8-related neutrophil chemotactic proteins in human dermal fibroblasts. J Immunol 144:2223–2232PubMedGoogle Scholar
  42. Selman M, Ruiz V, Cabrera S, Segura L, Ramírez R, Barrios R, Pardo A (2000) TIMP-1,-2,-3, and-4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment? Am J Physiol Lung Cell Mol Physiol 279:L562–L574PubMedGoogle Scholar
  43. Shan J, Huang J, Liao J, Robert R, Hanrahan JW (2011) Anion secretion by a model epithelium: more lessons from Calu-3. Acta Physiol (Oxf) 202:523–531CrossRefGoogle Scholar
  44. Song JA, Park HJ, Yang MJ, Jung KJ, Yang HS, Song CW, Lee K (2014) Polyhexamethyleneguanidine phosphate induces severe lung inflammation, fibrosis, and thymic atrophy. Food Chem Toxicol 69:267–275CrossRefPubMedGoogle Scholar
  45. Umezawa M, Sekita K, Suzuki K, Kubo-Irie M, Niki R, Ihara T, Sugamata M, Takeda K (2013) Effect of aerosol particles generated by ultrasonic humidifiers on the lung in mouse. Part Fibre Toxicol 10:64. doi: 10.1186/1743-8977-10-64 PubMedCentralCrossRefPubMedGoogle Scholar
  46. Val S, Belade E, George I, Boczkowski J, Baeza-Squiban A (2012) Fine PM induce airway MUC5AC expression through the autocrine effect of amphiregulin. Arch Toxicol 86:1851–1859CrossRefPubMedGoogle Scholar
  47. Wang BL, Tu YY, Fu JF, Zhong YX, Fu GQ, Tian XX, Wang LH, Gong L, Ren QY (2011) Unbalanced MMP/TIMP-1 expression during the development of experimental pulmonary fibrosis with acute paraquat poisoning. Mol Med Rep 4:243–248PubMedGoogle Scholar
  48. Warshamana GS, Corti M, Brody AR (2001) TNF-alpha, PDGF, and TGF-beta(1) expression by primary mouse bronchiolar-alveolar epithelial and mesenchymal cells: tnf-alpha induces TGF-beta(1). Exp Mol Pathol 71:13–33CrossRefPubMedGoogle Scholar
  49. Wilson MS, Wynn TA (2009) Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol 2:103–121PubMedCentralCrossRefPubMedGoogle Scholar
  50. Wynn TA (2011) Integrating mechanisms of pulmonary fibrosis. J Exp Med 208:1339–1350PubMedCentralCrossRefPubMedGoogle Scholar
  51. Zock JP, Plana E, Jarvis D, Antó JM, Kromhout H, Kennedy SM, Künzli N, Villani S, Olivieri M, Torén K, Radon K, Sunyer J, Dahlman-Hoglund A, Norbäck D, Kogevinas M (2007) The use of household cleaning sprays and adult asthma: an international longitudinal study. Am J Respir Crit Care Med 176:735–741PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ha Ryong Kim
    • 1
  • Kyuhong Lee
    • 2
    • 3
  • Chang We Park
    • 1
  • Jeong Ah Song
    • 2
  • Da Young Shin
    • 1
  • Yong Joo Park
    • 1
  • Kyu Hyuck Chung
    • 1
    Email author
  1. 1.School of PharmacySungkyunkwan UniversitySuwonKorea
  2. 2.Inhalation Toxicology Research CenterKorea Institute of ToxicologyJeongeupKorea
  3. 3.Human and Environment ToxicologyUniversity of Science and TechnologyDaejeonKorea

Personalised recommendations