Polyhexamethylene guanidine phosphate aerosol particles induce pulmonary inflammatory and fibrotic responses

Abstract

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.

Adverse health effects of humidifier disinfectants in Korea: lung toxicity of polyhexamethylene guanidine phosphate

“…In addition, TGF-β levels increase markedly in airway epithelial cells exposed to PHMG-P (Kim et al, 2016a), mirroring similar significant increases observed in cases associated with humidifier disinfectant exposure (Kim et al, 2016b). TGF-β is a growth factor produced in alveolar macrophages and epithelial cells, which stimulates fibroblasts to produce interstitial components such as fibronectin and collagen, and also promotes the proliferation and migration of pulmonary fibroblasts.…”

“…Moreover, inflammatory cell foci and collagen accumulation appeared in the interstitial, peribronchiolar, and perivascular areas in a concentration-dependent manner ( Fig. 2; Kim et al, 2016a).…”

“…Park et al (2015) calculated a different value, 0.945 mg/m 3 , assuming a bedroom air-volume of 27 m 3 and disinfectant-containing water consumption of 5 L of per day. Based on these figures, the majority of toxicity assessments for humidifier disinfectants have set the PHMG-P indoor air concentration at 0.1 mg/m 3 (Kim et al, 2016a;Park et al, 2014;Song et al, 2014). Another consideration is the length of exposure; most patients reportedly used humidifiers for 8 hr a day, over at least 4 months starting in October (Jeon and Park, 2012;Kim et al, 2014).…”

“…To clarify this discrepancy, Kim et al conducted animal experiments with a 3-week recovery period after PHMG-P exposure. PHMG-P-containing aerosol particles with a diameter of 90 nm were used, taking into account that particles shrink to the nanometer level following their release from humidifiers (Kim et al, 2016a). Table 4 summarizes the exposure conditions of various animal experiments with PHMG-P.…”

“…Adopting this woundhealing response model, we introduce here a putative mechanism for PHMG-P-induced lung fibrosis, based in large part on our research findings to date. We examined the toxic effects of PHMG-P by testing it on airway epithelial barriers we constructed from three human lung-derived cell lines using air-liquid interface cultures (Kim et al, 2016a). Increasing PHMG-P concentrations resulted in a dose-dependent rise in cytotoxicity, as well as increased membrane permeability to dextran, indicating cell membrane breakdown, and decreased levels of E-cadherin, a protein involved in cell adhesion.…”