Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells Ha-Na Jung 1 , Tamanna Zerin 1 , Biswajit Podder, Ho-Yeon Song ⇑ , Yong-Sik Kim ⇑ Department of Microbiology, College of Medicine, Soonchunhyang University, South Korea article info Article history: Received 25 October 2013 Accepted 15 February 2014 Available online 25 February 2014 Keywords: Polyhexamethylene guanidine Microarray ROS Apoptosis Cytotoxicity abstract In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 lg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 lg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 lg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding anti- oxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intra- cellular reactive oxygen species and alteration of gene expression. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction The guanidine family of antiseptics has been popular for many decades. One of the family members is polyhexamethylene guani- dine phosphate (PHMG), a cationic polymer with low toxicity (class III-toxicity) compared to currently used disinfectants. PHMG is widely-used as an antiseptic in the textile, agriculture, and lum- ber sectors; in medicine and in waste water treatment because of its water solubility and flavorless, odorless and colorless properties (Kuznetsov, 2004; McDonnell and Russell, 1999; Muller and Kramer, 2005). It can chemically bind to or coat various chemicals to prepare anti-microbial materials that avoid the problems of con- ventional anti-microbial compounds, such as leakage or elution (Guan et al., 2008; Lee et al., 2000). Even low doses of PHMG, such as 13 lg/mL, have an effective bactericidal effect (Zhou et al., 2010). Epidemiologically, the lethal dose of 50% mortality (LD 50 ) value for PHMG has been reported as 450 mg/kg in mice and 630 mg/kg in rats (Condrashov, 1992). In 2011, an incident in Korea linked to the use of sterilizers used in humidifier equipment caused 91 cases of illness, mostly involv- ing pregnant women and children, and 28 deaths due to pulmon- ary fibrosis. Four of those who died were pregnant women. The Korean Ministry of Health and Welfare ordered a mandatory recall following a warning from the Korea Centers for Disease Control and Prevention (KCDC) against the use of any brand of sterilizer for humidifiers. The warning was based on preliminary tests on mice that revealed the development of pulmonary fibrosis or scar- ring of lungs. The principal active ingredients among the recalled sterilizer formulations were PHMG and oligo (2-(2-ethoxy)-eth- oxyethyl)-guanidinium-chloride (PGH) (Park, 2011). In Russia, acute poisoning of over 12,500 patients, of whom 9.4% died, oc- curred from August 2006 to May 2007. Many of the victims were regular consumers of surrogate and elicit alcohol not intended for human consumption that contained diethyl phthalate (DEP) and PHMG (Ostapenko et al., 2011). Despite these incidences, PHMG has not garnered enough re- search attention in connection with adverse effects on human health. It is presumed that consumption of surrogate alcohol con- taining PHMG hinders lipid metabolism that ultimately results in li- ver injuries, particularly toxic hepatitis. A study assessing the http://dx.doi.org/10.1016/j.tiv.2014.02.004 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Address: Department of Microbiology, School of Medicine, Soonchunhyang University, Cheonan, Chungnam 330-090, South Korea. Tel.: +82 41 570 2412; fax: +82 041 577 2415 (H.-Y. Song). Tel.: +82 570 2413; fax: +82 041 577 2415 (Y.-S. Kim). E-mail addresses: songmic@sch.ac.kr (H.-Y. Song), yongsikkim@sch.ac.kr (Y.-S. Kim). 1 These authors contributed equally to this study. Toxicology in Vitro 28 (2014) 684–692 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit