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. 2019 Oct;32(4):311-317.
doi: 10.1293/tox.2019-0049. Epub 2019 Aug 8.

Immunohistochemical characterization of oxidative stress in the lungs of rats exposed to the humidifier disinfectant polyhexamethylene guanidine hydrochloride

Yong-Hoon Lee  1 , Dong-Seok Seo  1 , Mi Ju Lee  1 , Hyo-Geun Cha  1
Affiliations

Immunohistochemical characterization of oxidative stress in the lungs of rats exposed to the humidifier disinfectant polyhexamethylene guanidine hydrochloride

Yong-Hoon Lee et al. J Toxicol Pathol. 2019 Oct.

Abstract

Polyhexamethylene guanidine hydrochloride (PHMG-HCl), an antimicrobial additive in humidifier disinfectants, was associated with the pulmonary disease outbreak in South Korea. However, PHMG-mediated oxidative stress has only been studied in vitro. Here, we evaluated PHMG-induced oxidative stress in the lungs of rats exposed to PHMG-HCl. Male F344 rats were exposed to different concentrations of PHMG-HCl for 13-weeks via whole-body inhalation. Histopathological examination of the exposed rats showed the presence of lung lesions, including alveolar/interstitial fibrosis with inflammatory cell infiltration, bronchioalveolar hyperplasia, bronchiolar/alveolar squamous metaplasia, bronchial/bronchiolar epithelial detachment, and alveolar hemorrhage. Immunohistochemical analysis showed that 4-hydroxynonenal (4-HNE) was expressed in the bronchiolar epithelium, mainly in Clara cells and macrophages of the fibrotic tissue. The number of 4-HNE-positive cells increased significantly in a dose-dependent manner. This is the first in vivo study to report PHMG-induced oxidative stress. Our study provides clues to elucidate the mechanisms underlying PHMG-induced damage in patients affected by humidifier disinfectants.

Keywords: 4-hydroxynonenal (4-HNE); humidifier disinfectants; immunohistochemistry; inhalation toxicity; oxidative stress; polyhexamethylene guanidine hydrochloride (PHMG-HCl).

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Figures

Fig. 1.
Fig. 1.
Histopathology of the lungs of rats exposed to polyhexamethylene guanidine hydrochloride (PHMG-HCl). (A, B) No abnormal lesion was observed in the control and low-dose groups, respectively. (C, D) Fibrosis with inflammatory cell infiltration (arrow) was observed in the alveoli of the mid- and high-dose groups, respectively. (E, F) Hyperplasia of Type II pneumocytes was observed in the alveoli of the mid- and high-dose groups, respectively. (G, H) Epithelial detachment (arrow) and exudate (arrowhead) were observed in the bronchi or bronchiole of the mid- and high-dose groups, respectively. (I) Squamous metaplasia (arrow) was observed in the alveoli and bronchioles of the high-dose groups. (J) Hemorrhage (arrow) was observed in the alveoli of high-dose groups. Bars=100 μm, Magnification: ×200, H&E staining.
Fig. 2.
Fig. 2.
Characteristics of fibrosis in the lungs of rats exposed to polyhexamethylene guanidine hydrochloride (PHMG-HCl). (A, B) In the control and low-dose groups, no collagen deposition was found in the fibrotic tissues of the lung. (C, D) In the mid-dose and high-dose groups, collagen deposition was observed and stained blue in the lung fibrotic tissues (arrow). Bars=100 μm, Magnification: ×200, MT staining.
Fig. 3.
Fig. 3.
Immunohistochemical analysis of the lungs of rats exposed to polyhexamethylene guanidine hydrochloride (PHMG-HCl). (A) Control rats showed no 4-hydroxynonenal (4-HNE) positive cells. A positive reaction for 4-HNE was detected in the cytoplasm of Clara cells of bronchiolar epithelium (arrow) in the (B) low-dose, and (C) mid-dose groups. (D) A positive 4-HNE reaction was found in the cytoplasm of macrophages (arrow) of fibrotic tissue in the mid-dose group. (E) Positive 4-HNE reaction was observed in the cytoplasm of Clara cells of bronchiolar epithelium (arrow) and type II pneumocytes (arrowhead) of the alveoli in the high-dose groups. (F) Positive 4-HNE reaction was found in the cytoplasm of macrophages (arrow) of fibrotic tissue in the high-dose group. Bars=50 μm, Magnification: ×400.
Fig. 4.
Fig. 4.
Comparison of the mean number of 4-hydroxynonenal (4-HNE) positive cells per unit area among each groups. Significant differences; **P<0.01 compared with the control group; ††P<0.01 compared with the low-dose group; ‡‡P<0.01 compared with the mid-dose group.
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