Polyhexamethylene Guanidine Phosphate Enhanced Procoagulant Activity through Oxidative-Stress-Mediated Phosphatidylserine Exposure in Platelets.

Polyhexamethylene guanidine phosphate (PHMG-p) is a common biocidal disinfectant that is widely used in industry and household products. However, PHMG-p was misused as a humidifier disinfectant (HD) in South Korea, which had fatal health effects. Various health problems including cardiovascular diseases were observed in HD-exposed groups. However, the potential underlying mechanism of HD-associated cardiovascular diseases is poorly understood. Here, we examined the procoagulant activity of platelets caused by PHMG-p and clarified the underlying mechanism. PHMG-p enhanced phosphatidylserine (PS) exposure through alteration of phospholipid transporters, scramblase, and flippase. Intracellular calcium elevation, intracellular ATP depletion, and caspase-3 activation appeared to underlie phospholipid transporter dysregulation caused by PHMG-p, which was mediated by oxidative stress and mitochondrial dysfunction. Notably, antioxidant enzyme catalase and calcium chelator EGTA reversed PHMG-p-induced PS exposure and thrombin generation, confirming the contributive role of oxidative stress and intracellular calcium in the procoagulant effects of PHMG-p. These series of events led to procoagulant activation of platelets, which was revealed as enhanced thrombin generation. Collectively, PHMG-p triggered procoagulant activation of platelets, which may promote prothrombotic risks and cardiovascular diseases. These findings improve our understanding of HD-associated cardiovascular diseases.

Hematotoxic Effect of Respiratory Exposure to PHMG-p and Its Integrated Genetic Analysis.

Polyhexamethylene guanidine phosphate (PHMG-p), the main ingredient of humidifier disinfectants, circulates systemically through the lungs; however, its toxicological assessment has been primarily limited to pulmonary disease. Herein, we investigated the possible abnormalities in hematopoietic function 20 weeks after intratracheal instillation of PHMG-p in a rat model. Notable abnormalities were found out in the peripheral blood cell count and bone marrow (BM) biopsy, while RNA sequencing of BM tissue revealed markedly altered gene expression. Furthermore, signaling involved in hematopoietic dysfunction was predicted by analyzing candidate genes through Ingenuity Pathway Analysis (IPA) program. Respiratory PHMG-p exposure significantly decreased monocyte and platelet (PLT) counts and total protein, while significantly increasing hemoglobin and hematocrit levels in peripheral blood. Histopathological analysis of the BM revealed a reduced number of megakaryocytes, with no significant differences in spleen and liver weight to body weight. Moreover, PHMG-p exposure significantly activated estrogen receptor signaling and RHOA signaling, and inhibited RHOGDI signaling. In IPA analysis, candidate genes were found to be strongly related to 'hematological system development and function' and 'hematological disease.' Accordingly, our results suggest that PHMG-p could affect hematopoiesis, which participates in monocyte differentiation and PLT production, and may induce hematologic diseases via the respiratory tract.

Intratracheal exposure to polyhexamethylene guanidine phosphate disrupts coordinate regulation of FXR-SHP-mediated cholesterol and bile acid homeostasis in mouse liver.

A public health crisis in the form of a significant incidence of fatal pulmonary disease caused by repeated use of humidifier disinfectants containing polyhexamethylene guanidine phosphate (PHMG) recently arose in Korea. Although the mechanisms of pulmonary fibrosis following respiratory exposure to PHMG are well described, distant-organ effect has not been reported. In this study, we investigated whether intratracheal administration of PHMG affects liver pathophysiology and metabolism. Our PHMG mouse model showed a significant decrease in liver cholesterol level. An mRNA-seq analysis of liver samples revealed an alteration in the gene expression associated with cholesterol biosynthesis and metabolism to bile acids. The expression of genes involved in cholesterol synthesis was decreased in a real-time PCR analysis. To our surprise, we found that the coordinate regulation of cholesterol and bile acid homeostasis was completely disrupted. Despite the decreased cholesterol synthesis and low bile acid levels, the farnesoid X receptor/small heterodimer partner pathway, which controls negative feedback of bile acid synthesis, was activated in PHMG mice. As a consequence, gene expression of Cyp7a1 and Cyp7b1, the rate-limiting enzymes of the classical and alternative pathways of bile acid synthesis, was significantly downregulated. Notably, the changes in gene expression were corroborated by the hepatic concentrations of the bile acids. These results suggest that respiratory exposure to PHMG could cause cholestatic liver injury by disrupting the physiological regulation of hepatic cholesterol and bile acid homeostasis.

Non-Destructive Monitoring via Electrochemical NADH Detection in Murine Cells.

Nicotinamide adenine dinucleotide (NADH) is an important cofactor involved in metabolic redox reactions in living cells. The detection of NADH in living animal cells is a challenge. We developed a one-step monitoring method for NADH via an electrocatalytic reaction that uses a surface-modified, screen-printed electrode (SPE) having a redox active monolayer 4'-mercapto-N-phenlyquinone diamine (NPQD) formed by a self-assembled monolayer (SAM) of an aromatic thiol, 4-aminothiophenol (4-ATP). This electrode has a limit of detection (LOD) of 0.49 µM and a sensitivity of 0.0076 ± 0.0006 µM/µA in cell culture media, which indicates that it retains its selectivity. The applicability of this NADH sensor was demonstrated for the first time by cell viability monitoring via NADH-sensing in cell culture supernatants.

A New Murine Liver Fibrosis Model Induced by Polyhexamethylene Guanidine-Phosphate.

Liver fibrosis is part of the wound healing process to help the liver recover from the injuries caused by various liver-damaging insults. However, liver fibrosis often progresses to life-threatening cirrhosis and hepatocellular carcinoma. To overcome the limitations of current in vivo liver fibrosis models for studying the pathophysiology of liver fibrosis and establishing effective treatment strategies, we developed a new mouse model of liver fibrosis using polyhexamethylene guanidine phosphate (PHMG-p), a humidifier sterilizer known to induce lung fibrosis in humans. Male C57/BL6 mice were intraperitoneally injected with PHMG-p (0.03% and 0.1%) twice a week for 5 weeks. Subsequently, liver tissues were examined histologically and RNA-sequencing was performed to evaluate the expression of key genes and pathways affected by PHMG-p. PHMG-p injection resulted in body weight loss of ~15% and worsening of physical condition. Necropsy revealed diffuse fibrotic lesions in the liver with no effect on the lungs. Histology, collagen staining, immunohistochemistry for smooth muscle actin and collagen, and polymerase chain reaction analysis of fibrotic genes revealed that PHMG-p induced liver fibrosis in the peri-central, peri-portal, and capsule regions. RNA-sequencing revealed that PHMG-p affected several pathways associated with human liver fibrosis, especially with upregulation of lumican and IRAK3, and downregulation of GSTp1 and GSTp2, which are closely involved in liver fibrosis pathogenesis. Collectively we demonstrated that the PHMG-p-induced liver fibrosis model can be employed to study human liver fibrosis.

Adverse postnatal developmental effects in offspring from humidifier disinfectant biocide inhaled pregnant rats.

Inhalation exposure to polyhexamethylene guanidine phosphate (PHMG-P), one of the primary biocides used in humidifier disinfectants, caused a fatal pulmonary disease in Korea. Pregnant women were also exposed to PHMG-P, and subsequent studies showed that PHMG-P inhalation during pregnancy adversely affects their health and embryo-fetal development. However, the postnatal developmental effects after birth on prenatally PHMG-P-exposed offspring have not yet been investigated. Therefore, in this study, we aimed to examine the postnatal development of prenatally PHMG-P-exposed offspring. Pregnant rats (22 or 24 females per group) were exposed to PHMG-P during pregnancy in a whole-body inhalation chamber at the target concentrations of 0, 0.14, 1.60, and 3.20 mg/m3. After parturition, the prenatally exposed offspring were transferred to non-exposed surrogate mothers to minimize the secondary effects of severe maternal toxicities. Postnatal development of offspring was then examined with a modified extended one-generation reproductive toxicity study design. At 3.20 mg/m3 PHMG-P, increased perinatal death rates and decreased viability index (postnatal survival of offspring between birth and postnatal day 4) were observed. In addition, F1 offspring had lower body weight at birth that persisted throughout the study. PHMG-P-exposed pregnant rats also had severe systemic toxicities and increased gestation period. At 1.60 mg/m3 PHMG-P, a decreased viability index was also observed with systemic toxicities of PHMG-P-exposed pregnant rats. These results indicate that prenatal PHMG-P exposure adversely affects the offspring's future health and could be used for human risk assessment.

Causal relationship between humidifier disinfectant exposure and Th17-mediated airway inflammation and hyperresponsiveness.

In this study, we investigated whether humidifier disinfectants (HDs) induce asthmatic airway inflammation in an animal model and compared the features of HD-induced inflammatory symptoms with ovalbumin (OVA)-induced allergic asthma. Mice were intratracheally instilled three times with either the control or 0.1, 0.3, or 0.5 mg/kg of polyhexamethylene guanidine phosphate (PHMG-P). To characterize asthmatic features, the following parameters were analyzed: (i) differential cell counts and cytokine expression in the bronchoalveolar lavage fluid (BALF); (ii) presence of mucus-producing goblet cells and pulmonary eosinophilic infiltration in the lungs; (iii) serum immunoglobulin levels; and (iv) airway hyperresponsiveness (AHR). RNA-Seq and bioinformatics tools were used to investigate whether PHMG-P altered asthma-related gene expression in lung tissues. The PHMG-P exposure groups showed higher peribronchial/perivascular inflammation, elevated goblet cell hyperplasia, and inhaled methacholine-induced airway resistance. Additionally, IL-13 and IL-17 in BALF were significantly increased in the PHMG-P exposure groups. However, there were no significant differences in total serum IgE and BALF IL-4 and IL-5 levels in the PHMG-P exposure groups compared to the control group. PHMG-P exposure modulated the expression of genes related to Th17 signaling pathways including the IL-17A, IL-23, and STAT3 signaling pathways, but not the Th2 signaling pathway. Altogether, our results suggest that repeated exposure to low does PHMG-P induces asthma-like symptoms and is thus a possible risk factor for developing asthma. The PHMG-P-induced asthmatic airway inflammation showed a different pattern from that found in typical allergic asthma and may be related to irritant-induced airway inflammation and hyperresponsiveness characterized by Th2-low, Th17-related, IgE-independent, and mixed granulocytic features.

Problems with diagnostic criteria for humidifier disinfectant lung injury (HDLI): two cases of radiologically improved HDLI.

BACKGROUND: In Korea, to investigate the casual relationship between humidifier disinfectant and lung disease, four rounds of investigation and judgment were conducted. During this investigation, two adults who performed lung biopsy were recognized for their relevance between humidifier disinfectants and lung disease. At first, we did not think of the relationship to humidifier disinfectant because chest computed tomography (CT) finding of 2 cases were improved. However, they performed lung biopsy and it showed typical humidifier disinfectant lung injury (HDLI) pathologic findings, they could be recognized as HDLI. We report these cases here. CASE PRESENTATION: We selected 2 cases from the fourth-round investigation at Kangbuk Samsung Hospital. Patient of case 1 used humidifier disinfectants since September 2010. The patient was admitted 6 months later to the intensive care unit (ICU) due to severe dyspnea. Pathology following a lung biopsy revealed typical HDLI finding which was determined to be due to humidifier disinfectant exposure. Patient of case 2 used humidifier disinfectant from 2001 to 2008 for about 3 months each winter. The patient's cough and sputum production symptoms began in December of 2007. The patient was admitted to the respiratory medicine department due to worsening dyspnea. Pathology following a lung biopsy revealed typical HDLI finding. This was determined to have been caused by humidifier disinfectant exposure. CONCLUSIONS: Because the typical radiologic findings associated with HDLI can improve over time, it is necessary to consider the revision of current diagnostic criteria that the presence of radiologic findings is important.

Problems with diagnostic criteria for humidifier disinfectant lung injury (HDLI): two cases of radiologically improved HDLI

Reproductive and developmental toxicity screening of polyhexamethylene guanidine phosphate by oral gavage in rats.

Polyhexamethylene guanidine phosphate (PHMG-P) has effective antimicrobial activity against various microorganisms and has been widely used as a biocide in commercial products. However, its use as a humidifier disinfectant has provoked fatal idiopathic lung disease in South Korea, especially in pregnant or postpartum women and their young children. PHMG-P-related toxicological studies of reproduction and development in experimental animals have not been identified, and thus, we investigated the potential effects of early-stage oral exposure to PHMG-P by assessing its toxicological properties. PHMG-P was repeatedly administered by oral gavage at dose levels of 0, 13, 40 and 120 mg/kg to Sprague-Dawley rats during the pre-mating, mating, gestation and early lactation periods, and then general systemic and reproductive/developmental toxicities were investigated. At 120 mg/kg, PHMG-P-related toxicities including subdued behavior, thin appearance, decreased body weight, decreased food consumption and decreased F1 pup body weight were observed. Based on the results of this study, the no-observed-adverse-effect levels (NOAELs) of PHMG-P for both general systemic effects and development are considered to be 40 mg/kg/day.

Time-course transcriptomic alterations reflect the pathophysiology of polyhexamethylene guanidine phosphate-induced lung injury in rats.

Objective: Humidifier-disinfectant-induced lung injury is a new syndrome associated with a high mortality rate and characterized by severe hypersensitivity pneumonitis, acute interstitial pneumonia, or acute respiratory distress syndrome. Polyhexamethylene guanidine phosphate (PHMG-P), a guanidine-based antimicrobial agent, is a major component associated with severe lung injury. In-depth studies are needed to determine how PHMG-P affects pathogenesis at the molecular level. Therefore, in this study, we analyzed short-term (4 weeks) and long-term (10 weeks) PHMG-P-exposure-specific gene-expression patterns in rats to improve our understanding of time-dependent changes in fibrosis.Materials and methods: Gene-expression profiles were analyzed in rat lung tissues using DNA microarrays and bioinformatics tools.Results: Clustering analysis of gene-expression data showed different gene-alteration patterns in the short- and long-term exposure groups and higher sensitivity to gene-expression changes in the long-term exposure group than in the short-term exposure group. Supervised analysis revealed 34 short-term and 335 long-term exposure-specific genes, and functional analysis revealed that short-term exposure-specific genes were involved in PHMG-P-induced initial inflammatory responses, whereas long-term exposure-specific genes were involved in PHMG-P-related induction of chronic lung fibrosis.Conclusion: The results of transcriptomic analysis were consistent with lung histopathology results. These findings indicated that exposure-time-specific changes in gene expression closely reflected time-dependent pathological changes in PHMG-P-induced lung injury.

A review of current studies on cellular and molecular mechanisms underlying pulmonary fibrosis induced by chemicals.

Several studies showed that the inflammatory and fibrotic responses induced by polyhexamethylene guanidine phosphate (PHMG-p) were similar to those observed for idiopathic pulmonary fibrosis in South Korea in 2011. "Omic" technologies can be used to understand the mechanisms underlying chemical-induced diseases. Studies to determine the toxicity of chemicals may facilitate understanding of the mechanisms underlying the development of pulmonary fibrosis at a molecular level; thus, such studies may provide information about the toxic characteristics of various substances. In this review, we have outlined the cellular and molecular mechanisms underlying idiopathic pulmonary fibrosis and described pulmonary fibrosis induced by various chemicals, including bleomycin, paraquat, and PHMG-p, based on the results of studies performed to date.

A review of current studies on cellular and molecular mechanisms underlying pulmonary fibrosis induced by chemicals

Several studies showed that the inflammatory and fibrotic responses induced by polyhexamethylene guanidine phosphate (PHMG-p) were similar to those observed for idiopathic pulmonary fibrosis in South Korea in 2011. “Omic” technologies can be used to understand the mechanisms underlying chemical-induced diseases. Studies to determine the toxicity of chemicals may facilitate understanding of the mechanisms underlying the development of pulmonary fibrosis at a molecular level; thus, such studies may provide information about the toxic characteristics of various substances. In this review, we have outlined the cellular and molecular mechanisms underlying idiopathic pulmonary fibrosis and described pulmonary fibrosis induced by various chemicals, including bleomycin, paraquat, and PHMG-p, based on the results of studies performed to date.

A review of current studies on cellular and molecular mechanisms underlying pulmonary fibrosis induced by chemicals

Several studies showed that the inflammatory and fibrotic responses induced by polyhexamethylene guanidine phosphate (PHMG-p) were similar to those observed for idiopathic pulmonary fibrosis in South Korea in 2011. “Omic” technologies can be used to understand the mechanisms underlying chemical-induced diseases. Studies to determine the toxicity of chemicals may facilitate understanding of the mechanisms underlying the development of pulmonary fibrosis at a molecular level; thus, such studies may provide information about the toxic characteristics of various substances. In this review, we have outlined the cellular and molecular mechanisms underlying idiopathic pulmonary fibrosis and described pulmonary fibrosis induced by various chemicals, including bleomycin, paraquat, and PHMG-p, based on the results of studies performed to date.