Longitudinal long term follow up investigation on the carcinogenic impact of polyhexamethylene guanidine phosphate in rat models.

Polyhexamethylene guanidine phosphate (PHMG-p) is a major component in humidifier disinfectants, which cause life-threatening lung injuries. However, to our knowledge, no published studies have investigated associations between PHMG-p dose and lung damage severity with long-term follow-up. Therefore, we evaluated longitudinal dose-dependent changes in lung injuries using repeated chest computed tomography (CT). Rats were exposed to low (0.2 mg/kg, n = 10), intermediate (1.0 mg/kg, n = 10), and high (5.0 mg/kg, n = 10) doses of PHMG-p. All rats underwent repeated CT scans after 10 and 40 weeks following the first exposure. All CT images were quantitatively analyzed using commercial software. Inflammation/fibrosis and tumor counts underwent histopathological evaluation. In both radiological and histopathologic results, the lung damage severity increased as the PHMG-p dose increased. Moreover, the number, size, and malignancy of the lung tumors increased as the dose increased. Bronchiolar-alveolar hyperplasia developed in all groups. During follow-up, there was intergroup variation in bronchiolar-alveolar hyperplasia progression, although bronchiolar-alveolar adenomas or carcinomas usually increase in size over time. Thirty-three carcinomas were detected in the high-dose group in two rats. Overall, lung damage from PHMG-p and the number and malignancy of lung tumors were shown to be dose-dependent in a rat model using repeated chest CT scans during a long-term follow-up.

Evidence integration on health damage for humidifier disinfectant exposure and legal presumption of causation.

OBJECTIVES: Inhalation exposure to humidifier disinfectants has resulted to various types of health damages in Korea. To determine the epidemiological correlation necessary for presuming the legal causation, we aimed to develop a method to synthesize the entire evidence. METHODS: Epidemiological and toxicological studies are systematically reviewed. Target health problems are selected by criteria such as frequent complaints of claimants. Relevant epidemiologic studies are reviewed and the risk of bias and confidence level of the total evidence are evaluated. Toxicological literature reviews are conducted on three lines of evidence including hazard information, animal studies, and mechanistic studies, considering the source-to-exposure-to-outcome continuum. The confidence level of the body of evidence is then translated into the toxicological evidence levels for the causality between humidifier disinfectant exposure and health effects. Finally, the levels of epidemiological and toxicological evidence are synthesized. RESULTS: Under the Special Act revised in 2020, if the history of exposure and the disease occurred/worsened after exposure were approved, and the epidemiological correlation between the exposure and disease was verified, the legal causation is presumed unless the company proves the evidence against it. The epidemiological correlation can be verified through epidemiological investigations, health monitoring, cohort investigations and/or toxicological studies. It is not simply as statistical association as understood in judicial precedents, but a general causation established by the evidence as a whole, i.e., through weight-of-the-evidence approach. CONCLUSIONS: The weight-of-the-evidence approach differs from the conclusive single study approach and this systematic evidence integration can be used in presumption of causation.

Effects of stabilizer magnesium nirate on CMIT/MIT-induced respiratory toxicity.

Despite a humidifier disinfectant (HD) product containing chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) with approximately 22% magnesium nitrate as a stabilizer, no report on the effects of magnesium nitrate on the respiratory toxicity of CMIT/MIT is available. In this study, Kathon CG and Proclin 200, containing approximately 1.5% CMIT/MIT with different magnesium nitrate concentrations (22.6% and 3%, respectively), were used to compare respiratory effects after intratracheal instillation (ITI) in C57BL/6 mice. C57BL/6 mice were randomized into groups of saline control, magnesium nitrate, Kathon CG, and Proclin 200 with 1.14 mg/kg of CMIT/MIT as the active ingredient, and administration was performed 6 times in a 2-3 day-interval in 2 weeks in all groups. Differential cell count analysis, cytokine analysis, and histological analysis of lung tissue were performed to characterize the injury features. Both Kathon and Proclin 200 induced an increase in inflammatory cell levels in the bronchoalveolar lavage (BAL) fluid, in particular, eosinophils and type 2 T helper cell (Th2)-secreted cytokines. All histopathological changes including granulomatous inflammation, mixed inflammatory cell infiltration, mucous cell hyperplasia, eosinophil infiltration, and pulmonary fibrosis were induced with similar frequency and severity in Kathon CG and Proclin 200 groups. Our results suggested that magnesium nitrate did not affect CMIT/MIT-induced lung injury in the intratracheally instilled model. Further inhalation studies are needed to determine the distribution and toxicity differences of CMIT/MIT in the lungs according to the magnesium nitrate concentration.

Biodistribution and respiratory toxicity of chloromethylisothiazolinone/methylisothiazolinone following intranasal and intratracheal administration.

A variety of isothiazolinone-containing small molecules have been registered and used as chemical additives in many household products. However, their biodistribution and potential harmful effects on human health, especially respiratory effects, were not yet identified in sufficient detail. The purpose of this study was to investigate whether a biocide comprising a mixture of chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) could reach the lungs and induce lung injury when exposure occurs by two administration routes involving the respiratory tract: intratracheal and intranasal instillation. To investigate the biodistribution of CMIT/MIT, we quantified the uptake of 14C-labeled CMIT/MIT in experimental animals for up to seven days after intratracheal and intranasal instillation. In the toxicity study, lung injury was assessed in mice using total inflammatory cell count in bronchoalveolar lavage fluid (BALF) and lung histopathology. The results of the biodistribution study indicated that CMIT/MIT were rapidly distributed throughout the respiratory tract. Using quantitative whole-body autoradiogram analysis, we confirmed that following intranasal exposure, CMIT/MIT reached the lungs via the respiratory tract (nose-trachea-lung). After 5 min post intratracheal and intranasal instillation, the amount of radiotracer ([14C]CMIT/MIT) in the lungs was 2720 ng g-1 and 752 ng g-1 tissue, respectively, and lung damage was observed. A higher amount of the radiotracer resulted in higher toxicity. Both intratracheal and intranasal instillation of CMIT/MIT increased inflammatory cell counts in the BALF and induced injuries in the alveoli. The frequency and the severity scores of injuries caused by intratracheal instillation were approximately-four to five times higher than those induced by intranasal instillation. Therefore, we concluded that CMIT/MIT could reach the lungs following nasal and intratracheal exposure and cause lung injuries, and the extent of injury was dependent on the exposure dose.

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.

Analysis of lung cancer-related genetic changes in long-term and low-dose polyhexamethylene guanidine phosphate (PHMG-p) treated human pulmonary alveolar epithelial cells.

BACKGROUND: Lung injury elicited by respiratory exposure to humidifier disinfectants (HDs) is known as HD-associated lung injury (HDLI). Current elucidation of the molecular mechanisms related to HDLI is mostly restricted to fibrotic and inflammatory lung diseases. In our previous report, we found that lung tumors were caused by intratracheal instillation of polyhexamethylene guanidine phosphate (PHMG-p) in a rat model. However, the lung cancer-related genetic changes concomitant with the development of these lung tumors have not yet been fully defined. We aimed to discover the effect of long-term exposure of PHMG-p on normal human lung alveolar cells. METHODS: We investigated whether PHMG-p could increase distorted homeostasis of oncogenes and tumor-suppressor genes, with long-term and low-dose treatment, in human pulmonary alveolar epithelial cells (HPAEpiCs). Total RNA sequencing was performed with cells continuously treated with PHMG-p and harvested after 35 days. RESULTS: After PHMG-p treatment, genes with transcriptional expression changes of more than 2.0-fold or less than 0.5-fold were identified. Within 10 days of exposure, 2 protein-coding and 5 non-coding genes were selected, whereas in the group treated for 27-35 days, 24 protein-coding and 5 non-coding genes were identified. Furthermore, in the long-term treatment group, 11 of the 15 upregulated genes and 9 of the 14 downregulated genes were reported as oncogenes and tumor suppressor genes in lung cancer, respectively. We also found that 10 genes of the selected 24 protein-coding genes were clinically significant in lung adenocarcinoma patients. CONCLUSIONS: Our findings demonstrate that long-term exposure of human pulmonary normal alveolar cells to low-dose PHMG-p caused genetic changes, mainly in lung cancer-associated genes, in a time-dependent manner.

MTF1 Is Essential for the Expression of MT1B, MT1F, MT1G, and MT1H Induced by PHMG, but Not CMIT, in the Human Pulmonary Alveolar Epithelial Cells.

The inhalation of humidifier disinfectants (HDs) is linked to HD-associated lung injury (HDLI). Polyhexamethylene guanidine (PHMG) is significantly involved in HDLI, but the correlation between chloromethylisothiazolinone (CMIT) and HDLI remains ambiguous. Additionally, the differences in the molecular responses to PHMG and CMIT are poorly understood. In this study, RNA sequencing (RNA-seq) data showed that the expression levels of metallothionein-1 (MT1) isoforms, including MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X, were increased in human pulmonary alveolar epithelial cells (HPAEpiCs) that were treated with PHMG but not in those treated with CMIT. Moreover, upregulation of MT1B, MT1F, MT1G, and MT1H was observed only in PHMG-treated HPAEpiCs. The protein expression level of metal regulatory transcription factor 1 (MTF1), which binds to the promoters of MT1 isoforms, was increased in PHMG-treated HPAEpiCs but not in CMIT-treated HPAEpiCs. However, the expression of early growth response 1 (EGR1) and nuclear receptor superfamily 3, group C, member 1 (NR3C1), other transcriptional regulators involved in MT1 isomers, were increased regardless of treatment with PHMG or CMIT. These results suggest that MTF1 is an essential transcription factor for the induction of MT1B, MT1F, MT1G, and MT1H by PHMG but not by CMIT.

Exposure to environmental chemicals among Korean adults-updates from the second Korean National Environmental Health Survey (2012-2014).

National biomonitoring program can offer solid scientific evidence on exposure profiles of environmental chemicals at a national level, and provide a snapshot of changing exposure level over time. Therefore, several countries have maintained such programs for developing environmental health policies. The Korean National Environmental Health Survey (KoNEHS) was designed to understand the level of human exposure to environmental chemicals by time and location, and to identify possible sources of such exposure. The 2nd stage of KoNEHS, which was conducted between 2012 and 2014, examined a total of 6478 adult subjects over 19 years of age, and measured 21 environmental chemicals of major policy concern. Compared to the findings from the first stage monitoring (2009-2011), slightly higher levels of blood lead were observed, while those of mercury remained similar. Blood metal concentrations, however, were higher than those reported from national biomonitoring programs of United States, Germany and Canada. The urinary concentrations of phthalates metabolites were lower, but those of t,t-muconic acid and BPA were higher than those reported in the first stage survey. The urinary cotinine level decreased perhaps reflecting general declining patterns of first- and second-hand smoking. The results of the second stage survey were made available for public use since April 2016. Some policy efforts appear to be at least in part effective on mitigating chemical exposure among people, e.g., urinary phthalate metabolites and cotinine, while further confirmations are warranted. In-depth assessments will be conducted to identify vulnerable groups and important exposure pathways.

Investigation of disinfectants for foot-and-mouth disease in the Republic of Korea.

Disinfectants for foot-and-mouth disease were sprayed on livestock barns and roads from early February to May 2011. Although 90% of the disinfectant was concentrated on the roads, 10% was sprayed on cattle sheds and other sites where foot-and-mouth disease occurred. Since the outbreak of foot-and-mouth disease in November 2010, there has been a steady increase in disinfectant use. Consequently, its adverse environmental effects have prompted government officials to take preventive measures. The major chemical components of the disinfectants are citric acid, potassium sulfate base complex, quaternary ammonium compound, malic acid, and glutaraldehyde, ranging in amounts from tons to hundreds of tons. The exact amount of each component of the disinfectants could not be identified because the types of components used in the different commercial formulations overlapped. In this review, we obtained information on disinfectants that are widely used nationwide, including the types of major chemical components and their respective toxicities (both human and ecological).

Aquatic toxicity assessment of phosphate compounds.

OBJECTIVES: Tricalcium phosphate and calcium hydrogenorthophosphate are high production volume chemicals, mainly used as foodstuff additives, pharmaceuticals, lubricants, synthetic resin, and disinfectants. Phosphate has the potential to cause increased algal growth leading to eutrophication in the aquatic environment. However, there is no adequate information available on risk assessment or acute and chronic toxicity. The aim of this research is to evaluate the toxic potential of phosphate compounds in the aquatic environment. METHODS: An aquatic toxicity test of phosphate was conducted, and its physico-chemical properties were obtained from a database recommended in the Organization for Economic Cooperation and Development (OECD) guidance manual. An ecotoxicity test using fish, Daphnia, and algae was conducted by the good laboratory practice facility according to the OECD TG guidelines for testing of chemicals, to secure reliable data. RESULTS: THE RESULTS OF THE ECOTOXICITY TESTS OF TRICALCIUM PHOSPHATE AND CALCIUM HYDROGENORTHOPHOSPHATE ARE AS FOLLOWS: In an acute toxicity test with Oryzias latipes, 96 hr 50% lethal concentration (LC(50)) was >100 (measured:>2.14) mg/L and >100 (measured: >13.5) mg/L, respectively. In the Daphnia test, 48 hr 50% effective concentration (EC(50)) was >100 (measured: >5.35) mg/L and >100 (measured: >2.9) mg/L, respectively. In a growth inhibition test with Pseudokirchneriella subcapitata, 72 hr EC(50) was >100 (measured: >1.56) mg/L and >100 (measured: >4.4) mg/L, respectively. CONCLUSIONS: Based on the results of the ecotoxicity test of phosphate using fish, Daphnia, and algae, L(E)C(50) was above 100 mg/L (nominal), indicating no toxicity. In general, the total phosphorus concentration including phosphate in rivers and lakes reaches levels of several ppm, suggesting that phosphate has no toxic effects. However, excessive inflow of phosphate into aquatic ecosystems has the potential to cause eutrophication due to algal growth.

Assay-dependent effect of silver nanoparticles to Escherichia coli and Bacillus subtilis.

We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of dissolved silver ions were influenced profoundly by the microbial test method employed.

Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus.

In this study, the microbial toxicities of metal oxide nanoparticles were evaluated for Escherichia coli, Bacillus subtilis, and Streptococcus aureus in laboratory experiments. The nanoparticles tested were CuO, NiO, ZnO, and Sb(2)O(3). The metal oxide nanoparticles were dispersed thoroughly in a culture medium, and the microorganisms were cultivated on Luria-Bertani agar plates containing different concentrations of metal oxide nanoparticles. The bacteria were counted in terms of colony forming units (CFU). The CFU was reduced in a culture medium containing metal oxide NP, and the dose-response relationship was characterized. CuO nanoparticles were found to be the most toxic among the tested nanoparticles, followed by ZnO (except S. aureus), NiO, and Sb(2)O(3) nanoparticles. We determined that the intrinsic toxic properties of heavy metals are also associated with the toxicity of metal oxide nanoparticles. Ion toxicity was also evaluated to determine the effects of metal ions dissolved from metal oxide NPs, and the toxicity induced from the dissolved ions was determined to be negligible herein. To the best of our knowledge, this is the first study of the toxicity of NiO and Sb(2)O(3) NPs on microorganisms. We also discuss the implications of our findings regarding the effects of the intrinsic toxic properties of heavy metals, and concluded that the apparent toxicities of metal oxide NPs can largely be understood as a matter of particle toxicity.

Assessment of toxic heavy metals in urban lake sediments as related to urban stressor and bioavailability.

A suite of heavy metals was monitored at sediments and overlying water in three urban lakes located in Seoul, Korea during spring season 2006. Metals measured were zinc, arsenic, chromium, copper, nickel, and cadmium. All metal concentrations in urban lake sediments were much higher than those in natural lake sediments. Elevated metal levels in urban lake sediments are associated with urban runoff, including street dust polluted by heavy metals. Metals in sediments from urban lakes were extracted with a weak electrolyte solution (0.1 M Ca(NO(3))(2)) to predict the toxicity of metals. Among the six heavy metals studied, Cu was the most extractable, followed by Ni and Zn. Ca(NO(3))(2)-extractable metal recoveries has a good relationship with metal toxicity based on Chironomus riparius bioassay. This study showed that urban stressors such as vehicle emissions could increase the concentration of heavy metals in urban lake sediments. In addition, there is a positive relationship between sediment toxicity by using C. riparius bioassay and Ca(NO(3))(2)-extractability of heavy metals from sediments.