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Ambient particulate matter 2.5 (PM2.5) and total suspended particles (TSPs) are common airborne pollutants that cause respiratory and cardiovascular diseases. We investigated the differences of cytotoxicity and mechanism between PM2.5 and TSP activity in human alveolar epithelial A549 cells. Atmospheric samples from the central district of Seoul were collected and their chemical compositions were analyzed by inductively-coupled plasma mass spectrometry and ion chromatography. PM2.5 and TSP contained high concentrations of heavy metals (Cu, Fe, Zn, and Pb). The most abundant ions in PM2.5 were SO42−, NH4+, and NO3−. A549 cells were exposed to PM2.5 and TSP (25–200 µg/mL) for 24 h. TSP was more cytotoxic than PM2.5 per unit mass. PM2.5 induced oxidative stress, as evidenced by increased levels of a glutamate-cysteine ligase modifier, whereas low-concentration TSP increased hemeoxygenase-1 levels. PM2.5 and TSP did not affect c-Jun N-terminal kinase expression. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2) in PM2.5- and TSP-treated cells decreased significantly in the cytosol and increased in the nucleus. Thus, Nrf2 may be a key transcription factor for detoxifying environmental airborne particles in A549 cells. TSP and PM2.5 could activate the protective Kelch-like ECH-associated protein 1/Nrf2 pathway in A549 cells.

Recent research on in vitro systems has focused on mimicking the in vivo situation of cells within the respiratory system. However, few studies have predicted inhalation toxicity using conventional and simple submerged two-dimensional (2D) cell culture models. We investigated the conventional submerged 2-D cell culture model as a method for the prediction of acute inhalation toxicity. Median lethal concentration (LC50 ) (rat, inhalation, 4 h) and half maximal inhibitory concentration (IC50 ) (lung or bronchial cell, 24 h) data for 59 substances were obtained from the literature and by experiments. Cytotoxicity assays were performed on 44 substances with reported LC50 , but without IC50 , data to obtain the IC50 values. A weak correlation was observed between the IC50 and LC50 of all substances. Semi-volatile organic compounds (SVOCs) and non-VOCs (NVOCs) (16 substances) with a water solubility of ≥1 g/L were strongly correlated between 24-h IC50 and 4-h LC50 , and this had an excellent predictive ability to distinguish between Categories 1-3 and 4 (Globally Harmonized System classification for acute inhalation toxicity). Our results suggest that the submerged 2-D cell culture model may be used to predict in vivo acute inhalation toxicity for substances with a water solubility of ≥1 g/L in SVOCs and NVOCs.

Cetylpyridinium chloride (CPC), a quaternary ammonium compound and cationic surfactant, is used in personal hygiene products such as toothpaste, mouthwash, and nasal spray. Although public exposure to CPC is frequent, its pulmonary toxicity has yet to be fully characterized. Due to high risks of CPC inhalation, we aimed to comprehensively elucidate the in vitro and in vivo toxicity of CPC. The results demonstrated that CPC is highly cytotoxic against the A549 cells with a half-maximal inhibitory concentration (IC50 ) of 5.79 μg/ml. Following CPC exposure, via intratracheal instillation (ITI), leakage of lactate dehydrogenase, a biomarker of cell injury, was significantly increased in all exposure groups. Further, repeated exposure of rats to CPC for 28 days caused a decrease in body weight of the high-exposure group and the relative weights of the lungs and kidneys of the high recovery group, but no changes were evident in the histological and serum chemical analyses. The bronchoalveolar lavage fluid (BALF) analysis showed a significant increase in proinflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels. ITI of CPC induced focal inflammation of the pulmonary parenchyma in rats' lungs. Our study demonstrated that TNF-α was the most commonly secreted proinflammatory cytokine during CPC exposure in both in vitro and in vivo models. Polymorphonuclear leukocytes in the BALF, which are indicators of pulmonary inflammation, significantly increased in a concentration-dependent manner in all in vivo studies including the ITI, acute, and subacute inhalation assays, demonstrating that PMNs are the most sensitive parameters of pulmonary toxicity.

In water, sodium dichloroisocyanurate (NaDCC), a source for chlorine gas generation, releases free available chlorine in the form of hypochlorous acid, a strong oxidizing agent. NaDCC has been used as a disinfectant in humidifiers; however, its inhalation toxicity is a concern. Seven-week-old rats were exposed to NaDCC doses of 100, 500, and 2500 μg·kg−1body weight by intratracheal instillation (ITI) to investigate pulmonary toxicity. The rats were sacrificed at 1 d (exposure group) or 14 d (recovery group) after ITI. Despite a slight decrease in body weight after exposure, there was no statistically significant difference between the control and NaDCC-treated groups. A significant increase in the total protein level of the bronchoalveolar lavage fluid (BALF) was observed in the exposure groups. Lactate dehydrogenase leakage into the BALF increased significantly ( p < 0.01) in the exposure groups; however, recovery was observed after 14 d. The measurement of cytokines in the BALF samples indicated a significant increase in interleukin (IL)-6 in the exposure group and IL-8 in the recovery group. Histopathological examination revealed inflammatory foci and pulmonary edema around the terminal bronchioles and alveoli. This study demonstrated that ITI of NaDCC induced reversible pulmonary edema and inflammation without hepatic involvement in rats.

The guanidine family of antimicrobial agents, which includes polyhexamethylene guanidine phosphate (PHMG) and oligo(2-(2-ethoxy)ethoxyethyl) guanidinium chloride (PGH), and chlorophenol biocidal chemicals such as 2,4,4′-trichloro-2′-hydroxydiphenyl ether (triclosan) are used in various occupational and environmental biocidal applications. The excipient propylene glycol (PG) is used to dissolve the active ingredients. The skin sensitization (SS) potential of these substances has not been systemically investigated and is still debated. Moreover, mixtures of PHMG, PGH, or triclosan with PG have not been evaluated for SS potency. An in vivo assay known as the local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) was recently adopted as an alternative testing method and was used to address these issues. Via the LLNA: BrdU-FCM, PHMG, PGH, and triclosan were predicted to be sensitizers, while PG was predicted to be a nonsensitizer. In addition, d-limonene, which is used as a flavoring in various consumer products, was also predicted to be a sensitizer, although no unanimous conclusion has been reached regarding its SS potential. Mixtures of PHMG, PGH, triclosan, or d-limonene with PG at ratios of 9:1, 4:1, and 1:4 (w/w) were all positive in terms of SS potential, indicating that the PG excipient does not influence the SS predictions of these chemicals. Since humans can be occupationally and environmentally exposed to mixtures of excipients with active ingredients, the present study may give insight into further investigations of the SS potentials of various chemical mixtures.

The toxicity profiles of the widely used guanidine-based chemicals have not been fully elucidated. Herein, we evaluated the in vitro and in vivo toxicity of eight guanidine-based chemicals, focusing on inhalation toxicity. Among the eight chemicals, dodecylguanidine hydrochloride (DGH) was found to be the most cytotoxic (IC50: 0.39 &mu, g/mL), as determined by the water soluble tetrazolium salts (WST) assay. An acute inhalation study for DGH was conducted using Sprague-Dawley rats at 8.6 &plusmn, 0.41, 21.3 &plusmn, 0.83, 68.0 &plusmn, 3.46 mg/m3 for low, middle, and high exposure groups, respectively. The levels of lactate dehydrogenase, polymorphonuclear leukocytes, and cytokines (MIP-2, TGF-&beta, 1, IL-1&beta, TNF-&alpha, and IL-6) in the bronchoalveolar lavage fluid increased in a concentration-dependent manner. Histopathological examination revealed acute inflammation with necrosis in the nasal cavity and inflammation around terminal bronchioles and alveolar ducts in the lungs after DGH inhalation. The LC50 of DGH in rats after exposure for 4 h was estimated to be &gt, 68 mg/m3. Results from the inhalation studies showed that DGH was more toxic in male rats than in female rats. Overall, DGH was found to be the most cytotoxic chemical among guanidine-based chemicals. Exposure to aerosols of DGH could induce harmful pulmonary effects on human health.

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray‐form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1‐10 μg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 μg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC‐promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N‐acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 μg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 μg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 μg/kg) and TEG (1000 μg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF‐α), and interleukin 6 (IL‐6) in BALF. These results suggest that TEG can potentiate BAC‐induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray‐form products containing this chemical combination is potentially harmful to humans.

Many consumer products used in our daily lives result in inhalation exposure to a variety of chemicals, although the toxicities of the active ingredients are not well known; furthermore, simultaneous exposure to chemical mixtures occurs. Sodium metabisulfite (SM) and propylene glycol (PG) are used in a variety of products. Both the cytotoxicity and the sub-acute inhalation toxicity of each chemical and their mixtures were evaluated. Assays for cell viability, membrane damage, and lysosome damage demonstrated that SM over 100 μg/ml induced significant cytotoxicity; moreover, when PG, which was not cytotoxic, was mixed with SM, the cytotoxicity of the mixture was enhanced. Solutions of 1, 5, and 20% SM, each with 1% PG solution, were prepared, and the whole body of rats was exposed to aerosols of the mixture for 6 h/day, 5 days/week for 2 weeks. The rats were sacrificed 1 (exposure group) or 7 days (recovery group) after termination of the exposure. The actual concentration of SM in the low-, medium-, and high-exposure groups was 3.91 ± 1.26, 35.73 ± 6.01, and 80.98 ± 5.47 mg/m3, respectively, and the actual concentration of PG in each group was 6.47 ± 1.25, 8.68 ± 0.6, and 8.84 ± 1.77 mg/m3. The repeated exposure to SM and PG caused specific clinical signs including nasal sound, sneeze, and eye irritation which were not found in SM single exposure. In addition, the body weight of treatment group rats decreased compared to that of the control group rats in a time-dependent manner. The total protein concentration and lactate dehydrogenase activity in the bronchoalveolar lavage fluid (BALF) increased. Histopathological analysis of the lungs, liver, and nasal cavity was performed. Adverse effects were observed in the nasal cavity, with squamous cell metaplasia identified in the front of the nasal cavity in all high-exposure groups, which completely recovered 7 days after exposure was terminated. Whereas inhalation of SM for 2 weeks only reduced body weight in the high-dose group, inhalation of SM and PG mixtures for 2 weeks significantly decreased body weight and induced metaplasia of the respiratory epithelium into squamous cells in the medium- and high-dose groups. In conclusion, PG potentiated the toxicity of SM in human lung epithelial cells and the inhalation toxicity in rats.

Didecyldimethylammonium chloride (DDAC), an antimicrobial agent, has been reported to induce pulmonary toxicity in animal studies. DDAC is frequently used in spray-form household products in combination with ethylene glycol (EG). The purpose of this study was to evaluate the toxic interaction between DDAC and EG in the lung. DDAC at a sub-toxic dose (100 μg/kg body weight) was mixed with a non-toxic dose of EG (100 or 200 μg/kg body weight), and was administrated to rats via intratracheal instillation. Lactate dehydrogenase activity and total protein content in the bronchoalveolar lavage fluid (BALF) were not changed by singly treated DDAC or EG, but significantly enhanced at 1 d after treatment with the mixture, with the effect dependent on the dose of EG. Total cell count in BALF was largely increased and polymorphonuclear leukocytes were predominantly recruited to the lung in rats administrated with the mixture. Inflammatory cytokines, tumor necrosis factor-alpha and interleukin-6 also appeared to be increased by the mixture of DDAC and EG (200 μg/kg body weight) at 1 d post-exposure, which might be associated with the increase in inflammatory cells in lung. BALF protein content and inflammatory cell recruitment in the lung still remained elevated at 7 d after the administration of DDAC with the higher dose of EG. These results suggest that the combination of DDAC and EG can synergistically induce pulmonary cytotoxicity and inflammation, and EG appears to amplify the harmful effects of DDAC on the lung. Therefore pulmonary exposure to these two chemicals commonly found in commercial products can be a potential hazard to human health.

Benzalkonium chloride (BAC), a disinfectant, and triethylene glycol (TEG), an organic solvent/sanitizer, are frequently combined in commercially available household sprays. To assess the respiratory effect of this combination, Sprague-Dawley rats were exposed to an aerosol containing BAC (0.5%, w/v) and TEG (10%, w/v) for up to 2 weeks in a whole-body inhalation chamber. BAC (4.1–4.5 mg/m3, sprayed from 0.5% solution) promoted pulmonary cell damage and inflammation as depicted by the increase in total protein, lactate dehydrogenase, polymorphonuclear leukocytes, and macrophage inflammatory protein-2 in the bronchoalveolar lavage fluid, whereas TEG (85.3–94.5 mg/m3, sprayed from 10% solution) did not affect the lung. Rats exposed to the BAC/TEG mixture for 2 weeks showed severe respiratory symptoms (sneezing, wheezing, breath shortness, and chest tightness), but no lung damage or inflammation was observed. However, significant ulceration and degenerative necrosis were observed in the nasal cavities of rats repeatedly exposed to the BAC/TEG mixture. The mass median aerodynamic diameters of the aqueous, BAC, TEG and BAC/TEG aerosols were 1.24, 1.27, 3.11 and 3.24 μm, respectively, indicating that TEG-containing aerosols have larger particles than those of the aqueous and BAC alone aerosols. These results suggest that the toxic effects of BAC and BAC/TEG aerosols on the different respiratory organs may be associated with the difference in particle diameter, since particle size is important in determining the deposition site of inhaled materials.

Background Worldwide restrictions in animal use for research have driven efforts to develop alternative methods. Objective The study aimed to test the efficacy of the macrophage inflammatory protein-1beta (MIP-1beta) assay for testing chemicals' skin-sensitizing capacity. Methods The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC(50) (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1beta level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1beta production rates. The MIP-1beta production rate was expressed as the relative increase in MIP-1beta production in response to chemical treatment compared with vehicle treatment. Results and conclusion When the threshold MIP-1beta production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1beta production. These results indicate that MIP-1beta could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

Several air fresheners and deodorants that contain ethylene glycol were found (1-2% (w/v)) in commercially available products. Ethylene glycol is a liquid used as a surfactant or an emulsifier in household products. The aim of this study is to determine the inhalation toxicity of ethylene glycol. The effect of whole-body inhalation to EG was performed for an inhalation study of rat. Specific pathogen-free male Sprague-Dawley rats (7 weeks old) were exposed to ethylene glycol in a stainless steel whole body inhalation chamber with a capacity of 1 m3 (Sibata Model VT3-X15 Japan). For acute and sub-acute rat inhalation toxicities, the differences of body weights were not statistically significant comparing with those of control rats. In sub-acute rat inhalation toxicity, the relative kidney weights were significantly higher in high-exposure group (500 mg/m3) than those of the other groups. In blood biochemistry the values of TG (triglyceride) and BUN (urea nitrogen) were significantly decreased. Other hematological changes with toxicological relevance were not observed in exposed male rats when compared with the control animals. The histopathological findings were not observed in the lung and kidney tissues exposed to chemicals comparing with those of control tissues. The No Observed Adverse Effect Level (NOAEL) of EG in 28 day’s inhalation test was evaluated to be over 100 mg/m3.

Purpose: Allergic diseases are triggered by Th2-mediated immune reactions to allergens and orchestrated by various immunological factors, including immune cells and cytokines. Although many reports have suggested that childhood is the critical period in the onset of allergic diseases and aging leads to alter the susceptibility of an individual to allergic diseases, age-related changes in various immunological factors in healthy individuals as well as their difference between healthy and allergic children have not yet been established. Methods: We investigated the ratio of Th1/Th2 cells and the levels of 22 allergy-related cytokines across all age groups in individuals who were classified as clinically non-atopic and healthy. We also examined their differences between healthy and allergic children to evaluate immunological changes induced by the development of allergic diseases during childhood. Results: The Th1/Th2 ratio rose gradually during the growth period including childhood, reaching peak values in the twenties-thirties age group. Th1/Th2 ratios were significantly lower in allergic children than in healthy controls, whereas 14 of 22 cytokines were significantly higher in allergic children than in healthy controls. On the other hand, there were no differences in Th1/Th2 ratios and cytokines between healthy and allergic adolescents. Conclusions: In this study, age-related changes in Th1/Th2 ratios were found in normal controls across all age groups, and decreases in Th1/Th2 ratio were observed with increasing of 14 cytokines in allergic children. The results of this study may be helpful as reference values for both monitoring immunological changes according to aging in healthy individuals and distinguishing between normal and allergic subjects in terms of immune cells and soluble factors.

Purpose Childhood allergies are a serious problem, as they may lead to lifetime chronic disease. Determination of total and specific IgE levels is known to be a diagnostic tool for allergic sensitization; however, IgE levels are affected by various factors, such as age, sex, ethnicity, and geographic area. Thus, we evaluated the distribution of total and specific serum IgE levels against seven inhalant allergens in preschool children and examined their association with allergic diseases in Seoul, Korea. Methods Total/specific serum IgE determination and skin prick tests for seven common allergens were performed on 509 children aged 3 to 6 years from 16 child care centers in Seoul, Korea. Demographic characteristics were surveyed from parents using a modified International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. A diagnosis of atopic dermatitis was made by physicians. Results The geometric mean of total IgE was 80.48±3.80 kU/L in preschool children. IgE levels were higher in boys (boys, 102.34±3.52 kU/L; girls, 62.37±3.93 kU/L; P

Background Airway is one of the main routes for allergen exposure and entering airborne allergens through the airway can initiate allergic responses, resulting in development of allergic asthma. There are various environmental factors that also influence to the airway-mediated immune responses. Among the factors, respiratory syncytial virus (RSV) enables the condition of the airway to change because RSV infection is common in early life and induce the bronchiolitis. In addition, several studies have been reported that non-immune cells such as airway epithelial cells play a pivotal role in the initiation of allergic responses. The aim of our study is to determine the association between the sensitization of common airborne allergens and RSV infection in airway epithelial cells.

Introduction: A cell transformation assay (CTA) can detect the carcinogenicity of chemicals, but is classified into a category different from a genotoxicity assay which has been used for the prediction of chemical carcinogenicity. CTA detects non-genotoxic carcinogens as well as genotoxic ones. Most of non-genotoxic carcinogens are considered to be tumor-promoters and many of them have been detected as promoters in a two-stage BALB/c 3T3 CTA, which mimics a in vivo two-stage carcinogenesis test. The Bhas 42 cells were established from the BALB/c 3T3 cells through transfection of v-Ha-ras gene and regarded as initiated cells in the two-stage carcinogenesis theory. Using the Bhas 42 cells, a short-term CTA was developed. Bhas 42 CTA is superior to conventional CTAs in cost and labor performance. It consists of an initiation assay and a promotion assay to detect initiating activity and promoting activity, respectively. We applied this short-term CTA to 92 chemicals to characterize the assay and evaluate its performance for the prediction of chemical carcinogenicity. Methods: In the initiation assay, the Bhas 42 cells were seeded at a density of 2000 cells/mL onto 6-well microplates (Day 0), treated with test chemicals from Day 1, when the cells were sparse, to Day 4, and thereafter cultured in normal medium until Day 21. In the promotion assay, the cells were seeded at a density of 7000 cells/mL, treated with chemicals from Day 4, when the cells were sub-confluent, to Day 14, and maintained in normal medium until Day 21. The cultures were fixed and stained with Giemsa's solution. The transformed foci were judged on the basis of morphological characteristics. A statistical analysis for the number of transformed foci per well was performed by the multiple comparison using the Dunnett method. Results and Discussion: Our results of Bhas 42 CTA were compared with existing genotoxicity data. Bhas 42 CTA could detect Ames-negative and Ames-discordant chemicals, and the promotion assay detected most of them, confirming that the Bhas 42 cells act as initiated cells in the CTA. The concordance, sensitivity, specificity, positive predictivity, negative predictivity, false positive and false negative were calculated from the assay results for known carcinogens and non-carcinogens. Among these performance characteristics, the specificity and positive predictivity were high and exceeded 80%. The other performance characteristics were equivalent to those of genotoxicity tests. From these results, we considered that the accuracy of prediction for chemical carcinogenicity would be improved by introducing Bhas 42 CTA into the battery of in vitro assays. Acknowledgement: This work was supported by the New Energy and Industrial Technology Development Organization/the Ministry of Economy, Trade and Industry of Japan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4365.