Usefulness of myeloperoxidase as a biomarker for the ranking of pulmonary toxicity of nanomaterials.

BACKGROUND: In order to examine whether myeloperoxidase (MPO) can be a useful marker for evaluating the pulmonary toxicity of nanomaterials, we analyzed MPO protein in bronchoalveolar lavage fluid (BALF) samples obtained from previous examinations of a rat model. In those examinations we performed intratracheal instillation exposures (dose: 0.2-1.0 mg) and inhalation exposures (exposure concentration: 0.32-10.4 mg/m3) using 9 and 4 nanomaterials with different toxicities, respectively. Based on those previous studies, we set Nickel oxide nanoparticles (NiO), cerium dioxide nanoparticles (CeO2), multi wall carbon nanotubes with short or long length (MWCNT (S) and MWCNT (L)), and single wall carbon nanotube (SWCNT) as chemicals with high toxicity; and titanium dioxide nanoparticles (TiO2 (P90) and TiO2 (Rutile)), zinc oxide nanoparticles (ZnO), and toner with external additives including nanoparticles as chemicals with low toxicity. We measured the concentration of MPO in BALF samples from rats from 3 days to 6 months following a single intratracheal instillation, and from 3 days to 3 months after the end of inhalation exposure. RESULTS: Intratracheal instillation of high toxicity NiO, CeO2, MWCNT (S), MWCNT (L), and SWCNT persistently increased the concentration of MPO, and inhalation of NiO and CeO2 increased the MPO in BALF. By contrast, intratracheal instillation of low toxicity TiO2 (P90), TiO2 (Rutile), ZnO, and toner increased the concentration of MPO in BALF only transiently, and inhalation of TiO2 (Rutile) and ZnO induced almost no increase of the MPO. The concentration of MPO correlated with the number of total cells and neutrophils, the concentration of chemokines for neutrophils (cytokine-induced neutrophil chemoattractant (CINC)-1 and heme oxygenase (HO)-1), and the activity of released lactate dehydrogenase (LDH) in BALF. The results from the receiver operating characteristics (ROC) for the toxicity of chemicals by the concentration of MPO proteins in the intratracheal instillation and inhalation exposures showed that the largest areas under the curves (AUC) s in both examinations occurred at 1 month after exposure. CONCLUSION: These data suggest that MPO can be a useful biomarker for the ranking of the pulmonary toxicity of nanomaterials, especially at 1 month after exposure, in both intratracheal instillation and inhalation exposure.

Biopersistence of NiO and TiO2 Nanoparticles Following Intratracheal Instillation and Inhalation.

The hazards of various types of nanoparticles with high functionality have not been fully assessed. We investigated the usefulness of biopersistence as a hazard indicator of nanoparticles by performing inhalation and intratracheal instillation studies and comparing the biopersistence of two nanoparticles with different toxicities: NiO and TiO2 nanoparticles with high and low toxicity among nanoparticles, respectively. In the 4-week inhalation studies, the average exposure concentrations were 0.32 and 1.65 mg/m³ for NiO, and 0.50 and 1.84 mg/m³ for TiO2. In the instillation studies, 0.2 and 1.0 mg of NiO nanoparticles and 0.2, 0.36, and 1.0 mg of TiO2 were dispersed in 0.4 mL water and instilled to rats. After the exposure, the lung burden in each of five rats was determined by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES) from 3 days to 3 months for inhalation studies and to 6 months for instillation studies. In both the inhalation and instillation studies, NiO nanoparticles persisted for longer in the lung compared with TiO2 nanoparticles, and the calculated biological half times (BHTs) of the NiO nanoparticles was longer than that of the TiO2 nanoparticles. Biopersistence also correlated with histopathological changes, inflammatory response, and other biomarkers in bronchoalveolar lavage fluid (BALF) after the exposure to nanoparticles. These results suggested that the biopersistence is a good indicator of the hazards of nanoparticles.

Comparison between whole-body inhalation and nose-only inhalation on the deposition and health effects of nanoparticles.

OBJECTIVES: We performed the two inhalation exposures, whole-body inhalation and nose-only inhalation, to investigate the pulmonary deposition and health effects of the two inhalation methods. METHODS: In both methods, we exposed rats to the same TiO2 nanoparticles at almost the same exposure concentration for 6 h and compared the deposited amounts of nanoparticles and histopathological changes in the lungs. Rats were exposed to rutile-type TiO2 nanoparticles generated by the spray-dry method for 6 h. The exposure concentration in the whole-body chamber was 4.10 ± 1.07 mg/m(3), and that in nose-only chamber was 4.01 ± 1.11 mg/m(3). The particle sizes were 230 and 180 nm, respectively. A control group was exposed to fresh air. RESULTS: The amounts of TiO2 deposited in the lungs as measured by ICP-AES after acid digestion just after the exposure were: 42.6 ± 3.5 µg in the whole-body exposure and 46.0 ± 7.7 µg in the nose-only exposure groups. The histopathological evaluation was the same in both exposure groups: no infiltration of inflammatory cells in the alveolar space and interstitium, and no fibrosis. CONCLUSION: The two inhalation methods using the same material under the same exposure conditions resulted in the same particle deposition and histopathology in the lung.

[General background and practical implementation of the health management service institution in Korea].

The Korean Occupational Safety and Health Act requires an employer with more than 50 employees to assign a health manager or an occupational physician. However, there are many cases where it is difficult for medium-scale enterprises to perform occupational health practices autonomously because their financial base is weaker than that of large-scale enterprises. The Korean Occupational Safety and Health Act was amended in 1990 so that medium-scale enterprises could entrust a health management service institution with their health management tasks. This system is similar to the outsourcing of medical examinations, occupational physicians, or the measurement of the working environment in Japan, but its legal background and actual activities are korea-specific, and it has some different points. In particular, the quality control of health management service institutions by legal and administrative regulations, and the multidisciplinary provision of services contribute to the development of occupational health in medium-scale enterprises. This will be a good reference for occupational health services in small- and medium-scale enterprises in the future in Japan.

The efficacy of clinicopathological and imaging features in differentiating benign from malignant parotid micro- and minitumors: a retrospective analysis.

OBJECTIVE: To compare clinicopathological and imaging features of micro- and minitumors of the parotid gland and provide a reference for preoperative prediction of benign vs malignant status. STUDY DESIGN: Patients with parotid gland tumors treated surgically were selected. Relevant clinicopathological and imaging data were collected for patients with maximum tumor diameters ≤20 mm on preoperative computed tomography (CT). The lesions were divided into 2 groups, microtumors and minitumors, based on maximum tumor diameter. CT imaging features of benign and malignant tumors were compared through binary logistic regression analysis. RESULTS: Microtumors and minitumors were categorized by maximum diameters <10 mm (n = 74) and 10-20 mm (n = 611), respectively. Benign and malignant minitumors exhibited significant differences in boundary, tumor density, margin morphology, spiculation margin, and CT values in the plain and arterial phase (P ≤ .027), resembling those found in typical malignant parotid gland tumors. However, no significant differences were observed between benign and malignant microtumors. Logistic regression analysis identified boundary, margin morphology, and spiculation margin as independent predictors of malignancy. The prediction model excelled in identifying benign lesions but was less successful in identifying malignancies. CONCLUSION: Parotid gland minitumors had imaging features similar to typical larger malignant tumors. Active exclusion of the malignant risk and early surgical treatment is recommended for these tumors.

Analysis of pulmonary surfactant in rat lungs after intratracheal instillation of short and long multi-walled carbon nanotubes.

Multi-walled carbon nanotubes (MWCNTs) are interesting new materials, but there is some concern about their harmfulness due to their fibrous nature. To determine the difference in the biological effects of MWCMTs by fiber length, we prepared two MWCNT samples from one bulk sample. One consisted of cut up short fibers (Short; average length=0.94 µm) and the other was just dispersed (Long; average length=3.4 µm). The samples were administered to male Wistar rats by intratracheal instillation at doses of 0.2 mg and 1 mg/animal (Short) and 0.2 mg and 0.6 mg/animal (Long). The animals were sacrificed at time points from 3 d to 12 months after administration. Bronchoalveolar lavage fluid (BALF) was taken from the lungs and pathological specimens were prepared. The concentrations of phospholipids, total protein and surfactant protein D (SP-D) in the pulmonary surfactant of the BALF were determined, the surface tension of BALF was measured, and the inflammation score was determined by the point-counting method to assess pulmonary tissue inflammation. The present study suggests that inflammatory response in the lung was slightly higher for long MWCNTs than for short MWCNTs when compared at the same mass dose. The correlation between pulmonary surfactant components and BALF surface tension was also evaluated. The Spearman's rank correlation coefficients obtained for the phospholipid, total protein and SP-D concentrations were -0.068 (p=0.605), -0.360 (p=0.005) and -0.673 (p=0.000), respectively. Surface tension, measured by a simple method, should be reflected in the change of a surfactant protein, such as SP-D.

Comparison of the exposure assessment of di(2-ethylhexyl) phthalate between the PBPK model-based reverse dosimetry and scenario-based analysis: A Korean general population study.

Di (2-ethylhexyl) phthalate (DEHP), classified as a reproductive toxicant, is a ubiquitous pollutant in foodstuffs, dust, and commercial products. In this study, to provide a useful cross-check on the accuracy of the exposure assessment, the estimated daily intake of DEHP was compared using reverse dosimetry with a physiologically-based pharmacokinetic (PBPK) model and a scenario-based probabilistic estimation model for six subpopulations in Korea. For reverse dosimetry analysis, the concentrations of urinary DEHP metabolites, namely mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono (2-ethyl-5-oxohexyl)phthalate (MEOHP), from three human biomonitoring program datasets were used. For the scenario-based model, we evaluated the various exposure sources of DEHP, including diet, air, indoor dust, soil, and personal care products (PCPs), and also determined its levels based on the literature review and measurements of indoor dust. The DEHP exposure doses using both exposure assessment approaches were similar in all cases, except for the 95th percentile exposure doses in toddlers (1-2 years) and young children (3-6 years). The PBPK-reverse dosimetry estimated daily intakes at the 95th percentile ranged between 22.53 and 29.90 µg/kg/day for toddlers and young children. These exceeded the reference dose (RfD) of 20 µg/kg bw/day of the US Environmental Protection Agency (EPA) based on the increased relative liver weight. Although, food was considered the primary source of DEHP, contributing to a total exposure of 50.8-75.1%, the effect of exposure to indoor dust should not be overlooked. The occurrence of high levels of DEHP in indoor dust collected from Korean homes suggests the use of a wide variety of consumer products containing DEHP. Furthermore, more attention should be paid to the high exposure levels of DEHP, especially in young children. Therefore, it is necessary to perform continuous monitoring of the indoor dust, consumer products, and the body burden of children.

Assessment of Cytokine-Induced Neutrophil Chemoattractants as Biomarkers for Prediction of Pulmonary Toxicity of Nanomaterials.

This work determines whether cytokine-induced neutrophil chemoattractants (CINC)-1, CINC-2 and CINC-3 can be markers for predicting high or low pulmonary toxicity of nanomaterials (NMs). We classified NMs of nickel oxide (NiO) and cerium dioxide (CeO2) into high toxicity and NMs of two types of titanium dioxides (TiO2 (P90 and rutile)) and zinc oxide (ZnO) into low toxicity, and we analyzed previous data of CINCs in bronchoalveolar lavage fluid (BALF) of rats from three days to six months after intratracheal instillation (0.2 and 1.0 mg) and inhalation exposure (0.32-10.4 mg/m3) of materials (NiO, CeO2, TiO2 (P90 and rutile), ZnO NMs and micron-particles of crystalline silica (SiO2)). The concentration of CINC-1 and CINC-2 in BALF had different increase tendency between high and low pulmonary toxicity of NMs and correlated with the other inflammatory markers in BALF. However, CINC-3 increased only slightly in a dose-dependent manner compared with CINC-1 and CINC-2. Analysis of receiver operating characteristics for the toxicity of NMs by CINC-1 and CINC-2 showed the most accuracy of discrimination of the toxicity at one week or one month after exposure and CINC-1 and CINC-2 in BALF following intratracheal instillation of SiO2 as a high toxicity could accurately predict the toxicity at more than one month after exposure. These data suggest that CINC-1 and CINC-2 may be useful biomarkers for the prediction of pulmonary toxicity of NMs relatively early in both intratracheal instillation and inhalation exposure.

Behavioral characteristics to airborne particles generated from commercial spray products.

Commercial spray products are commonly used in daily life and airborne particles generated by these products may cause adverse health effects. Our study was aimed to characterize the behaviors of airborne particles from spray products and to determine the deposition loss rate. Four categories of spray products with highly frequent use - air fresheners, fabric deodorants, window cleaners, and a bathroom cleaner - were selected for the study. The products were applied in a cleanroom according to the instructions for use. Airborne particles (10-10,000 nm) were measured within the breathing zone of a user with a scanning mobility particle sizer and an optical particle spectrometer. Additionally, filter sampling was performed to examine the morphological characteristics of the particles using a field emission-scanning electron microscope (FE-SEM). The initial concentration and particle size distribution varied among different spray types and products. Two propellant-type air fresheners that we tested showed a high initial concentration of smaller sized particles. However, one of these and all hand-pressure type propellants showed a low initial concentration in all size ranges. We observed that particles in nucleation mode (10-31.6 nm) decreased and aggregated particles shifted to accumulation mode (100-1,000 nm) over time. The FS-SEM analysis confirmed the aggregation of nano-sized particles for all products. The deposition loss rates of various particle sizes depended on the initial concentration and distribution of particle sizes. For two air fresheners with high initial concentrations, the loss rate of small-sized particles was higher than that of the other products whereas the particle loss rate of large-sized particles was higher, regardless of initial concentration. The results of this study can give us useful information in the behaviors of airborne particles in the consumer spray products and resulting exposure assessment especially in the application to the exposure modeling of spray products.

Determining Exposure Factors of Anti-Fogging, Dye, Disinfectant, Repellent, and Preservative Products in Korea.

Reliable exposure factors are essential to determine health risks posed by chemicals in consumer products. We analyzed five risk-concerned product categories (anti-fogging, dye, disinfectant, repellent, and preservative products) for 13 products (three car anti-fogging products, a lens anti-fogging product, two car dye products, two drain disinfectants, an air conditioner disinfectant, a chlorine-based disinfectant, a fabric repellent, an insect repellent for food, and a wood preservative) considered to be of high risk in order to determine exposure factors via web surveys and estimation of amount of product. Among the 3000 participants (1482 (49%) men) aged ≥19 years, drain disinfectants were used most frequently (38.2%); the rate of usage of the other products ranged between 1.1-24.0%. The usage rates for the consumer products differed by sex, age, income, and education. Some consumer products such as car and lens anti-fogging products, chlorine-based disinfectants, fabric repellents, and drain disinfectants were regularly used more than once a month, while car dye products, air conditioner disinfectants, insect repellents for food, and wood preservatives were not regularly used owing to the specific product purposes and seasonal needs. Our results could be used for managing or controlling chemical substances in consumer products and conducting accurate exposure assessments.

Potentiation of Sodium Metabisulfite Toxicity by Propylene Glycol in Both in Vitro and in Vivo Systems.

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.

Assessment of Pulmonary Toxicity Induced by Inhaled Toner with External Additives.

We investigated the harmful effects of exposure to a toner with external additives by a long-term inhalation study using rats, examining pulmonary inflammation, oxidative stress, and histopathological changes in the lung. Wistar rats were exposed to a well-dispersed toner (mean of MMAD: 2.1 µm) at three mass concentrations of 1, 4, and 16 mg/m3 for 22.5 months, and the rats were sacrificed after 6 months, 12 months, and 22.5 months of exposure. The low and medium concentrations did not induce statistically significant pulmonary inflammation, but the high concentration did, and, in addition, a histopathological examination showed fibrosis in the lung. Although lung tumor was observed in one sample of high exposure for 22.5 months, the cause was not statistically significant. On the other hand, a persistent increase in 8-OHdG was observed in the high exposure group, indicating that DNA damage by oxidative stress with persistent inflammation leads to the formation of tumorigenesis. The results of our studies show that toners with external additives lead to pulmonary inflammation, oxidative stress, and fibrosis only at lung burdens beyond overload. These data suggest that toners with external additives may have low toxicity in the lung.

Analysis of pulmonary surfactant in rat lungs after inhalation of nanomaterials: Fullerenes, nickel oxide and multi-walled carbon nanotubes.

The health risks of inhalation exposure to engineered nanomaterials in the workplace are a major concern in recent years, and hazard assessments of these materials are being conducted. The pulmonary surfactant of lung alveoli is the first biological entity to have contact with airborne nanomaterials in inhaled air. In this study, we retrospectively evaluated the pulmonary surfactant components of rat lungs after a 4-week inhalation exposure to three different nanomaterials: fullerenes, nickel oxide (NiO) nanoparticles and multi-walled carbon nanotubes (MWCNT), with similar levels of average aerosol concentration (0.13-0.37 mg/m(3)). Bronchoalveolar lavage fluid (BALF) of the rat lungs stored after previous inhalation studies was analyzed, focusing on total protein and the surfactant components, such as phospholipids and surfactant-specific SP-D (surfactant protein D) and the BALF surface tension, which is affected by SP-B and SP-C. Compared with a control group, significant changes in the BALF surface tension and the concentrations of phospholipids, total protein and SP-D were observed in rats exposed to NiO nanoparticles, but not in those exposed to fullerenes. Surface tension and the levels of surfactant phospholipids and proteins were also significantly different in rats exposed to MWCNTs. The concentrations of phospholipids, total protein and SP-D and BALF surface tension were correlated significantly with the polymorphonuclear neutrophil counts in the BALF. These results suggest that pulmonary surfactant components can be used as measures of lung inflammation.

Comparison of the Pulmonary Oxidative Stress Caused by Intratracheal Instillation and Inhalation of NiO Nanoparticles when Equivalent Amounts of NiO Are Retained in the Lung.

NiO nanoparticles were administered to rat lungs via intratracheal instillation or inhalation. During pulmonary toxicity caused by NiO nanoparticles, the induction of oxidative stress is a major factor. Both intratracheal instillation and inhalation of NiO nanoparticles induced pulmonary oxidative stress. The oxidative stress response protein, heme oxygenase-1 (HO-1), was induced by the administration of NiO nanoparticles at both the protein and gene expression level. Additionally, certain oxidative-stress markers in the lung, such as 8-iso-prostaglandin F2α, thioredoxin, and inducible nitric oxide synthase were increased. Furthermore, the concentration of myeloperoxidase (MPO) in the lung was also increased by the administration of NiO nanoparticles. When the amount of NiO in the lung is similar, the responses against pulmonary oxidative stress of intratracheal instillation and inhalation are also similar. However, the state of pulmonary oxidative stress in the early phase was different between intratracheal instillation and inhalation, even if the amount of NiO in the lung was similar. Inhalation causes milder oxidative stress than that caused by intratracheal instillation. On evaluation of the nanoparticle-induced pulmonary oxidative stress in the early phase, we should understand the different states of oxidative stress induced by intratracheal instillation and inhalation.

Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles.

In order to examine whether intratracheal instillation studies can be useful for determining the harmful effect of nanoparticles, we performed inhalation and intratracheal instillation studies using samples of the same nanoparticles. Nickel oxide nanoparticles (NiO) and titanium dioxide nanoparticles (TiO2) were used as chemicals with high and low toxicities, respectively. In the intratracheal instillation study, rats were exposed to 0.2 or 1 mg of NiO or TiO2. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed from 3 days to 6 months following the single intratracheal instillation. In the inhalation study, rats were exposed to inhaled NiO or TiO2 (1.65, 1.84 mg/m(3), respectively) for 4 weeks. The same endpoints were examined from 3 days to 3 months after the end of exposure. Inhalation of NiO induced an increase in the number of neutrophils in BALF and concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and heme oxygenase (HO)-1. Intratracheal instillation of NiO induced persistent inflammation and upregulation of these cytokines was observed in the rats. However, inhalation of TiO2 did not induce pulmonary inflammation, and intratracheal instillation of TiO2 transiently induced an increase in the number of neutrophils in BALF and the concentrations of CINC-1, CINC-2 and HO-1. Taken together, a difference in pulmonary inflammation was observed between the high and low toxicity nanomaterials in the intratracheal instillation studies, as in the inhalation studies, suggesting that intratracheal instillation studies may be useful for ranking the harmful effects of nanoparticles.