Mechanisms of pulmonary disease in F344 rats after workplace-relevant inhalation exposure to cross-linked water-soluble acrylic acid polymers.

BACKGROUND: Recently in Japan, six workers at a chemical plant that manufactures resins developed interstitial lung diseases after being involved in loading and packing cross-linked water-soluble acrylic acid polymers (CWAAPs). The present study focused on assessing lung damage in rats caused by workplace-relevant inhalation exposure to CWAAP and investigated the molecular and cellular mechanisms involved in lung lesion development. METHODS: Using a whole-body inhalation exposure system, male F344 rats were exposed once to 40 or 100 mg/m3 of CWAAP-A for 4 h or to 15 or 40 mg/m3 of CWAAP-A for 4 h per day once per week for 2 months (9 exposures). In a separate set of experiments, male F344 rats were administered 1 mg/kg CWAAP-A or CWAAP-B by intratracheal instillation once every 2 weeks for 2 months (5 doses). Lung tissues, mediastinal lymph nodes, and bronchoalveolar lavage fluid were collected and subjected to biological and histopathological analyses. RESULTS: A single 4-h exposure to CWAAP-A caused alveolar injury, and repeated exposures resulted in regenerative changes in the alveolar epithelium with activation of TGFß signaling. During the recovery period after the last exposure, some alveolar lesions were partially healed, but other lesions developed into alveolitis with fibrous thickening of the alveolar septum. Rats administered CWAAP-A by intratracheal instillation developed qualitatively similar pulmonary pathology as rats exposed to CWAAP-A by inhalation. At 2 weeks after intratracheal instillation, rats administered CWAAP-B appeared to have a slightly higher degree of lung lesions compared to rats administered CWAAP-A, however, there was no difference in pulmonary lesions in the CWAAP-A and CWAAP-B exposed rats examined 18 weeks after administration of these materials. CONCLUSIONS: The present study reports our findings on the cellular and molecular mechanisms of pulmonary disease in rats after workplace-relevant inhalation exposure to CWAAP-A. This study also demonstrates that the lung pathogenesis of rats exposed to CWAAP-A by systemic inhalation was qualitatively similar to that of rats administered CWAAP-A by intratracheal instillation.

Carcinogenicity and testicular toxicity of 2-bromopropane in a 26-week inhalation study using the rasH2 mouse model.

2-Bromopropane (2-BP) is a colorless liquid at room temperature and is used in closed systems in factories, mainly as an intermediate for medicines, pesticides, and other chemicals. However, the carcinogenicity of 2-BP is still unknown. The CByB6F1-Tg(HRAS)2Jic (rasH2) transgenic mouse model has been established as an alternative to long-term studies (1.5 years-lifetime) to detect carcinogenicity in as short a time as six months. We performed a 26-week inhalation exposure study of 2-BP using the rasH2 mouse model. Male and female rasH2 mice were exposed to 0, 67, 200, or 600 ppm of 2-BP for 6 h/day, 5 days/week for 26 weeks. All tissues and blood were collected and subjected to biological and histopathological analyses. The results showed a concentration-dependent increase in lung tumor development in male and female rasH2 mice exposed by inhalation to 2-BP, which was significant by Peto's and Poly-3 trend tests. Furthermore, in male rasH2 mice, 2-BP was found to be a testicular toxin. This study is the first to demonstrate that 2-BP is carcinogenic in male and female mice and a testicular toxin in male mice using the rasH2 mouse model.

Pulmonary dust foci as rat pneumoconiosis lesion induced by titanium dioxide nanoparticles in 13-week inhalation study.

BACKGROUND: Most toxicological studies on titanium dioxide (TiO2) particles to date have concentrated on carcinogenicity and acute toxicity, with few studies focusing of pneumoconiosis, which is a variety of airspace and interstitial lung diseases caused by particle-laden macrophages. The present study examined rat pulmonary lesions associated with pneumoconiosis after inhalation exposure to TiO2 nanoparticles (NPs). METHODS: Male and female F344 rats were exposed to 6.3, 12.5, 25, or 50 mg/m3 anatase type TiO2 NPs for 6 h/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. After the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. RESULTS: Numerous milky white spots were present in the lungs after exposure to 25 and 50 mg/m3 TiO2 NPs. Histopathological analysis revealed that the spots were alveolar lesions, characterized predominantly by the agglomeration of particle-laden macrophages and the presence of reactive alveolar epithelial type 2 cell (AEC2) hyperplasia. We defined this characteristic lesion as pulmonary dust foci (PDF). The PDF is an inflammatory niche, with decreased vascular endothelial cells in the interstitium, and proliferating AEC2 transformed into alveolar epithelial progenitor cells. In the present study, the AEC2 in the PDF had acquired DNA damage. Based on PDF induction, the lowest observed adverse effect concentration for pulmonary disorders in male and female rats was 12.5 mg/m3 and 6.3 mg/m3, respectively. The no observed adverse effect concentration for male rats was 6.3 mg/m3. There was a sex difference in lung lesion development, with females showing more pronounced lesion parameters than males. CONCLUSIONS: Inhalation exposure to TiO2 NPs caused PDF, an air-space lesion which is an alveolar inflammatory niche containing particle-laden macrophages and proliferating AEC2. These PDFs histopathologically resemble some pneumoconiosis lesions (pulmonary siderosis and hard metal pneumoconiosis) in workers and lung disease in smokers, suggesting that PDFs caused by exposure to TiO2 NPs in rats are an early pneumoconiosis lesion and may be a common alveolar reaction in mammals.

No evidence for carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model.

With the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. We performed a 26-week inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) mice model for detecting carcinogenicity. Male and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 h/day, 5 days/week for 26 weeks. All tissues and blood were collected and subjected to biological and histopathological analyses. TiO2 NPs exposure induced deposition of particles in lungs in a dose-dependent manner in each exposure group. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of alveolar epithelial type 2 cells was examined, and it was not increased by exposure to TiO2 NPs. This is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration.

Dose-response relationship of pulmonary disorders by inhalation exposure to cross-linked water-soluble acrylic acid polymers in F344 rats.

BACKGROUND: In Japan, six workers handling cross-linked water-soluble acrylic acid polymer (CWAAP) at a chemical plant suffered from lung diseases, including fibrosis, interstitial pneumonia, emphysema, and pneumothorax. We recently demonstrated that inhalation of CWAAP-A, one type of CWAAP, causes pulmonary disorders in rats. It is important to investigate dose-response relationships and recoverability from exposure to CWAAPs for establishing occupational health guidelines, such as setting threshold limit value for CWAAPs in the workplace. METHODS: Male and female F344 rats were exposed to 0.3, 1, 3, or 10 mg/m3 CWAAP-A for 6 h/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. At 1 h, 4 weeks, and 13 weeks after the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. In a second experiment, male rats were pre-treated with clodronate liposome or polymorphonuclear leukocyte-neutralizing antibody to deplete macrophages or neutrophils, respectively, and exposed to CWAAP-A for 6 h/day for 2 days. RESULTS: CWAAP-A exposure damaged only the alveoli. The lowest observed adverse effect concentration (LOAEC) was 1 mg/m3 and the no observed adverse effect concentration (NOAEC) was 0.3 mg/m3. Rats of both sexes were able to recover from the tissue damage caused by 13 weeks exposure to 1 mg/m3 CWAAP-A. In contrast, tissue damage caused by exposure to 3 and 10 mg/m3 was irreversible due to the development of interstitial lung lesions. There was a gender difference in the recovery from CWAAP-A induced pulmonary disorders, with females recovering less than males. Finally, acute lung effects caused by CWAAP-A were significantly reduced by depletion of alveolar macrophages. CONCLUSIONS: Pulmonary damage caused by inhalation exposure to CWAAP-A was dose-dependent, specific to the lung and lymph nodes, and acute lung damage was ameliorated by depleting macrophages in the lungs. CWAAP-A had both a LOAEC and a NOAEC, and tissue damage caused by exposure to 1 mg/m3 CWAAP-A was reversible: recovery in female rats was less than for males. These findings indicate that concentration limits for CWAAPs in the workplace can be determined.

[Background data of spontaneous tumors in F344/DuCrlCrlj rats].

INTRODUCTION: We investigated the 2-year survival rate and incidence of spontaneous tumors in F344/DuCrlCrlj rats used in carcinogenicity studies of chemical substances. Records for animals used in the control groups of carcinogenicity studies which were conducted during the last 10 years were obtained from the database of the Japan Bioassay Research Center (JBRC). Six hundred ninety-nine males and 550 females were used in 14 and 11 inhalation studies, respectively, and 500 animals of each sex were used in 10 male and 10 female oral studies. METHODS: In each study, SPF (specific pathogen free) animals were housed for 2 years (104 weeks) as control groups in the carcinogenicity studies. All animals underwent necropsy and histopathological examination. Each study was conducted in accordance with the Good Laboratory Practice. RESULTS: The incidence of interstitial cell tumors was highest in both inhalation studies and oral studies (inhalation studies 86.1%, oral studies 68.6%). Tumors which had an incidence of 6% or higher were adenoma of the pituitary, C-cell adenoma of the thyroid, and mononuclear cell leukemia (LGL leukemia) of the spleen in male and female rats; fibroma of the subcutaneous tissue, adrenal pheochromocytoma, and islet cell adenoma of the pancreas in male rats; and endometrial stromal polyps and fibroadenoma of the mammary gland in female rats. Tumors other than the above had rare incidence rates. A clear difference in the incidence of spontaneous tumors was not observed between the inhalation and oral studies. The incidences of spontaneous tumors in control groups of previous oral studies are similar to our findings. There are no other reports of the spontaneous tumor incidence in the control groups of inhalation studies using F344/DuCrlCrlj rats. The 2-year survival rate was about 77% in both the inhalation and oral studies, and a gender difference was not observed. The F344/DuCrlCrlj rats used at JBRC had a higher 2-year survival rate than F344/N rats. This difference is possibly due to the low incidence of LGL leukemia in the F344/DuCrlCrlj rat. CONCLUSIONS: The incidences of spontaneous tumors in F344/DuCrlCrlj rats used in control groups of both inhalation and oral studies during the last 10 years at JBRC are similar to each other and similar to those reported in other studies. This is the first report on the incidence of spontaneous tumors in inhalation studies and contributes to the toxicological evaluation of studies using F344/DuCrlCrlj rats.

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats.

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m(3) MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m(3) MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m(3) MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m(3) MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m(3).

Thirteen-week inhalation toxicity of 1,4-dioxane in rats.

Thirteen-week inhalation toxicity of 1,4-dioxane was examined by repeated inhalation exposure of male and female F344 rats to 0 (control), 100, 200, 400, 800, 1600, 3200, or 6400 ppm (v/v) 1,4-dioxane vapor for 6 h/day and 5 days/wk. All the 6400-ppm-exposed males and females died during the first week. Terminal body weight decreased, and relative weights of liver, kidney, and lung increased. AST increased in the 200 ppm-and 3200-ppm-exposed females, and ALT increased in the 3200-ppm-exposed males and females. Nuclear enlargement of nasal respiratory epithelial cells occurring in the 100-ppm-exposed males and females was the most sensitive, followed by the enlarged nuclei in the olfactory, tracheal, and bronchial epithelia. 1,4-Dioxane-induced liver lesions occurred at higher exposure concentrations than the nasal lesions did, and were characterized by single-cell necrosis and centrilobular swelling of hepatocytes in males and females. Glutathione S-transferase placental form (GST-P) positive liver foci were observed in the 1600-ppm-exposed females and 3200-ppm-exposed males and females, which are known as a preneoplastic lesion in rat hepatocarcinogenesis. Plasma levels of 1,4-dioxane increased linearly with an increase in the concentrations of exposure to 400 ppm and above. The enlarged nuclei in the nasal epithelia and the GST-P-positive liver foci were discussed in light of the possible development of nasal and hepatic tumors by long-term inhalation exposure to 1,4-dioxane. A lowest-observed-adverse-effect level (LOAEL) was determined at 100 ppm for the nasal endpoint in both male and female rats.

Occurrence of two different types of glutathione S-transferase placental form-positive hepatocytes after a single administration of 2,3,7,8-tetrabromodibenzo-p-dioxin in rats.

Occurrence of glutathione S-transferase placental form (GST-P)-positive hepatocytes was examined, using 15 Wistar rats of both sexes each orally administered 2,3,7,8-tetrabromo-dibenzo-p-dioxin (TBDD) by gavage at a single dose of 0, 10, 30, 100 or 300 microg/kg body weight. Liver tissues were stained with anti-GST-P antibody. Two different types of GST-P-positive hepatocytes were found in the TBDD-dosed rat. One type was of the hepatocytes stained homogeneously with anti-GST-P antibody and clearly distinguishable from the surrounding normal tissue. The foci were composed of 2 to 60 hepatocytes exhibiting morphologically focal and clonal proliferation. The GST-P-positive hepatocellular foci occurred at two higher dose levels and only on Day 36 after the single administration. Another type was of the area occupied by the positively but heterogeneously stained hepatocytes appearing predominantly in the centrilobular region, at lower dose levels and persistently on Day 2 through 36. The stained hepatocytes appeared to be neither focally nor clonally proliferating. Females were more susceptible to formation of the two differently stained hepatocytes than males. It is suggested that the GST-P-positive foci represent an early stage of hepatocarcinogenesis, while the GST-P-positive area is associated with the induction of detoxifying Phase II GSTs.

Thirteen-week oral toxicity of 1,4-dioxane in rats and mice.

Subchronic oral toxicity of 1,4-dioxane was examined by administering 1,4-dioxane in drinking water at 6 different concentrations of 0 (control), 640, 1,600, 4,000, 10,000 or 25,000 ppm (wt/wt) to F344 rats and BDF(1)mice of both sexes for 13 weeks. Food and water consumption and terminal body weight were decreased dose-dependently in rats and mice. A dose-dependent increase in the relative weights of kidney and lung was noted in rats and mice, while the relative liver weight was increased only in rats. Increases in plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and a decrease in plasma glucose were noted primarily in the rats and mice dosed 25,000 ppm. Histopathological examination revealed that 1,4-dioxane affected the upper and lower respiratory tracts, liver, kidneys and brain in rats, while only the former two organs were affected in mice. Nuclear enlargement occurred in the respiratory, olfactory, tracheal and bronchial epithelia of the 1,4-dioxane-dosed rats and mice. The 1,4-dioxane-induced hepatic lesions were characterized by centrilobular swelling and necrosis in rats and mice and by glutathione S-transferase placental form (GST-P)-positive altered hepatocellular foci in rats, which are known as preneoplastic lesions. A no-observed-adverse-effect-level (NOAEL) was determined at 640 ppm for both rats and mice, since the nuclear enlargement in the nasal respiratory epithelium and the centrilobular swelling of hepatocytes in rats and the nuclear enlargement in the bronchial epithelium in mice were observed at 1,600 ppm. The NOAEL value corresponded to the estimated 1,4-dioxane intake of 52 mg/kg/day in rats and 170 mg/kg/day in mice.

Thirteen-week inhalation toxicity of carbon tetrachloride in rats and mice.

Subchronic toxicity of carbon tetrachloride (CCl4) was examined by inhalation exposure of F344 rats and BDF1 mice of both sexes to 0, 10, 30, 90, 270 or 810 ppm (v/v) CCl4 vapor for 13 wk (6 h/d and 5 d/wk). In the high exposure levels at 270 and 810 ppm, altered cell foci in the livers of both rats and mice, and fibrosis and cirrhosis in the rat liver were observed. Hematoxylin and eosin-stained altered cell foci of rats were recognized as glutathione-S-transferase placental form (GST-P) positive foci, which are preneoplastic lesions of hepatocarcinogenesis. The most sensitive endpoint of CCl4-induced toxicity was fatty change with large droplets in rats of both sexes and male mice, and cytoplasmic globules in male mice, as well as increased relative liver weight in male rats. Those endpoints were manifested at 10 ppm and the LOAEL was determined as 10 ppm for the hepatic endpoints in rats and mice. Enhanced cytolytic release of liver transaminases into plasma in rats and mice and its close association with hepatic collapse in mice were observed at medium and high levels of inhalation exposure. Both CCl4-induced hematotoxicity and nephrotoxicity were observed in both rats and mice, but those toxicities were manifested at higher exposure concentrations than hepatotoxicity. The LOAEL for the hepatic endpoint and the GST-P-stained altered cell foci provide relevant animal data for reconsidering the occupational exposure limit val1ue of 5 ppm for CCl4 and strengthen the evidence of CCl4-induced hepatocarcinogenicity which is used in its carcinogenicity classification.