Toxicity of Nicotine by Repeated Intratracheal Instillation to F344 Rats

In vivo, nicotine in cigarette smoke induces various effects not only on the respiratory system but also the central and peripheral nerve systems, circulatory organs and digestive organs, and there is a possibility of promotion of lung tumorigenesis. The present experiment was conducted to examine histopathological changes caused by nicotine in the lung with repeated intratracheal instillation (i.t.). Six-week-old male F344 rats were administered nicotine by i.t. at doses of 0.05, 0.1 and 0.2 mg nicotine/rat every 3 weeks beginning at week 4, for up to a total of 9 times and were then sacrificed at week 30. The total number of administrations, total dose of nicotine and effective number of rats were 9 times, 0.45 mg and 5 rats and 4 times, 0.20 mg and 5 rats for the 0.05 mg nicotine/rat group; 3 times, 0.30 mg and 5 rats and 4 times, 0.40 mg and 3 rats for the 0.1 mg group; and 3 times, 0.60 mg and 3 rats for the 0.2 mg group, respectively. As a control group, 5 rats were administered 0.2 ml saline/rat 9 times. Some rats administered 0.1 and 0.2 mg nicotine suffered convulsions just after administration. Histopathologically, though proliferative changes were not observed, neutrophil infiltration, edema and fibrosis in the lung were induced by nicotine. In conclusion, repeated treatment of nicotine promoted neurologic symptoms in the acute phase, and strong inflammation in the lungs in the chronic phase, even at a low dose. Toxicity of nicotine is suggested to depend not on total dose of nicotine in the experiment but rather on repeated injury with consecutive administration.     

Comparison of acute inhalation toxicity of sulfuric acid by the inhalation and intratracheal instillation methods

Recently, intratracheal instillation has been focused on as a simple, low-cost alternative to the inhalation method. In this study, intratracheal instillation of sulfuric acid, a typical acidic compound, was performed to compare the acute toxicity of acidic compounds that could cause damage to the respiratory system between intratracheal instillation and inhalation. Sulfuric acid was administered to male rats at doses of 0.7, 2, 7, 20, and 60 mg/kg by dividing the total dose into four doses. General condition and body weight were examined up to 14 days after administration, and macropathological and histopathological examinations were performed. The half-lethal dose was then estimated. All animals administered 20 and 60 mg/kg sulfuric acid and one animal administered 2 mg/kg sulfuric acid died within 4 h after administration. No abnormalities were observed in other animals. At 20 and 60 mg/kg, multiple red foci or diffuse red areas were macroscopically observed in the lungs. In these lesions, histopathologically, clefts between the mucosal epithelium and basement membrane and necrosis of the alveolar epithelium were observed. Deaths in these groups may have resulted from lung injury. No notable changes were observed in other animals. Therefore, the half-lethal dose of sulfuric acid by intratracheal instillation was estimated as 7–20 mg/kg. The acute toxicity by intratracheal instillation was evaluated with two-fold sensitivity since the exposure at the half-lethal sulfuric acid concentration in inhalation studies was calculated as 43.2 mg/kg.     

Lung toxicity of a vapor-grown carbon fiber in comparison with a multi-walled carbon nanotube in F344 rats

Carbon fibers have excellent physicochemical and electrical properties. Vapor-grown carbon fibers are a type of carbon fibers that have a multi-walled carbon tube structure with a high aspect ratio. The representative vapor-grown carbon fiber, VGCF^TM-H, is extremely strong and stable and has superior thermal and electrical conductivity. Because some high-aspect-ratio multi-walled carbon nanotubes (MWCNTs) have been reported to have toxic and carcinogenic effects in the lungs of rodents, we performed a 13-week lung toxicity study using VGCF^TM-H in comparison with one of MWCNTs, MWNT-7, in rats. Male and female F344 rats were intratracheally administered VGCF^TM-H at doses of 0.2, 0.4, and 0.8 mg/kg bw or MWNT-7 at doses of 0.4 and 0.8 mg/kg bw once a week for 8 weeks and then up to week 13 without treatment. The lung burden was equivalent in the VGCF^TM-H and MWNT-7 groups; however, the lung weight had increased and the inflammatory and biochemical parameters in the broncho-alveolar lavage fluid and histopathological parameters, including inflammatory cell infiltration, alveolar type II cells proliferation, alveolar fibrosis, pleural fibrosis, lung mesothelium proliferation, and diaphragm fibrosis, were milder in the VGCF^TM-H group than in the MWNT-7 group. In addition, the proliferating cell nuclear antigen (PCNA)-positive index in the visceral and pleural mesothelium was significantly higher in the MWNT-7 group than in the controls, but not in the VGCF^TM-H group. Thus, the results of this study indicate that the lung and pleural toxicities of VGCF^TM-H were less than those of MWNT-7.     

An intratracheal instillation bioassay system for detection of lung toxicity due to fine particles in f344 rats

It is an urgent priority to establish in vivo bioassays for detection of hazards related to fine particles, which can be inhaled into deep lung tissue by humans. In order to establish an appropriate bioassay for detection of lung damage after particle inhalation, several experiments were performed in rats using quartz as a typical lung toxic particle. The results of pilot experiments suggest that Days 1 and 28 after intratracheal instillation of 2 mg of fine test particles in vehicle are most appropriate for detection of acute and subacute inflammatory changes, respectively. Furthermore, the BrdU incorporation on Day 1 and the iNOS level on Day 28 proved to be suitable end-point markers for this purpose. An examination of the toxicity of a series of particles was performed with the developed bioassay. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, a ranking order could be clarified. The bioassay thus appears suitable for rapid hazard identification with a possible ranking of the toxicity of various particles at single concentrations.     

Case report of pulmonary metastasis in a male Wistar rat glioblastoma model

Glioblastoma (GBM) is a highly aggressive central nervous system cancer. Its extracranial metastases have rarely been reported in the past few decades. Moreover, the pathogenesis of extracranial GBM metastases remains unclear. Here, we report a case of pulmonary metastasis in a male Wistar rat of C6 GBM model. This reported Wistar male rat was one of the experimental control group without any other intervention except for C6 GBM cells orthotopic implantation. On postoperative day 15, the animal which was reported in this study showed highly cellular, pleomorphic, tumor with nuclear atypia in the brain (Ki67, approximately 65.7%) and lungs (Ki67, 49.5%). Tumor cells in the lung showed immunoreactivity for glial fibrillary acidic protein. Inflammatory CD68⁺ cell infiltration, weakly positive E-cadherin, and strongly positive staining for vimentin were observed both in tumors in the brain and lungs. Based on further morphological analysis, we speculate that the potential metastatic route into the lung might be hematogenous metastasis.     

Orbital malignant schwannoma and embryonal rhabdomyosarcoma in rats caused by leakage of locally injected nickel subsulfide to the orbit

Nickel subsulfide (Ni₃S₂) is known to induce intraocular neoplasms when injected intravitreally into the eyes of rats. Here, we found two extraocular orbital neoplasms in two different rat strains, presumably due to the leakage of locally injected Ni₃S₂ to the extraocular orbital tissues. In the F344/DuCrlCrlj rat, an orbital mass arose at 30 weeks after injection, and invaded into the cranium. Histologically, the orbital mass was composed of areas arranged in parallel bundles formed by densely packed elongated or spindle-shaped cells with indistinct cytoplasmic borders, and of areas of hypocellular arrangement consisting of round cells in eosinophilic myxoid-like substances. Metastases were observed in the right submandibular and cervical lymph nodes. The neoplastic cells were immunopositive for S-100 protein and vimentin. Transmission electron microscopy revealed that the neoplastic cells had cellular processes and pericytoplasmic basal laminae. In the RccHan^TM:WIST rat, an orbital mass arose at 36 weeks after injection. Histologically, the mass consisted of rhabdoid-like large round cells with proliferation of small round-to-polygonal cells, and these neoplastic cells infiltrated into the extraocular muscles. Immunohistochemically, the neoplastic cells were positive for desmin and vimentin. Transmission electron microscopy detected immature myofibrils with Z-band structures in the cytoplasm of these neoplastic cells. Consequently, the tumors were diagnosed as an orbital malignant schwannoma in an F344/DuCrlCrlj rat and an orbital embryonal rhabdomyosarcoma in a RccHan^TM:WIST rat. The results of this case report suggest that leakage of Ni₃S₂ to the orbit caused the induction of orbital malignant schwannoma or rhabdomyosarcoma in 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/m³ 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/m³ 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/m³ 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/m³ 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/m³.     

Acute Phase Pulmonary Responses to a Single Intratracheal Spray Instillation of Magnetite (Fe3O4) Nanoparticles in Fischer 344 Rats

Iron nanomaterials are of considerable interest for application to nanotechnology-related fields including environmental catalysis, biomedical imaging, drug delivery and hyperthermia, because of their superparamagnetic characteristics and high catalytic abilities. However, information about potential risks of iron nanomaterials is limited. The present study assessed pulmonary responses to a single intratracheal spray instillation of triiron tetraoxide nanoparticles (magnetite) in rats. Ten-week-old male and female Fischer 344 rats (n=5/group) were exposed to a single intratracheal spray instillation of 0 (vehicle), 5.0, 15.0 or 45.0 mg/kg body weight (BW) of magnetite. After 14 days, the rats were sacrificed, and biological consequences were investigated. The lung weights of the 15.0 and 45.0 mg/kg BW male and female groups were significantly higher than those of the control groups. The lungs of treated rats showed enlargement and black patches originating from the color of magnetite. The typical histopathological changes in the lungs of the treated rats included infiltration of macrophages phagocytosing magnetite, inflammatory cell infiltration, granuloma formation and an increase of goblet cells in the bronchial epithelium. The results clearly show that instilled magnetite causes foreign body inflammatory and granulating lesions in the lung. These pulmonary responses occur in a dose-dependent manner in association with the increase in lung weight.     

Evaluation of Caspase-3 and Ki-67 expression in squamous cell hyperplasia of the stomach induced by Platycodi radix water extract in Sprague–Dawley rats

Platycodi radix is widely used in traditional herbal medicine for the treatment of bronchitis, asthma, pulmonary tuberculosis, hypertension, hyperlipidemia, and diabetes. This study aimed to investigate cell proliferation (Ki-67) and apoptosis (Caspase-3) potential in squamous cell hyperplasia of the stomach induced by a Platycodi radix water extract in a subchronic toxicity study. One hundred formalin-fixed, paraffin-embedded stomach tissues of rats treated with Platycodi radix at doses of 0, 500, 1,000, and 3,000 mg/kg body weight/day were used for the analysis. They were conventionally stained using hematoxylin and eosin (H&E) and immunohistochemically (IHC) stained using caspase-3 and Ki-67 antibodies. The incidence of squamous cell hyperplasia was significantly increased in the 3,000 mg/kg b.w./day treatment group in both sexes (p<0.01). However, the hyperplastic change was completely repaired after 4 weeks of recovery period. Ki-67 expression was similar in all groups, with no statistically significant differences among the groups. Caspase-3 expression was significantly increased in both sexes in the 3,000 mg/kg b.w./day treatment group (p<0.01), compared with the vehicle control groups, and then reduced to normal levels in the recovery groups in both sexes. In conclusion, this study showed that squamous cell hyperplasia induced by the Platycodi radix water extract in the limiting ridge of the stomach is not considered to be abnormal proliferative change; as a result, squamous cell hyperplasia is considered to be a non-adverse effect when induced by the oral administration of the Platycodi radix water extract once daily for 13 weeks in rats.