Endocardial schwannomas in the Wistar rat.

Endocardial neoplasms were observed in 13 Wistar rats, 15-30 months old, from a 30-month inhalation study. Eight of the rats were dissected at 30 months and the other five between 15 and 29 months. Histologically, the lesions in the heart were diagnosed as endocardial schwannomas. Two of the 13 schwannomas were malignant, while the rest were benign. One malignant schwannoma metastasized to the thymus and the other metastasized to the aorta, oesophagus, liver and spleen. Some schwannomas appeared as early lesions limited to the endocardium consisting of round to ovoid cells interspersed with spindle cells, while other schwannomas appeared as advanced lesions consisting of a thin superficial layer of round to ovoid cells and a deeper layer of spindle cells characteristic of endocardial schwannomas. The histological appearance of the metastases was identical to that of the primary tumour. The diagnosis was confirmed by positive immunostaining for S-100 protein. The incidence of endocardial schwannomas in the Wistar rat strain was 1.8%.

Metastasizing seminal vesicle adenocarcinoma in a Wistar rat.

An adenocarcinoma in the seminal vesicles of a 15-month-old male Wistar rat from a 30-month inhalation study is described. The rat was killed because of cachexia, apathy and a large palpable mass in the abdominal cavity. Macroscopic examination of the abdominal cavity revealed a 3.8 cm x 3.2 cm yellow-grey to pink mass, firm to soft in consistency. The cut section revealed cystic spaces. Histologically, the mass consisted of epithelial cells arranged in glandular and solid patterns with abundant amounts of connective tissue. Epithelial tumour cells were round-to-cylindrical with round-to-oval basophilic nuclei and one or two prominent nucleoli and a distinct eosinophilic cytoplasm. The glandular structure contained clusters of macrophages in their lumen with eosinophilic cytoplasm and indented nuclei. Extensive necrosis and reactive inflammation were present. The histological features of the small nodules in the pancreas and on the surface of the liver, rectum and urinary bladder resembled those of the primary tumour in the seminal vesicles. Based on these criteria, the neoplasm (mass) was diagnosed as an adenocarcinoma of the seminal vesicles. The immunohistological examination confirmed the diagnosis, i.e. immunostaining was positive for cytokeratins (4, 7, 14, 15, 18, and 19), vimentin, PCNA, and ED(1).

Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate.

Lung tumors have been reproducibly induced in A/J mice exposed to a surrogate for experimental environmental tobacco smoke (ETSS) in a 5-mo inhalation period followed by 4 mo without further exposure. In order to increase our mechanistic understanding of this model, male mice were whole-body exposed for 6 h/d, 5 d/wk to ETSS with a particulate matter concentration of 100 mg/m(3). Food restriction regimens were included to model or exceed the ETSS-related impairment of body weight development. Half of the mice were pretreated with a single ip injection of urethane to study the effect of the above treatments on lung tumor development induced by this substance. At 5 mo, the tumor response was statistically the same for all groups of non-pretreated mice; however, the expected urethane-induced lung tumorigenesis was significantly inhibited by approximately 25% by ETSS and food restriction. This inhibition was accompanied by a threefold increase in blood corticosterone as a common stress marker for both ETSS and food restriction. At 9 mo, in mice not pretreated, the lung tumor incidence and multiplicity were significantly increased by twofold in the ETSS group; in the urethane-treated groups, the same high tumor multiplicity was reached regardless of previous treatment. The predominant tumor type in all groups was bronchiolo-alveolar adenoma. There was no induction of a specific K-ras mutation pattern by ETSS exposure. These data suggest a stress-induced inhibition of lung tumorigenesis in this model, explaining the need for the posttreatment period.

Toxicological evaluation of an electrically heated cigarette. Part 4: Subchronic inhalation toxicology.

The biological activity of mainstream smoke from an electrically heated cigarette (EHC) with controlled combustion and from the University of Kentucky Reference Cigarette 1R4F was determined in Sprague Dawley rats exposed nose-only for 90 days, 6 h a day, 7 days per week. For an equivalent response comparison between the two cigarette types, two doses were chosen for the EHC where the anticipated results were in the dynamic range of the 1R4F dose-response curve (four concentrations) for most end points. The number of cigarettes smoked per m(3) of diluted smoke resulted in total particulate matter concentrations of 40 and 90 microg l (-1) for the EHC and 40-170 microg l (-1) for the 1R4F. Biomonitoring indicated achievement of target doses. Mainstream smoke yields were lower for the EHC, with the exception of formaldehyde. No smoke-related mortality, remarkable in-life observations or abnormal gross pathological findings were observed. Smoke- and dose-related clinical pathology and organ weight changes included: increases in segmented neutrophils, some liver parameters and lung and adrenal weight relative to body weight; and decreases in lymphocytes, glucose concentration and spleen weight. Smoke-related histopathological findings in the respiratory tract included epithelial cell hyperplasia, squamous metaplasia, atrophy and accumulation of pigmented alveolar macrophages; they were mostly dose-dependent, more pronounced in the upper than lower respiratory tract and completely or partially reversed by 6 weeks post-inhalation. Qualitatively, the biological effects seen for the EHC and the 1R4F were comparable and similar to those observed in other mainstream smoke inhalation studies. Quantitatively, the biological activity of the EHC mainstream smoke was, on average, 65% lower than that of the 1R4F mainstream smoke on an equal cigarette basis and equivalent activity on an equal TPM basis.

Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity.

Mainstream smoke from blended research cigarettes with (test) and without (control) the addition of ingredients to the tobacco was assayed for inhalation toxicity. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was introduced at a low and a high level to the test cigarettes. Male and female Sprague-Dawley rats were exposed nose-only either to fresh air (sham) or diluted mainstream smoke from the test, the control, or the Reference Cigarette 1R4F at a concentration of 150 microg total particulate matter/l for 90 days, 6h/day, 7 days/week. A 42-day post-inhalation period was included to evaluate reversibility of possible findings. There were no remarkable differences in in-life observations or gross pathology between test and control groups. An increase in activity of liver enzymes, known to be due to the high smoke dose, revealed no toxicologically relevant differences between the test and control groups. No toxicological differences were seen between the test and control groups for smoke-related hematological changes, such as a decrease in total leukocyte count. The basic smoke-related histopathological effects, which were more pronounced in the upper respiratory tract than in the lower respiratory tract, were hyperplasia and squamous metaplasia of the respiratory epithelium, squamous metaplasia and atrophy of the olfactory epithelium, and accumulation of pigmented alveolar macrophages. There were no relevant qualitative or quantitative differences in findings in the respiratory tract of the rats exposed to the smoke from the control and test cigarettes. The data indicate that the addition of these 333 commonly used ingredients, added to cigarettes in three groups, did not increase the inhalation toxicity of the smoke, even at the exaggerated levels used.

Metastasizing fibrosarcoma in a Wistar rat--case report.

A 19-month-old male control Wistar rat from a 30-month inhalation study showed a subcutaneous greyish-white mass extending from the throat to the thoracic cavity. The rat had been euthanized because of its poor general condition. Histologically, the mass was diagnosed as a fibrosarcoma infiltrating the masseter muscle with metastases in the lungs, liver and heart. The primary tumour was characterized by fusiform spindle cells producing various amounts of interlacing bundles of collagen. The cells formed a characteristic herringbone pattern and mitotic figures were frequent. The histological parameters of the metastases were practically identical to those seen in the primary tumour. The diagnosis was confirmed by trichrome staining and positive immunostaining for vimentin and was differentiated from leiomyosarcomas by its negative immunostaining for desmin, from schwannomas by its negative immunostaining for S-100 and from malignant fibrous histiocytomas by the absence of giant cells. The incidence of fibrosarcomas in Wistar rats is very low (up to 3%) and metastasis is rarely observed.

Hepatocholangiocellular carcinoma in a rat--case report.

A mixed epithelial tumour in the liver of a 24-month-old male Wistar rat from a 30-month inhalation study is described. The rat, which was in a group exposed to low concentrations of diesel exhaust, was euthanized because of emaciation, forced respiration and abnormal gait. Macroscopic examination of the enlarged liver revealed multiple partly confluent beige-red nodules up to 1.5 cm in diameter. Small nodules up to 7 mm in diameter were seen in the spleen. Histologically, the tumour nodules in the liver consisted of hepatocellular and cholangiocellular components. The hepatocellular component consisted of moderately differentiated polygonal to round hepatocytes about twice as large as normal hepatocytes and having hyperchromatic, centrally located nuclei with prominent nucleoli and eosinophilic cytoplasm. Foci of haematopoiesis and focal necroses were prominent. The cholangiocellular component was moderately differentiated and consisted of tubular structures lined by low cuboidal to cylindrical cells showing cytoplasmic basophilia and small dark nuclei without prominent nucleoli. The histological features of the nodules in the spleen corresponded to those of the primary tumour in the liver. Based on these criteria, the tumour nodules were diagnosed as hepatocholangiocellular carcinoma. The immunohistological examination confirmed the diagnosis, i.e. immunostaining for cytokeratins was positive for eight and 18 (hepatocellular carcinoma) and for seven and 19 (cholangiocellular carcinoma) as well as for vimentin (dense fibrous stroma). This tumour is considered to be spontaneous because of its single occurrence.

Tobacco smoke-induced alterations of cytokeratin expression in the rat nasal activity cavity following chronic inhalation of room-aged sidestream smoke.

In a 12-month inhalation study on rats using room-aged sidestream smoke (RASS, 12 microg total particulate matter (TPM)/l) as an experimental surrogate for environmental tobacco smoke (ETS), we investigated differentiation changes, i.e. altered cytokeratin (CK) expression, in the epithelial lining at nasal cavity level 1 (NL1) (anterior portion of nasal cavity), and their correlation with histomorphological changes. In addition to conventional histopathological examination, routine paraffin sections were immunohistologically stained for various rat CK and evaluated. Changes in CK expression were observed in the nonciliated respiratory epithelium of maxilloturbinate, lateral wall, and nasoturbinate: in basal cells, increase of CK14 and CK18 and decrease of CK15; in nonciliated columnar cells, increase of CK15 and CK19. These CK changes had histomorphological correlates, i.e. reserve cell hyperplasia and squamous metaplasia. CK expression changes were also seen at sites without histomorphological changes, e.g. enhanced expression of CK14, CK18 in ciliated cells at the dorsal meatus, and CK15 at the septum. Most of the CK expression changes seen after 1 year of RASS exposure resembled the changes previously seen after 8 days of exposure.

Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. II. Changes in cytokeratin expression patterns following 8-day exposure to room-aged cigarette sidestream smoke.

The expression of specific cytokeratin (CK) polypeptide patterns is a sensitive marker of the cytoskeletal differentiation of epithelial cells. We developed an immunohistochemical method to assess CK expression patterns in the rat respiratory tract using serial paraffin-embedded sections from the nasal cavity, trachea, and lung. In the present study, this method was used to detect exposure-related differences in CK expression patterns in adult Wistar rats following inhalation of room-aged sidestream smoke (11 mg total particulate matter/m3 air, 8 days, 12 hr/day, whole body). In the anterior nasal cavity level 1 (NL1), changes in CK expression patterns were observed in the respiratory epithelium of the lateral wall and the maxilloturbinate (CK14, CK15, and CK18) and in the squamous epithelium of the ventral meatus (CK13). At nasal cavity level 2 (NL2), immediately behind NL1, changes were observed in the olfactory epithelium (CK13, CK14, and CK18) and in the respiratory epithelium of the septum (CK7 and CK19), the lateral wall (CK7 and CK13), and the lateral aspect of the maxilloturbinate (CK14). Changes were also observed in the submucosal glands, nasolacrimal duct, and vomeronasal organ. In the trachea only CK7 expression changed, and in the lung expression of CK7 (bronchioli) and CK8 (bronchus) changed; the expression of other CK polypeptides did not change. The observed changes in CK expression at NL1 correlated with the histomorphological changes, whereas CK expression changes were also seen in the olfactory and respiratory epithelia at NL2 and in the trachea and lung, where no histomorphological changes were seen. These findings indicate that changes in CK expression in respiratory tract epithelial cells are a sensitive marker for cellular stress response.

Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. I. Determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung.

Cytokeratin (CK) polypeptides constitute the intermediate filament cytoskeleton of epithelial cells. The patterns of CK expression can be regarded as specific markers for the epithelial differentiation status. Our objective was to map the cell type-specific CK expression patterns at all representative sites of the respiratory tract of untreated rats to use as a base for the detection of inhalation exposure-related differentiation changes. Using routine paraffin-embedded sections and a panel of well-characterized monoclonal antibodies for immunohistochemistry, we obtained CK staining patterns as follows. Nasal cavity: respiratory epithelium CK18, CK19 (basal, ciliated, nonciliated cells), CK14, and/or CK15 (basal and nonciliated cells); olfactory epithelium CK18 (basal, mid, apical zones and Bowman's glands), CK14, and CK15 (basal zone); squamous epithelium of ventral meatus CK14, CK15 (basal and suprabasal cells), CK1, 10/11, and CK13 (suprabasal cells); glands and columnar epithelia of vomeronasal organ and nasolacrimal duct CK7 and CK13 in addition to respiratory epithelial CK pattern. Trachea: similar to nasal respiratory epithelium with pronounced CK15 and additional CK7. Larynx: CK14, CK15 (basal, ciliated, nonciliated cells), CK8, CK18, CK19 (not in basal cells), CK4, and CK13 (cuboidal and squamoid cells of ventral half). Lung: bronchial epithelium CK14 and CK15 (basal cells only); bronchial and alveolar epithelium CK7, CK8, CK18, and CK19; bronchiolar epithelium similar but less CK8 and no CK7; pleural mesothelium CK7, CK8, and CK19. This inventory of complex CK expression patterns provides the basis for investigating test substance-related effects in inhalation toxicology, e.g., cigarette smoke-induced changes.

Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats.

Two experimental types of cigarette sidestream smoke (SS) were compared in a subchronic inhalation study on rats. Fresh SS (FSS) was generated continuously from the reference cigarette 2R1. Room-aged SS (RASS) was generated by aging FSS for 1.5 h in a room with noninert surfaces with materials typically found in residences or offices. Male Sprague-Dawley rats were head-only exposed to three dose levels of each SS type and to filtered, conditioned fresh air (sham-exposure) for 6 h/day, 7 days/week, for 90 days. Room-aging resulted in decreased concentrations of various SS components, e.g., total particulate matter (TPM) and nicotine, while other components, such as carbon monoxide (CO), were not affected. The CO concentrations were 6, 13, and 28 ppm for both SS types. TPM concentrations were between 0.6 and 8.7 micrograms/liter and thus up to 100-fold above the maximum of average concentrations of respiratory suspended particles reported for environmental tobacco smoke. Slight reserve cell hyperplasia in the anterior part of the nose as well as hyperplastic and metaplastic epithelial changes in the larynx were the only observed dose-dependent findings. The metabolism of benzo(a)-pyrene--as a proxy for polycyclic aromatic hydrocarbon metabolism--was induced in the nasal respiratory epithelium and in the lungs while no effect was seen in the nasal olfactory epithelium. The lowest-observed effect level was 6 ppm CO or 0.6 microgram TPM/liter. Most of the effects seen were less expressed in RASS-than in FSS-exposed rats when compared on the basis of the CO concentrations. When compared on the basis of TPM, these effects were equally pronounced for both SS types, suggesting a major role of particulate matter-associated compounds. All findings reverted to sham control levels following a 42-day postinhalation period.

Cytokeratin patterns of epithelial cells of the rat nasal cavity in vivo and in vitro.

Nasal epithelial cells are a primary target for the actions of inhaled substances. To enable the determination of alterations in cell differentiation in the rat inhalation model, we developed a methodology to assess cytokeratin expression in rat nasal tissue. A panel of commercially available antibodies was validated for specificity to defined rat cytokeratins by immunoblotting. The development of immunohistological procedures to enhance spatial resolution enabled mapping of cytokeratin patterns in various cell types at defined regions and levels of the rat nasal cavity using serial sections and a standardized evaluation schedule.