Assessment of the morphology and significance of the lymph nodal and hepatic lesions produced in rats by the feeding of certain mineral oils and waxes. Proceedings of a pathology workshop held at the Fraunhofer Institute of Toxicology and Aerosol Research Hannover, Germany, May 7-9, 2001.

A Panel of medical and veterinary pathologists reviewed published and unpublished reports dealing with studies of various white mineral oils and waxes in F344 and Sprague-Dawley rats. They also had available and studied histologic slides from both subchronic and chronic studies of certain mineral hydrocarbons (90-day oral study of low melting point wax (LMPW) in female Fischer 344 and Sprague-Dawley rats; 90-day studies of P15H* and P70H white oil and high melting point wax (HMPW) in male and female F344 rats and 24 month study of P70H white oil in male and female F344 rats. The Panel also reviewed mineral oil-induced alterations in tissues of human patients (liver, hepatic lymph node and spleen). The Panel agreed that certain of the mineral hydrocarbons produced lesions in the mesenteric lymph nodes and liver of the F344 rat and these lesions were best described as microgranulomas/granulomas. The lesions were fundamentally similar in both organs, although varying in severity with dose and type of mineral hydrocarbons. The Panel agreed that hepatic lesions with inflammatory cell infiltration, necrosis, and fibrosis were produced only by feeding of LMPW and the lesions were confined to F344 rats and not found in Sprague-Dawley rats. The most severe granulomatous lesions in the mesenteric lymph nodes were found in high dose LMPW-fed F344 rats. The microgranulomas were similar in subchronic and chronic studies. Also, little difference existed between controls and treated F344 rats in the incidence and severity of the lesions after 2 years of feeding P70H white oil. The Panel agreed that some slight reversibility existed for these lesions, but also agreed that complete resolution was unlikely as regression of the lesions in the rat would likely be slow. The Panel agreed that a minimal severity infiltrate of mononuclear inflammatory cells occurred in the base of the mitral valve in a slightly increased incidence in F344 rats fed LMPW. The Panel concluded that these mitral valve alterations had little if any toxicologic significance as the focal infiltrate was minimal in severity, occurred in controls, occurred in association with murine cardiomyopathy, and were unlike the responses in the liver and mesenteric lymph nodes. The Panel agreed that the lesions observed in the liver and mesenteric lymph nodes of F344 rats exposed to MHCs, especially the LMPW, were different morphologically from changes observed in lymph node, liver, and spleen of humans that were mineral oil-users. These changes in humans are usually found incidentally in tissues taken at biopsy or autopsy. The MHC-induced lesions can be considered incidental and inconsequential in humans.

Cystic squamous cell carcinomas in the lungs of Syrian golden hamsters induced by coal oven flue exhaust mixed with pyrolized tar pitch in combination with benzo(a)pyrene.

Among a variety of induced pulmonary tumours, cystic squamous cell carcinomas were observed in five Syrian hamsters that inhaled a mixture of pyrolized tar pitch with coal oven flue exhaust (PCE) and additionally received intratracheal injections of benzo(a)pyrene. The histological appearance of these particular tumours is described, compared to similar tumour types in rats and the susceptibility of both species to inert particles is discussed.

Pulmonary cystic keratinizing squamous cell lesions of rats after inhalation/instillation of different particles.

Cystic keratinizing squamous cell lesions from three inhalation studies (Study A, B, C) and one intratracheal instillation study (Study D) in rats were reclassified and a certain number of lesions examined immunohistochemically for PCNA (proliferating cell nuclear antigen) as a marker of cellular proliferation. The following classification was used: squamous cell metaplasia with marked keratinization, keratinizing cyst, cystic keratinizing epithelioma, cystic keratinizing squamous cell carcinoma, keratinizing squamous cell carcinoma and non-keratinizing squamous cell carcinoma. In study A (inhalation of coal oven exhaust and subcutaneous injection of a high dose of DB (ah)A) 49.3% of rats developed cystic keratinizing squamous cell carcinomas. Inhalation of coal oven exhaust gas together with intratracheal instillation of crocidolite or subcutaneous injection of a low dose DB(ah)A (dibenz(ah)anthracene) resulted in cystic keratinizing squamous cell carcinomas in 23% to 24% of the rats. High incidences of cystic squamous cell carcinomas in the range of 31.9% to 76.4% were observed in rats of Study B1 after a 10-months exposure to tar/pitch condensation aerosol (different B(a)P (benzo(a)pyrene) concentrations) with added carbon black in some groups. After a 20-months exposure period to the same inhalation atmospheres (Study B2) the incidence of squamous cell carcinomas was increased up to 95.8%. Exposure of rats to various concentrations of unfiltered diesel exhaust (Study C) resulted in incidences of cystic keratinizing epitheliomas ranging from 2.5% (2.5 mg/m3) to 10.7% (7.5 mg/m3). Epitheliomas were also observed in 16.2% of carbon black and 16.0% of titanium dioxide exposed rats. Only a few cystic keratinizing squamous cell carcinomas occurred. In the intratrachel instillation study (Study D) increased incidences of cystic keratinizing epitheliomas occurred in rats exposed to native diesel exhaust particles (16.7%), high dose of extracted diesel exhaust particles (14.6%), extracted printex 90-carbon black particles (18.8%), and extracted printex 90-carbon black particles + B(a)P (18.8%). High indicences of cystic keratinizing squamous cell carcinomas were noted in rats that received 15 mg B(a)P (14.6%) or 30 mg B(a)P (72.7%) intratracheally. Immunohistochemical labeling of nuclei with PCNA demonstrated proliferative activity in one or two (and focally more than two) peripheral cell layers of cystic keratinizing epitheliomas and in more than three peripheral cell layers of cystic keratinizing squamous cell carcinomas and keratinizing squamous cell carcinomas. The wall of keratinizing cysts showed no or a weak reaction.

Classification of cystic keratinizing squamous lesions of the rat lung: report of a workshop.

An international workshop of toxicologic pathologists reviewed cystic keratinizing squamous lesions of the rat lung. These lesions develop in response to the chronic inhalation of diverse particulate materials. Controversy exists over the biological significance of these changes and their relevance to humans. For the first time, in one place, a group of pathologists analyzed slides from all available studies. The workshop reached a consensus as to classification of these unique pulmonary tissue responses and offers diagnostic criteria for application. Although additional research is needed, this working classification scheme should serve as a practical interim approach for pathologists and regulatory agencies.

Neuroendocrine phenotype differentiation in a hamster lung epithelial cell line under low oxygen pressure or after transformation by diethylnitrosamine.

Oxygen pressure as low as 5% in the gas phase or 87 mmHg in the liquid phase induced various neuroendocrine cell (NEC) phenotypes in more than 80% of cells of a cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3). Further, cells from a number of colonies transformed in an anchorage-independent manner by diethylnitrosamine (DEN) demonstrated a NEC phenotype. Since the cell line used is of a pluripotent stem cell type, both hypoxia and DEN appear to possess a potency for NEC phenotype induction.

Effect of bovine respiratory syncytial virus infection on hypersensitivity to inhaled Micropolyspora faeni.

Respiratory syncytial virus causes mild-to-severe respiratory disease in human infants and young children; a closely related bovine respiratory syncytial virus causes a similar disease pattern in calves. Increased disease severity in atopic children suggests that allergic reactivity may enhance the severity of RSV-induced disease. To examine the association between bovine respiratory syncytial virus (BRSV) infection and allergic reactivity two groups of calves were exposed to aerosolized Micropolyspora faeni (Mf) during an experimental BRSV infection. One group exposed to Mf concurrent with BRSV was challenge-exposed to Mf while infected a second time with BRSV, while the other similarly sensitized and infected group was mock challenged. A control group was exposed only to Mf aerosol and another control group was infected with virus but not exposed to Mf aerosol. Parameters examined included: clinical signs, Mf-specific IgG and IgE, BRSV-specific antibody and IgE, leukotrienes C4 and B4 prostaglandins E2, F2 alpha and D2, and lung pathology. While the initial BRSV infection failed to enhance sensitization to inhaled Mf, a second BRSV infection exacerbated clinical signs resulting from Mf aerosol. Consideration of eicosanoid and antibody profiles together with clinical signs suggests that mechanisms of both type I and type III hypersensitivity were operative during Mf challenge of sensitized calves.

Irradiation of nonlymphoproliferative neoplasms of the nasal cavity and paranasal sinuses in 16 cats.

Sixteen cats with malignant tumors (10 carcinomas, 6 sarcomas) of the nasal cavity and paranasal sinuses were treated with curative intent by radiotherapy. Clinical stating was based on radiographic findings, using the tumor, node, metastasis classification system of the World Health Organization. Irradiation was done with a telecobalt-60 unit (13 cats) and an orthovoltage unit (3 cats). Fourteen cats were treated with irradiation alone, and 2 cats had incomplete surgical resections prior to radiotherapy. Treatment dose was 48 Gy (minimum tumor dose), administered by use of 4 Gy per fraction on a Monday/Wednesday/Friday basis over 4 weeks. Survival times after treatment ranged from 1 to 36 months. The 1- and 2-year overall survival rates were 44.3 and 16.6%, respectively. Histologic type and clinical stage did not have prognostic value. Most acute radiation reactions were mild and self-limiting. Chronic ocular complications were seen in 3 cats. These treatment responses compared favorably with those previously described in dogs and cats with intranasal neoplasms treated with teletherapy and provided a perspective for comparison of new treatment methods.

Morphological comparison between benign keratinizing cystic squamous cell tumours of the lung and squamous lesions of the skin in rats.

Approximately 700 cases of keratinizing cystic squamous lung lesions in rats were investigated by light microscopy in order to clarify the nomenclature and classification of these lesions. The structure of benign keratinizing cystic squamous cell tumours of the lung was compared to that of cystic squamous lesions in the skin of rats, with consideration of data from the literature. We conclude that the reviewed keratinizing cystic squamous cell lesions of the lung are true neoplasms and that the growth pattern of these cystic lesions is inconsistent with that of a simple cyst. In the development of squamous lung cancer, a continuum of proliferation from exaggerated metaplasia through benign cystic tumours to invasive squamous cell carcinomas can be observed.

Response of macaque bronchiolar epithelium to ambient concentrations of ozone.

Recently, we reported that exposure to ambient concentrations of ozone, near the U.S. National Ambient Air Quality Standard (0.12 ppm), induced significant nasal epithelial lesions in a non-human primate, the bonnet monkey. The present study defines the effects of ambient concentrations of ozone on the surface epithelium lining respiratory bronchioles and on the underlying bronchiolar interstitium in these same monkeys. Bonnet monkeys were exposed to filtered air or to 0.15 or 0.30 ppm ozone 8 hours/day for 6 or 90 days. At the end of exposures, monkeys were anesthetized and killed by exsanguination. Microdissected bronchiolar airways of infusion-fixed lungs were evaluated morphometrically by light microscopy and quantitatively by scanning and transmission electron microscopy for ozone-induced epithelial changes. Hyperplasia of nonciliated, cuboidal epithelial cells and intraluminal accumulation of macrophages characterized ozone-induced lesions in respiratory bronchioles. There were no significant differences in epithelial thickness or cell numbers among ozone-exposed groups. Ozone-exposed epithelium was composed of 80% cuboidal and 20% squamous cells compared with 40% cuboidal and 60% squamous cells in filtered air controls. In addition, the arithmetic mean thickness of the surface epithelium, a measure of tissue mass per unit area of basal lamina, was significantly increased in all of the ozone-exposed groups. The number of cuboidal epithelial cells per surface area of basal lamina was increased above control values by 780% after 6 days exposure to 0.15 ppm, 777% after 90 days to 0.15 ppm, and 996% after 90 days exposure to 0.30 ppm. There was also a significant ozone-induced increase in the thickness of the bronchiolar interstitium that was due to an increase in both cellular and acellular components. These results demonstrate that exposure to low ambient concentrations of ozone, near the current. National Ambient Air Quality Standard, induces pulmonary lesions in primates. The alterations do not appear to be concentration- or time-dependent, suggesting that the current National Ambient Air Quality Standard may be at or above the threshold for deep lung injury in primates.

Megavoltage irradiation of neoplasms of the nasal and paranasal cavities in 77 dogs.

Seventy-seven dogs with malignant tumors of the nasal and paranasal cavities were treated by use of radiotherapy. The tumors included carcinomas (58) and sarcomas (19). Radiographic findings, including site of involvement and tumor extension, were the basis of clinical staging. Staging was performed according to the tumor, node, metastasis staging of the World Health Organization, and a modified staging scheme based on prognostic factors that seemed to correlate best with response to treatment. All irradiations were done with a telecobalt 60 unit. Fifty-six dogs were treated with irradiation alone, and 21 had partial tumor resection prior to radiotherapy. Treatment dose was 48 Gy (minimal tumor dose) administered on a Monday-Wednesday-Friday basis at 4 Gy/fraction over 4 weeks. The irradiation technique emphasized rostral field with a generous treatment volume. Duration of follow-up after irradiation ranged from 1 month to 61 months. The 1- and 2-year overall survival rates were 60.3% and 25%, respectively, and the 1- and 2-year relapse-free survival rates were 38.2% and 17.6%, respectively. Results of histologic examination and our modified staging scheme were significant (P = 0.02 and P = 0.04, respectively) prognostic factors of relapse-free survival. Conversely, tumor site, tumor extension, World Health Organization clinical stage, and cytoreductive surgery prior to irradiation did not affect the outcome of treatment. According to our modified staging scheme, dogs with stage-2- disease have a poorer prognosis than dogs with stage-1 disease, with a relative risk of relapse 2.3-fold higher. Dogs with carcinoma had a poorer prognosis than dogs with sarcoma (predominantly chondrosarcoma) with a relative risk of relapse 3.3-fold higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology and histogenesis of squamous cell metaplasia of the rat lung after chronic exposure to a pyrolized pitch condensate and/or carbon black, or to combinations of pyrolized pitch condensate, carbon black and irritant gases.

Female Wistar rats were exposed to different concentrations of a pyrolized pitch condensate and/or carbon black particles and/or a combination of irritant gases for 18 hours/day, 5 days/week for 10 months, followed by a clean air period of up to 20 months. Bronchiolo-alveolar hyperplasia and squamous metaplasia were important components of the resulting lesions. Squamous metaplasia and associated hyperplasia was investigated by routine histology, scanning and transmission electron microscopy, and by immunohistochemical detection of various cytokeratins (CKs). Intensely CK positive squamous metaplasia lacking a distinct stratum spinosum was distinguishable from squamous metaplasia with a distinct stratum spinosum that reacted weakly CK positive or CK negative. The CK positive type was histologically characterized by narrow intercellular spaces, the weakly CK positive or CK negative type had markedly enlarged intercellular spaces. Differentiated hyperplastic epithelium and the normal lung parenchyma reacted CK negative. In poorly differentiated hyperplasia of the alveolar type associated with squamous metaplasia scattered cells with characteristics of squamous differentiation were detected. Ultrastructurally these cells showed increased amounts of filament bundles and immunohistochemically a positive reaction with the CK antibody. These cells were regarded as precursor stages of squamous metaplasia of the lung periphery in rats.

K-ras activation in non-small cell lung cancer in the dog.

To investigate the role of K-ras mutations in canine non-small cell lung cancer, we first determined the nucleotide sequence of the normal canine K-ras gene and then examined 21 canine lung tumors for activating K-ras mutations. Canine K-ras was analyzed by direct sequencing of polymerase chain reaction products generated with oligonucleotide primers derived from the human K-ras sequence. Four nucleotide differences were found between the canine and human K-ras sequence from position 5 to 211. The deduced amino acid sequence of the canine gene was identical to that of the human. Activated K-ras alleles were detected in 5 of the 21 canine lung tumors examined. The activating lesions were point mutations, predominantly in codon 12. Of the 14 adenocarcinomas examined, 2 (14%) had K-ras mutations. Two of 5 (40%) adenosquamous carcinomas and the only large cell carcinoma also contained activated alleles. The overall frequency of K-ras point mutation in non-small cell lung cancer (25%) is similar to that reported in human non-small cell lung cancer. We conclude that K-ras activation by point mutation is associated with, but not necessary for, non-small cell lung cancer development in the dog.

Induction and characterization of secretory differentiation in human fetal bronchial epithelial cell line (HFBE) cultured on collagen gel in growth hormone and vitamin A-supplemented medium.

A type of secretory differentiation was induced and characterized in a human fetal bronchial epithelial cell line (HFBE), which was grown on a collagen substratum in a basal differentiative medium (BDM) containing growth hormones and with supplementation of various concentrations of vitamin A (VA). HFBE cells grown on a collagen gel in BDM with or without VA assumed a spindle shape with thick cytoplasm arranged in strands running parallel to each other. Under a phase-contrast microscope, cells cultured in the absence of VA possessed a small number of bright inclusion bodies, which proved to be positive to PAS and almost negative to alcian-blue (AB) staining. Electron microscopy revealed well-developed rough endoplasmic reticulum (rER), enlarged Golgi apparatus and a small number of high-density granules resembling serous or Clara cell granules. HFBE cells treated with VA at levels higher than 6 mu/ml showed a remarkable increase of the secretory granules and contained amorphous material in the rER. Addition of a low concentration of VA (6 ng/ml) only stimulated the growth of HFBE cells. In contrast, higher concentrations of VA significantly inhibited the growth and 3H-thymidine incorporation into DNA in a dose-dependent manner. HFBE cells cultured on collagen gel with VA secreted products with 2 different molecular weights into the medium. A high molecular weight-product, consisting of void volume fractions from a Bio-gel A 15-m column, was identified as hyaluronic acid based on the results obtained from the DEAE-ion exchange chromatography and specific enzymatic digestion. A low molecular weight-product fractionated on the A 15-m was tentatively identified as mainly neutral glycoproteins containing N-linked glycans. While the secretion of hyaluronic acid was inhibited by VA in a dose-dependent manner, the secretion of the neural glycoproteins was most enhanced by VA in the range from the physiological concentration of 600 ng/ml to 6 micrograms/ml. These biochemical data on the secretory products, together with the morphological findings, demonstrate that the HFBE cell line serves as a new model for investigating the cellular differentiation of human lung epithelium.

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 micrograms/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glycoproteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. Moreover, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.

A comparison of terminal airway remodeling in chronic daily versus episodic ozone exposure.

This study compares centriacinar changes by ultrastructural morphology and morphometry following daily versus episodic ozone exposure in rats. Three groups of rats were exposed to air, 0.95 ppm ozone 8 hr daily for 90 days, and 0.95 ppm ozone 8 hr daily in seven successive 5-day episodes separated by 9-day recovery periods for a total of 89 days. Sections from the left lung and dissected acini from the right middle lobe were studied by light and electron microscopy. The centriacinar lesion following episodic exposure was similar but diminished in severity compared to that of rats exposed daily. Damage following episodic exposure appeared to be more than predicted by an exposure regimen which delivered 35% of the total ozone dose during daily exposure. The total volume of affected parenchyma was similar following both exposures. Respiratory bronchiole formation increased following both exposures but this was only statistically significant following daily exposure. The most severe epithelial damage was at the tips of alveolar septa in alveolar ducts distal to the respiratory bronchiole. Interstitial thickness in the injured respiratory bronchiole and proximal alveolar duct increased significantly and similarly following both exposures. Epithelium along the respiratory bronchiole of daily exposed rats was more differentiated. In the episodic group, respiratory bronchiole and alveolar duct epithelium consisted of a range of intermediate, less differentiated bronchiolar or alveolar epithelial cells. The episodic exposure resulted in a diminished lesion, but there appears to be some cumulative effect of repeated exposures with respiratory bronchiolar and alveolar duct epithelium in a more dynamic state of injury and repair.

Immunoglobulin E responses and lung pathology resulting from aerosol exposure of calves to respiratory syncytial virus and Micropolyspora faeni.

Bovine respiratory syncytial virus (BRSV) infection of calves has been associated with a type-I hypersensitivity syndrome not unlike respiratory syncytial virus-associated pulmonary symptomatology in humans. To study the mechanism of pulmonary pathology in calves and define the relationship with both viral-specific IgE response and IgE titers to concurrent aerosol of Micropolyspora faeni (Mf) we subjected groups of calves to inhalation of Mf during acute BRSV infection. The calves were divided into 4 groups: exposed to virus only (group 1); exposed to aerosolized Mf over a 24-day period and then challenged with Mf during BRSV infection (group 2); similarly exposed to aerosolized Mf and then challenged with Mf without BRSV infection (group 3) and exposed to aerosolized Mf, infected with virus but not challenged with Mf (group 4). All calves were followed for appearance of IgE-specific responses to both BRSV and Mf, clinical disease expression, and pulmonary pathology including viral and allergen localization by immunohistology. Our data indicate an association of BRSV-specific IgE with increased development of lung pathology and clinical disease expression and an enhancing effect of aerosolized Mf on induction of virus-specific IgE. The presence of BRSV-specific IgE was solely in calves with the most significant macroscopic and microscopic pneumonic lesions.