Donald L. Dungworth

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.

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.

Structural Evaluation of the Respiratory System

The theoretical and practical bases for morphological evaluation of the respiratory system useful for inhalation toxicology are reviewed. For most studies we recommend a comprehensive gross examination followed by in vitro tracheal infusion of a fixative containing both glutaraldehyde and formaldehyde in cacodylate buffer. Lungs fixed in this manner are suitable for LM, SEM, and TEM and lung volumes can be determined. The airway orientation of many lesions and the potential for gradients of damage are considered in the lung sampling plan. While LM of paraffin sections continues to be the basic method for evaluation, the SEM and TEM, especially when ancillary methods are used, provide valuable additional information. The use of backscattered electrons and energy-dispersive X-ray analysis in the SEM provides information concerning the localization and elemental analyses of particles. Cytochemical procedures characterize biological activities of specific cell types and are becoming more widely used. Morphometry permits correlation of quantified structure with physiological and biochemical data.

Assessment of Inhalation Hazards

Convert to machine form, etc. Identify needs for expertise Find and recruit experts Organize joint efforts, within constraints

In vitro evidence of cellular adaptation to ozone toxicity in the rat trachea

Abstract Adaptation to prolonged ozone (O3) exposure occurs in the tracheal epithelium of rats and is marked by the presence of ciliated cells with uniform short cilia but is not accompanied by shifts in cell populations, altered characteristics of epithelial secretory cell products, increased cell turnover, or elevated tracheal superoxide dismutase activity. The purpose of this study was to test the hypothesis that adaptation is a result of alterations intrinsic to epithelial cells or to the cells and their matrix, and not due to systemic or neural influences. Rats were preexposed to either filtered air (FA) or 0.96 ppm O3 for 8 hr/night for 60 days, and then their tracheae were removed and exposed to 3 ppm O3 in an explant culture system where behavioral, nasal, exudative, and secretory product influences can be eliminated. After exposure to 3 ppm O3 in vitro, quantitative electron microscopic evaluation demonstrated that the epithelia from the FA preexposure group had significantly more necrotic cells and sloughed cells and fewer ciliated cells than the epithelia from the O3 preexposure group. Thus previous exposure to ozone in vivo induces a change in tracheal epithelium which confers resistance to ozone-induced injury in the explant exposure system.

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

Summary 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.

Ozone-induced structural changes in monkey respiratory system

Abstract The principal sites of ozone-induced damage in the respiratory tracts of monkeys are the anterior nasal cavity and respiratory bronchioles. Intermittent exposures (8 h/day) for 6 or 90 days to 0.15 or 0.30 ppm ozone resulted in ciliated cell necrosis, shortened cilia, and secretory cell hyperplasia with less stored glycoconjugates in the nasal region. Respiratory bronchiolitis was also observed in these monkeys at 6 days and persisted to 90 days of exposure. Even at the lower concentration of 0.15 ppm O s nonciliated bronchiolar cells appeared hypertrophied and increased in abundance in respiratory bronchioles. The response of respiratory bronchioles to intermittent (8 h/day), long-term ozone exposure of 0.4 or 0.64 ppm included the following morphometric changes: 1) a thicker wall and narrower lumen, 2) thicker epithelial compartment and a much thicker interstitial compartment, 3) shifts in epithelial cell populations with many more nonciliated bronchiolar epithelial cells and fewer squamous type I epithelial cells, 4) larger nonciliated bronchiolar epithelial cells with a larger compliment of cellular organelles associated with protein synthesis, 5) greater volumes of interstitial fibers and amorphous ground substance and 6) greater numbers of interstitial smooth muscle cells, neutrophils, macrophages and mast cells per surface area of epithelial basal lamina. Following cessation of exposure there was persistence of some degree of the hyperplastic and metaplastic epithelial changes, but worsening of interstitial fibrosis. The principal lesion in response to intermittent (8 h/day) O s exposure of 0.25 ppm daily or to cyclical exposures (9 cycles of 1 month of O s followed by 1 month of filtered air) for 18 months was respiratory bronchiolitis. Cyclically exposed monkeys, but not those exposed daily, had significantly increased total lung collagen content, chest wall compliance and inspiratory capacity. The conclusions of this study are: 1) there is persistent epithelial injury in the anterior nasal cavity and respiratory bronchiole by as low as 0.15 ppm 0 3 , 2) there is worsening of the respiratory bronchiole lesion in monkeys in the post-exposure period and 3) cyclical exposures cause more severe injury than continued daily exposures.

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.

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.

Neuroendocrine Hyperplasias and Tumors, Larynx and Trachea, Syrian Hamster

The range of lesions described here was observed in hamsters of the strain Han:AURA from a chronic toxicity/carcinogenicity study involving inhaled particles. Since there was no difference in the frequency of the neuroendocrine proliferations between the exposure and control groups, all lesions were considered to be of spontaneous origin.