key: cord-0940674-q6scvymg
authors: Greaves, Peter
title: Respiratory Tract
date: 2012-01-13
journal: Histopathology of Preclinical Toxicity Studies
DOI: 10.1016/b978-0-444-53856-7.00006-3
sha: 3ca457d65e27b7ecf2f8ae642029f94144d2f198
doc_id: 940674
cord_uid: q6scvymg

This describes the pathology found in the respiratory system of laboratory animals and how this differs from pulmonary disease reported in humans which is greatly influenced by the use of tobacco products. It discusses the changes that can be induced by therapy by different routes of administration including agents administered by inhalation or the intranasal route. There is a discussion of inhalation technology and how species differences in anatomy and physiology of the lungs and airways can influence the disposition and pathological effects of administered drugs. It discusses the potential adverse effects of fine particles, particularly nanoparticles. Pathology of the pleural cavity is described including the effects of fibers and nanoparticles. Finally there is an analysis of the comparative pathology of neoplasms that occur in the respiratory tract and pleura of laboratory animals and humans. Keywords respiratory system, nose, nasal passages, trachea, larynx, bronchus, lungs, pleura, inhalation, drug safety, comparative pathology, carcinogenesis

By far and away the most important pulmonary diseases in humans are related to the smoking of tobacco. However, occupational lung diseases caused by inhalation of industrial chemicals, particulate matter and antigens are also important causes of morbidity and mortality. For this reason, considerable effort has been directed to the examination of airborne pollutants over recent years, including study of their effects in laboratory animals when administered by the inhalation route. Extensive study has shown that a complex array of defensive mechanisms protects the lung against the adverse effects of airborne substances and pathogenic organisms. Aerodynamic factors prevent access of particles larger than 10 μm diameter for these are deposited on the walls of the nasal passages. Particles measuring between 2 and 10 μm diameter tend to be trapped by the mucuscovered ciliated epithelium lining the bronchial tree and removed by mucociliary transport aided by the cough reflex. Smaller particles may reach the alveoli where they are ingested and transported by pulmonary macrophages. 1 So-called nanoparticles sized between 1 and 100 nm which exist in the environment as a result of dust storms, volcanic ash and other natural processes as well as a result of recent technological advancements appear to be less efficiently removed by pulmonary macrophage clearance than larger particles. 2 When inhaled these particles may translocate from the alveoli directly into the circulation. 3, 4 Some small inhaled particles reach the pleural cavity where they enter the lymphatic system via small round or oval openings from 2 to 10 μm diameter (stomata) in the parietal peritoneum. These stomata provide communication between the pleural space and the lymphatic vessels thus permitting the passage of cells, small particles and macromolecules into the lymphatic system. 5, 6 Consideration of airborne delivery to the lungs is also important in the development of therapies to be administered via the respiratory tract. While the inhalation route has been used for many years for volatile anesthetic gases, the respiratory tract is increasingly being employed for delivery of therapy. This is not only for asthma and other lung diseases but also for systemic delivery of polypeptides such as insulin and drugs to increase solubility, decrease immunogenicity and protect from excessive metabolism. 7 In contrast to the adverse pulmonary effects of cigarette smoke and industrial pollutants, therapeutic agents remain a relatively minor cause of pulmonary toxicity in humans although actual incidence is difficult to ascertain. However, drug-induced pulmonary disease appears to be an increasingly frequent clinical problem. The number of drugs associated with parenchymal pulmonary injury in humans continues to increase. In patients drug-induced pulmonary toxicity can occur through several mechanisms and take different forms. 8À10 Through their specific pharmacological action drugs can produce excessive effects on bronchial caliber or pulmonary function. Acute lung injury often manifests as pulmonary edema. This may be the result of cardiogenic or non-cardiogenic changes. For example, cocaine is believed to produce pulmonary edema as a consequence of an effect on pulmonary cardiovascular function. 8 Cytokines may alter vascular permeability. Drugs mediate allergic reactions in the bronchi or lungs. They may also produce a variety of obscure, diffuse pulmonary alveolar conditions including a pulmonary syndrome resembling systemic lupus erythematosis. As the respiratory tract is a major route by which microorganisms gain entry into the body, opportunistic pulmonary infections with bacteria, viruses, fungi or protozoa are consequences of immunosuppression or broad-spectrum antibacterial therapy. As in other organs, drugs that disturb coagulation may precipitate pulmonary thromboembolism or hemorrhage. Localized lung lesions also result from accidental, diagnostic or therapeutic inhalation of xenobiotics. Mucociliary clearance is also sensitive to therapeutic agents that affect the secretion of mucus and fluid, ciliary activity and transport. 11 Treatment with antacids or histamine H 2 blockers can also increase the risk of pneumonia developing in patients in intensive care units through increasing gastric pH. This leads to an overgrowth of Gram-negative bacteria in the stomach and retrograde pharyngeal colonization. 12 Anticancer therapy appears to be one of the main causes of serious pulmonary toxicity in patients. Although it is difficult to characterize because of the confounding effects of the underlying disease process, it has been suggested that about 10% of patients receiving well-established anticancer drugs develop various forms of pulmonary toxicity. 8 Some newer antineoplastic therapies and biological agents may have a similar liability. 10,13À15 Toxicity may be manifest as an early onset pulmonary edema or present after more than two months after therapy has been completed. 16 Bleomycin-induced toxicity is considered to be the typical form of late onset lung injury.

The nasal passages may also be a target of drug-induced toxicity mostly in the form of rhinitis characterized by inflammation of the nasal mucous membranes. Whereas there are two broad categories of rhinitis, allergic and non-allergic, drug-induced rhinitis is usually non-allergic in nature. 17 Although some causes are obscure, rhinitis medicamentosa denotes the inflammatory changes which occurs with persistent overuse of topical nasal decongestants. Aspirin and the non-steroidal anti-inflammatory drugs are examples of drugs that induce an acute inflammatory response in the nose probably via a mechanism involving the inhibition of cyclooxygenase-1. Clonidine, guanethidine and methyldopa are examples of α and β adrenergic antagonists that are believed to induce rhinitis through a neurogenic mechanism. 17 In preclinical safety studies, pathology of the respiratory system can be the result of intercurrent disease or be induced by drugs administered systemically by various routes. Intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and the technologies required to administer drug. The different anatomical and physiological characteristics of the airways also influence drug toxicity, disposition and metabolism. The development of drugs to be administered by inhalation or intranasal routes is particularly difficult because of the perceived risks of high local drug concentration in respiratory tissues and their use in potentially vulnerable patients with pulmonary disease. 18 

A complex technology has been developed to support the assessment of the effects of inhaled substances in rodent and non-rodent species and the extrapolation of the experimental findings to humans. 19, 20 In order to administer drugs by inhalation, it is necessary to generate aerosols (suspensions of particles in a gas) with a well-defined composition, particle size and shape. They must be delivered to the respiratory tract of laboratory animals in a way that parallels the likely human exposure. This should avoid non-respiratory pathways through the skin and food.

When aerosols are inhaled, various fractions of the particles are deposited at different locations in the respiratory tract. Site of deposition depends primarily on particle size, but variability in the sites of deposition occurs among different laboratory animal species and humans by virtue of the differences in the size and shape of the respiratory passages as well as breathing patterns. 21 In addition, there are different types of inhalers used in human therapy to consider: nebulizers, propellant-driven metered dose inhalers and dry powder inhalers. 22 The need to phase out ozone-depleting chlorofluorocarbons as propellants has also generated a need to assess new propellants and their impact on drug delivery. 7 The subsequent fate of inhaled particles depends not only on their size but also on their shape, chemical nature, and solubility in body fluids. Soluble substances are absorbed into the bloodstream and are removed by the pulmonary circulation. They may also undergo metabolism by enzymes present in the cell populations of the respiratory tract and reactive metabolites may cause local pulmonary damage. Insoluble, inert particles are removed primarily by the mucociliary transport system of the trachea and bronchi or through phagocytosis by macrophages. Overload of the lung by relatively inert, non-fibrous particles such as titanium dioxide or carbon black may impair alveolar macrophage-mediated particle clearance. 23 This may lead in turn to accumulation of dusts over time with eventual fibrotic and tumorigenic responses. 24 This has led to concerns that newer materials such as engineered long carbon nanotubes may accumulate in the lung and pleural cavity and induce inflammation and neoplasia. 25, 26 Unlike larger particles, it has been shown that nanosized particles are less easily phagocytized by macrophages and are less readily cleared from alveoli. 27 Nevertheless like larger particles effects of nanoparticles appear also to relate to levels of exposure, their chemistry, size, shape, state of agglomeration and electromagnetic properties. 2 Measurements of respiration rate, tidal volume, airway resistance, pulmonary gas exchange and the disposition of the inhaled substances have an important place in the evaluation of chemically induced lung damage in laboratory animals. 28, 29 Even though there are novel and very sensitive physiological methods for the characterization of edema following lung injury in rodents, light and electron microscopy of lung tissue sections provides vital qualitative evidence of the nature of any injury. 30 

The nasal chambers are the structures which are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. Although these chambers are not usually examined in great detail in conventional toxicity studies in which substances are administered orally or by parenteral routes, they are carefully examined histologically when drugs are administered by inhalation.

Study of nasopharangeal silicone rubber casts has shown considerable species differences in the anatomy of this part of the airway. 31À35 Relative to total nasal length, the nasopharynx is longest in rat and shortest in humans with the dog in an intermediate position.

Maxilloturbinates are relatively simple structures in humans and non-human primates but highly complex in dogs and rodents. As a consequence, regional nasal airflow and disposition patterns vary considerably and this influences the distribution of lesions produced by inhaled xenobiotics in the nasal cavity. 36 Comparison of the nasal cavity of rhesus monkey and humans using magnetic resonance imaging and nasal casts has shown that many similarities in structure exist between these two species. 37 The anterior nares are lined by stratified squamous epithelium. In other zones the sinuses are covered either by respiratory or olfactory epithelium with a zone of transitional epithelium at the junction between the two types. Respiratory epithelium is similar to that found elsewhere in the respiratory passages being composed of ciliated cells, serous and mucous cells, brush cells, intermediate cells and progenitor basal cells. It represents a cellular system engaged in mucociliary clearance carrying surface secretions to the nasopharynx to be cleared by swallowing. Although this epithelium is similar to that lining the other large airways, key differences are the particularly rich complement of secretory cells and the complex vasculature of the nose which can modulate capillary, arterial and venous blood flow through the mucosa. 38 Mucins may be particularly important. It has been postulated that they not only have a physical protective function but also possess antioxidant properties by virtue of the scavenging behavior of their high proportion of sugar groups. 39 The proportion of the nose lined by olfactory mucosa is variable between species being disposed over a much larger area in dogs and rodents than in primates. 32 However, it is structurally similar in humans and rodents. It is located in more dorsal or posterior regions of the nasal passages out of the direct line of airflow during normal respiration. Olfactory mucosa is a pseudostratified columnar epithelium composed of basal cells, sustentacular cells and sensory cells with mucus-secreting Bowman's glands situated in the lamina propria. Basal cells are composed of two distinct types, light and dark cells. The light type represents the primitive, stem cell population. Sustentacular or supporting cells are non-ciliated, columnar cells possessing microvilli that extend into the overlying layer of mucus.

Cell bodies of olfactory sensory neurons are situated in the middle layer of the epithelium between sustentacular and basal cells. Their dendritic processes extend above the epithelial surface to end in ciliated expansions referred to as the olfactory vesicles that are believed to be receptors of odor perception. Olfactory axons extend from the cell body, penetrate the basement membrane in bundles to become surrounded by Schwann cells and eventually join with the olfactory bulb.

The olfactory system is of importance in toxicology for it can be selectively damaged by xenobiotics, presumably as a result of its high metabolizing capacity. The superficial location of neural cells in the olfactory epithelium also provides a model system for the study of the effects of xenobiotics on neural cells.

Submucosal mucous glands have been well characterized in the rat, hamster and dog where they are divided into lateral nasal glands and maxillary recess glands. These are both situated in the posterior parts of the nasal cavity and composed of mucus-secreting cells. 40À42 Immunocytochemical study using antisera against the major isoenzymes of rat hepatic microsomal cytochromes P450 induced by β-naphthoflavone, 3-methylcholanthrene, phenobarbitone and pregnenolone-16-α-carbonitrile as well as NADPH-cytochrome P450 reductase, epoxide hydrolase and glutathione S-transferases B, C and E has shown their presence in rat nasal mucosal cells. 43 CYP2A enzymes appear to be expressed at high levels in the respiratory tract mucosa. 44À48 This suggests that the nasal mucosa has a capacity not only for metabolizing and activating xenobiotics by oxidation, but also for hydration and inactivation of potentially toxic epoxides and conjugating electrophilic, reactive metabolites with reduced glutathione. It has been shown that the distribution of immune-reactive enzymes is different in olfactory and respiratory mucosa. 49 Xenobiotics can be metabolized within both olfactory and respiratory mucosa but the olfactory regions appear to possess greatest capability for oxidative metabolism. Consequently, regional differences in nasal toxicity and tumor formation from inhaled materials may be a response not only to different water solubility and deposition patterns but also differences in the formation of reactive metabolites. 49 Another feature of this metabolizing activity is that it can be induced by systemically administered xenobiotics and this can alter the distribution of enzyme activity in the nasal mucosa. 43 Studies of the mouse olfactory mucosa have shown that while typical hepatic inducers of CYP2A5 do not significantly change its expression, olfactory toxicants can alter the pattern of enzyme distribution. 46, 48 Like many other tissues exposed to external environmental agents, the nasal mucosa possesses aggregates of lymphoid tissue in the underlying lamina propria. In rats these areas, characterized by follicles containing both T and B cell areas, are located in the ventral aspects of the lateral walls of the nasal airways at the opening of the nasopharyngeal duct. 32, 50 Like the gut-associated lymphoid tissue, these nasal follicles have been shown in the rat to be covered by specialized epithelium with islands of cells with microvilli, socalled M or membranous cells. Little is known of any toxicity occurring in this tissue despite its strategic position in the respiratory tract. 51

In rodents, the relatively small size of the nose and nasal sinuses facilitates histological examination. Usually this area is sectioned transversely into several standardized blocks following decalcification. 52 There have been a number of detailed publications describing the histological preparation and assessment and recording of pathology of the rodent nasal cavity. 52À56 Standardized histological sections, careful recording of lesions with the use of diagrams of the rodent nasal cavity are useful in the assessment of lesions in the nasal cavity found in inhalation studies. 57 In dogs and primates sectioning and blocking are more complex. Although dissection is required, a similar procedure following decalcification can be adopted. High-resolution three-dimensional magnetic resonance imaging has also been used to characterize the dimensions of the nasal cavities of cynomolgus monkeys and map the distribution of lesions induced by inhaled xenobiotics. 58, 59 Examination of hematoxylin and eosin-stained sections remains paramount in the assessment of the nasal cavity, although special stains may be helpful. Examination of cytokeratin expression in the respiratory mucosa has been used as a marker of epithelial differentiation in the respiratory tract. 60 A test system that relates to the innervation of the nasal mucosa is that proposed by Alarie. 61 The trigeminal nerve endings in the nasal mucosa of mice mediate the response to sensory irritants and this can be measured by a decrease in respiratory rate. It has been shown that a good correlation exists between the decrease in respiration rate in mice exposed to airborne chemicals and the nasal irritancy potential of the chemicals in humans. 62 This enables the detection of airborne sensory irritants and the prediction of acceptable levels of exposure to the upper respiratory tract in people.

Degeneration, inflammation, ulceration, (rhinitis, sinusitis)

Infectious agents cause inflammation in the nose and nasal sinuses and this may be associated with inflammation in the conjunctiva, middle ear and oral cavity. Murine pathogens may cause alterations in the respiratory tract that can confound the assessment of changes induced by xenobiotics. 63 In rats, microbiological agents implicated in the development of rhinitis and sinusitis include Corynebacterium kutscheri (pseudotuberculosis), Streptococcus pneumonia, Pasteurella pneumotropica, Klebsiella pneumoniae, Mycoplasma pulmonis and the sialodacryoadenitis virus or rat corona virus. 64 Rats infected with the sialodacryoadenitis virus show inflammation and necrosis of the upper respiratory epithelium as well as damage to salivary and lachrymal glands. The Sendai virus, a paramyxovirus, also has marked tropism for the respiratory tract, including the nasal cavity and is associated with systemic effects that can compromise studies in laboratory rodents. Occasionally, fungal infections of the airways with Aspergillus fumigatus are reported. 63 Strain differences in response to infectious agents have been observed among rats. Following housing of Lewis and Fischer 344 strains together to eliminate microbial and environmental differences it was shown that the Lewis strain developed a more severe rhinitis following inoculation with Mycoplasma pulmonis than Fischer 344 rats, although the reason for the difference was unclear. 65 Rats exposed to ammonia, a common pollutant of the air in laboratory animal cages, have also been shown to develop lesions of the dorsal meatus, dorsal nasal septum and prominence of the turbinates. 66 These lesions are characterized histologically by swelling 212 6. RESPIRATORY TRACT or mild degeneration of the epithelium. It is probable that ammonia exposure can potentiate the acute inflammatory response of the nasal cavity to microbiological pathogens.

A microorganism reported in the nasal cavity of rhesus monkeys employed in inhalation studies is the nematode of the genus Anatrichosoma. 67 Sections of this nematode are found in the squamous epithelium of the nasal vestibule and are associated with acanthosis and hyperkeratosis of the epithelium and a multifocal or diffuse granulomatous inflammation in the submucosa.

Administration of toxic or irritant substances to laboratory animals by the inhalation route produces degenerative, inflammatory and reactive changes in the nasal mucosa. The range of histological features is similar to those found in other mucosal surfaces altered by exogenous agents. While therapeutic agents administered by the inhalation route do not usually produce a severe degenerative or inflammatory response in the nasal mucosa, at least at therapeutic doses, the simple categories proposed by Hardisty and colleagues in recording of degenerative and reactive lesions following exposure to volatile chemicals are useful. 57 Categories suggested are: inflammation, degeneration, regeneration, atrophy (postdegenerative), respiratory metaplasia and basal cell hyperplasia. A far more detailed scheme along similar lines has been put forward in the context of the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) project. 68 Degeneration is usually the earliest morphological change characterized by loss of sensory and sustentacular cells resulting in a thinner mucosa. Individual cell necrosis may be seen in more severe cases. Regeneration is characterized by proliferation of basal cells associated with an epithelium that loses its regular structural features. Post-degenerative atrophy usually follows severe damage and is characterized by loss of sensory and sustentacular cells. Respiratory metaplasia is a process whereby the normal olfactory mucosa is replaced by pseudostratified epithelium of respiratory type often with cilia. Basal cell hyperplasia represents a longer-term effect where the proliferating basal cells form a distinct layer of cells below the respiratory epithelium.

An example of the type and distribution of the degenerative and inflammatory conditions which can be induced by inhaled irritants is provided by the study in which Swiss-Webster mice were given various irritants by inhalation for periods of six hours per day for five days at concentrations which produced a 50% decrease in respiratory rate (Alarie test) . Although the degree of histological changes varied with different agents, the changes were broadly similar in type and distribution. 69 Most agents examined produced little or no alteration in the squamous mucosa lining the anterior part of the nose apart from some mild increase in thickness of the squamous layers. Principal sites of damage were shown to be the anterior respiratory epithelium adjacent to the vestibule and the olfactory epithelium of the dorsal meatus. There was a distinct decrease in severity in posterior regions. Histologically, the lesions in respiratory epithelium ranged from mild loss of cilia and small areas of epithelial exfoliation to frank erosion, ulceration and necrosis of the epithelium and underlying tissues including bone. Variable polymorphonuclear cell infiltration was also found. In some cases, early squamous metaplasia developed on the free margins or the naso-maxillo-turbinates. Changes to the olfactory epithelium varied from focal to extensive loss of sensory cells associated with damage to sustentacular cells. In severe cases, complete loss of olfactory epithelium occurred. Although the degree of histological change was shown to vary with different agents, lesions induced by the more watersoluble chemicals tended to remain localized in the anterior part of the nasal cavity whereas agents with relatively low water solubility produced lung lesions in addition. It was suggested that these findings demonstrated the powerful 'scrubbing' action of the nasal cavity for water soluble, airborne xenobiotics. 70 Inflammatory alterations have been induced in the nasal cavity of rodents treated with therapeutic agents at high doses by inhalation. Significantly irritant substances do not make viable therapies but the precise relevance for humans of inflammatory changes that occur only at high exposures by the inhalation route is sometimes questionable.

In the case of tulobuterol, a β 2 adrenergic receptor agonist, it was argued that the nasal inflammation induced in rats in a one-month inhalation toxicity study was the result of a particularly high exposure of the nasal epithelium to drug, not representative of the likely human exposure to tulobuterol by inhalation, where little or no nasal exposure would occur. 71 RP73401 [3-(cyclopentyloxy)-N-(3,5-dichloro-4-pyridy)-4-methoxybenzamide], a novel type IV phosphodiesterase inhibitor which was being developed for the treatment of asthma and rheumatoid arthritis, was also reported to produce degeneration of the olfactory epithelium in rats but neither dogs nor mice after single and repeated oral doses and by inhalation. 72 Histologically, the olfactory epithelium showed necrosis of the superficial epithelial layers including the sustentacular and sensory cells with sparing of the basal cell layer. There was also damage to Bowman's glands. The development of proliferative lesions and ultimately tumors of principally neuroectodermal origin followed chronic treatment. As RP73401 was highly metabolized and the nasal lesions could be inhibited by treatment of rats with metyrapone, a non-specific inhibitor of cytochromes P450, it was postulated that the changes were the result of P450-mediated activation in the olfactory tissues, not linked to its pharmacological phosphodiesterase activity. 72 Nasal epithelial degeneration and necrosis has also been reported in both rats and dogs treated with another candidate anti-inflammatory drug CI-959 by the intranasal route. This agent effected olfactory epithelium more than respiratory mucosa suggesting that metabolism was important in the generation of this toxicity. 73 

Although the nasal cavity is not routinely examined histologically in great detail in toxicity studies conducted on drugs administered orally or by parenteral routes, damage to the nasal mucosa can be induced by drugs administered by these routes. One example is methimazole, a thioureylene antithyroid drug used in clinical practice where oral doses of 0.2 to 2 mg/kg/day are employed and abnormalities of taste and smell have been described. 74 Administration of methimazole at relatively high doses to Long-Evans rats by single oral (50 mg/kg) or intraperitoneal (25 mg/kg) routes was shown to produce damage to the sustentacular and sensory cells with sparing of the basal cells and basement membrane. 75 Bowman's glands were also involved. Methimazole is metabolized by the flavin-containing monooxygenase system and it is employed as a model substrate for this enzyme in vitro. The presence of flavin-containing monooxygenase isoforms in olfactory mucosa of Long-Evans rats suggested that reactive intermediates may be responsible for the nasal toxicity. 75 Similar changes have been reported in mice where depletion of 214 6 . RESPIRATORY TRACT glutathione in the olfactory mucosa has been demonstrated, also suggesting formation of local reactive metabolites. 76 Histological examination has also shown that intravenous administration of a single dose of vincristine to mice damages the olfactory epithelium. 77, 78 Vincristine is a vinca alkaloid derivative used in cancer therapy which has antimitotic activity and binds to tubulin. Cell death was noted in olfactory cells two to five days after dosing with a peak of cell proliferation at five days and repair after about ten days. These features resemble those that can be seen in other proliferating tissues after single doses of antimitotic drugs. Rats and marmosets have been shown to be less sensitive to these effects of vincristine, probably as a consequence of drug disposition differences. 79 The risk of damage to human olfactory cells from agents with these effects in rat nasal mucosa often remains uncertain because of a lack of understanding of relative exposure and metabolism in different species and the metabolic potential of human olfactory mucosa.

Docetaxel, another anticancer drug, has been shown to exert a transient neurological effect on olfactory epithelium of mice at doses similar to human doses. 80 This was demonstrated by electro-olfactograms from the chemosensory epithelium of the nasal septum and the endoturbinates.

A particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium and to a lesser extent in respiratory and glandular epithelial cells. 70, 81 These inclusions are PAS-negative and ultrastructural examination shows that they are membrane-bound, ellipsoid bodies containing homogenous electron dense matrix. Their significance remains uncertain.

A consensus classification for the variety of proliferative, non-neoplastic changes and atypical epithelial lesions and neoplasms found in the rat nasal cavity has been defined by Schwartz and colleagues. 82 The classification of the International Agency for Research on Cancer provides a similar perspective for rats and mice. 83, 84 A similar scheme has been devised in the context of the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) project. 68 Proliferative lesions may be occasionally seen in untreated rodents in carcinogenicity studies but are much more commonly induced by administration of xenobiotics in inhalation carcinogenicity studies. Spontaneous nasal tumors are uncommon but most often squamous in rats, whereas in mice spontaneous squamous tumors are extremely rare and hemangiomas and respiratory adenomas predominate. 85, 86 The generally agreed categories are described below.

Mucous (goblet) cell hypertrophy and hyperplasia affects the nasal respiratory epithelium and are characterized by the presence of enlarged mucus-filled goblet cells some of which form clusters suggestive of intraepithelial glands.

Squamous cell hyperplasia is seen in the stratified squamous epithelium of the nares and is characterized by a focal increase in the number of cell layers. Cells may show atypia with irregular enlarged, pleomorphic nuclei and nucleoli.

Squamous metaplasia occurs to respiratory epithelium under conditions of chronic damage. It is characterized histologically by the presence of three or more layers of epithelial cells with eosinophilic cytoplasm and clear cell boundaries whereas advanced lesions show typical keratinization and formation of intercellular bridges. Cellular atypia may also be seen and should be characterized when found.

Respiratory epithelial metaplasia (of the olfactory epithelium) represents atrophy and degeneration of the olfactory epithelium with loss of sensory cells and in advanced cases loss of sustentacular cells with replacement by ciliated and non-ciliated respiratory epithelium. It may be seen as a spontaneous focal lesion in aged rats.

Epithelial hyperplasia with cellular atypia (atypical hyperplasia, basal cell hyperplasia, dysplasia) is a term used to embrace proliferative lesions in the respiratory and olfactory mucosa in the nasal cavity in which there are varying degrees of altered differentiation and atypia. There is perturbation of the growth pattern of the epithelium such that the changes are not those found in the normal regenerative response to transient mucosal damage.

Adenomas (polypoid or villous adenoma, adenomatous or villous polyp) usually develop in the anterior part of the nasal cavity and are usually exophytic lesions that develop from respiratory epithelium or nasal glands. Adenomas of respiratory epithelium may be papillary in form but are by definition well circumscribed with minimal cellular pleomorphism and atypia. They may very occasionally occur spontaneously in aged rats. 85 Adenomas of nasal glands usually show an acinar pattern.

Squamous cell papillomas develop in the squamous epithelium of the nares or in areas of squamous metaplasia in respiratory or olfactory epithelium. They are exophytic lesions with limited connective tissue stroma. They may develop spontaneously in aged rats. 87 Carcinomas of either squamous or glandular differentiation develop in the nasal mucosa. Histologically, they have similar characteristics to those in other epithelial tissues. They are rare spontaneous lesions in aged laboratory rodents but may be induced by xenobiotics administered by inhalation, orally or by the parenteral route. Squamous carcinomas have been reported to develop in a small number of untreated Fischer 344 rats used in carcinogenicity studies in association with point mutations in the c-H-ras and c-K-ras gene. 87 Olfactory neuroblastoma (ethesioneuroblastoma, olfactory neuroepithelioma, olfactory neuroepithelial carcinoma) show olfactory differentiation and arise from olfactory epithelium. They do not seem to occur as spontaneous lesions in rats or mice and are only rarely induced. 82, 85 Cells are arranged in lobules or in solid sheets with scanty stroma. Cells are relatively uniform with scanty cytoplasm with round or oval hyperchromatic nuclei. True rosettes with lumens or pseudorosettes are also seen. Poorly differentiated tumors of this type may require ultrastructural study for diagnosis. Olfactory neuroblastomas typically show the presence of electron-dense neurosecretory granules, neurofilaments or axons. As there is no detailed understanding of the biological behavior of these neoplasms in laboratory rodents, the generic term olfactory neuroblastoma is usually preferred. They are almost always invasive tumors. 85 Olfactory carcinomas forming glands, follicles and rosettes have been occasionally reported in aged Syrian hamsters. 88, 89 Mesenchymal neoplasms may be seen in the nasal cavity, particularly after exposure to potent carcinogens. Their histological features are similar to those in the soft tissues and bone elsewhere in the body (see Chapter 2).

The mucosa lining the larynx and trachea becomes involved as part of an upper or lower respiratory tract infection. For instance, in rats, an acute laryngitis or tracheitis has been shown to accompany experimental infection with Mycoplasma pulmonis and the sialodycroadenitis virus. 65, 90 A spontaneous degenerative condition of tracheal and laryngeal cartilage of uncertain pathogenesis associated with granulomas has been reported in Fischer 344 rats. 91 The condition increases in severity and incidence with advancing age although it is seen in rats as young as six weeks of age. Tracheal cartilage rings may also show alterations in genetically engineered animals such as the C57BL/6J-TgN(C3-1-TAg) cJeg (TAg) mice that have generalized defects in cartilage development. 92 The larynx of rodents is also susceptible to the effect of inhaled substances, notably tobacco smoke but also pharmaceutical agents and propellants. 81, 93 In view of the localized nature of induced lesions in the larynx, standardized histological sectioning techniques have been proposed for rats, mice and hamsters using anatomical landmarks. 94À97 The target site is located on the ventral floor of the larynx near the base of the epiglottis cranial to the ventral laryngeal diverticulum. Lesions tend to occur in the ventrolateral region, which is covered by respiratory epithelium and the inner aspect of the arytenoid processes which is lined by squamous mucosa. The larynx responds to inhaled irritants by inflammatory, degenerative and regenerative changes in a manner similar to other regions of the respiratory tract. These include disruption of the epithelial cells, inflammatory cell exudates and infiltration, goblet cell hyperplasia and squamous metaplasia. 93 Squamous metaplasia represents a common adaptive response to inhaled irritants of diverse types. 98 These changes are not specific to inhaled irritants but also occur as a response to natural respiratory tract pathogens in conventionally housed rats. 99 An international expert group concluded that in the context of inhalation exposure to non-genotoxic chemicals squamous metaplasia in the rodent larynx does not generally represent a pre-neoplastic lesion. 100 The pseudostratified ciliated and non-ciliated mucosa of the trachea may also show pathological alterations in inhalations studies, although sites at the bifurcation (carina) are those often first affected. Consequently, the carina should be systematically included in examination of the respiratory tract for induced lesions. 82, 97 Neoplasia As in the nasal passages a range of proliferative lesions including squamous hyperplasia, mucous cell hyperplasia, as well as papilloma, carcinoma and mesenchymal tumors are occasionally reported in the airways in laboratory rodents and large species. 68, 101 

In humans and laboratory animals, the trachea terminates at the bifurcation giving rise to two main bronchi which serve left and right lungs. Depending on species, the main bronchi subdivide into further branches which enter the different lobes. Various forms of branching are recognized. Bronchi may arise as side branches from a parent or stem bronchus (monopodial). The parent bronchus can divide into two equal daughter bronchus (dichotomous) or several daughter bronchi (polychotomous). 102 Study of silicone rubber casts of the respiratory tract has shown that the bronchial trees of humans and non-human primates are essentially dichotomous, in contrast to the monopodial pattern of rodents. 34 The comparatively long trachea of the dog gives rise to dichotomous upper airways but monopodial branching develops peripherally within each lobe.

The size of the lungs is generally dependent on size and weight of the different species. Allometric studies have shown that lung volume, alveolar surface area and diffusing capacity increase proportionally with body weight across a broad range of mammalian species, although cell size and surface area appear to be more determined by cell function rather than species size. 103 Dogs have comparatively smaller body mass and higher airway dimensions compared to humans. 34 The number of lobes is species dependent. The human lung possesses an upper and lower left lobe and an upper, middle and lower right lobe. This contrasts with the upper, middle and lower left lobes and a fourth, azygos right lobe in rhesus monkeys and baboons. 34 The dog has three lobes on both right and left sides. Rats, mice and hamsters show cranial, middle, caudal and postcaval right lobes with a single, left lobe in mice and rats and a superior and inferior lobe on the left side in hamsters.

Cell types lining the bronchi are generally similar between species. 11 The majority of cells are ciliated cells that are accompanied by variable but relatively smaller proportions of basal cells, intermediate cells, mucous or goblet cells, serous cells, neuroendocrine and brush cells. In addition, mucous cells line the adjacent bronchial glands. 104 Unlike the tracheal mucosa, which is pseudostratified, the mucosa of intra-pulmonary bronchi is nonstratified.

Ciliated cells are tall, columnar cells attached to basal and intermediate cells by desmosomal junctions. Tight junctions exist between adjacent specialized cells at the apex. Each cell possesses 200 or more cilia that are engaged in mucociliary clearance. 105 The superficial cell surface also shows a pronounced glycocalyx. The cytoplasm of ciliated cells contains scattered profiles of rough endoplasmic reticulum, a supranuclear Golgi and numerous mitochondria particularly near the apex where a prominent cytoskeleton is also found. Mucous or goblet cells represent about 10% of the bronchial mucosa cell population in humans but less than 1% in pathogen-free rats. 11 The serous cell is a cylindrical or pyramidal cell containing small, round, closely packed serous granules. 104 The mucus-secreting and ciliated cells form the cellular basis for the mucociliary clearance mechanism of the main conducting airways. The epithelium is covered by a mucous blanket that is fairly complete in humans and rabbits but patchier in rats. 11 A deficient mucous barrier, excessive mucus or defective clearance contributes to the pathogenesis of all the common airway diseases. 106 The mucous layer is segregated into an upper layer or gel phase separated from epithelial cells by a serous, periciliary layer. The depth of the periciliary layer is uniform throughout the conducting airways and is vital for mucociliary clearance. However, the superficial gel layer increases in thickness from the distal to proximal airways. 106 Normal mucus is over 90% water and about 3% of solid matter comprising mucins, other proteins, salts, lipids, and cellular debris. Mucins are large glycoproteins with regions rich in serine and threonine residues linked by their hydroxyl side groups to sugar chains. They are highly anionic because most of their terminal sugars contain carboxyl or sulfate groups. 106 The complex carbohydrates of the glycocalyx and secreted mucosubstances show species-related differences in their sugar residues, which can be demonstrated histochemically by the use of labeled lectins. 107 Pathological changes in mucus occur through its excessive production, infiltration by inflammatory cells or processes that alter its hydration and biochemical constituents, notably abnormalities in secretion of salt and water.

Mucociliary clearance mechanisms are sensitive to the effects of many therapeutic agents, particularly those that alter mucins, fluid or electrolyte balance and ciliary activity. Anesthetic gases, barbiturates, narcotics and alcohol depress clearance function. By contrast, topical, oral or parenteral administration of β adrenergic agonists, isoprenaline and epinephrine (adrenalin), produce a dose-dependent stimulation of mucociliary transport by an effect on ciliary beat frequency, probably mediated by increasing levels of cyclic adenosine monophosphate in ciliated cells rather than through vascular changes. Although basal mucociliary function is dependent of normal vagal tone, parasympathomimetic agents can affect mucociliary transport. Acetylcholine and cholinergic agents stimulate ciliary activity whereas anticholinergic drugs, atropine and hyoscine, inhibit ciliary activity and mucociliary transport. These substances may alter deposition of inhaled particles in the lung. 11 Clara cells or non-ciliated bronchiolar cells located in the bronchiolar epithelium, first described by Clara in 1937, are small and cylindrical in shape with highly infolded nuclei, surface microvilli, well-developed Golgi, abundant smooth endoplasmic reticulum and characteristic oval, homogeneous electron-dense granules in the apical cytoplasm. In rats, rabbits and humans the granules are PAS positive, although they are usually considered PAS negative in hamster and mouse. 104 Clara cells have high metabolic activity. They contain cytochrome P450-dependent enzymes and secrete a variety of proteins. 108À110 Clara cell secretory protein is the major component of their cytoplasmic granules as well as surfactant proteins and they have been shown to produce mucin following antigen challenge. 110 In most laboratory rodents, the conducting airways terminate abruptly at the non-cartilaginous terminal bronchiole that opens directly into an alveolar-type airway, the alveolar duct which in turn communicates with the alveoli. 111 Squamous epithelial or type I cells form only about 10% of all lung cells but they line over 90% of the alveolar surface, by virtue of extremely long cytoplasmic extensions. The principal gas exchange takes place across this cell. In the rat, the typical thickness of this barrier is 20 nm for a cytoplasmic extension of a type I pneumocyte, 90 nm for basal lamina and 90 nm for an endothelial cell. 104 The type I cell contains juxtanuclear mitochondria and the long smooth cytoplasmic extensions contain many ribosomes and pinocytotic vesicles. The anatomical configuration and function of type I cells render them highly vulnerable to the adverse effects of inhaled gases and particles.

The other main alveolar lining cell is the granular pneumocyte or type II cell which constitutes about 10% of all lung cells, but which covers only about 5% of the alveolar surface. 112 This cell does not possess long cytoplasmic processes but it shows many microvilli on its luminal surface. The cell cytoplasm contains rough endoplasmic reticulum, Golgi apparatus, some mitochondria and characteristic oval, osmiophilic lamellar inclusions. Surfactant, a microaggregate of phospholipid and protein which modifies alveolar surface tension at low inflation volumes, is secreted by type II alveolar cells. Ultrastructural immunocytochemistry has shown the presence of surfactant apoproteins in the synthetic organelles and in the lamellar bodies of these cells, in agreement with the concept that the surfactant apoproteins are synthesized in the rough endoplasmic reticulum, glycosylated in the Golgi and are stored in lamellar bodies. 113 Type II cells are more resistant to the damaging effects of xenobiotics and unlike type I cells they retain the ability to undergo mitotic division. Following damage to type I cells, increased numbers of mitoses are evident in type II cells which results in the appearance of large undifferentiated epithelial cells which ultimately differentiate into type I and type II cells. 104 The lung also contains a dense neural network and a population of endocrine-like cells believed to be important in lung function. 114 These neurosecretory cells (Kultschitsky or APUD cells) are scattered sparsely in the epithelial surface of the larynx, trachea bronchi, bronchioles and alveoli. These cells are oval or cuboidal with oval nuclei, argyrophilic cytoplasm which electron microscopic examination shows to contain dense core granules. The role of neuroendocrine cells in the lung is uncertain but immunocytochemical study has shown them to contain a number of neuroendocrine substances including neuronespecific enolase, synaptophysin, chromogranin and a variety of other peptides similar to vasoactive intestinal peptide, bombesin, calcitonin, serotonin, leu-encephalin, β endorphin and ACTH. 114, 115 Cells lining the bronchi, bronchioles and alveolar walls are capable of metabolizing xenobiotics. Immunocytochemical study of the rat lung has shown the presence of immune-reactive cytochromes P450, NADPH cytochrome P450 reductase, epoxide hydroxylase and glutathione S-transferase in bronchial epithelial cells, ciliated bronchiolar cells, Clara cells, type II and possibly type I pneumocytes. 43 Different cell populations contain different amounts of enzymes, Clara cells containing the greatest concentrations of the phenobarbitone-inducible isoenzyme of cytochrome P450, NADPH-cytochrome P450 reductase and epoxide hydrolase. Studies of microsomal enzyme activities suggest that lung tissue contains fewer P450 isoenzymes than liver, principally forms CYP1A1, CYP2B1, CYP3A2 and CYP4B1. 116 Whereas P450 enzyme activity is highly concentrated in specific cell types, overall microsomal enzyme activity is low compared with liver on the basis of microsomal protein weight. 116 

Other important cells are the pulmonary alveolar macrophages and lymphocytes. Lymphocytes are found in the epithelium of the airways, in the interstitium of alveoli and as part of follicles in bronchial walls. Pulmonary macrophages form part of the specific immune defense system of the lung, involving, as elsewhere in the body, antigen presentation. An important point here with respect to drug delivery by inhalation is that phagocytosis is sensitive to particle size. It is thought that particles of 0.5À3 μm in diameter are taken up by macrophages and particles of less than 0.25 μm can escape from phagocytosis by macrophages. 27, 117 In the rat and mouse, distinctive populations of pulmonary macrophages have been described based on enzyme activities and reactivity to monoclonal antibodies against monocyte and macrophages surface determinates. 118, 119 Bronchus-associated macrophages in rat and mouse have more acid phosphatase and less non-specific esterase activity than the populations found in the pulmonary alveoli and interstitial tissues.

An important aspect of the immune system is the bronchus-associated lymphoid tissue or BALT, which forms part of the mucosal lymphoid system found in other epithelia. The morphology of BALT is a useful guide to the nature and degree of immune stimulus in the lung. BALT is organized in a way that is characteristic of other peripheral lymphoid organs. It is structurally similar in the laboratory rat, mouse, rabbit, guinea pig as well as in humans but its size and prominence is species and strain dependent as well as a function of the degree of antigenic stimulus. 119À121 In the rat, the BALT is composed of lymphoid aggregates or follicles located mostly between a bronchus and artery with a zone of lymphocytes situated immediately under the bronchial epithelium. As in other peripheral lymphoid tissue, BALT is organized into B and T cell zones but in no predetermined manner. Immunocytochemical staining has shown that B and T lymphocyte zones differ in location from one aggregate to another. There are about two T lymphocytes for every three B cells compared with a ratio of 2:5 in rat Peyer's particles. 122 The ratios may be different in other species. Quantitative observations of T cell subsets using monoclonal antibodies have also shown that rat BALT normally contains twice as many T helper as T suppressor/cytotoxic lymphocytes. 122 The T cells are confined to one or two discrete zones with a light scattering of T cells within the B cell zones and immediately under the bronchial epithelium. In common with lymph nodes, interdigitating cells are also found. The epithelium overlying BALT shows anatomical modifications. It is composed of ciliated and non-ciliated cells covered by microvilli.

In conventional, untreated laboratory rats, BALT shows little activity and germinal centers are usually absent, although BALT may be more prominent in some rat colonies in association with non-specific inflammatory lesions in lungs. 123, 124 In one colony of young Wistar rats germinal centers were not seen in BALT in untreated animals but they developed following the administration of a single intratracheal dose of lipopolysaccharide, a T cell-independent antigen. 125 Single intratracheal doses of T cell-dependent antigens such as horseradish peroxidase, bovine serum albumin and BCG have been shown to produce only minor morphological changes which include expansion of the zone of lymphocytes immediately under the epithelium and infiltration of the bronchial epithelium overlying BALT by lymphocytes. 126 In addition, perivascular, peribronchial or alveolar infiltrates of small and large lymphocytes and macrophages were observed in the lungs of rats given BCG.

Immunocytochemical study of the rat BALT following intratracheal challenge with horseradish peroxidase showed that the majority of cells that infiltrated the bronchial epithelium were T helper (CD4 positive) lymphocytes. 126 Furthermore, Ia antigen expression of the epithelial cells overlying the BALT was shown to increase, associated with an increase in the number and size of microvilli, a more pronounced glycocalyx and a decrease in number of cilia.

Immunocytochemical study of the BALT tissue in C57B1/6 mice using monoclonal antibodies to lymphoid and macrophage populations has demonstrated quite similar arrangements of cells to those in the rat with the majority of T cells belonging to the T helper (CD4 positive) class. 119 The pulmonary lymphatic system drains into mediastinal or cervical lymph nodes. Although among rat strains, differences in the location of lymph nodes and their drainage occur, tracer studies in the Fischer 344 rat using colloidal carbon have shown that the lung lymphatics drain mainly into posterior mediastinal lymph nodes and those in the tracheal wall drains primarily to the internal jugular and posterior cervical nodes. 127 

Integral to the surface of the lungs is the visceral pleura. There are species differences in pleural thickness and its blood supply. Rats, dogs and rabbits have a thin visceral pleura with blood supplied from the pulmonary circulation whereas in humans and pigs it is thicker with a blood supply from the bronchial (systemic) circulation. In all species the blood from the visceral pleura is drained by the pulmonary veins. 5 The visceral pleura is covered by a mesothelial cell layer which synthetizes a number of macromolecules including hyaluronan, surfactant, elastin and collagen. 5 There is a thin space between the visceral pleural layer and the parietal pleura lining the chest cavity. This space contains pleural fluid which is constantly being elaborated by subpleural capillaries with a contribution from mesothelial cells that secrete glycosaminoglycan. The fluid leaves the pleural cavity by virtue of stomata or lymphatic stomata situated in the caudal regions of the parietal pleura. These drain into the lymphatic vessels that reach the hilar, mediastianal and parasternal lymph nodes. 5, 6, 128 There has been renewed study of these stomata because of questions concerning the fate of and potential pathology produced by nanoparticles in the lungs. The stomata on the parietal pleural are 2 to over 10 μm in diameter and are found in association with accumulations of leukocytes, termed milky spots on the pleura. It appears that some inhaled particles reach the pleural fluid and drain into these stomata to eventually reach the hilar and other lymph nodes. It has been argued that long thin and durable fibers such as asbestos become impeded at these stomata thereby inducing localized inflammatory reactions, fibrosis and eventually neoplasia at these sites in the pleural cavity (see below). 25 

Although a variety of fixation, embedding and staining procedures are available for light and electron microscopic examination of lung tissue, there is no substitute for initial, careful visual inspection of the lungs at autopsy. Uneven collapse of lungs on opening the thoracic cavity, discoloration or alteration in texture of the pleural or cut surface, congestion and presence of fluid in the larger airways may indicate structural damage. In rodent lungs, small pulmonary adenomas may be detectable by inspection in good light.

Fresh lung weight is also a helpful measure in lung assessment, although passive vascular engorgement can significantly affect this value. Nevertheless, studies in the normal Fischer 344 rat have shown that after exsanguination, wet lung weights show a close relationship to body weight and that dry weight of lungs consistently represents about 20% of the wet weights regardless of age or body weight. 129 An increase in wet weight over dry weight appears to be a good index of pulmonary edema. 30 Various methods of fixation have been employed although simple immersion fixation in formalin for conventional light microscopy has the virtue of simplicity and it avoids the risk of translocation or removal of exudates from airways and alveoli. Mixtures of formaldehyde, paraformaldehyde and glutaraldehyde are used in initial fixation for electron microscopy. 53 The best overall appreciation of lung architecture is achieved by instillation of fixative via the trachea under an appropriate constant pressure or by perfusion fixation of the pulmonary arteries that is less liable to dislodge intra-alveolar exudate. In a review of methods employed routinely in rodent toxicity studies, instillation of fixative via the trachea was the preferred method in most laboratories because its advantages were seen to outweigh its disadvantages. 95 The sampling procedure is an important aspect of histological examination of the bronchi and lungs, particularly those of large laboratory animals. The extent of histological sectioning in conventional toxicity studies should be modulated to take account of lesions found by macroscopic examination, the type of study and the nature of the test substance. The bronchi should be carefully sampled to allow assessment of any alterations in bronchial epithelium.

Morphometric analysis represents a sensitive tool of value in the evaluation of druginduced lung changes, but it requires particularly rigorous sampling and evaluation procedures. 130, 131 A tiered, multiple stage or cascade sampling technique is normally considered the most appropriate for morphometric studies. 130 This involves dividing the lung into a series of homogeneous compartments or strata from which randomly selected samples can be examined by appropriate light or electron microscopic techniques. Magnetic resonance imaging and micro-computed tomography have also been tried as techniques for the study of pulmonary disease in small animals allowing correlation with conventional pathology techniques. 132, 133 Conventional special stains for reticulin and collagen as well as PAS and alcian blue for mucins are helpful in the characterization of lung damage and changes to the respiratory epithelium. Immunocytochemistry and enzyme cytochemistry are also useful in the study of the heterogeneous cell population of the lung. Xenobiotic metabolizing activity can be studied both by enzyme cytochemical methods as well as by immunocytochemical techniques using antisera specific for pulmonary monooxygenases and related enzymes. 48 Important structural components, particularly collagen and laminin, can be studied both at light and ultrastructural level with immunocytochemical methods. 134 Cytokeratin immunocytochemistry can be used as a method for the characterization of changes to epithelial cells. 60 Clara cells can be localized by the presence of Clara cell secretory protein and ciliated cells by the presence of tubulin. 110 They can also be visualized using immunohistochemical staining using antibodies for other cell constituents notably surfactant proteins and cytochromes P450. 108, 135 Endocrine cells are visualized by immunocytochemistry using antibodies to general neuroendocrine markers such as chromogranin and synaptophysin or regulatory peptides. 114 Other useful antigens, which can be demonstrated in the lung, include surfactants, lysozyme, immunoglobulins and those of microorganisms that infect the lung. 136 Electron microscopy is particularly useful for the detailed characterization of injury to the cells of the alveolar epithelium and endothelium (Figure 6 .1).

Pulmonary edema is a component of many inflammatory conditions of the lung including those induced by infections agents. However, the term edema is reserved for a poorly cellular exudate characterized by the presence of pale, homogeneous eosinophilic material in the alveoli, sometimes associated with a similar exudate in the lung septae and perivascular connective tissue.

It occurs in a number of spontaneous conditions such as in congestive cardiac failure, metastatic pulmonary neoplasms or as an agonal change in association with pulmonary congestion and hemorrhage. Drugs may induce cardiogenic pulmonary edema as a consequence of pulmonary hypertension or impaired ventricular contractility. Cardiogenic edema is often associated with vascular congestion and red blood cells and hemoglobin may leak into airspaces. This can give rise to the presence of hemoglobin crystals within the edema fluid in formalin-fixed tissue sections. Intra-alveolar edema, alveolar wall thickening and increased numbers of inflammatory and degenerate cells (plastic embedded, methylene blue 3600). Panel b: Electron micrograph illustrates the degenerative changes in type I and type II pneumocytes along with cellular debris and macrophages in the alveoli (31, 250) . Illustrations by courtesy of Dr N.G. Read.

Most importantly, pulmonary edema may be a manifestation of acute lung injury. Inhalation or systemic administration of toxic chemicals may produce acute pulmonary edema (Figure 6.1) . Some substances such as phenylthiourea and α-naphlythiourea produce massive pulmonary edema in laboratory animals when administered orally, principally as a result of damage to the endothelium of pulmonary capillaries and venules. 137 Over 30 drugs have been reported to produce non-cardiogenic pulmonary edema in humans either directly or through poorly understood immunogenic mechanisms. 8 Another form of pulmonary edema involves the main airways. Allergic reactions in sensitized airways of asthmatic individuals is believed to result from cross-linking of IgE and activation of mast cells that degranulate and release inflammatory mediators. 138 This has been reproduced in the main airways of rats sensitized to ovalbumin and then challenged with ovalbumin by the intratracheal route. 139 This treatment leads to rapid accumulation of bronchial exudate, degranulation of mast cells and the development of mucosal edema, most marked immediately below the respiratory epithelium.

Congestion and hemorrhage is a frequent finding in the lungs of laboratory animals where it is usually related to certain modes of death. It can be associated with administration of drugs and chemicals that have adverse effects on cardiac function or on the coagulation system. Administration of heparin to rats produces a characteristic extravasation of blood into the air spaces. 140 

Lower respiratory tract infection is generally not a major health hazard among laboratory animals but it is nevertheless an ever-present threat which can cause overt respiratory disease within a colony or develop following administration of xenobiotics. Subclinical pulmonary infections and infestations can also produce histological alterations in the bronchial airways or pulmonary parenchyma which mimic changes induced by inhaled irritants or systemically administered drugs. 123, 124 Furthermore, some respiratory pathogens alter immune defenses and exacerbate the effects of inhaled substances. 141 Focal nonspecific inflammatory pulmonary lesions may be also found in most laboratory animals, particularly primates without obvious causation. 142 A range of bacterial and viral pathogens may produce inflammatory lung changes. 63 Typically, bacterial pathogens such as Steptococcus pneumoniae produce acute bronchitis associated with a variable degree of acute inflammation of the lung parenchyma (bronchopneumonia) or a confluent lobar pneumonia. Viral agents are generally associated with histological features of bronchiolitis and interstitial pneumonia, characterized by an increase in mononuclear cells in the respiratory bronchioles and alveolar septa. The histological features are variable for they depend on the particular pathogen, species and strain, immune status, presence or absence of secondary infection and the particular stage at which the infection is examined. Respiratory infections are frequently mixed. Changes due to secondary bacterial infection are frequently superimposed on those induced by viruses.

Sequential histopathological examination of the lungs of laboratory animals following inoculation with respiratory tract pathogens has characterized the evolution of pathological changes produced by individual organisms. For instance, following inoculation with Mycoplasma pulmonis, one of the more important respiratory pathogens among laboratory rodents both Lewis and Fischer 344 rats were shown to develop upper and lower respiratory tract inflammation. In the Lewis strain this was characterized after 28 days by a variable acute inflammatory exudate in bronchi and bronchioles with focal bronchiectasis, inflammation and hyperplasia of the epithelium with a predominantly macrophage infiltration of the alveoli and alveolar walls. 65, 143 These changes were associated with marked hyperplasia of the bronchus-associated lymphoid tissue (BALT), which extended down the airways and blood vessels towards the periphery of the lungs. Although the lymphoid hyperplasia was also found in inoculated Fischer 344 rats, it was less marked and accompanied by little or no mucopurulent exudate or active inflammation of the bronchial walls. This disparity in response suggested that differences were related to the degree of lymphocyte activation in the two strains, an imbalance in regulation of lymphocyte proliferation in Lewis rats, or both. 143 Other studies have been conducted in both rats and mice infected with another important respiratory pathogen of laboratory rodents, the Sendai virus (parainfluenza type 1). Sequential studies showed that the initial damage to bronchial and bronchiolar epithelium is associated with polymorphonuclear and lymphocytic inflammation (bronchiolitis). Immunocytochemical and ultrastructural studies revealed the presence of viral antigen in the mucosa. 141 Hyperplastic and multinucleated syncytial epithelial cells develop in the hyperplastic terminal bronchiolar epithelium and the inflammatory process extended to involve peribronchial or peribronchiolar parenchyma with infiltration of alveolar walls by mononuclear cells, macrophages and neutrophils. A similar cell population accompanied by cell debris and edema fluid develops in air spaces. Pulmonary arteries show only minor involvement with inflammatory cells and focal reactive hyperplasia of the endothelium. Immunocytochemistry and ultrastructural examination suggested that virus replication takes place in alveolar type I and type II epithelial cells and macrophages but not in endothelial or interstitial cells of the alveolar septae. 144 It was shown that when repair occurs there may be residual distortion of bronchiolar and alveolar walls by collagen and hyperplastic cuboidal epithelium may line the thickened alveolar septa. Air spaces may also contain enlarged macrophages with pale vacuolated cytoplasm. 145 Strain differences in susceptibility have also been demonstrated to this virus. There is differential pulmonary interleukin 12 (IL-12) gene expression between virus-susceptible Brown Norway rats and resistant Fischer 344 rats, and IL-12 treatment provides protection from virus-induced chronic airway inflammation and remodeling. Moreover increased tumor necrosis factor α (TNFα) expression has been shown to be an important regulatory factor in the development of Sendai virus-induced bronchiolar fibrosis in infected rats. 146 Virus-inoculated Brown Norway rats had increased TNFα pulmonary mRNA levels and increased numbers of bronchiolar macrophages and fibroblasts expressing TNFα protein compared with virus-inoculated Fischer 344 rats. 147 The Corona virus, which causes sialodacryoadenitis in many rat colonies, also produces lower respiratory tract inflammation. This is characterized by acute bronchitis and bronchiolitis with focal extension into lung parenchyma. Thickened edematous, hypercellular alveolar walls infiltrated by monocytic cells are found. 90 Immunocytochemistry has shown the presence of viral antigen in bronchial and bronchiolar epithelial cells. There is also 226 6. RESPIRATORY TRACT peribronchial lymphocytic infiltration and increased prominence of BALT. Ultimately complete resolution occurs.

Viruses remain a potential source of spontaneous respiratory disease in laboratory dogs. Canine adenovirus type 2, parainfluenza SV5, canine herpes virus, coronavirus and parvovirus have all been isolated from laboratory dogs developing respiratory disease. 148 The syndrome of visceral larva migrans also incites focal inflammation, granulomas and fibrosis in the lungs of species such as dog and primate in which parasites may be prevalent. The syndrome of visceral larva migrans is usually defined as that which results from the migration of nematode larvae into the viscera. It has been well described in the beagle dog lung where it results from the larvae of toxocara species or metastrongyloid nematodes. 149, 150 The precise identification of parasites is not always possible in tissue sections. Histological appearances of infested lungs are highly variable. Nematodes surrounded by granulomas and granulomatous inflammation, mostly in a subpleural location, may be visible in sections. In affected lungs there may be perivasculitis and active arteriolitis, bronchiolitis and peribronchiolitis. Pleural involvement by the inflammatory process can be marked, particularly in regions overlying granulomas. Scarring develops and pleural and subpleural fibrosis is frequently associated with epithelial hyperplasia and squamous metaplasia of the associated airways ( Figure 6 .2a). 150 The lesions may be sufficiently severe to resemble those induced by high doses of anticancer drugs such as bleomycin (see below). Pulmonary acariasis is a common infestation of many species of non-human primates caused by various species of the mite Pneumonyssus. Reproduction of the mites appears to take place in the terminal bronchioles. Pneumonyssus simicola is the recognized form found in rhesus monkeys. 151 Although it is most prevalent in wild caught primates, the disease is not easily eliminated during breeding in captivity. 152 Even when eliminated by ivemectin the lesions of chronic bronchiolitis, bronchiectasis and pigmentation may persist as an incidental finding. 153 As the mite can produce significant destructive pulmonary pathology and render animals susceptible to secondary pulmonary bacterial infections, it can disrupt or confound the interpretation of toxicity studies performed in primates. The lesions are located most frequently in cranial lobes and are characterized by the presence of bullae distending the pleural surface, parenchymal cysts, nodules and scar tissue. 151, 152 Histologically, there is a wide range of inflammatory activity. Fully developed lesions are characterized by granulomatous bronchiolitis and peribronchiolitis with involvement of immediately adjacent alveoli. Cystic lesions involving the bronchiolar walls develop around the parasites giving rise to the appearance of walled-off cysts composed of highly cellular granulation tissue, associated with neutrophils, lymphocytes, macrophages, multinucleated giant cells and various pigments (see below). In less active lesions, dilated, cystic airways with walls composed of thick bands of smooth muscle and lined by squamous or cuboidal epithelium are found.

It is of note, however, that pulmonary inflammatory lesions may be seen in monkeys without evidence of infestation. Chamanza and colleagues reported highly variable incidences of focal inflammation, alveolar macrophage accumulation, focal pigmentation and focal pleural fibrosis in laboratory cynomolgus monkeys used in toxicity studies, but these changes were not seen in association with lung mites. 142 Pneumocystis carinii is an important cause of pneumonia in patients with the acquired immunodeficiency syndrome (AIDS) as well as in other immunocompromised patients including those treated with immunosuppressive drugs. 154 The natural habitat of Pneumocystis carinii is pulmonary alveoli and it is widely encountered in the human population without being associated with overt disease. Both clinical and experimental evidence suggests that impaired cellular immunity is much more important as a predisposing factor than impaired humoral immunity. 154 As in humans, laboratory animals may have latent pneumocystis infection that becomes clinically evident following immunosuppression. It has been shown in the rat that chronic administration of various regimens of corticosteroids, low protein diets, cyclophosphamide and other immunosuppressive drugs with concomitant antibiotic administration to prevent other infections gives rise to typical pneumocystis pneumonia. 155 Rodents with genetically deficient cellular immunity also develop pneumocystis pneumonia. The importance of pneumocystis pneumonia in toxicology is that it can be considered as a sentinel of chronic immune depression.

In hematoxylin and eosin-stained sections, pneumocystis pneumonia is characterized in both humans and rodents by the presence of alveoli filled with foamy eosinophilic material containing a few macrophages and indistinct nuclei of pneumocystis (Figure 6 .2b). Ovoid or crescent-shaped structures of the organisms become clearly visible with Gomori methenamine silver or toluidine blue stains. Ultrastructural study of rats with pneumocystis pneumonia shows that trophozoites attach themselves most frequently to type I 228 6 . RESPIRATORY TRACT pneumocytes by altering their morphology to the contours of the pneumocytes rather than by a process of invasion. 156 

Systemically administered therapeutic agents may produce histological changes within the lung parenchyma that mimic components of the normal response to respiratory pathogens. There is no sharp separation between agents that produce pulmonary edema and those that are associated with acute inflammatory changes, and histological features overlap because an acute inflammatory process is often accompanied by exudate within airspaces.

An example of drug-induced pulmonary inflammation in laboratory animals and humans is reported following the administration of interleukin 2 (IL-2). IL-2 is a glycoprotein lymphokine, molecular weight 15 kDa, which is normally produced by activated T cells and mediates immunoregulatory responses. It has been produced in large quantities by recombinant DNA technology for use in tumor immunotherapy. However, high doses have been associated with a number of adverse effects, notably the vascular leak syndrome, characterized clinically by pulmonary edema, pleural effusions and ascites. 157 The vascular leak syndrome has been reported in laboratory animals given high doses of this agent. Histological examination of the lungs of B6D2F mice developing this syndrome following administration of IL-2 showed infiltration of the alveolar walls with large lymphocytes and intra-alveolar proteinaceous exudate which contained large lymphocytes, macrophages and red blood cells. 158, 159 Pulmonary venules and arterioles showed the presence of lymphocytes attached to or lying beneath the endothelium, infiltrating vessel walls or in a perivascular location where they were accompanied by edema fluid or red blood cells. Similar, but less severe changes have been demonstrated in rats given IL-2. 158 In addition, treated rats showed an infiltration of pulmonary vasculature with eosinophils probably secondary to an eosinopoietic cytokine produced by IL-2-stimulated lymphocytes. Immunocytochemical evaluation of the lymphoid infiltrate in mice showed that most of the cells were Thy 1.2-positive (CD90) lymphocytes. Furthermore, co-administration of asialo GM1 (ganglio-n-tetrosyl-ceremide) with IL-2 not only abrogated the clinical signs but also reduced the number of asialo GM1-positive lymphocytes in the tissue sections.

As lymphoid cells expressing Lyt-2 (CD8, suppressor/cytotoxic T cells) were unaffected by asialo GM1 treatment, it was postulated that the vascular leak syndrome (but not antitumor efficacy) in these mice was mediated by an endogeneous subset of IL-2 stimulated lymphocytes or lymphokine-activated killer cells. 159 Corresponding changes were also observed in liver and lymphoid tissue. Immunocytochemical and detailed electron microscopic studies in rats have supported the concept that IL-2 induces cytotoxic vascular damage that is mediated both directly by lymphokine-activated killer cells and cytotoxic T lymphocytes with secondary release of inflammatory cytokines. 160 As in humans, severe chronic pulmonary inflammatory disease in laboratory animals may compromise pulmonary function and lead to secondary alterations in other organs. Although the mechanisms were not explored in detail, a diffuse interstitial pulmonary inflammatory process with lung hemorrhage was induced in rats treated for two years with prizidilol (SK&F 92657-A2), an antihypertensive agent with both vasodilator and β adenoceptor blocking properties. 161 Affected animals developed dyspnea associated with reduction in lung volume, deformity of the thoracic spinal column and marked cardiac hypertrophy.

Focal pulmonary inflammation, diffuse interstitial inflammation and pulmonary edema have also been described in cynomolgus monkeys as a consequence of multifocal pulmonary thromboemboli that can occur in prolonged intravenous infusion studies. 142 

Aggregates of foci of macrophages are commonly observed in older rodents and may be found as a response to a variety of inhaled substances as well as endogenous cellular debris and blood cells that leak into alveoli in chronic pulmonary congestion and hemorrhage. They also accumulate in lung tissue distal to bronchial lesions that impede clearance mechanisms. Phagocytic uptake of inhaled particles is one of the main mechanisms to remove insoluble small particles from the lung surfaces, particularly when mucociliary transport, cough, or sneezing fail or these protective systems are overwhelmed. 162 Thus it is a little surprising that macrophages accumulate in the alveoli of animals in toxicity studies where particulate therapeutic materials are given by inhalation or via the trachea in large doses.

Administration of cytokines such as interleukin 2 (IL-2) and the interferons may directly stimulate an increase in alveolar macrophages in association with other changes and these may contribute to lung damage. 163 

Inflammation with granulomas develops in the lungs of laboratory animals under a variety of different circumstances, which have been alluded to above. A common cause in rodents is granulomatous pulmonary inflammation resulting from aspiration of stomach contents or food particles (aspiration pneumonia). This is sporadically observed in aged rodents where it is associated with general ill-health, particularly resulting from pressure effects of large pituitary adenomas and subsequent disturbance of pharyngeal or laryngeal reflex mechanisms. 164 Histologically, the lungs show peribronchial and peribronchiolar granulomatous inflammation with macrophages and foreign body cells associated with fragments of refractive vegetable matter. The associated bronchial mucosa may also show reactive changes including goblet cell hyperplasia in long-standing cases. As dogs and primates are more liable to be infested by parasites, granulomatous inflammation in response to pulmonary larvae is more common in these species. However, tiny particles of inhaled food or plant materials may also be associated with foreign body granulomas in these species. 142 Pulmonary tuberculosis represents a potential problem among non-human primate colonies in view of its insidious onset and its liability for transmission from monkeys to humans. 165 Pathological findings are similar to those so well known in the human disease. The disease is characterized by the presence of granulomas in lung parenchyma and lymph nodes. In florid cases there may be caseation surrounded by epithelioid and multinucleated giant cells and variable numbers of lymphocytes, plasma cells and fibroblasts. Diffuse granulomatous pneumonia as a result of tuberculosis is also reported in nonhuman primates. Granulomatous pneumonitis is also produced in laboratory animals by the intravenous injection of bacille Calmette-Gue´rin (BCG). Twenty-eight days following intravenous injection of BCG, the lungs of C57B1/6 mice contained numerous granulomas composed of histiocytes and round cells which were surrounded by alveoli with thickened walls and associated with mild interstitial pneumonitis. 166 These histological changes were associated with an increase in the number of Thy 1.2-positive (CD90) cells, especially Lyt-1 (CD5) positive lymphocytes. The histological changes were abrogated by treatment with cyclosporin A suggesting an important role for CD5-positive lymphocytes in the development of the granulomas.

Discrete granulomas occur in the lungs of experimental animals in response to intra-tracheal or intravenous injection of certain relatively insoluble substances (Figure 6.3) . These include more recently developed potential therapeutic materials such as nanoparticulates. 167 Intra-tracheal administration of insoluble polymerized dextran and latex micro-particles to mice showed that the morphology and the systemic effects of granulomas depend on the nature of the injected substances. It has been shown that large granulomas develop rapidly in the pulmonary parenchyma around dextran particles that subsequently regress quickly, whereas latex particles produce small, discrete stable granulomas. 168 Although both forms of granulomas are of foreign body or non-immunological in type, those produced by dextran but not latex beads were shown to be associated with anergy-like immunosuppression probably caused by release of soluble factors from the granulomas.

It has been reported that granuloma formation after instillation of sephadex beads is associated with increases in interleukin 1 (IL-1)-like activity in the lung. 169 Studies comparing the effects of inhaled crystalline silica and titanium dioxide have shown a correlation between the release of the macrophage-derived cytokine IL-1 and granuloma formation, suggesting that IL-1 might be a useful biomarker for granuloma formation. 170 Localized, angiocentric granulomas of foreign body type, clustered around pulmonary arteries and arterioles and occasionally alveolar capillaries and venules also develop following intravenous injection of relatively insoluble polysaccharides or other polymers. 171 Characteristic epithelioid and large, foreign body-type giant cells efface the smaller vessels although overt necrosis is not usually observed (Figure 6.3) .

Hemosiderin-laden macrophages accumulate in the alveoli of laboratory animals in association with chronic pulmonary congestion and hemorrhage. Similar changes occur in patients in congestive cardiac failure where the hemosiderin-laden macrophages are termed heart failure cells.

The lungs of non-human primates are especially liable to contain alveolar, perivascular and peribronchial aggregates of macrophages laden with various brown or black pigments which also may be found in local lymph nodes. 142 Iron-containing pigments have been associated with the inflammatory changes produced by simian lung mites (Pneumonyssus simicola) which are prevalent in many non-human primates. In addition, lungs from some primate colonies may show perivascular and peribronchial collections of brown-gray macrophages containing highly refractive spicules and plates composed of high concentrations of silica. 172, 173 It has been shown that in Old World primates including rhesus and cynomolgus monkeys, this pigment contains fossil diatomaceous material, compatible with the concept that the animals inhale dusts containing diatoms and other silicon fragments to which they are exposed in their semi-arid, natural habitats. 172 

Chronic lung injury from a variety of different causes is frequently associated with the development of pulmonary fibrosis characterized by the replacement of the normal pulmonary structure by a thickened collagenous matrix with consequent reduction in the capacity for gas exchange.

In humans, conditions leading to pulmonary fibrosis vary widely. They include infections, shock lung syndrome, ionizing radiation, inhalation of irritant or immunogenic particulate matter, exposure to antigens or excessive amounts of oxygen as well as the results of the toxicity of paraquat and a range of both cytotoxic and non-cytotoxic therapeutic agents. 8,174À175 Laboratory animals also appear to develop a pulmonary fibrotic response to many of these inciting agents although species differences exist. One comparative study of the response to dusts between rats and humans suggested that although rats develop greater intra-alveolar inflammation and alveolar hyperplasia than humans the relative ranking of the fibrotic response is the same in both species. 176 Regardless of the inciting agent, the fibrogenic process appears to be generally characterized by disruption of the normal alveolarÀcapillary structure, leakage of exudate from the vascular compartment into the airspaces, subsequent invasion by inflammatory cells and fibroblasts associated with excess matrix formation. Studies in laboratory animals with different fibrogenic agents as well as in humans have suggested that central to pulmonary fibrogenesis increased production of tumor necrosis factor α (TNFα) by macrophages. 23, 146 ,177À179 This cytokine is not only a mitogen for fibroblasts but also a potent activator and chemo-attractant for macrophages, capable of stimulating release of other cytokines and inducing expression of adhesion molecule expression on endothelial cells. It has been shown that TNFα receptor knockout mice appear protected from the fibroproliferative effects of inhaled asbestos. 180 

The principal therapeutic agents which produce pulmonary fibrosis in both humans and laboratory animals are anticancer drugs. This form of fibrosis tends to present more than two months after therapy has commenced. 16 Bleomycin, a glycopeptide preparation derived from Streptomyces verticillus, is the best known example but pulmonary fibrosis is also associated with the clinical use of a number of other anticancer agents including 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU or carmustine), busulfan, mitomycin C and methotrexate as well as some of the newer agents. 8, 10, 16, 137 ,181À184 Therapeutic use of cyclophosphamide is also occasionally associated with the development of pulmonary interstitial fibrosis. 182, 183 Cyclophosphamide appears to be associated with two forms of pathology: an early onset pneumonitis and a late onset progressive pulmonary fibrosis. 183 The precise mechanisms involved in the induction of pulmonary fibrosis by antineoplastic drugs in humans are poorly understood. The true incidence for a particular drug is difficult to estimate because of confounding factors in cancer patients, such as concomitant administration of several drugs, radiation and oxygen therapy, diffuse pulmonary cancer and opportunistic infections. It is probable that drug-induced fibrosis is accentuated by concomitant administration of several antineoplastic agents, radiation therapy, hyperoxia, pre-existing pulmonary damage and age of the patient. Severity is often related to total dose of drug received. 137 Novel antineoplastic drugs may also produce this form of lung toxicity. 10 The iodinated benzofuran derivative amiodarone, a highly effective drug used in the treatment of cardiac arrhythmia, is an example of a non-cancer drug that has been associated with pulmonary fibrosis in patients although its etiology is clouded by the presence of systemic phospholipidosis (see below). However, it has been suggested based on clinicalÀpathological correlation that patients affected by treatment with amiodarone can be separated into two categories. One group are those with dyspnoea alone which correlates with the presence of foam cells or phospholipidosis. The second group of individuals are those with major changes in pulmonary function characterized by alterations in chest radiographs, a decline in diffusion capacity for carbon monoxide, vital capacity or total lung capacity. It is this latter group that shows pulmonary fibrosis as well as type II cell hyperplasia. 185 The bronchiolitis, alveolar septal inflammation and fibrosis induced by gold therapy in patients with rheumatoid arthritis is a distinctive syndrome characterized by presence of fever or skin rash, lymphocytosis in bronchoalveolar lavage fluid and alveolar opacities along the bronchovascular bundles on chest imaging studies. 186 It is probably immune mediated. This condition may be associated with peripheral eosinophilia and other druginduced alterations to the immune system. 174 

Bleomycin is associated with the development of interstitial pneumonia and pulmonary fibrosis in clinical use and this can be reproduced in experimental animals. However, the histopathological appearances of bleomycin-induced pulmonary fibrosis in patients are in many instances different from those seen in laboratory animals because the lungs of patients treated with beomycin are modified by the primary neoplastic disease, smoking, multiple drugs, radiation therapy and secondary pulmonary infections, interstitial pneumonitis and fibrosis. 187 It has been postulated that TNFα is an important mediator in the development of bleomycin-induced fibrosis. 178 In the preclinical evaluation of bleomycin beagle dogs were given cycles of drug by the intravenous route for periods of up to 26 weeks. 188 Dogs developed anorexia, weight loss, a variety of epithelial lesions as well as focal interstitial pneumonia and fibrosis. The focal lung lesions were characterized by increased elastic fibers, reticulin, collagen and acid mucosubstances. The lesions were situated predominantly in the pleural and subpleural zones, suggestive of a potentiating effect of friction between the pleural surfaces. Histologically the lesions resembled those produced by larvae migrans in the dog (Figure 6.2a) .

Similar histological changes have also been described in both rats and mice treated with bleomycin by both the intravenous and intratracheal route. 189, 190 As fibrosis is such a consistent change, bleomycin-treated rodents have been extensively employed as a model for pulmonary fibrosis. Early changes include mild, diffuse increases in interstitial lymphocytes, macrophages, polymorphonuclear cells and perivascular or interstitial edema. After about a week, interstitial infiltrates also comprise fibroblasts with early collagen deposition, associated with proliferation of macrophages and type II pneumocytes. 189, 190 Subsequently, the amount of interstitial collagen increases, with eventual scarring and collapse of lung tissue in proportion to the cumulative dose given. 191 Immunohistochemical and ultrastructural study of rats and mice treated with bleomycin shows a large accumulation of immune-reactive laminin and reduplication of the basement lamina within the thickened alveolar walls. 192 In bleomycin-treated rats three-dimensional scanning electron microscopy shows drug-induced capillary remodeling comprising irregular alveolar and pleural capillaries with increased diameter and decreased branching. 193 Certain strains of mice have been shown to possess greater sensitivity to bleomycin fibrogenesis. The C57BL/6 strain produces a greater fibroblastic response than DBA/2 and Swiss mice and the BALB/C strain demonstrates a particularly poor fibroblastic response. 194 Changes similar to those reported in patients with cyclophosphamide, an anticancer alkylating agent have been less easy to reproduce in laboratory animals. When mice were sequentially examined for periods of up to one year after a single intravenous dose of 100 mg/kg of cyclophosphamide, only slight pulmonary interstitial thickening and hypercellularity was observed in association with progressive multifocal accumulation of intra-alveolar macrophages. 195 However, these changes were also accompanied by a progressive increase in pulmonary hydroxyproline content and a decrease in pulmonary compliance with time in treated animals compared with controls. The changes were amplified by exposure to 70% ambient oxygen.

While amiodarone is capable of inducing phospholipidosis in laboratory animals, pulmonary fibrosis reported in patients does not seem to be reproducible in animal models. Pulmonary damage does not appear to occur in toxicity studies performed with gold therapies. Although a seven-year toxicity study of auranofin in dogs showed evidence of an immune-mediated thrombocytopenia similar to that occurring in patients, pulmonary fibrosis was not reported. 196 

Pleural fibrosis may result by extension of an inflammatory process from the lung parenchyma. It may also result from the presence of irritant substances within the pleural cavity. Poorly cellular collagenous pleural thickening is a common finding in humans exposed to asbestos fibers. It has been suggested that long fibers are impeded from leaving the pleural cavity through the lymphatic stomata in the parietal pleura so that fibers accumulate and induce localized inflammatory changes, fibrosis and eventually neoplasia (see below). 25 This fibrous thickening appears as discrete elevated gray or white plaques on the parietal pleura composed of laminated hyaline and poorly cellular collagen that may contain calcium. 197 Poorly cellular collagen may also form in rodents when the pleural cavity contains abundant long thin durable fibers or other irritant materials (Figure 6 .4). 

Emphysema is characterized by abnormal, permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious fibrosis. Causative mechanisms for the human disease are poorly understood although it has been postulated that important factors are persistent damage from proteolytic enzymes produced by increased numbers of neutrophils and macrophages, disruption of homeostatic repair processes or altered immune responses. 198 Three principal types, centriacinar, panacinar and distal acinar emphysema, are recognized in humans. Enlargement of air spaces as a result of congenital factors or fibrous scarring are grouped separately and not regarded as emphysema. 199 Emphysema has been reported as an age-related spontaneous change in laboratory rats. 200 However, several experimental rodent emphysema models have been developed, using intra-tracheal instillation of proteolytic enzymes papain, pancreatic and neutrophil elastase. This gives rise to histological appearances resembling panacinar emphysema in humans. 199 Irritant gases notably oxides of nitrogen are also capable of inducing changes in the lungs of laboratory rats and hamsters following long-term exposure which resembles mild human, centrilobular emphysema. 201, 202 Phospholipidosis (lipidosis, pulmonary alveolar proteinosis)

Phospholipidosis is the original name given in 1970 by Shikata and colleagues to the systemic disorder characterized by the presence of cytoplasmic vacuoles in a wide range of organs as well as foamy cells in the lung parenchyma which when seen under the electron microscope are dense myelin-like cytoplasmic bodies. 203, 204 These were described by these authors as concentric lamellar structures with a periodicity of 4À4.5 nm associated with an amorphous matrix in a membrane-bound space showing acid phosphatase activity typical of lysosomes. 205 These bodies were found in many organs in rats after treatment with 4-4 0 diethylaminoethoxy hexesterol dihydrochloride. However, the characteristic appearances at light microscopy of foamy macrophages in the alveoli as well as other organs were recognized over 20 years previously in rats treated with the antimalarial drug chloroquine. 206 A variety of different names have been applied to these cytoplasmic inclusions including myeloid bodies, myelinoid bodies, myelin figures or myelinosomes.

These lysosomal inclusions have been the focus of considerable study. Although they are seen in small numbers in a variety of normal cells, they have been shown to be increased in rodent organs following treatment by 50 or more xenobiotics including drugs in widespread use in humans. 8,207À213 Examples of drugs that induce systemic phospholipidosis include the anorectic drug chlorphentermine, tricyclic antidepressants, inhibitors of cholesterol biosynthesis such as triparanol, the antihistamine chlorcyclizine and its analogs, macrolide antibiotics, the selective estrogen receptor antagonist tamoxifen, chloroquine and the cardiovascular drugs amiodarone, 4,4 0 -diethylamino-ethoxyhexestrol and perhexiline. 210,214À217 Novel compounds that possess the property of producing phospholipidosis in laboratory animals with varying degrees of severity continue to be reported. 218À220 Many tissues and organs may develop the cytoplasmic inclusions including lymphoid cells, liver, pancreas, endocrine tissue, nervous system, muscle cells, eyes and particularly lungs. Aminoglycoside antibiotics are unusual as they may produce laminated phospholipids inclusions limited to renal tubular cells (see Chapter 10, Urinary Tract).

Many drugs that induce phospholipidosis share certain structural features, notably a hydrophilic cationic side chain, a primary, secondary or tertiary amine and a hydrophobic region that is usually an aromatic ring or ring system. As this structural pattern renders these molecules amphiphilic, it has been argued that these drugs bind with polar lipids by means of electrostatic and hydrophobic forces. 210 This leads to formation of drugÀlipid complexes which are poorly degraded by lysosomal enzymes. These complexes then accumulate in the cell cytoplasm to form phospholipid membrane inclusions. As the binding is not covalent, its reversibility depends on the dissociation rate constant under the particular intracellular conditions and drug concentration achieved. Predictions of this activity based on molecular structure have shown reasonably good correlation with the ability of compounds to produce phospholipidosis in cultured rat peritoneal macrophages. A number of in vitro cell-based systems have been proposed for screening for phospholipidosis. 221, 222 However, these perform less well in the prediction of in vivo potency, presumably because of differences in drug disposition in blood and tissues.

The lungs appear vulnerable to drug-induced phospholipidosis possibly because macrophages are in very close proximity to blood-borne agents. Phospholipidosis is also more clearly visible microscopically in alveoli whereas it can be easily overlooked in other organs. The continuous uptake of phospholipid-rich surfactant material from the alveoli by macrophages leads to excessive accumulation of phospholipids when their catabolism is impaired. 210, 211 Although the changes in the lungs are not specific for drug-induced phospholipidosis, an increase in the number of lipid-containing lung macrophages in treated animals compared with controls is relatively easy to detect and provides a simple way for the pathologist to screen for this effect.

In severe generalized phospholipidosis in rats, the lungs show irregular pale gray or yellowish patches of discoloration of the pleura and parenchyma. This is a result of patchy or confluent aggregates of large, pale, foamy macrophages ( Figure 6 .5a). They may be free lying or packed in alveoli and accompanied by granular, extracellular material. Their abundant cytoplasm shows a vacuolated appearance in which fine eosinophilic granules are sometimes visible. The nuclei are rounded and centrally located structures of variable size. Multi-laminated cells are also occasionally seen, as are vacuolated cells firmly attached to alveolar walls, probably pneumocytes. These foamy cells typically stain for phospholipids (e.g. acid hematin), although neutral lipids may also be present and stain with oil red O.

Semi-thin plastic embedded sections stained with toluidine blue allow better characterization of phospholipidosis in all organs including the lungs. The macrophages in the air spaces contain unmistakable dense, dark round cytoplasmic inclusions of variable size, some over 5 mm diameter. 223 Plastic-embedded sections also show the inclusions in other pulmonary cells including pneumocytes attached to the alveolar walls from which they can be seen discharging into the alveolar spaces.

As in other organs affected by phospholipidosis, ultrastructural examination reveals dense, multi-lamellar membranes and numerous heterogeneous dense bodies of lysosomal origin (Figure 6 .5b). These bodies need to be distinguished from membranous bodies that form as a result of fixation for ultrastructural study. Lipids tend to leach out and become hydrated to form myelinoid membranes during glutaraldehyde fixation. These structures are subsequently fixed by osmium to give rise to electron-dense membranous figures both outside and inside cells particularly in mitochondria where they may be mistaken for pathological lesions. 224 The lamella patterns seen in phospholipidosis may be simple alternating dense and clear lines spaced at 4 to 4.5 nm or more complex arrangements of clear and dense lines. The other typical crystalloid inclusions of hexagonal aggregates of tubular subunits seen in other organs are not usually found in the lungs. The significance of these various forms is uncertain but they probably represent the various phases in which phospholipids exist and are influenced by proportions of lipids present. Electron microscopic examination reveals that in addition to pulmonary macrophages inclusions may be present in pneumocytes types I and II, pulmonary capillary endothelial cells, smooth muscle cells, bronchiolar epithelium and occasionally neutrophils. 225À227 The changes are typically still visible several weeks after withdrawal from treatment with the offending agent.

Although the extent of pulmonary phospholipidosis in the lungs varies between dosage regimen and animal species, studies with 4,4 0 -diethylaminoethoxyhexestrol, amiodarone and chlorphentermine indicate that similar cytological and ultrastructural changes occur in most laboratory animal species studied including rats, mice, hamsters, guinea pigs, rabbits and dogs. 225, 226, 228, 229 

What are the implications for humans of drugs that induce systemic phospholipidosis in laboratory animals? Although not all drugs that produce phospholipidosis in animals have been studied in humans, only very few drugs which produce phospholipidosis in animals have been shown capable of inducing significant phospholipidosis in human clinical practice. 8 However, interpretation of the relevance of phospholipidosis is complicated by drugs such as chloroquine, 4,4 0 -diethylamindethoyhexestrol and amiodarone which have been shown in patients to produce phospholipidosis and also cellular damage in the same organs.

A well-studied example is the iodinated benzofuran derivative amiodarone, which remains an important and highly effective drug used in the treatment of cardiac arrhythmia. Lung toxicity characterized by a diffuse cellular infiltrate in the alveoli and interstitium typical of interstitial pneumonitis in addition to bronchiolitis obliterans continues to be a problem in a few patients treated for cardiac arrhythmias with this drug. 230 Phospholipidosis occurs not only in a wide variety of organs in laboratory animals treated with amiodarone but also in liver, peripheral nerve cells, skin, lymphoid cells and lungs in patients treated with therapeutic doses. 226,229,231À233 However, these inclusions are not prominent and do not appear to be closely associated with cell damage. Moreover, although pulmonary interstitial fibrosis occurs in association with phospholipidosis in patients, amiodarone-induced phospholipidosis in rodents is not associated with pulmonary fibrosis or significant functional alterations. Several theories have been proposed for the pulmonary alveolitis and interstitial fibrosis in humans. 234 The weight of evidence suggests that the accumulation of lipid-laden histiocytes is not causally related to the alveolitis or pulmonary fibrosis. 235 Cytotoxicity, possibly through the metabolite desethylamiodarone or an immune-mediated mechanism, has been postulated, possibly favored by the binding of drug to components of pulmonary tissue. 233 Free radical formation or indirect influences on inflammatory mechanisms may also be involved. 236 It is also possible that pulmonary disease results from an interaction of several mechanisms and metabolic factors unique to particular patients.

Overall there is little evidence that the mere presence of phospholipids packaged within lysosomes is deleterious to the organism. Indeed the weight of evidence suggests that drug-induced phospholipidosis is largely an adaptive phenomenon and does not in itself have functional or deleterious consequences unless excessive. 237 Nevertheless, dose, drug disposition, metabolism and elimination and the degree of tissue exposure to drug are important considerations in safety assessment of drugs that produce phospholipidosis in laboratory animals. Although phospholipidosis is more likely to occur at high doses employed in toxicity studies than at lower therapeutic doses used in patients, it has been suggested that this may be offset by faster elimination of the drug, characteristic of small laboratory animals. 210 The potential for drugs to accumulate in critical tissues such as eye and heart is especially important when drugs are administered for long periods of time particularly as tissue/plasma ratios of some amphiphilic drugs may exceed 100, following repeated administration. 238 Consequently, although phospholipidosis may not have direct functional consequences, any implications for humans of drugs that induce phospholipidosis in laboratory animals can only be appropriately assessed on a case-by-case basis, with due consideration of mechanism, drug disposition and clinical riskÀbenefit analysis.

It should be noted that the accumulation of foamy macrophages has long been recognized as a spontaneous alteration in aging rats (see also macrophage accumulation above). 239 It may also be found in lung tissue distal to bronchial lesions that impede clearance mechanisms. In contrast to drug-induced changes, the spontaneous accumulation of alveolar foam cells occurs sporadically in older rats and is observed in both controls and treated animals. Drug-induced phospholipidosis occurs within a period of several months during which lungs of control animals remain fairly free of foam-cell accumulation.

Similar morphological changes due to the increased presence of phospholipids in lysosomes can also result from treatment with compounds that are not cationic amphiphilic structures. Mechanisms include direct or indirect inhibition of lysosomal enzyme activity. This re-enforces the need to understand the mechanism of any chemically induced increase of phospholipids in the lungs of laboratory animals.

For example, it has been shown that when glycosaminoglycans accumulate in inherited human lysosomal disorders they inhibit other lysosomal enzymes, thereby inducing lysosomal phospholipid inclusions. 240 This is reflected by administration of high doses of the trypanocidal drug suramin to rats which induces intracellular storage of glycosaminoglycans associated with phospholipid inclusions in diverse organs including lungs. 241 Although at light microscopy clear vacuoles are typically seen, electron microscopic examination shows the presence of both clear vacuoles containing glycosaminoglycans and lamellar phospholipid inclusions. A similar effect seems to have been produced in rats by Elmiron s , a semi-synthetic heparin-like macromolecular carbohydrate derivative, chemically and structurally similar to glycosaminoglycans used clinically for anticoagulant effects and interstitial nephritis. 242 Hook reviewed other agents such as oxidant gases and insoluble particles including silica that can also increase phospholipid levels and histological appearances that resemble those seen in systemic phospholipidosis. 243 Some of these agents inhibit phospholipid catabolism in the lungs giving rise to accumulation of surfactant protein A and surfactant lipoproteins and a clinical and pathological picture similar to pulmonary alveolar proteinosis in humans. Foamy macrophages associated with inhaled silica in the rat appear to be sparser than those in drug-induced phospholipidosis. 68 Studies from humans with pulmonary alveolar proteinosis have shown that three clinically distinct forms of this condition occur: congenital, secondary or acquired. The congenital disease is caused by a diverse range of mutations in the genes encoding surfactant proteins or the β c chain of the receptor of granulocyteÀmonocyte colony stimulating factor (GM-CSF). The secondary form occurs in association with conditions where there is functional impairment or reduced numbers of alveolar macrophages such as in hematological cancers, following immune suppression or inhalation of silica or toxic fumes. Acquired or idiopathic alveolar proteinosis that accounts for over 90% of all cases (0.37 per 100,000 persons) has been an enigma until recently. Patients are at risk from infections, particularly nocardia, and the five-year survival rate appears to be about 75%. Studies from transgenic mouse models and in humans have shown that autoantibodies against GM-CSF are important in the development of the acquired form of the disease as this antibody causes a defect in macrophage function which impairs the catabolism of surfactant lipids and proteins. 244, 245 Eosinophilic inclusions

Large eosinophilic cytoplasmic inclusions are occasionally seen at an apical location in a small proportion of pulmonary Clara cells in normal rats. An increased number of these have been reported in rats treated with inhaled corticosteroids for periods of up to two years. 135 These inclusions are homogeneous, round, dense, eosin-stained globules in the cytoplasm of bronchiolar epithelial cells. They also stain with PAS. They have been shown to be more common at the level of distal terminal bronchioles than in proximal airways. Immunohistochemical staining has shown that they contain surfactant protein B and to a lesser extent Clara cell secretory protein. Electron microscopy shows no evidence of cell degeneration or adverse effects on Clara cells. Although the reason for increase in these secretary products in Clara cells following treatment with corticosteroids is unclear, they appear to have no pathological significance. Goblet cell hyperplasia is a well-recognized response of the mucosa of conducting airways to chronic inflammation and inhalation of irritant substances such as cigarette smoke and sulfur dioxide. 84, 93, 246, 247 The degree of goblet cell hyperplasia is dictated by the severity and duration of the irritation or inflammatory process. Florid cases of goblet cell hyperplasia are characterized by thickening and pseudostratification of the tracheal or bronchial mucosa by a population of tall, mucus secreting cells with abundant pale cytoplasm. In addition, goblet cells extend further down the airways than in normal animals and mucus may fill or distend the airways or impact in the alveoli. In less florid cases, a simple increase in the number of goblet cells may be found without other structural change. 93 Goblet cell hyperplasia of the lining epithelium may be accompanied by an increase in size of the underlying submucosal glands. This has clearly been demonstrated in patients with chronic bronchitis and in rats where submucosal glands are normally quite prominent. 246, 248 Species differences may exist because the airways of laboratory animals are variably endowed with goblet cells and submucosal mucous glands. The normal rat has more goblet cells lining the airways than either mouse or hamster. 246 The factors controlling these alterations are uncertain but is has been long suggested that increased mitotic activity as well as cell conversion, probably by metaplasia of serous or Clara cells to mucous cells is involved. 249 It has more recently been shown in mice sensitized to ovalbumin and subject to a single antigen challenge by aerosol that Clara cells in the proximal airways show great plasticity and become mucin-secreting cells. 110 Pharmacological agents can induce goblet or mucous cell hyperplasia. Rats given six or 12 daily injections of isoprenaline, a non-selective β receptor agonist, showed a dose and time-dependent increase in the number and size of alcian blue-positive goblet (mucous) cells as well as serous cells in the tracheal and bronchial mucosa. This was associated with an increase in length, width and depth of submucosal glands. 250 Similar changes were produced by pilocarpine. Both alcian blue and PAS positive cells were increased in number following administration of this agent, suggesting that pilocarpine induced both acid and neutral glycoprotein secretion. Comparison of the distribution of these changes in the rat following isoprenaline, with those of salbutamol, pilocarpine and tobacco smoke, showed that there were regional differences in the distribution of these changes in the airways. 251 Isoprenaline produced a greater increase in secretory cells in peripheral airways than tobacco smoke which itself produces a greater increase in mitotic activity. Isoprenaline and pilocarpine produced a more diffuse change than the more selective β agonist, salbutamol. The changes induced by these therapeutic agents are presumably the result of their pharmacological activity. 251 Sturgess and Reid showed that the changes in the rat were accompanied by hypertrophy of the pancreas, submaxillary and parotid salivary glands. 250 (See Digestive System, Chapter 8.)

Unlike the rat and mouse, the hamster appears predisposed to develop minor multifocal epithelial hyperplasia of the tracheal and bronchial mucosa spontaneously with advancing age. These changes are flat or polypoid in nature and are composed of clear cells and goblet cells. 88, 89 SQUAMOUS HYPERPLASIA, SQUAMOUS METAPLASIA

The epithelium of the bronchi shows squamous metaplasia in response to chronic irritation or injury. It is characterized by three or more layers of epithelial cells with abundant eosinophilic cytoplasm with prominent cell boundaries. It may be associated with degenerative alterations to the mucosa or goblet cell hyperplasia. Squamous metaplasia can also develop in the alveolar parenchyma as a response to prolonged damage such as produced by large burden of inhaled irritant or insoluble dusts. The metaplasia is also characterized by the presence of several layers of flattened epithelial cells showing squamous differentiation. The term pulmonary keratinizing cyst has been recommended for pulmonary cystic lesions lined by non-neoplastic squamous epithelium without excessive proliferative change. 252 These keratinizing lesions appear to be a particular response of the rat lung to inhaled irritant materials. Similar lesions are very uncommon in humans. 177 

Hyperplasia may involve the lining epithelium of the alveoli or bronchioli. This form of hyperplasia has been termed alveolar hyperplasia, adenomatosis, alveolar bronchiolization or epithelialization. It occurs spontaneously but can be induced by infections and administration of irritant xenobiotics in rats, mice and hamsters. 64,84,253À254 Histologically, the lesions consist of localized but unencapsulated foci of hyperchromatic regular, cuboidal or columnar cells investing airspaces without appreciable distortion of alveolar walls.

Neuroendocrine hyperplasia is also seen in various laboratory species including rats and mice. Haworth and colleagues reported pulmonary neuroendocrine cell hyperplasia in a small percentage of most strains of untreated two-year-old rats. The hyperplastic zones were clearly demarcated using immunohistochemistry with antibodies for protein G product 9.5 and calcitonin gene-related peptide. 258 It has also been reported in rats given silica particles by the intra-tracheal route. 259 In both rats and humans it has been seen in association with hypoxic conditions, although it has been suggested that these changes might be a result of increased peptide content rather than cell proliferation. 114, 260 Neuroendocrine hyperplasia has been well described in hamsters. Although scattered or small aggregates of neuroendocrine cells (neuroepithelial bodies) are found at various levels of the bronchi and bronchioli in normal hamsters, administration of nitrosamines and 4-nitroquinoline 1-oxide produces neuroendocrine hyperplasia. 261À263 Hyperplastic lesions are recognizable as clusters of non-ciliated cuboidal, oval or columnar cells located in the bronchial or bronchiolar epithelium. They contain argyrophilic granules that show immunoreactivity for corticotrophin (ACTH) and neuron-specific enolase. Ultrastructural examination reveals the presence of dense-core cytoplasmic granules of APUD (Amine À Precursor Uptake Decarboxylase) type.

The most frequently diagnosed neoplasm worldwide is lung cancer where it is usually caused by smoking tobacco. 264 Bronchogenic squamous carcinoma is generally the most common subtype in men but in North America the incidence of adenocarcinoma now exceeds that of squamous cell tumors for reasons not fully understood. It may be related to improved diagnosis of peripheral tumors, an increasing proportion of ex-smokers or changes in cigarette composition favoring greater exposure of the lung periphery to inhaled carcinogens. 265 Some of the most aggressive subtypes are small and large cell neuroendocrine lung cancers, defined as small or large tumor cells with greater than ten mitoses per two square mm (10 high power microscopic fields). 265 These seem to be seen almost exclusively in heavy cigarette smokers. Bronchogenic carcinoma and malignant mesothelioma have also been linked to exposure to asbestos fibers. The less carcinogenic serpentine fibers (e.g. chrysotile) are curly stranded structures, whereas the more potent are the amphiboles (e.g. crocidolite, amosite, tremolite) which have straight, rod-like fibers. 266 In contrast to findings in people, squamous cell lung tumors are only occasionally seen arising spontaneously in laboratory animals. Even laboratory animals, rats, mice, hamster, monkeys and dogs exposed to tobacco smoke for long periods and at high doses fail to develop significant increases in lung tumors. 267, 268 While some have argued that pulmonary proliferative changes seen in rodents exposed to tobacco smoke correlate with human tobacco-induced cancer, these rodent lesions are quite different from those seen in people. 269 Moreover, there is no good experimental model for neuroendocrine lung cancer linked to smoking that has such a poor prognosis in patients. Thus, particular caution is merited if using animal models for prediction of lung tumorigenic potential of inhaled substances.

By far the most common primary pulmonary neoplasms found in laboratory rats, mice and hamsters are adenomas and adenocarcinomas. These appear to develop from the bronchiolar or alveolar epithelium, although their precise histogenesis is somewhat disputed. For this reason they are usually called bronchiolo-alveolar or alveolar/bronchiolar adenomas and carcinomas. 270 Although spontaneous squamous neoplasms are uncommon in rodents, cystic keratinizing lesions can be induced in rats by high burdens of particulate material in the lungs. 252 Few of these appear to progress to invasive squamous carcinomas in long-term rat studies with particulate materials. Pleural mesotheliomas and mesenchymal neoplasms also occur in these species but are uncommon. In contrast to squamous tumors, mesothelial neoplasms can be induced in rodents by administration of asbestos and other long and durable mineral fibers. 271 Mesenchymal tumors have similar histological features to those in soft tissues and mesotheliomas may show either epithelial or mesenchymal differentiation or both.

In most rat strains alveolar or bronchiolar neoplasms occur spontaneously in relatively small numbers, but morphologically identical neoplasia can be induced by administration of chemical carcinogens. 272 The most common are classified as bronchiolo-alveolar adenoma (pulmonary adenoma) and bronchiolo-alveolar carcinoma. The National Toxicology Program database on control Fischer 344 rats used in carcinogenicity studies indicates an overall percentage of less than 3% of animals with bronchiolo-alveolar adenomas and less than 1% with bronchiolo-alveolar carcinomas. 86 However, the range of bronchiolo-alveolar adenomas in different studies was between 0 and 14% in this series.

Histologically, bronchiolo-alveolar tumors are mostly small, discrete, rounded nodules located in the lung parenchyma and composed of fairly uniform cells with moderately hyperchromatic nuclei arranged in solid (alveolar), tubular, papillary or mixed growth patterns. They usually compress surrounding tissues without infiltration or metastatic spread (adenoma) although loss of differentiation, infiltration and spread to adjacent tissues can occur (adenocarcinoma). Ultrastructural study of bronchiolar-alveolar neoplasia in Fischer 344 rats has shown the presence of osmiophilic, lamellated inclusion bodies similar to those found in alveolar type II cells. Therefore it has been suggested that the neoplasms may be derived from this cell type. 272 Bronchiolar-alveolar adenomas and carcinomas are usually considered together in the evaluation of carcinogenicity. 273 Pulmonary squamous carcinoma occurs but is a very uncommon spontaneous neoplasm in the rat. 82 The large proliferative but benign cystic lesions found in the lungs of rats following accumulation of large amount of particulate matter have been termed pulmonary cystic keratinizing epitheliomas for they have been regarded as benign neoplasms. When these lesions show evidence of tissue invasion they are regarded as pulmonary squamous cell carcinomas. Similar lesions are very occasionally reported as spontaneous lesions. 274 

Analogous neoplasms are found more commonly in most strains of laboratory mice used in carcinogenicity bioassays although considerable variation in incidence is reported. They are common in strain A mice where they are observed in low frequency at three to four months of age and incidences reach nearly 100% by 24 months of age. 275 Fewer but significant numbers are found in B6C3F 1 mice, although there is considerable inter-laboratory variation. 276 The National Toxicology Program database on control B6C3F 1 mice used in carcinogenicity studies indicates an overall percentage of about 16% of males and 6% of females with bronchiolo-alveolar adenomas but only about 5% and 2.5% respectively with bronchiolo-alveolar carcinomas. 86 However, the range of bronchiolo-alveolar tumor varied considerable between studies in this series. Even in the same laboratory, mice housed under similar conditions can show large variation in incidence in these neoplasms with time. The incidence of lung adenomas and adenocarcinomas occurring in CD-1 mice used as controls in 18-month carcinogenicity bioassays conducted under similar conditions over a period of three years in one laboratory varied from between 19 to 36% in males and from 6 to 16% in females. 277 By contrast, some strains of mice such as the C5781/10J strain show a very low predisposition to the development of lung adenomas. 278 Although these mouse pulmonary adenomas and adenocarcinomas do not resemble the common lung tumors in humans, strain differences have been exploited to study genetic susceptibility and resistance to pulmonary adenomas and carcinomas. 269, 279, 280 Histologically, pulmonary tumors of this type in mice are generally small, sharply circumscribed nodules composed of fairly uniform, closely packed columns of cuboidal or columnar cells arranged in tubular or papillary structures with scanty fibrovascular stroma (Figure 6 .6). They may be less well differentiated with cellular pleomorphism and show intrabronchial growth, invade lung parenchyma and produce metastatic deposits.

The histogenesis of mouse pulmonary adenomas and adenocarcinomas is disputed. On the basis of sequential light and electron microscopic study of pulmonary adenomas induced in Bagg-Webster Swiss mice by transplacental exposure to ethylnitrosourea, it has been suggested that they develop from either alveolar type II cells or Clara cells. 281, 282 Careful, stepwise analysis using light microscopic and electron microscopic examination has suggested that adenomas can be divided into three principal groups. Some are composed of solid growths of uniform cuboidal cells with expanding margins limited to alveolar septae (alveolar pattern). These cells contained concentrically arranged cytoplasmic lamellar bodies and abundant, large mitochondria similar to mitochondria found in alveolar type II cells. Tubular or papillary patterns are composed of cuboidal cells showing histological and ultrastructural features of Clara cell differentiation. 281 However, immunocytochemical studies of chemically induced and spontaneous pulmonary neoplasia in B6C3F1, BALB/c or A strain mice have shown that the majority of adenocarcinomas, including those showing papillary patterns, contain surfactant apoprotein, typical of type II antigens suggesting that most neoplasms show alveolar type II differentiation. 283 However, in view of the plasticity of Clara cells, this does not exclude a Clara cell origin of the tumors. Immunocytochemistry of specific Clara cell secretory protein expression in a transgenic mouse model of lung carcinomas developing from Clara cells has shown that the protein is lost during tumor cell progression. 284 It has also been shown in strain A mice that the proportion of tumors with papillary and solid/alveolar growth patterns varies with the inducing agent. 285 This also suggests biological differences exist between histological subtypes.

Very few squamous carcinomas are reported in most mouse studies. A chemically induced mouse model of squamous cell carcinoma has been generated by administration of N-nitroso-tris-chloroethylurea. Strain differences in susceptibility to squamous cancer development have been demonstrated in this model, with NIH Swiss, A/J and SWR/J being highly susceptible, AKR/J and C57BL/6J being resistant and FVB/J and BALB/CJ mice showing an intermediate response to carcinogen. 286 

Hamsters develop lung adenomas spontaneously in small numbers with advancing age. They are composed of uniform cylindrical cells similar to those found in bronchial epithelium or goblet cells showing distinct mucus production. 88, 89, 287 An immunohistochemical study of similar pulmonary neoplasms induced in hamsters by N-nitrosodiethylamine has shown the presence of Clara cell antigen in early phase of development but as the tumors developed they became more squamous in type and showed immunoreactivity for cytokeratins. 257 A Clara cell origin was suggested for most of these neoplasms.

The high incidence and the inherent variability of pulmonary adenomas and adenocarcinomas in conventional mouse carcinogenicity bioassays sometimes gives rise to statistically significance differences between control and treatment groups. There is considerable risk in overinterpretation of such group differences in conventional mouse bioassays. In the analysis of group differences, consideration needs to be given to tissue sampling procedure, age standardization, historical control incidence, effects on food intake as well as the results of mutagenicity studies and carcinogenicity bioassays in other rodent species. Indeed, a considerable number of widely employed therapeutic agents of different classes have produced an increase in benign or malignant pulmonary tumors in carcinogenicity studies performed in mice without this proving of any significance to humans. Davies and Monro counted at least 17 drugs in the 1994 Physicians' Desk Reference of the United States. 288 In the two-year rodent carcinogenicity studies conducted in the National Toxicology Progam, the lung is second to the liver as the most common site of neoplasia in male and female mice and common in female rats. 270 One example of the difficulties is the carcinogenicity bioassay in which CF1 mice were treated for 80 weeks with the synthetic analgesic tilidine fumarate. Here a statistically significant difference (p , 0.01) was reported in the incidence of lung adenocarcinomas between the top dose female group (24%) and concurrent controls (10%). 289 It was argued that group differences did not indicate tumorigenic potential of tilidine fumarate on the basis that the incidence in the high dose group was within the historical control range (27%) and that there was no tumorigenic effect in the parallel 104-week rat carcinogenicity study.

A more difficult evaluation concerned metronidazole, a nitroimidazole, which is an important therapeutic agent active against anaerobic organisms and trichomonas species. Administration of this compound led to an increased incidence of pulmonary adenomas and carcinomas in three separate mouse carcinogenicity bioassays. 290, 291 The analysis of these findings was somewhat complicated by evidence that metronidazole shows mutagenic activity in bacterial assays using some strains of Salmonella typhimurium. It was argued that the risk to human patients was slight because the increase in prevalence in pulmonary tumors was likely to be a result of changes in nutritional status of the mice through the effect of metronidazole on gut flora, as similar differences could occur between ad libitum-fed mice and those fed the same but restricted diet. 291 It was also postulated that the positive findings in bacterial mutagenesis assays were an inherent part of the antibacterial activity of metronidazole as a result of nitroreduction that does not occur in normal mammalian tissues. This conclusion was supported by negative effects in hamster carcinogenicity bioassays as well as lack of excess cancer risk in women followed up for 10 years or more. 291 

The common occurrence of lung adenomas in strain A mice has been utilized in the development of a quantitative bioassay for carcinogenic activity. This followed the demonstration that administration of genotoxic carcinogens such as 3-methylcholanthrene to this strain could significantly increase the incidence of pulmonary adenomas within periods of up to six months. 292 Over many years the strain A mouse pulmonary tumor assay has been used to test a large number of chemicals of different classes including polycyclic hydrocarbons, nitrosamines, food additives, alkyl halides, metals and chemotherapeutic agents. 275, 285 However, as with many test systems, correlation of results in the strain A test with two-year carcinogenicity study data and genotoxicity results have been shown to be poor so prudence is needed in the use of this test. 293 

A variety of focal proliferative changes may be seen in the pleural cavity in laboratory animals. Usually these are localized zones of focal thickening or villous projections covered by cuboidal cells with little or no stratification. They may possess a fibrovascular core or stalk and fibrosis or inflammation may accompany the changes.

Malignant mesotheliomas of the pleural cavity are rare spontaneous lesions in rodents. However, they can be induced in rodents by the presence of large numbers of persistent durable fibrous materials of certain geometric dimensions. 294 Decades of experimental study have shown that the presence of durable fibers longer than 10 μm is important in this form of solid state carcinogenesis. 295 In people carcinogenicity of asbestos is also related to fiber length but other factors are also important, particularly smoking, which may have a multiplicative effect. 266,296À298 The adverse effect of long fibers has been a focus of renewed study because of concerns about the toxic and carcinogenic potential of engineered nanotubes which may have similar dimensions to fibers that have shown tumorigenic potential in rodents. 25, 26, 167 The morphology of these tumors appears to be similar in rodents and humans. 265, 299, 300 They can be divided into two principal histological types, one showing principally epithelial differentiation, the other showing a sarcomatous appearance, although mixed types also occur. 82 The epithelial or epitheloid type contains fronds or papillary structures, poorly formed glandular structures of rounded cells with round vesicular nuclei (Figure 6 .7a). Cells are PAS negative and infiltrate into adjacent connective tissue.

The sarcomatous or sarcomatoid type is typically composed of relatively uniform spindleshaped cells with elongated nuclei, interlacing bundles similar to fibrosarcomas or leiomyosarcomas or forming whorls suggestive of pleomorphic sarcomas or malignant fibrous histiocytomas ( Figure 6 .7b). They may contain an abundant connective tissue (desmoplastic) component.

In the mixed or biphasic type both epitheloid and sarcomatous components coexist. They occur not only in the pleural cavity but also in the peritoneum. One of the most frequent sites in the rat is the tunica vaginalis of the testis from which they may spread to the peritoneal cavity. 301 Potassium bromate, used in cosmetics and food products and a by-product of water disinfection by ozonization, is an example of a chemical that has been reported to induce mesotheliomas in the tunica vaginalis and peritoneum in long-term studies in Fischer 344 rats (see Male Genital Tract, Chapter 11). 302, 303 

Lung defenses against infection: a clinical correlation

Nanomaterials and nanoparticles: sources and toxicity

Translocation pathway of the intratracheally instilled C60 fullerene from the lung into the blood circulation in the mouse: possible association of diffusion and caveolaemediated pinocytosis

Translocation pathway of the intratracheally instilled ultrafine particles from the lung into the blood circulation in the mouse

Pleural liquid and its exchanges

Ultrastructure and three-dimensional study of the lymphatic stomata in the costal pleura of the rabbit

Inhalation aspects of therapeutic aerosols

Drug-induced lung toxicity

Drug-induced diseases. Drug-induced respiratory disease

Pulmonary toxicity from novel antineoplastic agents

Toxicology of inhaled materials. general principles of inhalation toxicology

Risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation

Noninfectious pneumonitis after everolimus therapy for advanced renal cell carcinoma

Pulmonary toxicities of biologics: a review

Therapyinduced toxicity of the lungs: an overview

Pulmonary toxicity of chemotherapy

Drug-induced rhinitis

Considerations for toxicology studies of respiratory drug products

Overview of inhalation toxicology

Inhalation exposure technology, dosimetry, and regulatory issues

Pulmonary deposition: determinants and measurement techniques

Inhalation therapy: technological milestones in asthma treatment

Contemporary issues in fiber toxicology

The relevance of the rat lung response to particle overload for human risk assessment: a workshop consensus report

Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma

Toxicology of engineered nanomaterials À a review of carcinogenic potential

Nanomedicine in pulmonary delivery

Interpretation of new techniques used in determination of pulmonary function in rodents

Measurement of respiratory patterns in rodents using wholebody plethysmography and a pneumotachograph

Evaluation of lung injury in rats and mice

Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants

Comparative aspects of nasal airway anatomy. Relevance to inhalation toxicology

Comparative anatomy, physiology, and function of the upper respiratory tract

Comparative anatomy of mammalian respiratory tracts: the nasopharangeal region and the tracheobronchial region

The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium

Airflow, gas deposition, and lesion distribution in the nasal passages

A comparative analysis of primate nasal airways using magnetic resonance imaging and nasal casts

The upper airways. 1. Nasal physiology and defence of the lungs

Antioxidant protection: a function of tracheobronchial and gastrointestinal mucus

The histochemical and microscopical differentation of the respiratory glands around the maxillary sinus of the rat

Hamster nasal glands: their structure, sialic acid content, and vulnerability to actinomycin D

The lateral nasal gland of the dog, its structure and secretory content

Sites for xenobiotic activation and detoxication within the respiratory tract: implications for chemically induced toxicity

Cytochrome-P450 2A of nasal epithelium À regulation and role in carcinogen metabolism

Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes

Differential xenobiotic induction of CYP2A5 in mouse liver, kidney, lung, and olfactory mucosa

Nasal cytochrome P450 2A: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen

Cell-specific expression of CYP2A5 in the mouse respiratory tract: effects of olfactory toxicants

Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach

Nasal lymphoid tissue in the rat

Toxicity to nasal associated lymphoid tissue

Histopathologic examination of the rat nasal cavity

Structural evaluation of the respiratory system

Normal histology of the nasal cavity and application of special techniques

Approaches to the identification and recording of nasal lesions in toxicology studies

Nasal diagrams À a tool for recording the distribution of nasal lesions in rats and mice

Histopathology of nasal olfactory mucosa front selected inhalation toxicity studies conducted with volatile chemicals

Determination of surface areas, volumes, and lengths of cynomolgus monkey nasal cavities by ex vivo magnetic resonance imaging

Three-dimensional mapping of ozone-induced injury in the nasal airways of monkeys using magnetic resonance imaging and morphometric techniques

Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. 1. Determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung

Irritating properties of airborne materials to the upper respiratory tract

Bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in man

Infectious diseases of the upper respiratory tract: implications for toxicology studies

Respiratory tract

Murine respiratory mycoplasmosis in LEW and F344 rats: strain differences in lesion severity

Evaluation by scoring and computerized morphometry of lesions of early Mycoplasma pulmonis infection and ammonia exposure in F344/N rats

One year inhalation toxicity study of chlorine in rhesus monkeys (Macaca mulatta)

Proliferative and nonproliferative lesions of the rat and mouse respiratory tract

Respiratory tract lesions induced by sensory irritants at the RD 50 concentrations

The toxicity of diethylamine in F-334 rats and B6C3F1 mice following 1 year inhalation exposure

One-month inhalation study of tulobuterol hydrochloride in rats and dogs

Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats

Nasal toxicity of CI-959, a novel anti-inflammatory drug in Wistar rats and beagle dogs

The side effects of antithyroid drugs

Olfactory toxicity of methimazole: doseÀresponse and structureÀactivity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa

Methimazole-induced damage in the olfactory mucosa: effects on ultrastructure and glutathione levels

Olfactory epithelium as a novel toxic target following an intravenous administration of vincristine to mice

Investigation of initial changes in the mouse olfactory epithelium following a single intravenous injection of vincristine sulphate

Species and sex differences in susceptibility to olfactory lesions among the mouse, rat and monkey following an intravenous injection of vincristine sulphate

Peripheral olfactory sensitivity in rodents after treatment with docetaxel

The respiratory system

Proliferative lesions of the rat respiratory tract, R-1. In: Guides for toxicologic pathology

Respiratory system. In: International classification of rodent tumours, Part 1, The rat. Lyon: International Agency for Research on Cancer

International classification of rodent tumours. The mouse

Proliferative and neoplastic lesions in the rodent nasal cavity

Spontaneous neoplasm incidences in Fischer 344 rats and B6C3F1 mice in two-year carcinogenicity studies. A National Toxicology Program update

Spontaneous proliferative lesions in the nasopharyngeal meatus of F344 rats

Spontaneous tumors and common diseases in three types of hamsters

Spontaneous tumors and common diseases in two colonies of Syrian hamsters. II Respiratory tract and digestive system

Sialodacyoadenitis virus-associated lesions in the lower respiratory tract of rats

Oropharyngeal granulomas and tracheal cartilage degeneration in Fischer-344 rats

Articular chondromatosis and chrondroid metaplasia in transgenic TAg mice

Cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases

Histological sectioning of the rodent larynx for inhalation toxicity testing

Histologic methods and interspecies variations in the laryngeal histology of F344/N rats and B6C3F1 mice

Interspecies variations in the histology of toxicologically important areas in the larynges of CRL:CD rats and Syrian golden hamsters

Revised guides for organ sampling and trimming in rats and mice À Part 2 À A joint publication of the RITA and NACAD groups

Toxicologic significance of histologic change in the larynx of the rat following inhalation exposure: a critical review

A comparison of the pathology of the larynx from SPF, germ-free, conventional, feral and myoplasma-infected rats

International ESTP Expert Workshop: Larynx squamous metaplasia. A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment

Spontaneous neoplasms observed in cynomolgus monkeys (Macaca fascicularis) during a 15-year period

Modelling of biological tree structures

Allometric relationships of cell numbers and size in the mammalian lung

The ultrastructure of various cell types in the lung of the rat

Length and distribution of cilia in human and canine airways

Airway mucus function and dysfunction

Lectin-binding affinities of the respiratory tract. A light microscopical study of ciliated epithelium in rat, guinea pig and hamster

Uteroglobin

Identification, cellular-localization, isolation, and characterization of human Clara cell-specific 10-kD protein

Mucin is produced by Clara cells in the proximal airways of antigen-challenged mice

Morphology of the terminal bronchiolar region of common laboratory mammals

Morphological changes in the lung during the life span of Fischer 344 rats

Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells and alveolar macrophages of rat lung

Quantitative microscopical methods for the identification and localisation of nerves and neuroendocrine cell markers in mammalian lung

What can the biology of small cell cancer of the lung teach us about the endocrine lung?

The role of metabolism in chemical-induced pulmonary toxicity

Influence of particle size on drug delivery to rat alveolar macrophages following pulmonary administration of ciprofloxacin incorporated into liposomes

Characterization of pulmonary macrophages and bronchus-associated lymphoid tissue (BALT) macrophages in the rat. An enzyme-cytochemical and immunocytochemical study

Subpopulations of lymphoid and non-lymphoid cells in bronchus-associated lymphoid tissue (BALT) of the mouse

Bronchial lymphoid tissue. 1. Morphologic characteristics

The specificity of the high endothelial venule in bronchus-associated lymphoid tissue (BALT)

T cells and T cell subsets in rat bronchus associated lymphoid tissue (BALT) in situ and in suspension

Have you seen this?' Inflammatory lesions in the lungs of rats

Inflammatory lesions in the lungs of Wistar rats

Histological changes in rat bronchus-associated lymphoid tissue after administration of five different antigens

Changes occurring in the epithelium covering the bronchus-associated lymphoid tissue of rats after intracheal challenge with horseradish peroxidase

Patterns of lymphatic drainage to individual thoracic and cervical lymph nodes in the rat

Recent advances in the research of lymphatic stomata

Age-body weight relationships to lung growth in the F344 rat as indexed by lung weight measurements

Application of morphometric methods to study diffuse and focal injury in the lung caused by toxic agents

Morphometric assessment of pulmonary toxicity in the rodent lung

Imaging techniques for small animal models of pulmonary disease: MR microscopy

Imaging techniques for small animal imaging models of pulmonary disease: Micro-CT

The connective tissue of the rat lung: electron immunohistochemical studies

Eosinophilic inclusions in rat Clara cells and the effect of an inhaled corticosteroid

Immunohistochemical techniques and their applications in the histopathology of the respiratory system

Systemically applied chemicals that damage lung tissue

Bronchial mucosal mast cells and their implications in the pathogenesis of asthma

Morphological-changes in rat tracheal mucosa immediately after antigen challenge

Comparative studies of heparin and heparin fragments: distribution and toxicity in the rat

Interactions between Sendai virus and bacterial pathogens in the murine lung: a review

Incidences and range of spontaneous findings in control cynomolgus monkeys (Macaca fascicularis) used in toxicity studies

Murine respiratory mycoplasmosis in F344 and LEW rats À evolution of lesions and lung lymphoid-cell populations

Pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (Sendai) virus

Respiratory tact lesions in weanling outbred rats infected with Sendai virus

Increased tumor necrosis factor-alpha (TNFalpha) gene expression in parainfluenza type 1 (Sendai) virus-induced bronchiolar fibrosis

IL-12 reduces the severity of Sendai virus-induced bronchiolar inflammation and remodeling

Studies of respiratory disease in random source laboratory dogs: viral infections in unconditioned dogs

Visceral larva migrans in the dog

Lesions produced by a new lung worm in beagle dogs

Diagnostic exercise: macaque with dyspnoea

Treatment of pulmonary acariasis in rhesus macaques with ivermectin

A primer of primate pathology: lesions and nonlesions

Attachment of microbes to host cells: relevance of Pneumocystis carinii

Animal model: Pneumocystis carinii pneumonia in the immunosuppressed rat

Attachment of Pneumocystis carinii to rat pneumocytes

A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin 2 or high-dose interleukin 2 alone

Toxicity of human recombinant interleukin-2 in rats. Pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement

Toxicity of human recombinant interleukin-2 in the mouse is mediated by interleukin-activated lymphocytes. Separation of efficacy and toxicity by selective lymphocyte subset depletion

Characterization of the pulmonary lesions induced in rats by human recombinant interleukin-2

Dyspnoea and thoracic spinal deformation in rats after oral prizidilol (SK&F 92657-A2)

Update on macrophage clearance of inhaled micro-and nanoparticles

Characterization of the pulmonary lesions induced in rats by human recombinant interleukin-2

Diagnostic exercise: pneumonia in a rat

Multidrug chemotherapy of tuberculosis in rhesus monkeys

Granulomatous pneumonitis induced by bacille Calmette-Guérin in the mouse and its treatment with cyclosporin A

Mechanisms of pulmonary toxicity and medical applications of carbon nanotubes: two faces of Janus?

Anergy-like immunosuppression in mice bearing pulmonary foreign body granulomatous inflammation

Direct evidence for granuloma-inducing role of interleukin-1

Cytokine and growth factor release by alveolar macrophages: potential biomarkers of pulmonary toxicity

Animal model: pulmonary granulomatous vasculitis induced in rats by treatment with glucan

Naturally occurring diatomaceous pneumoconiosis in subhuman primates

Ultrastructural and micropulse analysis of simian lung mite pigments

Gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease

Environmental, inhaled and ingested causes of pulmonary fibrosis

Comparative pathology of environmental lung disease: an overview

Lung cytokine production in bleomycin-induced pulmonary fibrosis

Treatment of human recombinant soluble TNF receptor of pulmonary fibrosis induced by bleomycin or silica in mice

The physiology of transforming growth factor-a

TNF-a receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos

Methotrexate pneumonitis induced by intrathecal methotrexate therapy. A case report with pharmacokinetic data

Cytotoxic drug-induced pulmonary disease: update 1980

Lung toxicity associated with cyclophosphamide use. Two distinct patterns

Interstitial pneumonitis associated with bleomycin therapy

Amiodarone pulmonary toxicity: cytopathology, ultrastructure, and immunocytochemistry

Gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease

Bleomycin lung toxicity: who are the patients with increased risk?

Preclinical toxicologic evaluation of bleomycin (NSC 125 066), a new antitumor autibiotic

Bleomycin-induced pulmonary toxicity in the rat

ntratracheal versus intravenous administration of bleomycin in mice: acute effects

An investigation of possible models for the production of progressive pulmonary fibrosis in the rat. The effects of repeated intraatracheal instillation of bleomycin

Extensive laminin and basement membrane accumulation occurs at the onset of bleomycin-induced rodent pulmonary fibrosis

Capillary remodeling in bleomycininduced pulmonary fibrosis

The role of strain variation in murine bleomycin-induced pulmonary fibrosis

Pulmonary toxicology of cyclophosphamide: a 1-year study

The role of conventional pathology and toxicology in evaluating the immunotoxic potential of xenobiotics

Hyaline and calcified pleural plaques as an index of exposure to asbestos. A study of radiological and pathological features of 100 cases with a consideration of epidemiology

Current concepts in mechanisms of emphysema

Animal models of emphysema

Lung scleroproteins in young and adult rats and in rats with spontaneous emphysema: comparative studies by biochemical and histochemical approach

A quantitative study of stenosis in the respiratory bronchiole of the rat in NO2-induced emphysema

Long-term sequelae of bronchiolitis induced by nitrogen dioxide in hamsters

Phospholipid fatty liver. A proposal of a new concept and its electron microscopical study

Drug-induced generalized phospholipidosis

Electron microscopic observation of experimental phospholipidosis induced by 4-4 0 -diethylaminoethoxy hexesterol dihydrochloride in the rat

Chloroquine: pathological changes observed in rats which for two years had been fed various proportions

Morphological and biochemical changes in liver of various species in experimental phospholipidiosis after diethyaminoethoxyhexestrol treatment

Intra alveolar foam cells associated with lipidosis-like alterations in lung and liver of rats treated with tri cyclic psychotropic drugs

Cationic amphiphilic drug-induced phospholipidosis

Reasor MJ. Drug-induced lipidosis and the alveolar macrophage

Drug-induced lysosomal disorders in laboratory animals: new substances acting on lysosomes

Pulmonary and generalized lyosomal storage induced by amphiphilic drugs

Tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses

Fine structural alterations in the lungs of iprindole-treated rats

Foam cell response in the lung and lymphatic tissues during long-term high-level treatment with erythromycin

Interactions of macrolide antibiotics (erythromycin A, roxithromycin, erythromycylamine [dirithromycin], and azithromycin) with phospholipids: computer-aided conformational analysis and studies on acellular and cell culture models

Epididymal and systemic phospholipidosis in rats and dogs treated with the dopamine D3 selective antagonist PNU-177864

Myopathy related to administration of a cationic amphiphilic drug and the use of multidose drug distribution analysis to predict its occurrence

Phospholipidosis in neurons caused by posaconazole, without evidence for functional neurologic effects

A cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development

Validation of an in vitro screen for phospholipidosis using a high-content biology platform

The induction of pulmonary phospholipidosis and the inhibition of lysosomal phospholipases by amiodarone

Collecting and processing tissues for diagnostic electron microscopy

Chlorphentermine-induced lipidosis like ultrastructural alterations in lungs and adrenal glands of several species

Amiodarone lung toxicity: a human and experimental study

Generalized phospholipidosis induced by amphiphilic cationic psychotropic drug

Morphological and biochemical changes in the liver of various species in experimental phospholipidosis after diethylaminoethoxyhexestrol

Recovery from amiodarone-induced lipidosis in laboratory animals. A toxicological study

Case 12-2003

Amiodarone induced phospholipidosis. Biochemical, morphological and functional changes in the lungs of rats chronically treated with amiodarone

Granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study

Amiodarone-associated pulmonary fibrosis. Evidence of an immunologically mediated mechanism

Amiodarone-induced pulmonary toxicity: an under-recognized and severe adverse effect?

An evaluation of possible mechanisms underlying amiodarone-induced pulmonary toxicity

An 82-year-old man with dyspnea and pulmonary abnormalities

Drug-induced phospholipidosis: are there functional consequences?

Lipidosis induced by amphiphilic cationic drugs

Multifocal histiocytosis in the lungs of rats

Pathological discussion. Case Record of the Massachusetts General Hospital

Organomegaly and histopathology in an animal model of mucopolysaccharidosis induced by suramin

Lysosomal-storage disorder induced by Elmiron following 90-days gavage administration in rats and mice

Alveolar proteinosis and phospholipidosis of the lungs

Pulmonary alveolar proteinosis

Pulmonary alveolar proteinosis, a primary immunodeficiency of impaired GM-CSF stimulation of macrophages

An experimental study of hypersecretion of mucus in the bronchial tree

Goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: the effect of tobacco smoke with or without the anti-inflammatory agent phenylmethyloxadiazole

Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis

Mitotic activity of airway epithelium after short exposure to tobacco smoke and the effect of the anti-inflammatory agent phenylmethyloxadiazole

The effect of isoprenaline and pilocarpine on (a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen

Experimental chronic bronchitis

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

Pathological changes during aging in barrier-reared Fischer 344 male rats

Neoplastic and nonneoplastic lesions in aging F344 rats

Histopathological profile of a Wistar rat stock including a survey of the literature

Naturally occurring Sendai disease of mice

Immunohistochemical demonstration of Clara cell antigen in lung tumors of bronchiolar origin induced by N-nitrosodiethylamine in Syrian golden hamsters

Pulmonary neuroendocrine cell hyperplasia: identification, diagnostic criteria and incidence in untreated ageing rats of different strains

Hyperplasia of alveolar neuroendocrine cells in rat lung carcinogenesis by silica with selective expression of proadrenomedullin-derived peptides and amidating enzymes

Increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine-cells of hypoxic rats

Sequential morphologic alterations in the bronchial epithelium of Syrian golden hamsters during N-nitrosomorpholine-induced pulmonary tumorigenesis

Lung endocrine À like cells in hamsters treated with diethylnitrosamine: alterations in vivo and in cell culture

4-nitroquinoline 1-oxide-induced pulmonary endocrine cell hyperplasia in Syrian golden hamsters

The 2004 World Health Organization classification of lung tumors

Pathology and genetics of tumours of the lung, pleura, thymus and heart

Asbestos-induced lung diseases: an update

A review of chronic inhalation studies with mainstream cigarette smoke in rats and mice

A review of chronic inhalation studies with mainstream cigarette smoke, in hamsters, dogs, and nonhuman primates

Comparative oncology of lung tumors

Summary of chemically induced pulmonary lesions in the National Toxicology Program (NTP) toxicology and carcinogenesis studies

Animal models of of mesothelioma induced by mineral fibers: implications for human risk assessment

Morphology of spontaneous and induced tumors in the bronchiolo-alveolar region of F344 rats

Guideline for combining neoplasms for evaluation of rodent carcinogenicity studies

Spontaneous cystic keratinising epithelioma in the lung of a Sprague-Dawley rat

Strain A mouse lung tumor bioassay

Variability in the rates of some common naturally occuring tumors in Fischer 344 rats and (C57BL/6NxC3H/HeN)F1 (B6C3F1) mice

A carcinogenicity study in mice of a b-adrenegic antagonist, primidolol; increased total tumour incidence without tissue specificity

Effect of diet on spontaneous disease in the inbred mouse strain C57B1/10J

Pol iota is a candidate for the mouse pulmonary adenoma resistance 2 locus, a major modifier of chemically induced lung neoplasia

Cancer susceptibility in the mouse: genetics, biology and implications for human cancer

Histogenesis of the papillary Clara cell adenoma

Histologic and ultrastructural features of the Clara cell adenoma of the mouse lung

Immunocytochemical localization of the surfactant apoprotein and Clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice

Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis

Strain A/J mouse lung adenoma patterns vary when induced by different carcinogens

A chemically induced model for squamous cell carcinoma of the lung in mice: Histopathology and strain susceptibility

Animal model: spontaneous carcinoma of the lung in hamsters

Marketed human pharmaceuticals reported to be tumorigenic in rodents

Evaluation of chronic toxicity and carcinogenesis in rodents with the synthetic analgesic, tilidine fumarate

Induction of lung tumors and malignant lymphomas in mice by metronidazole

Toxicologic evaluation of metronidazole with particular reference to carcinogenic, mutagenic, and teratogenic potential

Induced pulmonary tumors in mice. II. Reaction of lungs of strain A mice to carcinogenic hydrocarbons

Strain A mouse pulmonary tumor test results for chemicals previously tested in the National Cancer Institute carcinogenicity tests

Proliferative lesions of the rat respiratory tract

The inhalation toxicology of p-aramid fibrils

Asbestos fibre dimensions and lung cancer mortality among workers exposed to chrysotile

Mesothelioma epidemiology, carcinogenesis, and pathogenesis

Assessing the combined effect of asbestos exposure and smoking on lung cancer: a Bayesian approach

International classification of rodent tumors, The mouse

International Classification of Rodent Tumours. Part 1 The Rat. Lyon: International Agency for Research on Cancer

Proliferative lesions of the testes in rats with selected examples from mice

Time-and dose-dependent development of potassium bromate-induced tumors in male Fischer 344 rats

Origin and distribution of potassium bromateinduced testicular and peritoneal mesotheliomas in rats