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A I if"! , 73”! 4'4 I 1,", . 1/531! ‘ .,‘l "I” I , [$531]? a", N..” A ...,,,,A,, // ~47 5/? 5mm / W” 5. aim/3 A .?.'A 19..."; A ’1’ .21.? fl/ €92.55 LIBRARY Michigan State University This is to certify that the dissertation entitled EFFECT OF CARBON TETRACHLORIDE 0N HAMSTER TRACHEAL EPITHELIAL CELLS presented by Massumeh Ahmadizadeh has been accepted towards fulfillment of the requirements for Ph.D. Environmental Toxicology- Anatomy dwmfiv a Major professor degree in Date /c0 Feb MN MS U i: an Aflirmau've Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES .— ‘ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EFFECT OF CARBON TETRACHLIRIDE (Ii HMSTER TRACHEAL EPITI-IELIAL CELLS By Hassuneh Ainadizadeh A DISSERTAIIOI Subnitted to Michigan State University in partial fulfillment of the requ'lr-ents for the degree of DOCTOR (I: PHILOSOPHY Depart-ant of Anatony and Center for Enviromental Toxicology EFFECT or CARBGI momma on "MISTER TRACHEAL EPITHELIAL CELLS By nassmeh Al-adizadeh This investigation was designed to assess cytotoxicity of carbon tetrachloride (CCl4) in hamster trachea. Adult, male, Syrian golden hamsters were treated with 2.5 ml/kg CCl4, and controls received the vehicle only. Animals were sacrificed at various intervals following treatment. Tracheas were fixed and embedded for light and electron microscopy. Cell damage was evaluated by light and electron microscopy. Glycoprotein-containing cells were evaluated by light microscopy using Alcian blue/Periodic acid Schiff (AB-PAS). In control hamsters secretory cells were columnar, and contained abundant smooth endoplasmic reticulum. Cytochemically, most secretory cells were PAS positive and cOntained neutral glycoproteins. CCl4 produced injury in secretory cells and ciliated cells. Injury was characterized by loss of staining, dilatation of the nucleus and other intracellular organelles. However, the extent of injury varied in different regions and levels of tracheal epithelium. This chemical also caused alterations in glycoprotein-containing cells. For example CCl4 produced transient shift from neutral to acidic glycoproteins. Cells containing homogenous, PAS positive material were absent in control, MASSUMEH AHMADIZADEH Page 2 but were obvious after C014. In other experiments, hamsters were fed Q51 l_il; or fasted for 24 hrs or were pretreated with 0.6 ml/kg diethylmaleate (DEM) for 30 min prior to administration of CCl4. All hamsters received 1.0 ml/kg CCl4 or vehicle. Fasted animals were further starved after administration of CCl4 or vehicle. Animals were killed 24 hrs later. Tissues were again studied with light and electron microscopy. Fasting alone produced injury in secretory cells and ciliated cells. Fasting potentiated CCl4-induced injury. DEM produced injury in both secretory cells and ciliated cells, however, the extent of injury varied in different regions and levels of the epithelium. Since CCl4-induced cell injury is dependent on metabolism of this agent by intracellular NADPH dependent cytochrome P-450 monoxygenases, this study suggests that secretory cells and ciliated cells of hamster trachea have the potential to bioactivate CCl4. Depletion of glutathione and/or induction of the microsomal enzyme system by fasting may have potentiated CCl4-induced injury. The observation that DEM produced injury in hamster trachea rendering its effect on CCl4 toxicity difficult to ascertain from these studies. DEDICATION In memory of my Father, Seyed Mohamed Tagi ‘Ahmadizadeh and my advisor Dr. Robert Echt, who would have delighted in sharing the joy of this achievement. ii ACKNOHLEDGEMENTS There are many individuals who contributed to the successful completion of this project. All of them have my heartfelt appreciation. I would like to thank Drs. Robert Roth and Lawrence Ross for serving as co-chairs for my committee. I express sincere gratitude to the members of my committee for their encouragement, support, and advice on the many diverse aspects of this project. Dr. Robert Roth provided expertise necessary in the area of toxicology. Drs. Karen Klomparens, Lawrence Ross, and Al Stinson were invaluable consultants on light and electron microscopy. Dr. William Heusner provided a vital input in the area of statistics. A special thank you is extended to Dr. Robert Roth for his financial assistance and I am also grateful for his patience during critical analysis of my work. My appreciation is extended to Dr. Ernest Moore, Professor and Chairperson, of the Department of Audiology and Speech Sciences for his support and concern for my well-being and success during my graduate research program. I am highly appreciative of his patience during computer analyzation of my data in his laboratory. I extend my sincere thanks to Dr. Ester Roege, Ms. Inge Taubitz, and Mrs. Barbara Wheaton for their skilled technical assistance. My heartfelt thank you is extended to my wonderful friends for their understanding and support during times of frustrations. I offer many thanks to Ms. Jackie Schartzer for typing my dissertation. iii Finally, I am deeply indebted to my Mother, brothers and sisters. Though geographically distant from me, they have always been with me in spirit and their love and support made it all possible for me, so that I might attain greater fulfilment and happiness in my career. iv TABLE OF CONTENTS Page CHAPTER I GENERAL INTRODUCTION ' Conducting Airways of the Lungs ................ l Microsomal Mixed-Function Oxidases . . ............ 6 Components of the Mixed Function Oxidase (MED) System ..... 7 Pulmonary Drug Metabolism ................... 9 Cellular Localization of Cytochrome P-450 Enzyme System in Airway Epithelium . . ......... . ...... l0 Role of Metabolism and Covalent Binding in Lung Injury. . . . ll Role of Glutathione. . . . . . . . ............. ll The Airway Epithelial Cells as Potential Targets for Chemicals Requiring Metabolic Activation 4'Ip0mean01 o o o o o o o e o o e o o o oooooooooo 14 Carbon Tetrachloride. . . . . . . . ............ 16 Benzo(a)pyrene. . . . . .............. ‘ . . . . l8 Effect of Chemical Irritants on Glyc0protein Cells of the Airways of Lung ..................... 20 PURPOSE ............ 5 ................. 2l REFERENCES. . . .. ........................ 23 CHAPTER II EFFECT OF CARBON TETRACHLORIDE 0N HAMSTER TRACHEAL EPITHELIAL CELLS ABSTRACT ............................. 3l INTRODUCTION. . . ........................ 32 MATERIALS AND METHODS ...................... 33 RESULTS ............................. 35 DISCUSSION ............................ 46 REFERENCES ............................ 52 TABLE OF CONTENTS Continued: Page CHAPTER III EFFECT OF CARBON TETRACHLORIDE 0N GLYCOPROTEIN CONTAINING CELLS OF HAMSTER TRACHEA ABSTRACT ............................. 55 INTRODUCTION. . . . . ...................... 56 MATERIALS AND METHODS ...................... 58 RESULTS . ............................ 60 DISCUSSION ............................ 76 REFERENCES. . . . . ...... . .......... . ..... 80 CHAPTER IV EFFECT OF FASTING 0R DIETHYLMALEATE 0N CARBON TETRACHLORIDE INDUCED INJURY IN HAMSTER TRACHEAL EPITHELIAL CELLS ABSTRACT. . . .......................... 83 INTRODUCTION. . . ........................ 84 MATERIALS AND METHODS ...................... 85 RESULTS ............................. 87 DISCUSSION ............................ llS REFERENCES ............................ 119 CHAPTER V GENERAL CONCLUSION ......................... l22 vi CHAPTER II: Table CHAPTER III: CHAPTER IV: LIST OF TABLES Number of damaged cells in various levels and regions of tracheal epithelia of hamsters treated with CCl4 ..... . . . . . ...... Number of glycoprotein-containing cells (GP) of various types in levels of dorsal regions of trachea of hamsters treated with CCl4 for various times. . ....... . . . ...... Number of glycoprotein-containing cells (GP) of various types in levels of lateral regions of trachea of hamsters treated with CCl4 for various times ........ . . ..... . . . . Number of glycoprotein-containing cells (GP) of various types in levels of ventral regions of trachea of hamster treated with CCl4 for various times. .......... . . . . . . . Effect of diethylmaleate (DEM) or fasting on CCl -induced injury in tracheal epithelial cel s ...................... Effect of diethylmaleate (DEM) or fasting on CCl4-caused alterations in glycoprotein- containing cells (GP) of various levels of dorsal regions of tracheal epithelia ...... Effect of diethylmaleate (DEM) or fasting on CCl4—caused alterations in glycoprotein- containing cells (GP) of various levels of lateral regions of tracheal epithelia ...... Effect of diethylmaleate (DEM) or fasting on CCl4-caused alterations in glycoprotein- containing cells (GP) of various levels of ventral regions of tracheal epithelia ...... vii Page . . 45 . 74 . . 75 . .lll . .llZ . .ll3 . .ll4 CHAPTER I: Figure CHAPTER II: I LIST OF FIGURES Page A simplified scheme showing the major components of xenobiotic-metabolizing enzyme system .................... 8 Schematic representation of relationships between toxification and detoxification pathways for xenobiotic agents ........... l3 Light microgrpah of ventral region of upper trachea of hamster treated with vehicle only (control) . . . . . . . . . . . . . . . . . . . 38 Light micrograph of laterial region of lower trachea of l hr CCl4 treated hamster . ....... 39 Transmission electron micrograph of ventral region of control hamster trachea. . . . ...... 40 Transmission electron micrograph of laterial region of tracheal epithelial cells of hamster treated l hr previously with CCl4 ...... 4l Transmission electron micrograph of laterial regions of tracheal epithelial cells of hamster treated 4 hrs previously with CCl4 ..... 42 Transmission electron micrograph of laterial region of tracheal epithelial cells of hamster treated l2 hrs previously with CCl4 ..... 43 Transmission electron micrograph of ventral region of tracheal epithelial cells of hamster treated 24 hrs previously with CCl4 ..... 44 viii LIST OF FIGURES (Continued): CHAPTER III: Figure l 10 Light micrograph of lateral region of upper trachea of hamster treated l2 hrs previously With CC14. O C I I O O O O I O O O O O O O O O O 0 Light micrograph of ventral region of upper trachea of hamster treated 4 hrs previously with CCI4. . ..... . . . . ......... Light micrograph of dorsal region of upper trachea of hamster treated l2 hrs previously With CCI4. . ...... . . . . . . . . . . . . . . Light micrograph of dorsal region of lower trachea of hamster treated 4 hrs previously With cc14. O O O O C O O O O O O O O O O O O O O 0 Light micrograph of lateral region of lower trachea of hamster treated 24 hrs previously With CCI4. o o o o o O o o o e o o o o ooooo Transmission electron micrograph of lateral region of tracheal epithelial cells of control hamster O O O O O O O O O I O O O O O O O O O O O 0 Transmission electron micrograph of lateral region of tracheal epithelium of hamster treated l hr previously with CCl4. . . . . . . . Transmission electron micrograph of lateral region of tracheal epithelium of hamster treated 4 hrs previously with CCl4 . . . . . . . Transmission electron micrograph of ventral region of tracheal epithelium of hamster treated 12 hrs previously with CCl4. . ..... Transmission electron micrograph of lateral region of tracheal epithelial cells of hamster treated 24 hrs previously with CCl4. . . . . . . . ix Page . 63 . . 64 65 . 66 . . 67 . 68 . . 69 . . 7O . . 71 . 72 LIST OF FIGURES (Continued): CHAPTER IV: Figure l 10 ll 12 13 Page Light micrograph of ventral region of upper trachea of peanut oil treated hamster fed gg‘lflb . . . . ................... 93 Light micrograph of lateral region of upper trachea of peanut oil treated fasted hamster .............. . ........ 94 Light micrograph of ventral region of upper trachea of peanut oil treated hamster pretreated with DEM ........ . ........ 95 Light micrograph of ventral region of upper trachea of CCl4 treated hamster pretreated with DEM ........ . . . . . . . . . . . . . . 96 Light micrograph of dorsal region of upper trachea of CCl4 treated hamster fed ag_lib. ..... 97 Light micrograph of lateral region of lower trachea of CCl4 treated fasted hamster ....... 98 Light micrograph of dorsal region of lower trachea of CCl4 treated hamster pretreated with DEM ....... . .............. 99 Transmission electron micrograph of ventral region of trachea of peanut oil treated hamster fed ag_lib ................. lOO Transmission electron micrograph of lateral region of CCl4 treated hamster fed ad lflb ...... lOl Transmission electron micrograph of lateral region of peanut oil treated hamster ........ l02 Transmission electron micrograph of lateral region of trachea of CCl4 treated fasted hamster ....................... l03 Transmission electron micrograph of lateral region of trachea of peanut oil treated hamster treated with DEM .............. lO4 Transmission electron micrograph of lateral region of trachea of peanut oil treated hamster treated with DEM .............. lOS LIST OF FIGURES (Continued): CHAPTER IV Continued: Figure 14 15 16 17 18 Transmission electron micrograph of lateral region of trachea of CCl4 treated hamster treated with DEM . . . ..... . . . . . Light micrograph of lateral region of upper trachea of peanut oil treated, fasted hamster .............. . . . . Light micrograph of lateral region of upper trachea of a peanut oil treated hamster treated with DEM . . . . . . . . . . . . . Light micrograph of dorsal region of lower trachea of a peanut oil treated fasted hamster. . . . . . . ...... . . . . . Light micrograph of lateral region of upper trachea of CC14 treated hamster treated with DEM . . . ....... . . . . . . . xi Page ..... 106 ..... 107 ..... 108 ..... 109 ..... 110 CHAPTER I GENERAL INTRODUCTION Conducting Airways of the Lungs The conducting airways are a system of branching tubes of regular, cylindrical, or somewhat irregular cross sections that extend from the trachea to the respiratory bronchioles. The conducting airway complexity has only been recognized recently. It comprises an epithelial lining, a mechanical and an immunological defense organ, and an exocrine and endocrine gland. The tracheobronchial epithelial lining is tall-columnar in which at least eight cell types have been identified by electron microscopy. These cell types are: basal, intermediate, brush, goblet, serous, Clara, neurosecretory and ciliated cells. However, based on species variation one or more cell types may be absent in the respiratory airway. Furthermore, the population of each cell type varies among different species. A description of each of these cell types follows: 1) Basal cells. The basal cells rest on the epithelial basement membrane, but they do not reach the airway lumen. These cells are found in the airway epithelium as distally as bronchioles, but they are more numerous in the trachea. They characteristically contain large nuclei that fill most of the cell. The cytoplasm is 2) 3) electron-dense and contains many tonofilaments, numerous ribosomes, small Golgi zones, and a few nfitochondria. Basal cells are generally' regarded as the progenitors of the: other cell types (Blenkinsopp, 1967; Breeze et al., 1976; Breeze and Truck, 1984). Intermediate cells. The intermediate cells seem to represent stages between basal cells and fully differentiated cells. These cells are found in proximal and distal airways. Intermediate cells are roughly Spindle shaped and usually, but not always, reach the airway lumen. The abundant cyt0plasm contains mitochondria and profiles of rough endoplasmic reticulum. They have large oval nuclei. .As the intermediate cell differentiates, it becomes more electron dense as it starts to accumulate mucin granules. Conversely, it becomev more electron lucent with onset. of ciliogenesis (Breeze et al., 1976; Breeze and Hheeldon, 1977). Brush cells. The brush cells are columnar, rest on a basement membrane and reach the airway lumen. These cells are found in the epithelia of trachea, bronchi, and bronchioles. The cytoplasm of a brush cell contains many free ribosomes and a few profiles of rough endoplasmic reticulum. The nucleus is not lobulated and sometimes contains a nucleolus. The presence of a pronounced luminal brush border of microvilli is the distinguishing characteristic of these cells. The function of brush cells is unknown (Breeze and Nheeldon, 1977). 4) 5) Goblet cells. The goblet cells have chalice-like shapes. These cells are abundant in the trachea and bronchi of man, horse, guinea pig, cat and dog, but they are relatively sparse in the rat, mouse, hamster and rabbit. The base of the goblet cell contains an elongated nucleus which has moderately condensed euchromatin. The surrounding cytoplasm is packed with rough endoplasmic reticulum, and a few mitochondria are present. A well developed Golgi apparatus is usually found above the nucleus. Many mucous granules are present in the apical cell cytoplamn. The protein moiety of mucus is synthesized in the rough endoplasmic reticulum and passes to the Golgi apparatus where it combines with carbohydrate and is sulfated before release from the cell. Within the goblet cells, neutral or acidic glycoprotein granules can be identified by the Alcian blue/Periodic acid schiff (AB-PAS) technique at pH 2.6. The PAS positive granules stain red and contain neutral glycoproteins, while AB positive granules stain blue and contain acidic glycoproteins (Jones et al., 1973). Goblet, serous and Clara cells are thought to produce the mucous coating of the tracheobronchial tree (Breeze and Nheeldon, 1977; Reid and Jones, 1980). Goblet cells also contribute lining material in proximal airways (trachea and bronchi). Serous cells. Epithelial serous cells were described in the trachea and extrapulmonary bronchi of rat (Jeffery and Reid, 1975). These cells contain nuclei with irregular outlines and abundant rough endoplasmic reticulum in the perinuclear cytoplasm. The membrane bound, electron dense vesicles in the apical cytoplasm and 6) basal nuclei are characteristic features of these cells. Epithelial serous cell granules contain neutral glycoproteins and resemble those of the serous cells of the submucosal glands. The function of the serous epithelial cells is not known, although it has been suggested that they may contribute to the periciliary fluid layer beneath the mucus. Serous cells produce a secretion of lower viscosity than that of mucous cells (Meyrick and Reid, 1975). Serous cells are believed to secrete neutral glycoproteins, lysozyme, and the epithelial transfer component of IgA (Breeze and Truk, 1984). Clara cells. Nonciliated bronchiolar epithelial cells were first described by Kolliker (1881). Later, Clara (1937) described their structure in more detail; therefore, these cells are now termed Clara cells. They are numerous in bronchioles (Azzopardi and Thurlbeck 1969; Cutz and Conen, 1971; Smith et al., 1979; Plopper et al., 1980 a,b,c), but are found also in bronchi and tracheas of rats, mice, hamsters, and rabbits (Hansell and Moretti, 1969; Pack et al., 1980, 1981; Plopper et al., 1983). Clara cells contain membrane-bound, electron dense secretory granules in the apical cytoplasm. The most characteristic features of these cells is the abundant smooth endoplasmic reticulum (SER) which fills the majority of the cytoplasm in the apical area (Plopper et al., 1980 a,b,c). This abundant SER, which is present in many species is believed to be the site of cytochrome P-450 dependent mixed function oxidase (MFO). 7) 5 There is general agreement that the cytoplasmic granules reflect a secretory function of Clara cells, although the secretory function and its mechanism are not completely understood. The Clara cells granules have been shown to be PAS-positive (Azzopardi and Thurlbeck, 1969; Cutz and Conen, l97l; Pack et al., 1980). An apocrine secretory' mechanisni was originally suggested by Clara (1937). This suggestion was later supported by Etherton et al. (1973) and by Smith et al. (1974); however, the work of other investigators supports a merocrine secretory mechanism (Kuhn et al., 1974; Yoneda, 1977; Yoneda and Birk, 1981). Morphological evidence, using transmission and scanning electron microscopy indicates that the secretory activity of tracheal nonciliated Clara cells in mice may be either apocrine or merocrine (Pack et al., 1980) . Neurosecretory ELLE; Neurosecretory cells are commonly demonstrated in the airways by three methods: 1) electron microscopic identification of the Specific granules. 2) the localization of intracytoplasmic, fluorogenic monoamines using Falck technique and 3) their argyrophilic properties after Grimelus silver nitrate staining. These cells are found at all levels of the tracheobronchial and bronchiolar epithelium. The characteristic features of neurosecretory cells include the existence of small, basal granules with a electron dense core. The cytoplasm contains abundant Golgi apparatus, smooth endoplasmic reticulum and free ribosomes. Neurosecretory cells have a capacity for amine precursor uptake, decarboxylation (APUD) and storage. The cells have a triangular shape and a round or oval nucleus. Pulmonary 8) 6 endocrine cells may be single or occur in groups called neuroepithelial bodies. The functions of neurosecretory cells are not known. Suggested roles include a stretch receptor or a C02 chemoreceptor, with possible involvment in regulation of the pulmonary circulation in the neonatal period or under hypoxic conditions (Breeze and Turk, 1984). Ciliated cells. The luminal surface of ciliated cells is characterized by numerous erect and free standing cilia that are visible with light microscopy. Approximately 250 cilia are found on the luminal surface of each cell. The ciliated cells are present throughout the airway epithelium. These cells are columnar, and they have a relatively electron lucent cytoplasm containing scattered ribosomes, rough endoplasmic reticulum and many apical mitochondria. The cilia beat about 1000 times per minute (Breeze and Truk, 1984). The effective beat in the upper or lower resiratory tract is always toward the pharynx. Functionally, cilia beat in waves over the surface of the epithelium to move material across the epithelium on a layer of fluid. Cilia are particulary important in removing inhaled particulate matter (Breeze and Hheeldon, 1977; Breeze and truk, 1984). Microsolal Mixed-Function Oxidases The cytochrome P-450 dependent, microsomal mixed-function oxidase (MFO) system occurs in smooth endoplasmic reticulum (SER) of the liver and other organs and metabolizes steroids, hormones, vitamins, and fatty acids as well as a multitude of organic compounds. These include drugs, pesticides, food additives and environmental chemicals such as polycyclic hydrocarbons and industrial by-products. Metabolism by MFO invariably results in products that are more polar than the parent compound. In most cases, the metabolites are also less toxic. However, microsomal xenobiotic metabolism does not necessarily result in substrate detoxification. Many cases are known where the metabolite is more toxic than the parent compound. For many years, major emphasis has focused on the xenobiotic metabolizing enzymes of manmalian liver, because the liver of most species contains a large concentration of these enzymes. It is widely recognized, however, that several extrahepatic tissues (e.g. lung, kidney, skin) also possess microsomal enzyme systems. The hepatic microsomal enzyme system is quantitatively' more important for the biotransformation of most orally administered drugs than are extrahepatic enzymes. However, xenobiotic metabolizing enzymes of tissues in direct contact with the environment (e.g. lung or skin) appear to play an important role in determining local fate and toxicity of certain chemicals. Components Of The Mixed Function Oxidase (MED) Syste- The MFO enzyme systems located in SER membranes consist of cytochrome P-450, NADPH cytochrome P-450 reductase and lipids and requires NADPH and molecular oxygen for activity (Fig. 1). a) Cytochrome P-450: The terminal oxidase of the MFO system is a hemoprotein (or group of proteins) called cytochrome P-450. The name cytochrome P-450 is derived from the fact that the cytochrome (or pigment) exhibits an absorbance maximum at 450 nm when reduced xenobiotic (A) Reduced NADP+ 3 flavoprotein _ a oxidized I” H [A F° H A + H20 cytochrome cytochrome N50 N50 _ reductase e [A-Fe’TI-Oz ' [a-Fezfl . ' oxidized N ADPH flavoproteln 02 Figure l: A simplified scheme showing the major components of the xenobiotic-metabolizing enzyme system. A foreign compound (A) binds with the oxidized form of cytochrome P-450. The resulting xenobiotic-cytochrome complex then combines with molecular oxygen. Cytochrome P-450 reductase transfers an electron from NADPH to the heme iron, reducing the iron ++ H 4. from Fe to Fe . The complex then binds molecular oxygen, and a second electron reduces the 02, forming a radical which is now able to oxidize compound A bound to I cytochrOme‘P-460, 9 and complexed with carbon monoxide. b) Cytochrome P-450 reductase: Cytochrome P-450 reductase is a flavin enzyme which carries electrons from NADPH to the heme iron of cytochrome P-450. The 'flavoprotein is sometimes referred to as NADPH-cytochrome C reductase. c) Lipids: Phospholipids are essential for activity of the reconstituted system. It has been suggested that lipids may be required for substrate binding, facilitating electron transfer or providing a I'template“ for the interaction of cytochrome P-450 and NADPH-cytochrome P-450 reductase. Pulmonary Drug Metabolism The lung, like the liver, possesses a system containing cytochrome P-450 that metabolizes exogenous substrates. This system is similar in many respects to that in the liver. As with liver, the xenobiotic metabolizing activity is concentrated in the microsomal fraction of the lung (Hook et al., 1972), and cytochrome P-450 is apparently required for the oxidation of a number of xenobiotic agents (Bend et al., 1972). Oxidative xenobiotic metabolism in the lung is thought to involve the same electron transport system as the liver (Matsubara and Tochino, 1971). In addition, the lung also contains multiple forms of cytochrome P-450 (Liem et al., 1980). Thus, as far as major constituents are concerned, the system closely resembles that of the liver. A number of drugs and carcinogens induce the synthesis of microsomal enzymes in rodent liver. However, several studies have shown induction of these enzymes in the lung (Grover et al.,1974; 10 Grover, 1974; Matsubara et al.,1974; Burk and Prough, 1976). Pretreatment of the rat with 3-methylcholanthrene (3-MC) or phenobarbital (PB) caused induction of hepatic microsomal activities. 3-MC induced the pulmonary microsomal enzyme system; however, PB had no effect on the lung enzymes (Matsubara et al. (1974). Like 3-MC, pretreatment of mouse and rabbit with 2,3,7,8- tetrachlorodibenzo-p- dioxin (TCDD) caused induction of both pulmonary and hepatic MFO activity (Poland et al., 1974; Liem et al., 1980). Agents such as cytochrome c, SKF 525A, and others inhibit microsomal mixed-function oxidase activities in lung as well as in liver jg_vitro (Bend et al., 1972; Litterst et al., 1977). These have provided useful tools with which to study P-450-dependent xenobiotic metabolism. Cellular Localization of Cytochrome P450 Enzyle System in Airway Epitheliu- The cytochrome P-450 dependent monooxygenase enzyme system occurs in smooth endoplasmic reticulum and provides a major pathway for metabolism of xenobiotic agents. The most characteristic feature of Clara cells is the abundant smooth endoplasmic reticulum. There is much evidence to suggest that these cells are a primary site of cytochrome P-450 dependent monooxogenases reactions. Cytochrome P-450 was identified in pulmonary Clara cells of rats, guinea pigs and rabbits by using immunoflorescence techniques (Dees et al., 1980; Serabjit-Singh et al., 1980; Devereux et al., 1981). However, microsomal enzyme systems in other epithelial cells of airway have not been reported. 11 Role of Metabolisn and Covalent Binding in Lung Injury During the past several years it has become evident that the carcinogenicity, Inutagenicity and cytotoxicity produced by' certain, chemically inert, foreign compounds are due to their reactive metabolites (Gillette et al., 1974; Gillette, 1974). The term “reactive metabolite" suggests that the molecule in question is chemically unstable and reacts quickly with suitable, nearby molecules to form a more stable configuration. These alkylation and arylation reactions generally involve electron sharing and covalent bond formation. The reactive metabolites are thought usually to be formed in close proximity to the molecular alkylation sites. Thus, the presence of covalently bound metabolites within an organ suggests that the activation occured directly in the target tissue. The relationship between the reactive metabolite formation and acute tissue injury has been studied in the rodent liver, where positive correlations between the covalent binding of reactive metabolite to hepatic macromolecules and hepatotoxicity have been demonstrated (Ilett et al., 1973). More recently, correlations between metabolite binding to lung and chemically induced lung injury have been reported (Boyd, 1977; Boyd et al., 1978; Devereux et al., 1982). The presence of covalently bound metabolites within cells with high SER content in the lung suggests that the activation occurs within the lung tissue. Role of Glutathione Glutathione (GSH), a sulfhydryl tripeptide, is found in high concentration in the liver and may play a key role in protecting the liver from the effect of toxic metabolites of foreign chemicals (Fig. 12 2). It has been suggested that one of the physiological functions of GSH is to protect cellular components from attack by electrophilic chemicals or their metabolites that may cause tissue damage by reaction with macromolecules (Boyland and Chasseaud, 1969; Jakoby, 1978; Meister and Anderson, 1983). Various drugs and environmental chemicals have been shown to deplete the liver of GSH. This appears to occur by a reaction of GSH at an electrophilic position in the compound or one of its metabolites. This reaction can be either nonenzymatic or catalyzed by a group of GSH-dependent enzymes known as the gluthione S- transferases (Jakoby, l978; Meister and Anderson, 1983). The product of the reaction is either secreted into bile or further metabolized and excreted into the urine as the N-acetylcysteine derivative, more commonly known as mercapturic acid. Consequently, it is believed that one of the physiological functions of GSH is to scavenge electrophilic compounds which can potentially cause tissue damage by reacting covalently with macromolecules. Previous studies have demonstrated that depletion of GSH in lungs of rats by diethylmaleate (Richardson and Murphy, 1975) enhances covalent binding and toxicity of 4-ipomeanol in target tissue (Boyd and Burka, 1978). Thus, it seems likely that GSH protects the lung against certain electrophilic, toxic metabolites. 13 1 parent ”F0 reactive covalent binding stable metabolite toxicity compound ’ electrophilic “" to cellular ‘ ( metabolite . macromolecules MFO Spontaneous GSH nontoxic, conjugated metabolite 1 l excret ion l Fimne 2: Schematic representation of relationships between toxification and detoxification pathways for agents. xenobiotic 14 The Airway Epithelial Cells as Potential Targets for Chemicals Requiring Metabolic Activation 4-1poneanol A derivative from moldy sweet potatoes, 4-ipomeanol, is highly toxic to the lungs of experimental animals (Boyd et al., 1973). Boyd et al. (1978) demonstrated that rat pulmonary microsomes mediated the covalent binding jn_yjtrg of this pulmonary toxin when radiolabeled 4- ipomeanol (14C-4-ipomeanol) was incubated in the presence of NADPH and oxygen with various subcellular fractions of rat lung. No binding occured in the absence of NADPH or oxygen. Boyd et al. (1978) found that 4-ipomeanol is not sufficiently reactive to alkylate tissue components without prior metabolism. The effects of various inhibitors and inducers of MFO on the metabolism, covalent binding and toxicity of 4-ipomeanol were investigated. Pretreatment with agents that reduce MFO activity, such as pyrazole, piperonyl butoxide and cobaltous chloride, markedly reduced covalent binding and pulmonary injury from 4-ipomeanol (Boyd, 1977; Boyd and Burka, 1978; Boyd et al., 1978). Interestingly, SKF 525A prevented the covalent binding of 4-ipomeanol jg_vitro, but had no effect on the covalent binding or pulmonary toxicity of 4-ipomeanol in rats in vivo (Boyd and Burka, 1978). One possible explanation for this observation is that the concentration of SKF 525A jg_yivg_was too low to inhibit the metabolic activation of this toxin. Effects of the MEG inducers, 3-methylcholanthrene (3M0) and phenobarbital (PB), on covalent binding and pulmonary toxicity of 4~ ipomeanol were investigated. Pretreatment of rats with 3MC decreased 15 covalent binding of 4-ipomeanol 1_ y_i_v_q in the lung, but actually increased it in the liver. This was associated with centrilobular necrosis, but pulmonary damage was relatively minimal (Boyd, 1975; Boyd and Burka, 1978). These findings demonstrated a positive correlation between 4-ipomeanol toxicity and the level of' covalent binding to target tissue. Pretreatment of rats with P8 increased covalent binding of 4- ipomeanol to liver microsomes in_vitro, but no change occured in binding to lung microsomes j_ vitro. Boyd et al. (1978) concluded that 4-ipomeanol can be metabolized by forms of cytochrome P-450 induced by 3MC or PB. Interestingly, covalent binding of 4-ipomeanol was reduced in vivo in lung and liver of rats pretreated with PB. This was paralleled by decreased toxicity of 4-ipomeanol in the lung and liver of rats. Thus, it appears that PB induced detoxifying pathways lg 1112 rather than ones that result in activation (Boyd, 1976). Previous studies demonstrated that the reactive metabolite formed by 4—ipomeanol metabolism is highly electrophilic (Boyd and Burka, 1978; Boyd et al., 1978; 1979; Buckpitt and Boyd, 1980). Addition of glutathione (GSH) markedly reduced covalent binding of 4-ipomeanol i vitro. GSH did not prevent reactive metabolite formation, but it did inhibit its covalent binding by acting as alternate nucleophile (Boyd et al., 1978; Buckpitt and Boyd, 1980). Pretreatment of rats with diethylmaleate (DEM), which depleted pulmonary GSH (Richardson and Morphy, 1975), markedly enhanced 4-ipomeanol covalent binding jn_yivg (Boyd and Burka, 1978). Thus, it appears that GSH may play an important role in protecting the lung from the toxic, reactive 4-ipomeanol metabolite(s). The pulmonary cellular specifity for the metabolic activation of 4-ipomeanol 1_,vivo has been studied by autoradiography (Boyd, 1977). 16 4-ipomeanol was covalently bound preferentially to the intrapulmonary Clara cells of rat, mouse, and hamster. 4-ipomeanol also caused necrosis in these cells. To determine whether metabolism of 4- ipomeanol was required to develop radioactive binding to Clara cells, animals were pretreated with piperonyl butoxide prior to administering 4-ipomeanol. Pretreatment of animals with this MFO inhibitor markedly decreased covalent binding of 4-ipomeanol to Clara cells. Histological examination of the lungs from these animals revealed an absence of necrosis. The metabolic activation of 4-ipomeanol was also studied in several cell types isolated from rabbit lung (Devereux et al. 1981). The highest rates of cytochrome P-450 mediated meabolism of 4-ipomeanol was observed in isolated Clara cells. A considerable amount of activity, but much less than that in Clara cells, was also seen in isolated alveolar type II cells. In summary, biochemical and morphological studies have demonstrated that 4-ipomeanol is metabolized in _s_i_tg by pulmonary microsomal enzymes to a highly reactive metabolite which covalently binds to cellular macromolecules. 4-ipomeanol produced injury preferentially to pulmonary Clara cells. It has been suggested that Clara cells are susceptible to 4-ipomeanol-induced injury due to their capacity to activate this chemical metabolically. Carbon Tetrachloride Carbon tetrachloride (CCl4) induced injury has been extensively studied in the liver (Recknagel, 1967: Recknagel and Glende, 1973). However, a few studies have focused on the pulmonary toxicity of this chemical (Valdivia and Sonnad, 1966; Chen et al., 1977; Willis and 17 Recknagel, 1979; Boyd et al., 1980; Ahmadizadeh and Echt, 1985; Ahmadizadeh et al., 1987). It is generally agreed that CCl4 is metabolized by the cytochrome P-450 locus in smooth endoplasmic reticulum of liver to the trichloromethyl free radical (CClé), which is capable of producing injury either by stimulating lipid peroxidation or by binding covalently to tissue macromolecules (Reynolds, 1967; Recknagel and Glende, 1973; Comporti, 1985). Chen et al. (1977) found pulmonary cytochrome P-450 to be markedly decreased in rat lung 4 hrs after oral administration of 2.5 ml/kg CCl4. These authors also found that the concentration of cytochrome P- 450 was decreased in rat lung microsomes 1 vitro 30 min after the addition of CCl4 in the presence of NADPH. Similarly, Boyd et al. (1980) reported that the oral administration of 2.5 nfl/kg of CCl4 to rats caused a marked reduction in rat pulmonary cytochrome P-450. Boyd et al. (1980) also noted that the metabolism of 4-ipomeanol i vitro was impaired in lung preprations from mice or rats treated jfl_yiyg_with 2.5 ml/kg CCl4. These results led to the conclusion that pulmonary cytochrome P-450 dependent monooxygenase activities participate in the bioactivation of CCl4 in lung. However, the mechanism by which CCl4 causes lung injury is a subject of controversyu For example, Willis and Recknagel (1977) reported that CCl4 stimulated the production of thiobarbituric acid (TBA) reactive material in rat lung microsome preparations, suggesting lipid peroxidation. In contrast, Villarruel et al. (1977) found no alteration in the ultraviolet absorbance of lipid from lungs of CCl4- treated rats, but they found significant amounts of 14C irreversibility bound to lung lipids 3 hours after oral administration of 1 ml/kg of 18 14CCl4 to rats. These authors suggested that the irreversible binding to cellular components rather than lipid peroxidation is the major mechanism in CCl4-induced lung injury. It appears that CCl4 is metabolized by pulmonary a cytochrome P-450 to electrophilic, toxic metabolite(s) capable of causing injury by stimulating lipid peroxidation and/or binding covalently to tissue macromolecules. Studies have shown that CCl4 produced injury to type II pneumocytes of guinea pigs and rats (Valdivia and Sonnad, 1966; Gould and Smuckler, 1971; Chen et al., 1977). Boyd et al. (1980) reported that oral administration of 2.5 ml/kg of CCl4 to mice or rats caused injury in pulmonary Clara cells. Boyd et al. (1980) found that CCl4 induced injury that was strikingly selective to Clara cells, whereas adjacent ciliated airway epithelial cells appeared to be normal. These authors suggested that pulmonary Clara cells are susceptible to CCl4rinduced injury due to their capacity to activate this chemical metabolically. However, the finding of CCl4-induced injury in type II pneumocytes raises the possibility that these cells also contain a microsomal, P- 450 MFO system. This is further supported by the studies of Devereux and Fouts (1981) and of Jones et al. (1983) suggesting the presence of cytochrome P-450 system in alveolar type II cells of rabbit and rat. Benzo(a)pyrene Polycyclic aromatic hydrocarbons are ubiquitous pollutants of air, soil and water. One of the most abundant of this class of compounds is benzo(a)pyrene (BP). This chemical is known to be metabolized to epoxides, phenols, and quinones by cytochrome P-450 dependent monooxygenases and by epoxide hydrases. The resultant metabolites are then conjugated to form more hydrophilic metabolites by various 19 conjugating enzyme systems. Most of the metabolites are excreted. However, the MFO enzyme system will also convert BP to more reactive forms that can bind covalently to cellular macromolecules (Gelboin, 1980). Many studies have indicated that the carcinogenic, mutagenic and toxic effects of BP depend on its enzymatic conversion to active ferms. Arylhydrocarbon hydroxylase (AHH), also called benzpyrene hydroxylase, is an example of a mixed function of oxygenase enzyme system that metabolizes BP. AHH has a central role in detoxification as well as inactivation of polycyclic hydrocarbons. AHH activity has been demonstrated in many tissues and many species (Hiebel et al., 1971, 1973; Nebert and Gelboin, 1969; Nebert and Gielen, 1972; Ahmadizadeh et al., 1984). The activity has been found in both Clara cells and type II cells of rabbit lung (Devereux et al., 1981). Stowers and Anderson (1984) showed that treatment of mice or rabbits with BP resulted in the binding of BP metabolites to DNA in lungs of mice and rabbits. In another study, binding of BP metabolites was determined in Specific cell populations from lungs of rabbits treated with BP (Horton et al., 1985). Binding of radioactivity was found in Clara cells and type 11 cells. Kaufman et al. (1973) reported covalent binding of BP-3H in tracheal epithelial cells of hamster after tracheas were isolated from untreated hamsters and incubated i vitro as Short-term organ cultures containing radiolabeled BP. These authors found that incubation of the trachea in a medium containing 7,8- benzoflavone, an inhibitor of AHH activity (Niebel, 1971), prevented the covalent binding of BP-3H. They suggested that the hamster tracheal epithelium can metabolize BP and that the metabolism is 20 necessary for the binding of BP to tissue. Reznik-Schuller and Mohr (1974 a,b; 1975) demonstrated injury in airway epithelial cells of hamsters treated with BP _i_r_1_ vivo. It appears that the lesions were associated with the binding of BP metabolites to bronchiolar and tracheal epithelial cells. Effect of Chemical Irritants on Glycoprotein Cells of the Airways of Lung AS noted above, several different epithelial cell types contribute to glycoprotein secretion in the respiratory tract. These cells are Clara, goblet and serous cells (Azzopardi and Thurlbeck, 1969; Lamb and Reid, 1969; Jeffery and Reid, 1975, 1977; Becci et al., 1978a; Spicer et al., 1980; Al-Ugaily et al., 1980; Pack et al., 1980, 1981). Many studies have demonstrated that acute and chronic inhalation of certain chemical irritants increased the secretion of glycoproteins in the airwayu Exposure of animals to chemical irritants such as sulfur dioxide, ammonia vapor and tobacco smoke caused structural and functional changes in the secretory elements of the airways. Lamb and Reid (1968) reported that exposure of rats to sulfur dioxide ($02) increased the number of glycoprotein-containing cells in the trachea. Seltzer et a1. (1984) found hypersecretion of mucus in dogs after exposure to $02. Freeman and Haydon (1964) reported that exposure of rats to nitrogen dioxide (N02) increased the population of glyc0protein-containing cells in the respiratory airway. Exposure of rats to tobacco smoke increased mitotic activity and increased the number of goblet cells in tracheas of rats (Jones et al., 1973; Jones and Reid, 1978). 21 Becci et al. (19786) observed hypertrophy of glyc0protein cells in hamster tracheobronchial epithelium 24 hours after intratracheal instillation of mixture of benzo(a)pyrene and ferric oxide (BP-Fe203). These authors found that glycoprotein became more acidic 1T1 BP-Fe203 treated animals when compared to that of unexposed animals. These effects were attributed to the BP and not to the Fe203 particles. Since BP requires metabolic activation by the microsomal enzyme system, it seemed likely that glchprotein cells of trachea can be altered by chemicals that are bioactivated by the mixed function oxidases. After exposure to irritants, the origin of new goblet cells in trachea and bronchi of rats was investigated. Under conditions of exposure to irritants such as tobacco smoke and sulphur dioxide, serous cells transformed to goblet cells (Jeffery et al., 1977). In the distal airways, the Clara cells transformed to goblet cells (Meyrick, 1977). In summary, exposure of animals to chemical irritants caused alterations in glyc0protein cells of airways. Modification of glycoprotein-containing cells of airways after exposure to a chemical that is bioactivated by microsomal enzyme system was also reported. Thus, it appears that glyc0protein-containing cells of airways can be altered by chemicals that are bioactivated by MFOs. PURPOSE Increasing evidence suggests that epithelial cells of airways have potential for metabolizing xenobiotic compounds. Carbon tetrachloride induced cell injury is dependend on metabolism of this agent by intracellular NADPH-dependent cytochrome P-450 monooxygenases. CC14 22 produced injury in intrapulmonary airway epithelial cells. 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Early pathological alterations of the bronchial epithelium in Syrian golden hamsters after intratracheal instillation of benzo(a)pyrene. I. Morphological studies from semithin sections. ZbI. Bakt. Hyg., I. Abt. Orig. B 159: 493-502. Reznik-Schuller, H. and Mohr, U. (1975). Investigations on the carcinogenic burden by air pollution in man. XII. Early pathological alterations of the bronchial epithelium in Syrian golden hamsters after intratracheal instillation of benzo(a)pyrene. 2. Further ultrastructural studies. Zbl. Bakt. Hyg.,I. Abt. 0rig.B 160: 108-129. Richardson, R.J. and Murphy, 5.0. (1975). Effect of glutathione depletion on tissue deposition of methylmercury in rats. Toxicol. Appl. Pharmacol. 31: 505 -519. Seltzer, J., Scanlon, P.D., Drazen, J.M., Ingram, R.H. Jr. and Reid, L. (1984). Morphological correlation of physiologic changes caused by SO -induced bronchitis in dogs. The role of inflammation. Am. Rev. Respir. Dis. 129: 790-797. Serabjit-Singh, C.J., Nolf, C.R., Philpot, R.M., and plopper, C.G. (1980). Cytochrome P-450: localization in rabbit lung. Science 207: 1469-1470. Smith, P., Health, 0. and Moosavi, H. (1974). The Clara cell. Thorax 29: 147-163. Smith, M.N., Greenberg, 5.0. and Spjut, H.J. (1979). The Clara cell: A comparative ultrastructural study in mammals. Am. J. Anat. 155: 15- 30. Spicer, S.S., Mochizuki, 1., setser, M.E., and Martinez, J.R. (1980). Complex carbohydrates of rat tracheobronchial surface) epithelium visualized ultrastructurally. Am. J. Anat. 158: 93-109. Stowers, S.J., Anderson, M.R. (1984). Ubiquitous binding of benzo(a)pyrene metabolites to DNA and protein in tissues of mouse and rabbit. Chem. Biol. Interact. 51: 151-166. Valdivia, E. and Sonnad, J. (1966). Fatty changes of the granular pneumocyte in CC14 intoxication. Arch. Path. 81: 514-519. 30 Villarruel, M.D.C., DeToranzo, E.G.D., and Castro, J.A. (1977). Carbon tetrachloride activation, lipid peroxidattion and the mixed function oxygenase activity of various rat tissues. Toxicol. Appl. Pharmacol. 41: 337-344. Hiebl, F. J., Leutz, J. C., Diamond, L., and Gelboin, H. V. (1971). Aryl hydrocarbon (benzo(a)pyrene) hydroxylase in microsomes from rat tissues: Differential inhibition and stimulation by benzoflavones and organic solvents. Archs. Biochem. Biophys. 144: 78-86. Hiebl, F.J., Leutz, J.C., and Gelboin, H.V. (1973). Aryl hydrocarbon (benzo(a)pyrene) hydroxylase: inducible in extrahepatic tissues of mouse strains not inducible in liver. Arch. Biochem. Bi0phys. 154: 292-294. Hillis, R.J. and Recknagel, R.0. (1979). Potentiation of carbon tetrachloride of NADPH-dependent lipid peroxidation in lung microsomes. Toxicol. Appl. Pharmacol. 47: 89-94. Yoneda, K. (1977). Pilocarpine stimulation of the bronchiolar Clara cell secretion. Lab. invest. 37: 447-452. Yoneda, K. and Birk, M.G. (1981). The mode of secretion of the Clara cell in rat bronchiole: a freeze- fracture study. Exp. lung Res. 2:177-185. CHAPTER II THE EFFECT OF CARBON TETRACHLORIDE ON HAMSTER TRACHEAL EPITHELIAL CELLS ABSTRACT This study was designed to assess cytotoxicity of carbon tetrachloride (CC14) 'hi hamster tracheal epithelium. Adultg male, Syrian golden hamsters were treated with 2.5 ml/kg C014 and controls received the vehicle (peanut oil) only. Animals were sacrificed after 1, 4, 12, and 24 hours. Tissue samples from upper and lower trachea were fixed and embedded in glycol methacrylate for light microscopy. One or two tracheal rings were fixed in formaldehyde/91utaraldehyde cacodylate buffer for transmission electron microscopy. For histopathologic evaluation of the tracheal epithelial cells, each tracheal level was cut transversely at 3 um and stained with toluidine blue. CC14 produced ciliated and nonciliated cell injury in the various levels and regions of hamster trachea. The severity of CC14- induced injury differed in various levels and regions of tracheal epithelium. For example, in trachea of hamsters sacrificed 1 hour after CC14 treatment, the number of damaged cells was considerably higher in the dorsal region of lower trachea when compared to those of ventral region of upper trachea. The number of damaged cells markedly increased after 4 and 12 hours in the ventral region of lower trachea. By 24 hours, the number of injured cells had decreased so that no 31 32 significant difference from control was evident. The ultrastructural alterations of epithelial cells were obvious as early as 1 hour post CC14 treatment. Intracellular organelles, including smooth and rough endoplasmic reticulum, mitochondria and Golgi apparatuses, were damaged by this chemical. Since CC14 induced cell injury is dependent on metabolism of this agent by intracellular NADPH-dependent cytochrome P- 450 monooxygenases, this study suggests that hamster tracheal epithelial cells have the potential to bioactivate C014. INTRODUCTION The mixed function oxidases (MFOS) occur in smooth endoplasmic reticulum (SER) and provide a major pathway for metabolism of xenobiotic agents. Generally, the MFO system produces metabolites that are less toxic than the parent compounds. However, in many cases, MFO metabolism leads to the formation of metabolites more toxic than their parent chemicals. For example, carbon tetrachloride (CC14) is metabolized by MFO components to a free radical(s) that is highly cytotoxic. Cellular localization of cytochrome P-450-dependent monooxygenases in the respiratory system has generated considerable interest due to the fact that many environmental chemicals gain access to the body through the respiratory system. In this respect, detoxification or toxification of a chemical by the pulmonary MFO system may in part determine its site and mechanism of action. Clara cells have been suggested as a primary Site of MFO enzyme activity in the lung (Boyd et al., 1977; Serabjit-Singh et al., 1980). 33 The presence of Clara-like cells have been identified in 'tracheal epithelium of several species, including hamster, mouse, and rabbit (Hansell and Moretti, 1969; Pack et al., 1980; Al-Ugaily et al., 1980; Plopper et al., 1983). 0014 produced injury in intrapulmonary Clara cells of mice and rats (Boyd et al., 1980). It has been suggested that these cells are susceptible to 0014 injury because of their capacity to metabolize this chemical to a toxic intramediate(s). Since tracheal Clara cells contain abundant amounts of smooth endoplasmic reticulum Similar to intrapulmonary Clara cells, it seemed likely that a xenobiotic agent such as 0014 might produce injury in tracheal epithelial cells. To test this hypothesis, I examined the effect of 0014 on various regions and levels of tracheal airway epithelium. MATERIALS AND METHODS Adult, male, Syrian golden hamsters (100-140g) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) and housed in groups of three in clear polypropylene cages in a light cycle (12 hr light and 12 hr dark) and temperature controlled room. Animals were allowed food (Hayne Lab Blox, Chicago, IL) and tap water ad libitum. Carbon tetrachloride (0014) was obtained from Sigma Chemical Company (St. Louis, MO), dissolved in peanut oil and administered i.p. at dose of 2.5 ml/kg body weight. Control animals received vehicle only. 1, 4, 12, or 24 hrs after treatment, animals were sacrificed with 100-300 mg/kg of sodium pentobarbital (i.p.). Three hamsters were used for each group. 34 Light Microscopy: Tracheal tissues were fixed in 4% paraformaldehyde in phosphate buffer (pH 7.2). Each Specimen was divided into upper and lower levels and embedded in glycol methacrylate (GMA, Polyscience, Inc.). Tracheas were transversely sectioned at 3 um from proximal to distal ends with glass knives on a JB-4 microtome. Three histological sections, each at least 15 um apart, were taken from each tissue block and stained with 1% toluidine blue. The luminal epithelium was subdivided into dorsal, right and left lateral, and ventral regions.' The total number of injured cells/mm/region was counted at 400x magnification and compared statistically. Only those cells with distinct nuclei were counted. The criteria for cell injury included: nuclear dilation, loss of staining capacity and obvious cellular swelling. Data were expressed as mean 1 standard error. The results were analyzed by analysis of variance, completely randomized design, and treatment differences were identified by the method of Newman-Keuls (Steel and Torrie, 1960). P <0.05 was used as the criterion for significance. Since the data were nonsymmetrical and the sample size at each time period was small (n=3 animals) a standard 10910 trans- formation was used to compensate for the nonsymmetry, while preserving the relative pattern among groups. The transformed data in each morphological level and region of trachea were then subjected to a one- way analysis of variance across time. 35 Transmission Electron Microscopy: One or two rings of trachea from each animal were cut into pieces approximately 1 mmz, and 1-2 blocks from each region (dorsal, lateral or ventral) were prepared for electron microscopy. Tissues were fixed in formaldehyde-91utaraldehyde cacodylate buffer (Karnovsky, 1965). The tissues were washed in 0.2 M cacodylate buffer, post-fixed in 2% osmium tetroxide, dehydrated with ethanol and embedded in epon-araldite (Mollenhauer, 1964). Sections 1 um thick were cut from selected areas and stained with toluidine blue for examination by light microscopy. Thin sections (approximately 90 nm) were cut from selected areas. The sections were stained with uranyl acetate and lead citrate and examined with a Philips 201 transmission electron microscope. RESULTS Administration of peanut oil vehicle alone did not produce detectable injury to hamster tracheal epithelial cells (Fig. 1). However, cell injury was observed in the various morphological levels and regions of the trachea following 0014 treatment. Light microscopy revealed that both ciliated and nonciliated tracheal epithelial cells were swollen, had loss of staining capacity, and nuclei appeared to be dilated. 0014 produced injury in tracheal epithelial cells as early as 1 hour following 0014 treatment (Fig. 2). However, the degree of injury varied in different levels and regions of tracheal epithelium. 36 Quantitative Analysis by Light Microscopy: l) Dorsal region a) Upper trachea: In this region, 0014 did not produce a significant effect by '1 hour' post-treatment. However, the number of damaged epithelial cells was markedly increased at 4 and 12 hours. By 24 hours, the number of injured cells had decreased so that no significant difference from control was evident. Lower trachea: 0014 produced a Significant effect by 1 hour post treatment. However, the number of damaged cells was considerably decreased at 4, 12, and 24 hours so that no significant differences from control were evident. 2) Lateral region a) D) Upper trachea: In this region, tracheal epithelium remained essentially normal in appearance at 1 hour after 0014 treatment. However, the number of damaged epithelial cells was increased at 4, 12, and 24 hours when compared to those of control animals. Lower trachea: Numbers of damaged epithelial cells were increased at 1 and 12 hours 0014 post-treatment when compared with control values. However, number of damaged cells decreased at 4 and 24 hours so that no Significant differences from control were evident. 37 3) Ventral region a) Upper trachea: In this region, 0014 did not produce a significant effect at l, 12, and 24 hours. However, the number of damaged epithelial cells was elevated 4 hours post 0014 treatment. b) Lower trachea: In lower trachea, the population of damaged epithelial cells was larger at l, 4, and 12, but not 24 hours after 0014 when compared with control values. Transmission Electron Microscopy: In control hamster tracheal epithelium, tall columnar, nonciliated cells were observed that contained abundant smooth endoplasmic reticulum, numerous mitochondria, well-developed rough endoplasmic reticulum (RER), and prominent Golgi complexes. In addition, the apical cytoplasm contained membrane bound, electron lucent, secretory granules (Fig. 3). Cfiliated cells were columnar, and numerous mitochondria, RER, and Golgi complexes were seen in electron lucent cytoplasm (Fig. 3). Ultrastructural alterations in nonciliated and ciliated cells were noted as early as 1 hour following 0014 treatment (Fig. 4). Damage in intracellular organelles of nonciliated cells was characterized by dilation of SER, ribosomal disaggregation, swelling of mitochondria and numerous cytoplasmic vacuoles. The ultrastructural features of ciliated cells injured by 0014 were dilation of RER, swelling of mitochondria, disaggregation of ribosomes and numerous cytoplasmic vacuoles (Figs. 4-7). However, the degree of intracellular injury varied in different regions of hamster trachea in a manner similiar to the light microscopic observations. 38 Figure 1: Light micrograph of ventral region of upper trachea of hamster treated with vehicle only (control). The ciliated cells (open arrow) and nonciliated cells (solid arrows) are intact. Note pseudostratified appearance of tracheal epithelium. X 400. 39 Figure 2: Light micrograph of laterial region of lower trachea 1 hr 0014 treated hamster, showing injury (loss of staining capacility and dilatation of cytoplasm and nucleus) to ciliated cells (open arrow) and nonciliated cells (solid arrow). X 400. 40 o . o '0 o o O 4’ I ' ¢»’ 9 a 94 ('0' “ a 9 o 1” 0.. . "I a geoeg. “ méég'fleii 0 , 3, 0 “ ' " p1. Figure 3: Transmission electron micrograph of ventral region of control hamster trachea, showing ciliated (CC) and nonciliated (N0) cells. Note electron lucent, membrane bound secretory granules (g). Mitochondria (M), rough endoplasmic reticulum (er), smooth endoplasmic reticulum (ser), Golgi complex (G) and nucleus (N). X 6272. Figure 4: 41 Transmission electron micrograph of laterial region of tracheal epithelial cells of hamster treated 1 hr previously with 0014. Vacuoles (V) are evident in nonciliated (N0) cells. Note ciliated cells (00) with cytoplasmic bleb (arrow). X 15000. 42 Figure 5: Transmission electron micrograph of laterial region of tracheal epithelial cells of hamster treated 4 hrs previously with 0014. Severe damage (dilatation of intracellular organelles including mitochondria, rough endoplasmic reticulum and numerous vacuoles) is Shown in both ciliated (CC) and nonciliated (NC) cells. Nucleus (N). vacuole (V). X 5732. Figure 6: 43 H., W o. cfi‘.‘f. ‘ , if ’- ‘ \I . .1) Transmission electron micrograph of laterial region of tracheal epithelial cells of hamster treated 12 hrs previously with 0014. Note injury (dilatation of intracullular organelles including mitochondria, rough endoplasmic reticulum and numerous vacuoles) in both ciliated (CC) and nonciliated (NC) cells. Nucleus (N). X 5732. Figure 7: 44 Transmission electron micrograph of ventral region of tracheal epithelial cells of hamster treated 24 hrs previously with 0014. Vacuoles (arrows) are evident in ciliated (CC) and nonciliated (NC) cells; however cells look less injured than at earlier times. Nucleus (N). X 8624. 45 Table 1: Numbers of damaged cells in various levels and regions of tracheal epithelia of hamsters treated with 0014.* CCT4'Treatment Control i‘T hr 456? 12Thr 24 hr DEFSaT d UT 0:0 2.27:1.31Cd 18. 50:4. 729” 13. 32:1 .783” 2.22:0.63g LT 0:0 18.12:8.30a 1.31:0.37 4.25:0.92 0.74:0.13 Lateral UT 0:0 1.30:0 . 54" 14. 96:5 . 24ab 12. 81:0 . 90ab 3. 24:0. 553de LT 0:0 11.69:4.38a 3.18:1 .09d 18.49:5.31ac 2.98:0.40 Ventral d b b 1n 0:0 0.94:0.48c 7.74:1 .98a 5.96:3.98 2.00:0.54b 8 LT 0:0 9.68:4.27a 8.92:1.62a 17.77:2.74a 1.56:0.66 C Adult, male, SyFian golden hamsters were treated’with 0014 (2.5 ml/kg). Control animals were given vehicle only. Animals were killed 1, 4, 12, or 24 hrs later. Tracheal tissues were removed and subdivided into upper (UT) and lower (LT) levels, and prepared for histological determination of number of damaged cells as described in Methods. Three hamsters were used for each group. *Number of damaged cells per 1 mm length of tracheal epithelium. Control values are given for comparison. a=Significantly different from that of the control group (p<0.05). b=Significantly different from that of the group killed 1 hr after 0014 (p<0.05). c=Significantly different from that of the group killed 4 hrs after 0014 (p<0.05). d=Significantly different from that of the group killed 12 hrs after 0014 (p<0.05). DISCUSSIINI The use of 2-3 um glycol methacrylate histologic sections allows for an accurate identification of cell types in tracheal epithelium and, therefore, permits reliable quantitative analyses of cellular structural reSponses after exposure to a toxicant such as 0014. This study demonstrated that intraperitionial administration of 0014 produces differential injury. I observed that the extent of 0014- induced injury varied in different levels and regions of hamster trachea. Since the cytotoxicity produced by 0014 requires metabolic activation, this finding suggests that MFO metabolic activity of hamster airway tracheal epithelial cells varied 'hi different morphological regions. Gabridge et al. (1977) used a nitroblue tetrazolium assay for overall metabolic activity of airway tracheal epithelial cells in hamster and reported that the metabolic activity of tracheal epithelial cells was different in various rings of trachea. These regional differences point out the importance of specifying the location of a cellular response to toxic chemicals both in lilQ and la 11559. It is widely accepted that 0014 is metabolized to a toxic Species by a cytochrome P-450 dependent monooxygenase enzyme system. Boyd et al. (1980) reported that 0014 produced injury in intrapulmonary Clara cells of mice and rats. This suggested that these cells were susceptible to injury by 0014 due to their capacity to activate the chemical metabolically. 46 47 I observed an abundance of smooth endoplasmic reticulum (SER) in nonciliated hamster tracheal epithelial cells. This raised the possibility that these cells may have the potential for bioactivation of 0014. Recently, Plopper et a1. (1983) reported that hamster tracheal Clara cells were structurally similiar to intrapulmonary Clara cells and contained large amounts of SER, with electron dense granules in the apical cytoplasm. In the present study nonciliated cells containing electron lucent granules in the apical portions of cytoplasm were observed. These findings confirmed those of others (Reznik- Schuller and Mohr, 1974; Becci et al., 1978; Kennedy et al., 1978), who have reported the presence of electron lucent granules. Many studies have demonstrated that chemical carcinogens produced injury in hamster tracheal epithelial cells. These chemicals are known to be metabolized by a microsomal enzyme system, resulting in the generation of alkylating intermediates that are reSponsible‘ for the mutagenic, toxic and carcinogenic effects of the parent compound (Health, 1962; Malling, l97l; Magour and Nievel, 1971; Kaufman, et al., 1973; Czygan et al., 1973; Bartsch et al., 1975; Reznik-Schuller and Tomaszewski, 1980; Reznik-Schuller and Hague, 1981). For example, studies conducted by Reznik-Schuller and Mohr (1974) demonstrated that benzo(a)pyrene is carcinogenic for the tracheal epithelium of Syrian golden hamster. In this reSpect, the observation that 0014-induced cell injury suggests the presence of a NADPH-dependent monooxygenase enzyme system in epithelial cells of hamster trachea. The finding that the extent of 0014-induced cell injury was different among various levels and regions of hamster tracheal epithelium raised the possibility that some tracheal epithelial cells 48 may be more capable than others of bioactivating 0014. Alternatively, cells of other regions and levels of trachea also might be capable of activating 0014, but the cells which appeared to be less sensitive may more effectively detoxify the reactive 0014 metabolite and thereby prevent toxicity. The mechanism(s) by which 0014 causes cell injury is not completely resolved. However, a large body of evidence suggests that 0014 iS bioactivated by a NADPH-dependent cytochrome P-450 locus in SER of hepatocytes to' the trichloromethyl free radial (0013), which then interacts with intracellular organelles leading to an alteration of cellular integrity. 0014 may cause injury to essential macromolecules by stimulating lipid peroxidation and/0r binding covalently to them (Recknagel, 1967; Recknagel and Glende, 1973; Comporti, 1985). Biochemical mechanisms exist which protect cells from reactive metabolites. For example, substances such as glutathione (GSH) can react with free radicals and prevent cell necrosis (Orrenius and Jones, 1978; Meister and Anderson, 1983). Aldehyde dehydrogenases (ALDH) are capable of detoxifying a variety of aldehydes (Heiner, 1980); several investigators suggested that products of toxic aldehydes generated during 0014-induced lipid peroxidation inhibit ALDH activity in livers. Inhibition of cytosolic and mitochondrial ALDH may be important in the hepatotoxic effects of 0014 (Hjelle et al., 1981; Hjelle and Petersen, 1981; Hjelle et al., 1983). ALDH is present in hamster tracheal epithelium (Ahmadizadeh and Echt, unpublished results). 'Thus, the extent of 0014-induced epithelial cell injury may also be related to the intracellular concentration of GSH and/or ALDH activity. In addition to 0014-induced damage to nonciliated cells, I observed injury to ciliated tracheal epithelial cells in various 49 regions and levels of hamster trachea as early as 1 hour after 0014 treatment” ‘This finding raises several possibilities. First, the ciliated cells may possess microsomal enzyme systems and may be capable of bioactivating this chemical. Gabridge et al. (1977), using the nitroblue tetrazolium assay, suggested that hamster~ ciliated airway epithelial cells have a high metabolic activity. Harris et al. (1973) reported radioactive binding of benzo(a)pyrene (BP) in ciliated cells of hamster trachea after tracheas were isolated from untreated hamsters and incubated j_n_ v_it_r_g as short-term organ cultures containing radiolabeled BP. These authors found that binding was markedly reduced by the addition of 7,8-benzoflavone, an inhibitor of arylhydrocarbon hydroxylase (AHH) activity. Thus ciliated cells appear to have the capacity to metabolize foreign chemicals in the hamster. Interestingly, Castro et al. (1983) reported that highly purified rat liver mitochondria were capable of metabolizing 0014. These authors suggested that cytochrome P-450 of mitochondria participates in bioactivation of reactive metabolites. This finding raised the possibility that not only SER, but intracellular organelles, such as mitochondria, may be associated with an MFO enzyme system. Ciliated cells of trachea of hamsters contain numerous mitochondria. However, whether this organelle can participate in activation of 0014 to a toxic, reactive metabolite remains to be determined. A second possible explanation for 0014-induced injury in ciliated tracheal epithelial cells is that bioactivation of 0014 may occur in nonciliated_ tracheal epithelial cells, and toxic metabolites translocate to ciliated cells via cell-cell communication. Alterations of the intercellular junction have been associated with pathological 50 changes in various tissues. James et al. (1986) observed that 0014 increased gap junction size in rat hepatocytes. The presence of gap junctions in guinea pig tracheal airway epithelial cells was reported by Inoue and H099 (1974). In this way, translocation of toxic metabolite(s) from nonciliated to ciliated cells might be responsible in part for ciliated cell injury. It is generally accepted that toxic aldehydes generated during 0014-induced lipid peroxidation have relatively long life spans and, therefore, can have an effect on subcellular targets distant from the production of a toxic metabolite (Comporti, 1985). A third possible mechanism for 0014-induced ciliated cell injury is that the generation of a toxic metabolite may occur in the liver, and subsequent translocation of the toxic intermediate(s) to the lung may account at least in part for tracheal epithelial cell injury. However, further studies will be needed to clarify these hypotheses. Nith light microscopy, the number of injured cells decreased in all regions and levels of hamster trachea 24 hours after 0014 treatment. This finding suggests that 0014-induced tracheal epithelial cell injury is reversible. This observation is consistent with the results of Boyd et al. (1980), who reported that 0014 produced reversible injury in intrapulmonary Clara cells in mice and rats. 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McGraw-Hill, New York. Heiner, H. (1980). Aldehyde oxidizing enzymes. In enzymatic basis of detoxication (N.B. Jakoby, ed.), pp. 261-280. Academic Press, New York. CHAPTER III EFFECT OF CARBON TETRACHLORIDE ON GLYCOPROTEIN- CONTAINING CELLS IN HAMSTER TRACHEAL EPITHELIUH ABSTRACT This study was designed to examine the distribution of glyc0protein containing cells in hamster trachea and their histochemical response to carbon tetrachloride (0014). Adult, male, Syrian golden hamsters were treated with 2.5 ml/kg 0014 and controls received a vehicle (peanut oil) only. Animals were killed after 1, 4, 12 or 24 hrs. Tracheal tissues from upper (UT) and lower (LT) tracheal levels were fixed and embedded in glycol methacrylate for light microscopy. One (N‘ two tracheal rings were 'fixed ‘hi formaldehyde- glutaraldehyde cacodylate buffer and processed for transmission electron microscopy. For cytochemical evaluation of glycoprotein- containing cells, each tracheal level was cut transversely at 3 um and stained with Alcian blue/Periodic acid-Schiff (AB-PAS) at pH 2.6. Granules of acid glycoproteins were AB positive, and those of neutral glycoproteins were PAS positive. In control hamster most of the glycoprotein cells were PAS- positive. A few contained AB-PAS-positive granules. However, the frequency distribution of the glycoprotein-containing cells varied in different regions and levels. The secretory cells were columnar and 55 56 contained electron lucent, membrane bound secretory' granules. Intracellular organelles, including abundant smooth and rough endoplasmic reticulum, mitochondria and Golgi complexes, were observed in these cells. 0014 produced numerous changes in glycoprotein-containing cell numbers and cytochemistry depending upon the time elapsing since treatment. For example, cells appeared that contained PAS-positive glycoprotein that was distributed homogenously in the cell rather than discrete granules. Numbers of these cells markedly increased after 12 and 24 hrs post 0014 treatment. However, numbers of cells containing acidic glycoproteins (AB-PAS-positive) increased 1 and 4 hrs after 0014 treatment. Ultrastructural alterations of the secretory cells were obvious as early as 1 hr after 0014 treatment. Intracellular organelles, including smooth and rough endoplasmic reticulum, mitochondria, Golgi complexes, were damaged by 0014. This chemical also produced injury in electron lucent secretory granules. Since 0014-induced cell injury depends on metabolimn of this agent by intracellular, NADPH-dependent cytochrome P-450 monooxygenases, this study suggests that glycoprotein containing cells in hamster trachea have the potential to bioactivate this chemical. INTRODUCTION One of the most important defense barriers in the tracheobronchial tree is mucus, consisting primarily of secreted glycoproteins. The goblet, serous and Clara cells contribute to glycoprotein secretion in the respiratory tract (Spicer et al., 1971; 1974; Jeffery and Reid, 57 1977; Spicer et al., 1980; Pack et al.,1980; 1981). Numerous studies have demonstrated that acute or chronic inhalation of chemical irritants increases secretion of glycoproteins in the airway. Exposure of rats to chemical irritants such as sulfur dioxide (Ried, 1963; Spicer et a1, 1974; Freeman and Haydon, 1964 Lamb and Reid, 1968) and tobacco smoke (Lamb and Reid, 1969; Jones et al., 1973; Jeffery and Reid, 1977; Jones and Reid, 1978; Coles et al., 1979) causes hypertr0phy and hyperplasia of glyc0protein cells in airways. Modification of glycoprotein-containing cells in the trachea after exposure to a chemical carcinogen was also reported. Becci et al. (1978a) found that treatment of hamsters with a mixture of benzo(a)pyrene and ferric oxide (BP-Fe203) caused hypertr0phy and hyperplasia of glycoprotein cells in hamster trachea. These authors reported that the glycoprotein became more acidic in treated animals when compared to that of unexposed hamsters. These effects were attributed to BP and not to the Fe203. Benzo(a)pyrene is metabolized by mixed function oxidases (MFO) to an electrophile that is highly cytotoxic; thus, it appears that glycoprotein cells can be altered by chemicals that are bioactivated by MFO. Carbon tetrachloride (0014) induced cell injury is dependent on metabolism of this agent by intracellular NADPH-dependent cytochrome P- 450 monooxygenases. Since BP requires metabolic activation by MFO and Since it altered glyc0protein-containing cells, it seems likely that a xenobiotic agent such as 0014 might also alter glycoprotein-containing cells of respiratory airways. To test the hypothesis, I examined the effect of 0014 administration i vivo on glyc0protein-containing cells of various regions and levels of hamster trachea. MATERIALS AND METHODS Adult, male, Syrian golden hamsters (100-140g) were obtained from Harlan Sprague Dawley (Indianapolis, Ind.) and housed in groups of three in clear polypropylene cages in a light cycle (12 hr light and 12 hr dark) and temperature controlled room. Animals were allowed food (Hayne Lab Blox, Chicago, IL) and tap water 3: libitum. Carbon tetrachloride (0014) was obtained from Sigma Chemical Company (St. Louis, MO), dissolved in peanut oil and administered i.p. at a dose of 2.5 ml/kg body weight. Control animals received the vehicle only. At 1, 4, 12, or 24 hrs after treatment, animals were sacrificed with 100- 300 mg/kg of sodium pentobarbital (i.p.). Three hamsters were used for each group. Light Microscopy: Tracheal tissues were fixed in 4% paraformaldehyde in phosphate buffer (pH 7.2). Each Specimen was divided into upper and lower levels and embedded in glycol methacrylate (GMA, Polyscience, Inc.). Tracheas were sectioned transversely at 3 um from proximal to distal ends with glass knives on a JB-4 microtome. Three histological sections at least 15 um apart were taken from each tissue block and stained with Alcian blue/ Periodic acid-Schiff (AB-PAS) stain at pH 2.6. The luminal epithelium was subdivided into dorsal, right and left lateral, and ventral regions. The total number of glycoprotein 58 59 containing cells/mm/region was counted at 4OOX magnification and compared statistically. Data were expressed as mean :_standard error. The results were analyzed by analysis of variance and treatment differences were identified by the method of Newman-Keuls (Steel and Torrie, 1960). P<0.05 was used as the criterion for significance. Since the data were nonsynlnetrical and the sample size at each time period was small (n=3 animals) a standard 10910 transformation was used to compensate for the nonsymmetry while preserving the relative pattern among comparison groups. The transformed data in each morphological level and region of the trachea were subjected to a one-way analysis of variance. Transmission Electron MicroscOpy: One or two rings of trachea from each animal were cut into pieces approximately 1 mm2 , and 1-2 blocks from each region (dorsal, lateral or ventral) were prepared for electron microscooy. Tissues were fixed in formaldehyde-91utaraldehyde cacodylate buffer (Karnovsky, 1965). The tissues were washed in 0.2 M cacodylate buffer, post-fixed in 2% osmium tetroxide, dehydrated with ethanol and embedded in epon-araldite (Mollenhauer, 1964). Sections 1 um thick were cut from selected areas and stained with toluidine blue for examination by light microscopy. Thin sections (approximately 90 nm) were cut from selected areas. The sections were stained with uranyl acetate and lead citrate and examined with a Philips 201 transmission electron microscope. RESULTS Within the glycoprotein containing cells, the presence of granules of acid and neutral glycoproteins were identified by the Alcian blue/Periodic acid Schiff (AB-PAS) stain at pH 2.6. Granules of acid glyc0proteins are AB positive, and those of neutral glycoproteins are PAS positive (Jones et al., 1973). Based on the amount of intracellular granules, the gly00protein containing cells were described as small (S) or large (L). A small glyc0protein-containing cell was one containing granules at its apex, and a large cell was one containing granules throughout its cytoplasm (Figs. 1-4). Glycoprotein cells containing PAS-positive, material that was distributed homogenously rather than in discrete granules was observed in some groups. These cells were described as small (PAS-H-S) or large (PAS-H- L). A small cell was one containing homogenous PAS-positive material only at its apex; and a large was one containing homogenous, PAS- positive material throughout its cytoplasm (Fig. 5). Glycoprotein Containing Cells In Control Animals: In tracheas from control animals, most glycoprotein-containing cells were PAS-positive (Tables 1-3). The small PAS-positive (PAS-G- S) cells were more predominant in the lateral region of the upper trachea (UT) and ventral region of the lower trachea (LT). However, the large PAS-positive (PAS-G-L) cells were more numerous in the 60 61 lateral region of In} The glycoprotein cells containing both AB and PAS (AB-PAS) positive granules were found infrequently. No cells were apparent with only AB positive granules. Similarly, glyc0protein cells containing homogenous, PAS-positive material in the cyt0plasm were absent. Transmission electron microscopy of trachea from peanut oil treated hamsters revealed that the secretory cells were columnar, and the cyt0plasm was electron-dense and contained electron lucent, membrane bound, secretory granules in the apical region or throughout the cytoplamn (Fig. 6). Intracellular organelles, including abundant smooth and rough end0plasmic reticulum, mitochondria and Golgi complex, were observed in these cells. Changes In Glycoprotein Cells After 0014 Treatment: The effect of 0014 on intracellular glyc0protein is shown in tables 1-3. Unlike controls, in 0014- treated hamster trachea, glyc0protein cells containing homogenous, PAS-positive material were observed. hi the dorsal region, the number of these cells markedly increased in upper trachea (UT) and lower trachea (LT) 12 or 24 hrs after 0014 treatment. In lateral regions, glycoprotein cells containing homogenous, PAS-positive material increased in UT at 1 hr, LT at 4, 12, and 24 hrs after 0014-treatment. In ventral regions, the cells containing PAS-positive, homogenous material increased in UT at 1, 4, 12, 24, and in LT at 24 hrs post 0014 treatment. In contrast, the glycoprotein cells containing PAS-positive granules decreased in trachea of hamsters treated with 0014. In dorsal regions, these cells decreased in UT and LT at 1 , 4 and 24 hrs after 0014 treatment. In lateral regions, the glycoprotein cells containing 62 PAS-positive granules decreased in UT at l, 24, and in LT at l, 4, and 24 hrs after 0014 treatment. In ventral regions, the PAS-positive granule-containing cells decreased in LT at 1, 4, and UT at 12 hrs and UT and LT at 24 hrs after 0014 treatment. Glyc0protein-containing cells with AB-PAS-positive granules were rarely seen in control animals. However, these cells were predominant in all regions of UT and LT at 1 and 4 hrs after 0014 treatment. Cells containing AB-positive granules were absent in control hamster, but were observed infrequently in 0014 treated hamsters. Transmission electron microscopy revealed that 0014 produced injury in secretory cells as early as 1 hr 0014 post-treatment. Damage in intracellular organelles was characterized by rupture of membrane bound secretory granules, dilation of Golgi apparatuses, smooth and rough endoplasmic reticulum, swelling of mitochondria and numerous cyt0plasmic vacuoles (Figs. 7-10). Figure l: 63 Light micrograph of lateral region of upper trachea of hamster treated 12 hrs previously with 0014. Note cells containing PAS-positive granules in the apical cytoplasm (solid arrow) or throughout the cytoplasm (open arrow). X 400. Figure 2: 64 Light micrograph of ventral region of upper trachea of hamster treated 4 hrs previously with 0014. Note cells containing AB-PAS positive granules in the apical cytoplasm (solid arrow) or throughout the cytoplasm (open arrow). X 400. 65 Figure 3: Light micrograph of dorsal region of upper trachea of hamster treated 12 hrs previously with 0014. Note cell containing AB-positive granules in the apical cytoplasm (arrow). X 400. 66 Figure 4: Light micrograph of dorsal region of lower trachea of hamster treated 4 hrs previously with 0014. Note cell containing AB-positive granules throughout cytoplasm (arrow). X 400. 1. 67 Figure 5: Light micrograph of lateral region of lower trachea of hamster treated 24 hrs previously with 0014. Note cells containing homogenous, PAS-positive material in the apical cytoplasm (solid arrow) or throughout the cytoplasm (open arrow). X 400. Figure 6: 68 Transmission electron micrograph of lateral region of tracheal epithelial cells of control hamster. Note electron lucent, membrane bound, secretory granules (g) in apical cytoplasm (center) or throughout the cytoplasm (left) of nonciliated cells. X 6272. 69 Figure 7: Transmission electron micrograph of lateral region of tracheal epithelium of hamster treated 1 hr previously with 0014. Note rupture of membrane bound secretory granules (arrow). Secretory granules (g). X 7165. Figure 8: 70 Transmission electron micrograph of lateral region of tracheal epithelium of hamster treated 4 hr previously with 0014. Note injury (dilatation of intracellular organelles including mitochondria, rough endoplasmic reticulum, Golgi apparatuses and many vacuoles) in both ciliated and nonciliated cells. Vacuole (arrow), Secretory granule (g), nucleus (N). X 8624. Figure 9: 71 ' n v'\ M ' I‘I‘T' -_ ;_ V - . . ‘51.,» :J“ . . ' ..- V L ‘. 16?.- 7‘5: Transmission electron micrograph of ventral region of tracheal epithelium cells of hamster treated 12 hrs previously with 0014. Note many vacuoles (arrows) in the cyt0plasm of the nonciliated cell. Nucleus (N), ciliated cells (00). X 8624. Figure 10: 72 Transmission electron tracheal epithelial previously with 0014. micrograph of lateral region of cells of hamster treated 24 hrs Note many vacuoles (arrows) in the cytoplasm of nonciliated cell. Nucleus (N). X 8820. Table l: 73 Number of glycoprotein-containing cells (GP).of various types in levels of dorsal regions of trachea of hamsters treated with 0014 for various times.* 0014 treatment.“ GP Control ’1 hr 4'hr lZThr 524 hr PAS-G-S d d b UT 9.11:1.60 0.56:0.25a 1.75:1.01a 12.82:5.26bc 2.53:0.79 d LT 4.00:0.71 0.32:0.06ad 1.77:0.79 5.91:1.77 0.83:0.60a PAS'G‘L I d b b bd UL 7.55:2.30, 0.52:0.1eag 3.39:0.51d 8.00:2.75b 1.38:0.35"d LT 2.48:0.32 0.39:0.05a 0.23:0.0a 3.05:0.69 C 0.45:0.22a PAS-H-S b 01 0 1.27:0.99 3.74:1.15 3.72:2.86 l4.32:4.453b LT 0 2.56:0.24 3.09:0.83 8.08:2.82a 17.28:7.73a ° PAS-H-L b d UT 0 2.52:1.00 4.54:1.89 5.53:1.28a 3293:3869;d LT 0 4.14:1.40 2.71:0.87 6.58:2.12a 24.55:s.33a C AB-PAS-G-S b UT 0.78:0.23 11.27:3.85a 6.24:2.52 7.70:2.87a 0521:o.21 LT 0.09:0.09 7.94:1.52a 7.96:3.35a 1.7:1.08 0 c AB-PAS-G-L b 01 0.12:0.12 4.41:1.93a 4.60:1.27a 0 C 0.83:0.83 LT 0.09:0.09 3.95:1.14 2.60:0.14 0.93:0.81 0 AB-G-S UT 0 0.11:0.10 0 0.69:0.68 0 LT 0 0 0.12:0.06abd 0 0c AB-G-L UT 0 0.19:0.19 0 0 0 LT 0 0.29:0.14 0.25:0.12 0 0 Adult, male, Syrian golden hamsters were treated with 0014 (2.5 ml/kg)} Control animals were given vehicle only. Animals were killed 1, 4, 12, or 24 hrs later. Tracheal tissues were removed and subdivided into upper (UT) and lower (LT) levels and prepared for cytochemical determination of glycoprotein-containing cells as described in Methods. Three animals were used in each group. *Number of glyc0protein-containing cells in 1 mm length of tracheal epithelium. Values are mean + standard error. a=Significantly different from that of the control 4roup (p<0.05). b=Significantly different from that of the group ki led 1 hr after 0014 (p<0.05). E=Significantly different from that of the group killed 4 hrs after 0014 p