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A practical model of bronchogenic carcinoma in Camm-Hartley guinea pigs and Golden Syrian hamsters Bainbridge, Terry Cyril 1986

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A PRACTICAL MODEL OF BRONCHOGENIC CARCINOMA IN CAMM-HARTLEY GUINEA PIGS AND GOLDEN SYRIAN HAMSTERS _ By TERRY CYRIL BAINBRIDGE B.M.L.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF PATHOLOGY) We accept t h i s t h e s i s as conforming t o the r e a u i r e d standard The U n i v e r s i t y o f B r i t i s h Columbia May, 1986 (cp Terry C y r i l B a i n b r i d g e , 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 - i i -ABSTRACT Bronchogenic carcinoma (lung cancer) i s a major source of morbidity and m o r t a l i t y i n i n d u s t r i a l i z e d n a t i o n s . Although bronchogenic carcinoma i s l a r g e l y a preventable neoplasm, i t w i l l undoubtedly remain a major medical concern throughout t h i s century, and c o n s i d e r a b l e e f f o r t needs t o be d i r e c t e d towards i t s e a r l y d e t e c t i o n , e s t a b l i s h i n g e f f e c t i v e treatment, and understanding the n e o p l a s t i c process. The o b j e c t i v e of t h i s study was to develop a p r a c t i c a l and r e l i a b l e model of l o c a l i z e d bronchogenic carcinoma i n l a b o r a t o r y rodents. This was done by impregnating c o t t o n threads with the potent carcinogen, benzo(a)pyrene (BP). These BP-impregnated threads were then coated w i t h a s i l i c o n e rubber sheath to c o n t r o l the release of BP from the threads. The prepared threads were sewn around four c a r t i l a g e r i n g s i n the v e n t r a l t r a c h e a l w a l l of guinea p i g s and hamsters. The animals were s a c r i f i c e d p e r i o d i c a l l y , and two h i s t o p a t h o l o g i s t s graded the t r a c h e a l e p i t h e l i u m adjacent t o the thread. The f i r s t experiment c o n s i s t e d of 94 Camm-Hartley guinea p i g s : 48 experimental animals with BP-impregnated threads and 46 c o n t r o l animals w i t h non-impregnated threads. The second experiment c o n s i s t e d of 70 Golden S y r i a n hamsters: 54 experimental and 16 c o n t r o l animals. The data showed that the i m p l a n t a t i o n of the thread i n the trachea induced a regenerative h y p e r p l a s i a of the e p i t h e l i u m , and that the BP i n i t i a t e d c a r c i n o g e n e s i s . Squamous metaplasia and p r o g r e s s i v e i n t r a e p i t h e l i a l n e o p l a s i a (IEN) was evident p r i o r to the development of squamous c e l l carcinoma (CA). In the experimental guinea p i g s , only one guinea p i g developed an i n v a s i v e CA at 265 days. In the - i i l -experimental hamsters, the f i r s t CA was seen at 55 days and a f t e r 120 days, 65% of the animals showed h i s t o p a t h o l o g i c evidence of CA. Most of the hamsters w i t h CA a l s o had s p i n d l e c e l l tumors i n the t r a c h e a l stroma. The c o n t r o l hamsters and guinea p i g s d i d not develop IEN, and the mature r e s p i r a t o r y e p i t h e l i u m was r e c o n s t i t u t e d . We conclude that t h i s method produced l o c a l i z e d , r e a d i l y a c c e s s i b l e p r e n e o p l a s t i c and n e o p l a s t i c l e s i o n i n the trachea of hamsters, and t o a l e s s e r extent i n guinea p i g s . The model should prove u s e f u l i n the study of tumor u l t r a s t r u c t u r e , the immunologic response t o cancer, and the r e l a t i o n s h i p of d i e t t o cancer. - i v -TABLE OF CONTENTS - • — Page ABSTRACT 1 1 TABLE OF CONTENTS - i v LIST OF TABLES • v i i LIST OF FIGURES v i- i : L LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS x i DEDICATION ^ i i INTRODUCTION 1 A. Bronchogenic Carcinoma 1 B. Bronchogenic Carcinoma and C i g a r e t t e Smoking 2 C. C i g a r e t t e Smoke 4 D. M u l t i s t a g e Theory of Carcinogenesis 5 E. Benzo(a)pyrene Metabolism... 6 F. Animal Models of Bronchogenic Carcinoma 10 MATERIALS AND METHODS .... 14 A. Animals 14 B. Threads 14 C. S u r g i c a l Procedures 15 D. Experiments 16 E. Pr e p a r a t i o n of the Tracheas 16 F. E v a l u a t i o n of the Tracheas 17 "-2J>"= G. L i v e r s , Lungs and Lymph Nodes 18 H. Animal S u r v i v a l 19 RESULTS 20 A. Benzo(a)pyrene Content of the Threads 20 B. Histopathology of the Tracheas 20 C. Histopathology of the Tracheal E p i t h e l i u m i n the C o n t r o l Animals 21 D. Histopathology of the Tracheal E p i t h e l i u m i n the Experimental Animals 21 E. Spin d l e C e l l Tumors . 24 F. E v a l u a t i o n of the Histopathology of the Tracheal E p i t h e l i u m . . . 25 G. E f f i c a c y of D i f f e r e n t Thread Options t o Induce Sguamous C e l l Carcinoma 27 H. Inflammation and K e r a t i n i z a t i o n of the Tracheal E p i t h e l i u m 27 I. Complications of the Model.... 28 J. Lymph nodes, L i v e r and Lung 28 K. S u r v i v a l Data 30 FIGURES AND TABLES 31 DISCUSSION 49 A. C o n s i d e r a t i o n of the H i s t o p a t h o l o g i c R e s u l t s 49 B. E f f i c a c y of D i f f e r e n t Thread Options t o Induce Squamous C e l l Carcinoma 52 C. Tissue O r i g i n of the Sp i n d l e C e l l Tumor 54 D. The Role of Inflammation, Regenerative H y p e r p l a s i a and K e r a t i n i z a t i o n of the Tracheal E p i t h e l i u m i n Tumor Promotion.. 57 - v i -E. Ascorbic A c i d and Cancer 58 F. Complications of the Model. 61 G. Animal S u r v i v a l 62 CONCLUSION. ._ 64 REFERENCES , 67 — v i i -LIST OF TABLES Table Page 1 Assessment of the O v e r a l l Interobserver E r r o r i n the H i s t o p a t h o l o g i c Grading of the Tracheal E p i t h e l i u m 42 2 E f f i c a c y of the D i f f e r e n t Thread Options t o Induce Squamous C e l l Carcinoma i n Hamsters 45 LIST OF FIGURES Figure" " " Page 1. Impregnation of Cotton Threads with Benzo(a)pyrene 31 2. P r e p a r a t i o n of the S i l i c o n e Rubber Sheath ...31 3. Apparatus used t o Coat the Threads with a S i l i c o n e Rubber Sheath.31 4. Diagram of the S u r g i c a l Procedure Performed on the Rodents 33 5. Diagram of a Tumor Forming Around the Thread w i t h i n the V e n t r a l Tracheal Wall 33 6. Low Power Photomicrograph of a Thread w i t h i n the Tracheal Wall...33 7 & 8. Low and High Power Photomicrographs of the P r o l i f e r a t i n g E p i t h e l i u m i n a Guinea P i g C o n t r o l , 4 Days Post-Operation ....35 9. Regenerating Squamous E p i t h e l i u m i n a Guinea P i g C o n t r o l , 20 Days Post-Operation 35 10 & 11. Low and High Power Photomicrographs of Mature Tracheal E p i t h e l i u m i n a Guinea P i g C o n t r o l , 183 Days Post-Operation.. 35 12. Normal Guinea P i g Tracheal E p i t h e l i u m 37 13. Sguamous Me t a p l a s i a and K e r a t i n i z a t i o n i n an Experimental Hamster 37 14. M i l d I n t r a e p i t h e l i a l Neoplasia i n an Experimental Hamster 37 15. Moderate I n t r a e p i t h e l i a l Neoplasia i n an Experimental Hamster 37 16 & 17. Marked I n t r a e p i t h e l i a l Neoplasia i n an Experimental Hamster 3.7/38 - i x - -18 & 19. Low and High Power Photomicrographs of a Squamous C e l l Carcinoma i n a Guinea P i g Trachea AO 20. Invasive Squamous C e l l Carcinoma i n a Hamster Trachea 40 21. Squamous C e l l Carcinoma Merging with a S p i n d l e C e l l Tumor i n a Hamster Trachea 40 22. High Power Photomicrograph of a S p i n d l e C e l l Tumor i n a Hamster Trachea. 40 23. Gross Appearance of a Tr a c h e a l Tumor i n a Hamster. 40 24. H i s t o p a t h o l o g i c Data o f Guinea Pig Tracheal E p i t h e l i u m 43 25. H i s t o p a t h o l o g i c Data of Hamster Tracheal E p i t h e l i u m 43 26. Bar Graph of Guinea P i g S u r v i v a l . . . 47 27. Bar Graph of Hamster S u r v i v a l 47 LIST OF ABBREVIATIONS a r y l hydrocarbon hydroxylase benzo(a)pyrene c a r t i l a g e squamous c e l l carcinoma c o n t r o l animals (not exposed to BP) d o r s a l t r a c h e a l w a l l experimental animals (exposed to BP) f i b r o s i s i n t r a e p i t h e l i a l k e r a t i n i n t r a e p i t h e l i a l n e o p l a s i a (1-mild, 2-moderate & 3-marked) k e r a t i n ( s u p e r f i c i a l ) l i g h t microscopy mature e p i t h e l i u m p o l y c y c l i c aromatic hydrocarbons polymorphonuclear leukocytes stroma (submucosa, c a r t i l a g i n o u s l a y e r & a d v e n t i t i a o f trachea) s p i n d l e c e l l tumor squamous metaplasia s i l i c o n e rubber sheath thread t r a n s m i s s i o n e l e c t r o n microscopy t r a c h e a l lumen t r a c h e a l tumor v e n t r a l t r a c h e a l w a l l ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r , Dr. J.C. Hogg, f o r p r o v i d i n g the opportunity to work i n h i s l a b o r a t o r y , f o r o f f e r i n g h i s time and c o n s t r u c t i v e , c r i t i c i s m s , and f o r h i s i d e a of i n s e r t i n g c a r c i n o g e n - impregnated threads i n t o rodent tracheas to produce tumors. Next, I would l i k e t o thank the s t a f f of the Pulmonary Research Laboratory f o r t h e i r a s s i s t a n c e : Dr. D.C. Walker f o r h i s h e l p f u l comments and e l e c t r o n microscopy s k i l l s , Andre MacKenzie f o r h i s uncanny procurement a b i l i t y , S t u a r t Greene f o r h i s photographic e x p e r t i s e , Lee Kowk, Sheree Strange and Joan Dixon f o r t y p i n g t h i s manuscript, Dean E n g l i s h f o r feeding the animals, Barry Wiggs and Joseph Comeau f o r the computer a s s i s t e d a n a l y s i s of the data, and Dr. A. Autor f o r her i n s i g h t f u l d i s c u s s i o n s of oxygen f r e e r a d i c a l biochemistry. I am indebted t o the anatomical pathology s t a f f at S t . Paul's H o s p i t a l . Judy Tanch and her t e c h n o l o g i s t s prepared s e v e r a l h i s t o p a t h o l o g i c specimens. S p e c i a l thanks are extended t o Dr. J.L. Wright & Dr. N. Chan f o r t h e i r h i s t o p a t h o l o g i c i n s t r u c t i o n and i n t e r p r e t a t i o n . Dr. A. Churg and Edgar Chong h e l p f u l l y o f f e r e d t o type the inter m e d i a t e f i l a m e n t s on the hamster s p i n d l e c e l l tumors. Two medical students, Suzie Tevendale and E r i c Webber, each spent a summer he l p i n g out with v a r i o u s phases of the study. L a s t l y , I would l i k e t o express thanks f o r funding t o Dr. D.F. Hardwick, the d i r e c t o r of Pathology at the U n i v e r s i t y of B r i t i s h Columbia, t o the F a c u l t y of Medicine at the U n i v e r s i t y of B r i t i s h Columbia and t o the Med i c a l Research C o u n c i l of Canada. — x i i -To the l a t e pathology P r o f e s s o r C.F.A. C u l l i n g , who was i n s t r u m e n t a l i n c r e a t i n g the Bachelor of Medical Laboratory Science program, t h a t provided a unique opportunity f o r medical l a b o r a t o r y t e c h n o l o g i s t s t o develop i n t o s c i e n t i s t s . - 1 -INTRODUCTION Bronchogenic Carcinoma Bronchogenic carcinoma (lung cancer) i s a major source of morbidity and m o r t a l i t y i n i n d u s t r i a l i z e d n a t i o n s . P r e s e n t l y , i t i s the most common cause of c a n c e r - r e l a t e d death i n males and w i t h i n the next decade, i t i s pr o j e c t e d to exceed breast carcinoma as the most common cause o f ca n c e r - r e l a t e d death i n women; the o v e r a l l f i v e year s u r v i v a l r a t e of bronchogenic carcinoma i s l e s s than 10% ( S i l v e r b e r g , 1983). The major problem i s that the l o c a l i z e d b r o n c h i a l l e s i o n s metastasize e a r l y t o the r e g i o n a l lymph nodes and other organs o f the body. Due t o the r a p i d p r o g r e s s i o n o f the dis e a s e , m e t a s t a t i c carcinoma i s seen i n 50% of p a t i e n t s at the time of the i n i t i a l p r e s e n t a t i o n (Van Houtte et a l . , 1983). This tumor can be p o t e n t i a l l y cured by surgery, yet the o v e r a l l r a t e of cure has improved l i t t l e s i n c e the i n t r o d u c t i o n of s u r g i c a l treatment ( W i l k i n s et a l . , 1978). Radiotherapy and chemotherapy may prolong s u r v i v a l i n some p a t i e n t s , but are not c u r a t i v e (Van Houtte et a l . , 1983). The f a c t that t h i s cancer passes q u i c k l y through the stage where i t i s l o c a l i z e d and operable t o the stage where i t i s i n v a s i v e and inoperable has f r u s t r a t e d attempts at e a r l y d e t e c t i o n and treatment. Since most lung cancers are bronchogenic carcinomas, (accounting f o r 90-95% of lung c a n c e r s ) , the two terms are o f t e n used interchangeably. The remaining 5 t o 10% of lung cancers are b r o n c h i a l c a r c i n o i d s ( o r b r o n c h i a l adenomas), mesenchymal and other miscellaneous neoplasms. Bronchogenic carcinomas are c l a s s i f i e d i n t o the f o l l o w i n g h i s t o l o g i c t ypes: squamous c e l l (epidermoid) carcinoma ( v a r i a n t - s p i n d l e c e l l carcinoma), adenocarcinoma ( a c i n a r and p a p i l l a r y carcinomas, and b r o n c h i o l o a l v e o l a r carcinoma), small - 2 -c e l l carcinomas (oat c e l l and s p i n d l e or polygonal s m a l l c e l l ) , l a r g e c e l l carcinoma ( v a r i a n t s - g i a n t c e l l and c l e a r c e l l carcinoma) and adenosquamous carcinoma (combined squamous c e l l carcinoma and adenocarcinoma). Not a l l bronchogenic carcinomas are of b r o n c h i a l o r i g i n , as the term suggests. For example, adenocarcinomas are u s u a l l y p e r i p h e r a l l y l o c a t e d ( b r o n c h i o l a r or b r o n c h i o l o a l v e o l a r o r i g i n ) , as opposed to the more c e n t r a l h i l a r ( b r o n c h i a l ) l o c a t i o n of most squamous c e l l and small c e l l carcinomas. (More d e t a i l e d i n f o r m a t i o n about human bronchogenic carcinomas i s a v a i l a b l e i n Shimosato et a l . , 1981, and C a r t e r et a l . , 1980). In the model of bronchogenic carcinoma reported here, the trachea serves as the c a r c i n o g e n i c s u b s t r a t e . In humans, malignant t r a c h e a l tumors are comparatively r a r e , most represent d i r e c t e x t e n s i o n from b r o n c h i a l or esophageal carcinoma. Yet primary t r a c h e a l tumors are reported, u s u a l l y squamous c e l l or adenoid c y s t i c carcinomas (Houston, 1969). The t r a c h e a l e p i t h e l i u m i s i n c o n t i n u i t y w i t h the b r o n c h i a l e p i t h e l i u m and has a s i m i l a r array of c e l l s : b a s a l c e l l s t o regenerate the e p i t h e l i u m , c i l i a t e d columnar c e l l s to sweep the intrapulmonary d e b r i s up and out, and goblet c e l l s t o provide the mucous l a y e r which coats the t r a c h e o b r o n c h i a l t r e e . Although the anatomical l o c a t i o n of bronchus and trachea d i f f e r , the e p i t h e l i u m i s s i m i l a r , and the c e l l u l a r response to carcinogens i s s i m i l a r i n human bronchus and hamster trachea (Autrup et a l . , 1980). Bronchogenic Carcinoma and C i g a r e t t e Smoking I t i s w e l l recognized that a c a u s a l r e l a t i o n s h i p e x i s t s between c h r o n i c c i g a r e t t e smoking and bronchogenic carcinoma. This f a c t i s based on p r o s p e c t i v e and r e t r o s p e c t i v e e p i d e m i o l o g i c a l s t u d i e s , c l i n i c a l s t u d i e s , - 3 -autopsy s e r i e s and experimental s t u d i e s i n animals (United S t a t e s Department of Health, 1979). " E p i d e m i o l o g i c a l s t u d i e s show th a t the r i s k of developing bronchogenic carcinoma i s , on the average, 10 times g r e a t e r f o r smokers than f o r non-smokers (Hammond and Horn, 1958). T h i s r i s k i n c r e a s e s w i t h heavy c i g a r e t t e consumption, and with o c c u p a t i o n a l exposure t o chromates, n i c k e l , asbestos and uranium (Frank, 1982). The c a u s a l r e l a t i o n s h i p between smoking and bronchogenic carcinoma has been strengthened by d e l i n e a t i n g dose-response r e l a t i o n s h i p s : the r i s k f o r bronchogenic carcinoma i n c r e a s e s w i t h the number of c i g a r e t t e s per day, and the d u r a t i o n of smoking (Hammond and Horn, 1958). The r i s k of developing lung cancer does d e c l i n e f o r e x - c i g a r e t t e smokers compared t o c o n t i n u i n g c i g a r e t t e smokers (Wynder, 1972). The c l i n i c a l evidence suggesting that c i g a r e t t e smoking causes lung cancer cen t e r s around s t u d i e s of the r e s p i r a t o r y e p i t h e l i u m i n smokers compared to non-smokers. The r e s p i r a t o r y e p i t h e l i u m of c h r o n i c c i g a r e t t e smokers i s b e l i e v e d t o proceed through a p r o g r e s s i v e s e r i e s of p r e n e o p l a s t i c changes before the development of bronchogenic carcinoma (Auerback et a l . , 1962). These p r e n e o p l a s t i c changes ( e p i t h e l i a l d y s p l a s i a o r i n t r a e p i t h e l i a l n eoplasia) have been e x t e n s i v e l y s t u d i e d i n human c e r v i c a l e p i t h e l i u m ( R i c h a r t et a l . , 1982 & Buckley et a l . , 1982). C l i n i c a l s t u d i e s show th a t the e a r l y antecedants of bronchogenic carcinoma, e p i t h e l i a l h y p e r p l a s i a and d y s p l a s i a , are seen i n a gr e a t e r p r o p o r t i o n of smokers than i n comparable non-smokers (Auerbach et a l . , 1979). Experimental animal models of tobacco smoke induced c a r c i n o g e n e s i s c o n s t i t u t e a c r u c i a l l i n k i n the c h a i n o f evidence supporting the c a u s a l r e l a t i o n s h i p of c i g a r e t t e smoking t o bronchogenic carcinoma. The e a r l i e s t - 4 -animal models i n v o l v e d repeated s k i n p a i n t i n g of tobacco t a r and some of the condensate s u b f r a c t i o n s o f tobacco smoke ( O r r i s et a l . , 1958, and Murphy and Sturm, 1925). Then various f r a c t i o n s of c i g a r e t t e smoke condensate were i n o c u l a t e d i n t o the lungs of r a t s , w i t h only moderate success ( B l a c k l o c k and Burgan, 1962, and B l a c k l o c k , 1961). L a t e r , more s o p h i s t i c a t e d models of c i g a r e t t e smoke induced lung cancer were conducted i n dogs (Auerback et a l . , 1970). In gen e r a l , although bronchogenic carcinoma can be induced i n animal models with c i g a r e t t e smoke or tobacco t a r condensates, the y i e l d s are low (Kuschner and L a s k i n , 1971). C i g a r e t t e Smoke The combustion of tobacco generates s e v e r a l hundred compounds by a v a r i e t y of chemical and p h y s i c a l processes (Wynder and Hoffmann, 1967). To a i d the a n a l y s i s of tobacco smoke, i t s c o n s t i t u e n t s have been separated i n t o a gas phase (defined as those substances which w i l l pass through a Cambridge f i l t e r pad which i s 99.9% e f f i c i e n t f o r p a r t i c l e s more than 0.1 microns i n diameter) and a p a r t i c u l a t e phase (d e f i n e d as those substances which c o l l e c t on the f i l t e r ) . The gas phase c o n t a i n s carbon monoxide, carbon d i o x i d e , n i t r o g e n oxides, ammonia, v o l a t i l e N-nitrosamines and hydrogen cyanide. The p a r t i c u l a t e phase c o n s i s t s of water, n i c o t i n e and t a r . The t a r i s p r i m a r i l y composed of a myriad of p o l y c y c l i c aromatic hydrocarbons (PAH), t o which c a r c i n o g e n i c i t y i s a t t r i b u t e d . Some of the PAH i n c l u d e benzopyrenes, chrysenes, and anthracenes. The t a r a l s o c o n t a i n s n o n - v o l a t i l e N-nitroamines, aromatic amines, i s o p r e n o i d s , benzenes, naphthalenes, aza-arenes, phenols, c a r b o x y l i c a c i d s , m e t a l l i c i o n s , and r a d i o a c t i v e compounds (potassium-40, lead-210, polonium-210 and rad.ium-226). - 5 -The PAH are only a s m a l l f r a c t i o n of a very complex mixture. Nevertheless, f r a c t i o n a t i o n s t u d i e s with tobacco t a r have shown t h a t those f r a c t i o n s enriched i n PAH induce tumors i n animals ( O r r i s et a l . , 1958, and B l a c k l o c k and Burgan, 1962). Over 100 i n d i v i d u a l PAH have been i d e n t i f i e d , but the c l a s s i c PAH, benzo(a)pyrene (BP) and dibenz(a,h)anthracene, stand out as being major carcinogens i n c i g a r e t t e smoke. Yet other c o n s t i t u e n t s c o n t r i b u t e to the c a r c i n o g e n i c p o t e n t i a l of c i g a r e t t e smoke. For example, n o n - v o l a t i l e N-nitroamines are formed during c u r i n g , fermentation and combustion of tobacco; these chemicals are known carcinogens (Hecht et a l . , 1978, and H i l f r i c h et a l . , 1977). I t must be appreciated that the amount of these carcinogens i n c i g a r e t t e smoke i s very low. There i s about 25 ng of BP per c i g a r e t t e smoked (Wynder and Hoffman, 1967), n e c e s s i t a t i n g repeated exposures f o r many years before bronchogenic carcinoma develops. M u l t i s t a g e Theory of Carcinogenesis When the chemical components of c i g a r e t t e smoke are t e s t e d i n i s o l a t i o n , many are not a c t i v e as complete carcinogens (able to induce cancers by themselves). Rather, c e r t a i n chemical agents play i n t e r a c t i v e r o l e s i n c a r c i n o g e n e s i s , and o f t e n a set of chemicals i s r e q u i r e d t o produce experimental cancers. To e x p l a i n t h i s phenomenon, the m u l t i s t a g e theory of c a r c i n o g e n e s i s was devised, i n which at l e a s t two d i s t i n c t independent steps are necessary to transform a normal c e l l to-a malignant c e l l ( P i t o t , 1982 and Farber, 1981). A tumor i n i t i a t o r i s an agent which induces a dormant tumor c e l l , l i k e l y by a d i r e c t a t t a c k of the i n i t i a t i n g agent on the DNA of the c e l l . I n i t i a t i o n i s g e n e r a l l y considered to be completed r e l a t i v e l y r a p i d l y and to be e s s e n t i a l l y i r r e v e r s i b l e . A tumor promoter i s an agent which a l l o w s - 6 -expression of the dormant tumor c e l l by s e l e c t i v e p r o l i f e r a t i o n of the i n i t i a t e d c e l l s . Tumor promoters are e f f e c t i v e only i f a p p l i e d t o the t i s s u e a f t e r a p r i o r treatment w i t h the tumor i n i t i a t o r . In c o n t r a s t t o i n i t i a t i n g agents, promoting agents e x h i b i t a r e v e r s i b i l i t y i n t h e i r a c t i o n , and t h i s may be modulated by d i e t , hormonal and other environmental f a c t o r s . T his has important i m p l i c a t i o n s f o r human cancers, as tumor promotion can p o t e n t i a l l y be manipulated a f t e r p r e v e n t i v e s t r a t e g i e s t o avoid tumor i n i t i a t i o n have f a i l e d . A co-carcinogen i s a chemical which i s n e i t h e r a tumor i n i t i a t o r , a tumor promoter, nor a complete carcinogen, but i t i s capable of augmenting the ca r c i n o g e n i c response of t i s s u e to a minimal dose of carcinogen. A l a t e r stage i n tumor development i n which the tumor mass grows, begins t o invade, and p o t e n t i a l l y metastasizes i s o f t e n d e l i n e a t e d , r e f e r r e d t o as tumor p r o g r e s s i o n . The various chemicals i n c i g a r e t t e smoke probably operate i n concert t o produce carcinomas, each operating at d i f f e r e n t stages i n c a r c i n o g e n s i s . BP'S tumor i n i t i a t i n g p o t e n t i a l i s enhanced by promoting agents. Other PAH, such as pyrene and benzo(g,h,i)fluoranthene, are thought t o be co-carcinogens; whereas the weakly a c i d i c f r a c t i o n of c i g a r e t t e smoke c o n t a i n s phenols which are tumor promoters, a k i n t o the tumor promoter prototype, phorbol e s t e r , i n mouse s k i n (Slaga and K l e i n - S z a n t o , 1983 and Slaga et a l . , 1982). Benzo(a)pyrene Metabolism Benzo(a)pyrene (BP) i s not a carcinogen i n i t s n a t i v e form, i t must undergo metabolism i n the microsomal (smooth endoplasmic r e t i c u l u m ) monooxygenase (mixed f u n c t i o n oxidase or cytochrome P-450) enzyme system. T h i s enzyme system i s u s u a l l y r e s p o n s i b l e f o r d e t o x i f i c a t i o n o f f o r e i g n - 7 -chemicals, but with some chemical carcinogens, t h i s same system c r e a t e s a c t i v e chemical carcinogens " ( M i l l e r , 1978). Two metabolic routes of c a r c i n o g e n i c i t y are p o s s i b l e with BP. The most i n t e n s i v e l y i n v e s t i g a t e d i s the d i r e c t mechanism of BP c a r c i n o g e n e s i s i n which the proximate carcinogen ( n a t i v e BP) i s metabolized by monooxygenase enzymes t o a simple epoxide; t h i s epoxide i s hydrolyzed to a d i h y d r o d i o l by epoxide hydrolase (epoxide hydratase), and f i n a l l y the u l t i m a t e carcinogen i s formed, a d i o l - e p o x i d e d e r i v a t i v e o f BP, by another a c t i v a t i o n step by monooxygenase enzymes (Conney, 1982, Sims, 1980 and Heidelberger, 1977). S e v e r a l d i f f e r e n t d i o l - e p o x i d e s are formed, but i t i s b e l i e v e d t h a t the (+)-anti-BP-7,8-diol-9,10 epoxide i s the most b i o l o g i c a l l y a c t i v e (Slaga et a l . , 1979). This e l e c t r o p h i l i c reactant then e x e r t s i t s c a r c i n o g e n i c e f f e c t by covalent i n t e r a c t i o n w i t h c e l l u l a r macromolecules, namely by forming an adduct with guanine i n DNA ( P h i l l i p s , 1983). How t h i s i n t u r n r e s u l t s i n a malignant c e l l u l a r phenotype i s not understood. The other,metabolic route which BP may f o l l o w i s the i n d i r e c t mechanism of c a r c i n o g e n i c i t y i n which f r e e r a d i c a l intermediates are generated when BP i s s h u t t l e d through the monooxygenase enzyme system (Menger et a l . , 1976, Wilk and G i r k e , 1972, and Tso et a l , 1977). These BP f r e e r a d i c a l i n t e r m e d i a t e s engender oxygen-free r a d i c a l s which can then damage c e l l u l a r macromolecules and lead t o c a r c i n o g e n e s i s ( C a v a l i e r i et a l . , 1976, Ide et a l . , 1983 and C e r u t t i and Remsen, 1977). The r e l a t i v e r o l e o f the d i r e c t and i n d i r e c t mechanism of BP c a r c i n o g e n i c i t y remains to be e l u c i d a t e d . Furthermore, each of these two metabolic pathways generate a spectrum of products, which may have a d d i t i v e , s y n e r g i s t i c and perhaps even opposing a c t i o n s . BP i s known to be a complete carcinogen; that i s , i t i s i n h e r e n t l y - 8 -able to produce both tumor i n i t i a t i o n and promotion. The a c t i v a t i o n o f BP l e a d i n g t o DNA adduct formation ( d i r e c t mechanism), i f coupled with one c y c l e of c e l l p r o l i f e r a t i o n (a property tumor promoters), w i l l r e s u l t i n tumor i n i t i a t i o n (Farber, 1981). On the other hand, oxygen f r e e r a d i c a l s (formed i n the i n d i r e c t mechanism) are b e l i e v e d t o r e s u l t i n tumor promotion ( C e r u t t i , 1985 and G o l d s t e i n et a l . , 1983). Perhaps the two d i f f e r e n t mechanisms of BP c a r c i n o g e n i c i t y play separate r o l e s i n the m u l t i s t a g e i n d u c t i o n of cancer, but t h i s may be o v e r l y s i m p l i s t i c and has yet t o be proven. The metabolism of BP i n human lungs has r e c e i v e d much a t t e n t i o n i n the past. This i s because BP has been i m p l i c a t e d as one of the major carcinogens i n c i g a r e t t e smoke causing bronchogenic carcinoma. Although t h i s r e l a t i o n s h i p i s g e n e r a l l y accepted, one of the enigmas about c i g a r e t t e smoking and lung cancer i s t h a t only a s u b - f r a c t i o n of smokers develop lung cancer. The reason f o r t h i s i s not known, but i n the e a r l y 1970's an appealing theory was proposed, which s t a t e d t h a t s u s c e p t i b i l i t y to bronchogenic carcinoma i s a s s o c i a t e d w i t h higher l e v e l s o f i n d u c i b l e a r y l hydrocarbon hydroxylase (AHH) a c t i v i t y . The p o l y c y c l i c aromatic hydrocarbons (PAH) i n g e n e r a l , and BP i n p a r t i c u l a r , , are metabolized i n human c e l l s by a complex of microsomal enzymes (monooxygenase and epoxide hydrolase or AHH) t o products capable of inducing n e o p l a s t i c t r a n s f o r m a t i o n ( M i l o et a l . , 1978). AHH a c t i v i t y i s known t o be induced by c i g a r e t t e smoke and by p u r i f i e d PAH (Welch et a l . , 1971). F u r t h e r , i t has been shown th a t v a r i a t i o n i n the extent of AHH i n d u c t i o n i s under genetic c o n t r o l ; AHH i n d u c i b i l i t y i s determined by a s i n g l e gene with a d d i t i v e e x p r e s s i o n of two a l l e l e s (Kellermann et a l . , 1973a). Kellermann reported (Kellermann et a l . , 1973b) th a t p a t i e n t s w i t h bronchogenic carcinoma show a s e l e c t i o n i n gene frequencies which a l l o w s higher l e v e l s o f i n d u c i b l e AHH - 9 -a c t i v i t y when compared t o c o n t r o l s . Subsequent s t u d i e s r e f u t e d t h i s evidence (Paigen e t a l . , 1977)."The prototype t i s s u e f o r determination o f AHH a c t i v i t y i n man has been c u l t u r e d p e r i p h e r a l blood lymphocytes. The i n c o n s i s t e n c i e s i n the reported human AHH a c t i v i t y i n p a t i e n t s w i t h bronchogenic carcinoma might be due t o sampling e r r o r s , day t o day v a r i a t i o n i n lymphocyte AHH a c t i v i t y , d i f f e r e n t lymphocyte c u l t u r e c o n d i t i o n s and d i f f e r e n t AHH assays. In an e f f o r t t o provide a more r e p r o d u c i b l e measure o f AHH a c t i v i t y , human pulmonary a l v e o l a r macrophages from lavage i n combination w i t h c u l t u r e d human lymphocytes have been assayed, and these s t u d i e s a l s o showed t h a t lung cancer p a t i e n t s have higher l e v e l s of i n d u c i b l e AHH a c t i v i t y than p a t i e n t s without lung cancer but w i t h comparable smoking h i s t o r i e s (McLemore et a l . , 1981). Because these s t u d i e s support an a t t r a c t i v e theory, namely t h a t s u s c e p t i b i l i t y t o bronchogenic carcinoma i s i n h e r i t e d as an augmented a b i l i t y t o metabolize chemical carcinogens, i t i s tempting t o accept the aforementioned evidence. But p e r i p h e r a l blood lymphocytes and a l v e o l a r macrophages are an i n d i r e c t means of assessing the c a p a c i t y o f the b r o n c h i a l e p i t h e l i u m t o a c t i v a t e chemical carcinogens. P r o s p e c t i v e i n v e s t i g a t i o n of the AHH a c t i v i t y i n human bronchus, i n p r o p e r l y c o n t r o l l e d p a t i e n t groups, would be r e q u i r e d before t h i s theory gains general acceptance. Given that carcinogens such as BP .must be metabolized by the mono-oxygenase enzyme system before producing pulmonary neoplasms, knowledge o f the d i s t r i b u t i o n of AHH a c t i v i t y w i t h i n the t r a c h e o b r o n c h i a l t r e e may help e x p l a i n why pulmonary neoplasms occur at p a r t i c u l a r s i t e s i n the r e s p i r a t o r y system. The o v e r a l l pulmonary AHH a c t i v i t y i s about 3% of human and 10% of rodent h e p a c t i c AHH a c t i v i t y (Autrup et a l . , 1981). However, the pulmonary AHH a c t i v i t y appears to" r e s i d e i n c e r t a i n c e l l l i n e s . Boyd showed t h a t the - 10 -pulmonary n o n - c i l i a t e d b r o n c h i o l a r ( C l a r a ) c e l l i n rodents possesses high AHH a c t i v i t y (Boyd, 1977). The r a t a l v e o l a r type I I c e l l i s a l s o capable of met a b o l i z i n g BP (Jones et a l . , 1982). Others ( H a r r i s e t a l . , 1978) have presented evidence that human pulmonary a l v e o l a r macrophages can metabolize BP to the u l t i m a t e carcinogen and then r e l e a s e t h i s product. Because the ma j o r i t y of human bronchogenic squamous c e l l carincomas form i n the bronchus, i t i s of p a r t i c u l a r relevance t h a t human b r o n c h i a l e p i t h e l i u m can metabolize BP (Prough et a l . , 1979, Autrup et a l . , 1980, Grover et a l . , 1976, and H a r r i s et a l . , 1982). Animal Models of Lung Cancer Animal models of bronchogenic carcinomas can be produced, provided the carcinogen i s held i n contact w i t h the e p i t h e l i u m f o r s u f f i c i e n t p e r i o d s of time (Kuschner, 1968, and Shabad et a l . , 1964). S e v e r a l d i f f e r e n t means of che m i c a l l y inducing r e s p i r a t o r y t r a c t tumors i n animals have been t r i e d w i t h varying degrees of success: i n h a l a t i o n s t u d i e s , systemic a d m i n i s t r a t i o n of carcinogens, i n t r a t r a c h e a l i n s t i l l a t i o n , t r a c h e a l e x p l a n t s a n d . t r a n s p l a n t s , and implant techniques. I n h a l a t i o n techniques are valuable i n environmental s t u d i e s when examining p o t e n t i a l l y noxious r e s p i r a t o r y agents, as t h i s best approximates man's exposure. However, t h i s i s a poor means of inducing tumors,because chemical carcinogens are c l e a r e d from the r e s p i r a t o r y system^and the tumor y i e l d s are low (Kuschner et a l . , 1957). I n t e r e s t i n g l y , some chemical carcinogens can be introduced s y s t e m i c a l l y and pulmonary tumors w i l l form. For example, subcutaneous i n j e c t i o n of d i e t h y l n i t r o s a m i n e w i l l induce r e s p i r a t o r y t r a c t tumors i n Golden S y r i a n - 11 -hamsters (Stenback et a l . , 1973, and R e z n i k - S c h u l l e r , 1980). The organotropy of d i e t h y l n i t r o s a m i n e f o r the r e s p i r a t o r y t r a c t i s presumed t o be due t o the a b i l i t y of pulmonary c e l l s to take up and metabolize the nitrosamines (Reznik-S c h u l l e r , 1980). I t i s important to note t h a t d i e t h y l n i t r o s a m i n e can a l s o induce l i v e r tumors ( P i t o t et a l . , 1978). The problem w i t h these animal models i s that the tumors may not be l o c a l i z e d e x c l u s i v e l y t o the r e s p i r a t o r y system nor l o c a l i z e d w i t h i n the r e s p i r a t o r y t r e e , and i t i s d i f f i c u l t t o c o n t r o l the dose of carcinogen reaching the r e s p i r a t o r y system. I n t r a t r a c h e a l i n s t i l l a t i o n i s a popular method of c h e m i c a l l y i n d u c i n g r e s p i r a t o r y t r a c t tumors. S a f f i o t t i produced an animal model of lung cancer by i n s t i l l i n g a s a l i n e suspension of BP adsorbed t o hematite ( f e r r i c oxide) i n t o the t r a c h e o b r o n c h i a l t r e e of Golden S y r i a n hamsters ( S a f f i o t t i et a l . , 1968). This i n t e r v e n t i o n was c a r r i e d out once a week f o r 15 weeks. He was able to b r i n g about t r a c h e a l and b r o n c h i a l carcinomas, mainly squamous c e l l , i n a l a r g e percentage of the s u r v i v i n g hamsters. This model of lung cancer c l o s e l y simulated the c e l l type, the d i s t r i b u t i o n and the mode of carcinogen d e l i v e r y seen i n humans h a b i t u a l l y exposed t o c i g a r e t t e smoke. This method has, to a l a r g e extent, remained the mainstay i n c h e m i c a l l y induced models of lung cancer. There have been many manipulations made of the same b a s i c method, such as type of c a r r i e r dusts, v e h i c l e o f carcinogen a d m i n i s t r a t i o n , and species of rodent (Yashimoto et a l . , 1980, Shabad et a l . , 1964, Stenback et a l . , 1975 and Henry et a l . , 1973). Although t h i s method i s e f f e c t i v e , i t s u f f e r s two major disadvantages. F i r s t , the r e p e t i t i v e a d m i n i s t r a t i o n of the carcinogen i s time consuming, and second, the carcinomas which form are not l o c a l i z e d , h i n dering s t u d i e s of the p r e n e o p l a s t i c l e s i o n s . In order to g a i n access to the p r e n e o p l a s t i c l e s i o n s , S c h r e i b e r (et - 12 -a l . , 1975) devised a method t o induce carcinomas i n a c i r c u m s c r i b e d r e g i o n of the hamster trachea. He d i d t h i s by using a s p e c i a l c a t h e t e r to apply N-nitroso-N-methylurea t o the t r a c h e a , twice weekly. A f t e r 30 repeated exposures of the carcinogen and w a i t i n g f o r another 20 weeks, squamous c e l l carcinoma developed i n 80% o f the hamsters. This method lends i t s e l f to the study of p r e n e oplasia because i t i s l o c a l i z e d , but again i t i s time consuming, and the i n d u c t i o n time i s r e l a t i v e l y long. An ingenious method of studying l o c a l i z e d t r a c h e a l tumors was devised by Kendrick (et a l . , 1974). He e x c i s e d tracheas, f l u s h e d them w i t h c u l t u r e media, and g r a f t e d the donor trachea i n t o the deep subcutaneous t i s s u e o f i s o g e n i c hosts, i n r a t s , mice and hamsters. A f t e r a 4 week recovery, BP or 3-methycholanthrene, i n g e l a t i n plugs, was i n s e r t e d i n t o the t r a c h e a l lumen of the g r a f t . Although t h i s method i s reasonably e f f e c t i v e , i t c e r t a i n l y i s not t e c h n i c a l l y f e a s i b l e f o r l a r g e s c a l e s t u d i e s . More r e c e n t l y , t r a c h e a l explant models have become a v a i l a b l e (Chopra and Cooney, 1985, and Moossman et a l . , 1984) which enable i n v e s t i g a t o r s t o study c h e m i c a l l y induced b a s a l c e l l h y p e r p l a s i a and squamous metaplasia i n c u l t u r e c o n d i t i o n s , but carcinomas have not been produced by t h i s method. Implant techniques of inducing r e s p i r a t o r y t r a c t tumors are p r e f e r a b l e because the a d m i n i s t r a t i o n of the carcinogen i s accomplished i n one o p e r a t i v e procedure, and thus the animals do not have to be r e p e t i t i v e l y a n e s t h e t i z e d and manipulated. They a l s o induce l o c a l i z e d tumors. As e a r l y as 1937, Andervo.nt induced lung tumors i n 10 t o 20% of mice t h a t had dibenz(a,h)anthracene-coated threads i n s e r t e d i n t o t h e i r chest c a v i t i e s . Other i n v e s t i g a t o r s (Shors et a l . , 1978 & 1980) i n c o r p o r a t e d BP i n t o a c y l i n d e r i c a l plug of s i l i c o n e rubber; t h i s carcinogen-laden plug was then - 13 -secured i n the b r o n c h i a l lumen of Golden S y r i a n hamsters. Bronchogenic carcinomas,"representative o f the c e l l types seen i n humans, were produced at the s i t e of the implant i n the hamsters. Nevertheless, these implant techniques are e i t h e r t e c h n i c a l l y d i f f i c u l t and time consuming or are a s s o c i a t e d with r e l a t i v e l y high r a t e s of morbidity and m o r t a l i t y . In summary, animal models of lung cancer have been developed, but there are s e v e r a l shortcomings i n the e x i s t i n g models. F i r s t and foremost, most animal models r e q u i r e a labour i n t e n s i v e p r o t o c o l t o i n i t i a t e and maintain the d e l i v e r y of the carcinogen t o the r e s p i r a t o r y e p i t h e l i u m . Furthermore, most models do not produce l o c a l i z e d carcinomas; t h e r e f o r e , the p r e n e o p l a s t i c and e a r l y n e o p l a s t i c l e s i o n s are d i f f i c u l t t o study, because they are d i f f i c u l t t o l o c a t e . The models which produce l o c a l i z e d tumors are t e c h n i c a l l y cumbersome and not s u i t a b l e f o r l a r g e s c a l e s t u d i e s . These and other disadvantages of the c u r r e n t l y a v a i l a b l e models prompted our search f o r a b e t t e r model. The o b j e c t i v e of t h i s study was t o develop a p r a c t i c a l and r e l i a b l e model of l o c a l i z e d bronchogenic carcinoma i n l a b o r a t o r y rodents, so t h a t one may i n v e s t i g a t e the n e o p l a s t i c process and i n t e r v e n t i o n s - which i n f l u e n c e r e s p i r a t o r y t r a c t c a r c i n o g e n e s i s . - 1 4 -MATERIALS & METHODS Animals Female Camm-Hartley guinea p i g s (Cavia p o r c e l l u s ) , weighing 350 t o 600 g, were used i n t h i s study. They were fed P u r i n a Guinea P i g Chow #5025 ad. l i b i t u m (Ralston Purina Co., S t . L o u i s , M i s s o u r i ) . T h e i r d r i n k i n g water was supplemented with a s c o r b i c a c i d , approximately 0.75 g/1. Female Golden S y r i a n hamsters (Mesocricetus a u r a t u s ) , weighing 90 t o 125 g, were a l s o used. These animals were fed P u r i n a Rodent Laboratory Chow #5001 ad l i b i t u m . Both rodents were obtained from the Charles R i v e r Breeding Laboratory I n c o r p o r a t i o n , W i l l i n g t o n , Massachusetts. A l l animals were kept i n metal cages w i t h mesh f l o o r s over rock s a l t ; the cages were steam cleaned at l e a s t t wice a week. Four to s i x guinea p i g s were housed per cage; the hamsters r e s i d e d i n i n d i v i d u a l cages. Threads Benzo(a)pyrene (3,4-benzopyrene) was obtained from A l d r i c h Chemical Co. Inc..Milwaukee, Wisconsin (catalogue #B1,008-0) and Sigma Chemical Co., St. L o u i s , M i s s o u r i (catalogue #8-1760). The benzo(a)pyrene (BP) was 98% pure, w i t h a melting p o i n t of 175-177°C. Small amounts of BP were placed i n a 1cm x 1cm x 0.5cm m i l l e d c a v i t y i n an aluminum block ( F i g u r e 1) which was placed i n an oven a t 190-210°C. Cotton threads, secured t o an a p p l i c a t o r device, absorbed the molten BP f o r 5 t o 15 minutes i n the oven. At l e a s t twenty threads could be impregnated w i t h BP i n one hour. Large diameter (3.0) and s m a l l diameter (5.0) c o t t o n threads were obtained from E t h i c o n Suture L t d . , Peterborough, Ontario (catalogue #G862H and C182H r e s p e c t i v e l y ) . - 15 -Some threads were coated w i t h a t h i n s i l i c o n e rubber sheath (SRS), composed of 100 p a r t s S i l a s t i c 382 medical grade elastomer t o one p a r t stannous octoate c a t a l y s t , obtained from Dow Corning C o r p o r a t i o n , Midland, Michigan. This was done by slowly dragging and t w i s t i n g BP-impregnated and non-impregnated threads through a pool of c a t a l y z i n g s i l i c o n e rubber on dis p o s a b l e g l a s s s l i d e s ( F i g u r e 2 ); the threads were allowed t o dry, hanging f r e e l y ( F i g u r e 3 ) , to ensure t h a t the SRS remained t h i n and smooth. A s m a l l 3/8 c i r c l e c u t t i n g needle was t i e d t o the prepared threads and t r a n s f e r r e d t o 12 x 75 mm g l a s s t e s t tubes f o r steam s t e r i l i z a t i o n . The amount of p o t e n t i a l carcinogen exposure i n the animals was as c e r t a i n e d g r a v i m e t r i c a l l y by weighing s i m i l a r l y oven t r e a t e d c o t t o n threads, one impregnated with the carcinogen, and one not impregnated with carcinogen. The l e n g t h of the thread sewn i n t o the trachea was a l s o recorded. S u r g i c a l Procedures The animals f a s t e d f o r 24 hours p r i o r t o the o p e r a t i o n . The guinea p i g s were anesthetized i n t r a p e r i t o n e a l l y with 30 t o 35 mg/kg of Somnotol (sodium p e n t o b a r b i t a l ) , obtained from MTC Pharmaceuticals, Hamilton, O n t a r i o . The hamsters received 65 t o 90 mg/kg of i n t r a p e r i t o n e a l Somnotol. With the rodent secured to the o p e r a t i o n board, the v e n t r a l neck was shaved and swabbed with the a n t i b a c t e r i a l s k i n c l e a n s e r , H i b i t a n e (Ayerst L a b o r a t o r i e s , Montreal, Quebec). Using s t e r i l e drapes and blades, and wearing gloves and masks, a 2.0 to 3.0 cm long m i d l i n e s k i n i n c i s i o n was made over the c e n t r a l p o r t i o n o f the v e n t r a l neck. I f r e q u i r e d , 0.1-0.5 ml of 2% x y l o c a i n e ( l i d o c a i n e h y d r o c h l o r i d e ) , from A s t r a Pharmaceuticals, M i s s i s s a u g a , O n t a r i o , was i n j e c t e d l o c a l l y . The underlying subcutaneous t i s s u e and f a s c i a were teased a p a r t , and the two s t r a p (sternohyoid) muscles were separated t o expose the t r a c h e a - 16 -( F i g u r e 4 ) . In the m i d - t h i r d of the t r a c h e a , a thread was sewn around 4 c a r t i l a g e r i n g s i n the v e n t r a l ' w a l l , i n ~ t h e s a g i t t a l plane, and t i e d on the e x t e r i o r surface of the trachea ( F i g u r e s 4 and 5 ) . The wound was i r r i g a t e d w i t h s t e r i l e s a l i n e and the s k i n i n c i s i o n was c l o s e d w i t h three to f i v e s t i t c h e s of 6.0 s i l k s uture. The l e s i o n was then r i n s e d w i t h 3% hydrogen peroxide. Up t o f o u r guinea p i g s and s i x hamsters could be s u c c e s s f u l l y operated on i n one hour. Experiments The f i r s t experiment c o n s i s t e d of 94 female Camm-Hartley guinea p i g s . Of the 48 experimental guinea p i g s , 20 r e c e i v e d BP-impregnated c o t t o n threads and 28 r e c e i v e d BP-impregnated c o t t o n threads w i t h a SRS. Of the 46 c o n t r o l guinea p i g s , 19 r e c e i v e d non-impregnated c o t t o n threads and 27 r e c e i v e d non-impregnated c o t t o n threads w i t h a SRS. The second experiment c o n s i s t e d of 70 female Golden S y r i a n hamsters. Of the 54 experimental hamsters, 17 r e c e i v e d BP-impregnated c o t t o n threads, 37 r e c e i v e d BP-impregnated c o t t o n threads w i t h a SRS. Of the 16 c o n t r o l hamsters, 7 r e c e i v e d non-impregnated c o t t o n threads and 9 r e c e i v e d non-impregnated c o t t o n threads w i t h a SRS. P r e p a r a t i o n of the Tracheas At autopsy, the tracheas were removed and f i x e d f o r 4 t o 24 hours i n 2.5% glutaraldehyde made up i n 0.1M sodium cacodylate b u f f e r (pH 7.3). A f t e r g l u t a r a l d e h y d e f i x a t i o n , the trachea was cut s a g i t t a l l y , i n the plane of the thread. The f i x e d trachea was stored i n 0.1M cacodylate b u f f e r (pH 7.3), dehydrated through ascending grades of a l c o h o l and f i n a l l y embedded i n g l y c o l -- 17 -methracrylate (JB-4 Embedding K i t , P o l y s c i e n c e Inc., Warrington, P e n n s y l v a n i a ) . The embedded trachea was sect i o n e d at 2.0 t o 3.5 microns on a S o r v a l l JB-4 microtome. A minimum of 8 s l i d e s , at 15 t o 30 micron increments, were prepared on at l e a s t one h a l f of the trachea near the thread i n s e r t i o n s i t e . U s u a l l y 12 to 16 s l i d e s were prepared from each h a l f of the tra c h e a . These t r a c h e a l s l i d e s were s t a i n e d i n H a r r i s ' Hematoxylin f o r 10 t o 20 minutes at 60 t o 70°C, washed i n running tap water f o r 10 minutes, p a r t i a l l y dehydrated i n e t h a n o l , c o u n t e r s t a i n e d i n 0.5% e o s i n f o r 2 t o 5 minutes at room temperature, and d e c o l o u r i z e d by quick d i p s i n absolute ethanol followed by acetone. A f t e r immersion f i x a t i o n i n 2.5% glu t a r a l d e h y d e , tumor specimens f o r TEM were p o s t - f i x e d i n 1% osmium t e t r o x i d e , s t a i n e d en bloc w i t h s a t u r a t e d u r a n y l acetate and embedded i n Spurr's l o w - v i s c o s i t y embedding media ( K i t #1916, P o l y s c i e n c e Inc., Warington, Pennsylvania) as p r e v i o u s l y d e s c r i b e d (Hulbert et a l . , 1981). S e c t i o n s were cut w i t h g l a s s knives on a R e i c h e r t U l t r a c u t ultramicrotome, mounted on 100 mesh copper g r i d s , s t a i n e d w i t h u r a n y l acetate and lead c i t r a t e (Hulbert et a l . , 1981) and observed w i t h a P h i l i p s 400 TEM. Four blocks o f hamster SCT were observed f o r the presence or absence of desmosomes and to n o f i l a m e n t s by TEM. The f o u r hamster tracheas had been exposed to. BP f o r 88,91,119 and 215 days. E v a l u a t i o n of the Tracheas To be c o n f i d e n t t h a t BP exposed e p i t h e l i u m was assessed, one s l i d e of the trachea t h a t showed remnants of the threads was s e l e c t e d f o r each animal. These s l i d e s were then i n t e r p r e t e d independently by two p a t h o l o g i s t s (observers A & B) i n a s i n g l e b l i n d f a s h i o n . The most advanced h i s t o p a t h o l o g i c l e s i o n observed was recorded from a continuum of e p i t h e l i a l - 18 -c a t e g o r i e s : mature c i l i a t e d r e s p i r a t o r y e p i t h e l i u m (ME), squamous metaplasia (SM),"three p r o g r e s s i v e "degrees of i n t r a e p i t h e l i a l n e o p l a s i a ((IEN) mi l d --comparable to m i l d e p i t h e l i a l d y s p l a s i a , moderate comparable t o moderate e p i t h e l i a l d y s p l a s i a , and marked comparable t o marked e p i t h e l i a l d y s p l a s i a and carcinoma i n s i t u ) , and squamous c e l l carcinoma (CA). The t r a c h e a l stroma (submucosa, c a r t i l a g i n o u s l a y e r and a d v e n t i t i a ) was f u r t h e r assessed f o r the presence or absence of a s p i n d l e c e l l tumor (SCT). The o v e r a l l i n t e r o b s e r v e r e r r o r between the two p a t h o l o g i s t ' s grading of the t r a c h e a l e p i t h e l i u m was assessed by c o n s t r u c t i n g a 2-sided contingency t a b l e and determining the degree of c o r r e l a t i o n between the observers on 6 e p i t h e l i a l c a t e g o r i e s . The c o r r e c t e d (Yates c o r r e c t i o n ) contigency c o e f f i c i e n t was c a l c u l a t e d . The e f f i c a c y of the d i f f e r e n t thread options t o induce squamous c e l l carcinoma i n hamsters was a l s o evaluated. To determine i f the SRS s i g n i f i c a n t l y i n c r e a s e d the tumor y i e l d , the f r a c t i o n o f hamsters developing carcinoma with a BP-impregnated thread was compared t o the f r a c t i o n developing carcinoma with a BP-impregnated thread w i t h a SRS. The author graded the degree of e p i t h e l i a l inflammation and recorded the presence or absence of k e r a t i n i z a t i o n , i n a l l rodent tracheas. L i v e r , Lungs and Lymph Nodes Samples of l i v e r , lung, and v e n t r a l neck lymph nodes (only i n hamsters) were f i x e d i n 10% f o r m a l i n , prepared by r o u t i n e h i s t o l o g i c methods ( p a r a f f i n wax), s e c t i o n e d at 7 microns and s t a i n e d w i t h H a r r i s ' s Hematoxylin and E o s i n . - 19 -The histopathology of the l i v e r s was assessed i n 10 c o n t r o l guinea p i g s (ranging Trom 55 to"298 "days post-operation) and i n 12 experimental guinea p i g s (ranging from 83 to 265 days p o s t - o p e r a t i o n ) . The histopathology of the lungs was assessed i n 14 c o n t r o l guinea p i g s (ranging from 55 t o 348 days post-operation) and 19 experimental guinea p i g s (ranging from 15 t o 342 days p o s t - o p e r a t i o n ) . The upper and lower lobes o f both the r i g h t and l e f t lung were evaluated. S i m i l a r l y , the l i v e r s were assessed i n 12 c o n t r o l hamsters (ranging from 1 t o 250 days post-operation) and 11 experimental hamsters (ranging from 31 t o 209 days p o s t - o p e r a t i o n ) . Twenty-five hamster lungs were evaluated: 15 c o n t r o l s (ranging from 1 to 250 days post-operation) and 10 experimental animals (ranging from 31 to 180 days p o s t - o p e r a t i o n ) . In a d d i t i o n , the lymph nodes were assessed i n 21 experimental hamsters (ranging from 84 t o 209 days) and i n 5 c o n t r o l hamsters (ranging from 34 t o 210 days p o s t - o p e r a t i o n ) . Animal S u r v i v a l Animals t h a t remained healthy were s a c r i f i c e d at pre-determined times f o r morphology by p e n t o b a r b i t a l overdose, and the animals t h a t developed s t r i d o r were s a c r i f i c e d when i t became severe. Some animals died spontaneously. The mode and date of death were recorded f o r a l l animals and c o n s t i t u t e d part of the s u r v i v a l data. The hamsters e i t h e r died or were s a c r i f i c e d w i t h i n 250 days of the ope r a t i o n and the guinea p i g s w i t h i n 350 days. - 20 -RESULTS Benzo(a)pyrene Content of the Threads Cotton threads were used because they are non-absorbable and were . found to absorb c o n s i d e r a b l y more BP than nylon, s i l k and other s y n t h e t i c suture threads. The 5.0 c o t t o n thread absorbed an average of 106 ug of BP per cm of thread (range of 44 t o 155 ug/cm); whereas, the 3.0 c o t t o n thread absorbed an average of 382 ug of BP per cm of thread (range of 145 t o 865 ug/cm). In the hamsters, an average of 1.4 cm o f thread was i n s e r t e d i n t o the trachea (range of 1.3 t o 1.6 cm), and the guinea p i g s r e c e i v e d an average of 2.1 cm of thread (range of 2.0 t o 2.2 cm). The e x t e r n a l diameter of a 5.0 thread with a SRS approximated t h a t of a 3.0 thread without a SRS. Histopathology of the Tracheas Figure 5 shows the course of the thread i n the v e n t r a l t r a c h e a l w a l l . Note t h a t the thread encompassed a l l l a y e r s of the t r a c h e a : mucosa, submucosa, c a r t i l a g i n o u s l a y e r and a d v e n t i t i a . A low power photomicrograph of a thread w i t h i n the t r a c h e a l w a l l of an experimental hamster ( F i g u r e 6) i s provided f o r o r i e n t a t i o n . The e p i t h e l i u m ( i n d i c a t e d by arrowheads) can be seen along the s u r f a c e of the t r a c h e a l lumen (TL) and a l s o extends down i n t o the stroma(S) along the thread (TD). Areas "B" and "C" are where the p r o l i f e r a t i n g e p i t h e l i u m was i n c l o s e contact w i t h the thread; the most advanced e p i t h e l i a l changes were seen i n t h i s area, and as such, t h i s i s the e p i t h e l i u m which was assessed i n the study. Most of the subsequent photomicrographs are of p o r t i o n s of areas "B" and "C", e s p e c i a l l y the s i t e where the thread p i e r c e d through the t r a c h e a l w a l l i n t o the lumen. Area "A" - 21 -serves as an i n t e r n a l c o n t r o l , as i t d i d not undergo much change other than e p i t h e l i a l h y p e r p l a s i a and inflammation. Histopathology of the Tracheal E p i t h e l i u m i n the C o n t r o l Animals The c e l l u l a r response of guinea p i g trachea t o non-impregnated threads (the c o n t r o l animals) i s shown i n Fi g u r e s 7 through 11. W i t h i n 4 days of the o p e r a t i o n , the t r a c h e a l e p i t h e l i u m adjacent t o the thread showed a regenerative b a s a l c e l l h y p e r p l a s i a (Figures 7 & 8 ) . The normal c i l i a t e d p s e u d o s t r a t i f i e d columnar t r a c h e a l e p i t h e l i u m ( r e f e r t o Figure 12) has been i n s u l t e d by the o p e r a t i o n . In response, the b a s a l c e l l s p r o l i f e r a t e t o r e p a i r the damaged and denuded e p i t h e l i u m . Arrows i n d i c a t e c e l l s undergoing m i t o s i s . The e p i t h e l i u m was a l s o moderately i n f i l t r a t e d w i t h acute inflammatory c e l l s . F i g ure 9 shows an attenuated l a y e r of regenerating squamous e p i t h e l i u m beginning t o cover the thread (TD) w i t h i n the t r a c h e a l lumen (TL), at 20 days po s t - o p e r a t i o n . By 6 months the t r a c h e a l wound was completely healed i n the c o n t r o l guinea p i g s , and the mature t r a c h e a l e p i t h e l i u m was r e c o n s t i t u t e d ( F i g u r e s 10 & 11). The c o t t o n thread (TD) was not resorbed. I t remained i n the t r a c h e a l stroma sheathed by g i a n t c e l l s , macrophages and c o l l a g e n ( F ) . The same sequence of c e l l u l a r changes were seen i n the hamster c o n t r o l s , but i n g e n e r a l , the l e s i o n s healed i n about h a l f the time r e q u i r e d f o r the guinea p i g s . Histopathology of the Tracheal E p i t h e l i u m i n the Experimental Animals The h i s t o p a t h o l o g i c response of the t r a c h e a t o BP-impregnated threads was much d i f f e r e n t than to the non-impregnated threads. In the experimental animals, the regenerating e p i t h e l i u m grew along the thread i n t o . t h e stroma - 22 -c o n s i d e r a b l y more than i n the c o n t r o l animals, and the thread w i t h i n the t r a c h e a l lumen was u s u a l l y only p a r t i a l l y r e - e p i t h e l i z e d . The t r a c h e a l e p i t h e l i u m showed a p r o g r e s s i v e i n c r e a s e i n the degree of i n t r a e p i t h e l i a l n e o p l a s i a , which culminates, i n many of the experimental animals, as squamous c e l l - c a r c i n o m a . The o r d e r l y sequence of mature r e s p i r a t o r y e p i t h e l i u m (ME), t o squamous metaplasia (SM), to m i l d , moderate and marked i n t r a e p i t h e l i a l n e o p l a s i a (IEN) i s shown i n Fi g u r e s 12 t o 17. These f i g u r e s served as the standards from which the observers graded the t r a c h e a l e p i t h e l i u m i n a l l animals i n the study. F i g u r e 12 shows the morphologic appearance of normal guinea p i g t r a c h e a l e p i t h e l i u m . This c i l i a t e d p s e u d o s t r a t i f i e d columnar e p i t h e l i u m c o n s i s t s of three c e l l t ypes: b a s a l c e l l s (arrowheads), c i l i t a t e d columnar c e l l s (curved arrows), and goblet c e l l s (arrowheads). The normal hamster t r a c h e a l e p i t h e l i u m was s i m i l a r ; although, the hamster e p i t h e l i u m tended t o be s l i g h t l y f l a t t e r and the columnar c e l l s more c u b o i d a l . The subsequent e p i t h e l i a l c a t e g o r i e s of squamous metaplasia and i n t r a e p i t h e l i a l n e o p l a s i a were v i r t u a l l y i d e n t i c a l i n the two s p e c i e s of animals. F i g u r e 13 shows the p o l a r i z e d b a s a l c e l l s (arrowheads) and the s t r a t i f i e d squamous e p i t h e l i u m (arrows), t y p i c a l of squamous metaplasia. This was an e a r l y f i n d i n g i n both the experimental and c o n t r o l animals. The squamous metaplasia e v e n t u a l l y r everted back t o normal t r a c h e a l e p i t h e l i u m i n the c o n t r o l s (Figure 10 and 11). But w i t h i n c r e a s i n g exposure to BP, the experimental animals showed p r o g r e s s i v e l y more d y s p l a s i a w i t h i n t h i s squamous m e t a p l a s t i c e p i t h e l i u m . Figure 14 shows an example of m i l d i n t r a e p i t h e l i a l n e o p l a s i a . The b a s a l c e l l s (arrowheads) are reasonably w e l l p o l a r i z e d , but there are a few immature e p i t h e l i a l c e l l s (arrows) i n the s u p e r f i c i a l e p i t h e l i u m . As the b a s a l c e l l s - 23 -lo s e t h e i r o r d e r l y arrangement along the basement membrane and as more immature e p i t h e l i a l c e l l s move t o the s u p e r f i c i a l l a y e r of the e p i t h e l i u m , the d e s i g n a t i o n of moderate i n t r a e p i t h e l i a l n e o p l a s i a i s warranted (Fi g u r e 15). Fig u r e 16 & 17 show examples of marked i n t r a e p i t h e l i a l n e o p l a s i a ; there are immature e p i t h e l i a l c e l l s throughout the height of the e p i t h e l i u m . Numerous a t y p i c a l e p i t h e l i a l c e l l s (arrows), as w e l l as immature e p i t h e l i a l c e l l s , and marked un d u l a t i o n of the b a s a l aspect of the e p i t h e l i u m are evident i n F i g u r e 17. This photomicrograph represents carcinoma i n - s i t u which i s b e l i e v e d t o be the precursor t o i n v a s i v e squamous c e l l carcinoma. We d i d not s p e c i f i c a l l y grade f o r carcinoma i n - s i t u , but i n s t e a d i n c l u d e d i t w i t h i n the c o n f i n e s of the broader category of marked i n t r a e p i t h e l i a l n e o p l a s i a . F i g u r e s 18 t o 21 i l l u s t r a t e the r e s u l t a n t squamous c e l l carcinomas (CA). An example of a l o c a l l y i n v a s i v e squamous c e l l carcinoma, i n a guinea p i g exposed t o BP f o r 265 days, i s shown i n Figur e s 18 & 19. The c e l l u l a r f e a t u r e s . o f a moderately d i f f e r e n t i a t e d squamous c e l l carcinoma are ev i d e n t i n Fig u r e 19: prominent n u c l e o l i , pleomorphism ( v a r i a t i o n i n c e l l u l a r shape), a n i s i o c y t o s i s ( v a r i a t i o n i n c e l l u l a r s i z e ) and m i t o t i c f i g u r e s (the presence of which u s u a l l y suggests i n c r e a s e d r a t e of c e l l d i v i s i o n ) . F i g u r e 20 shows a w e l l d i f f e r e n t i a t e d squamous c e l l carcinoma w i t h tongues o f malignant e p i t h e l i u m p r o j e c t i n g i n t o the un d e r l y i n g stroma, i n a hamster exposed t o BP f o r 139 days. The o v e r a l l morphologic p a t t e r n o f the s o l e guinea p i g squamous c e l l carcinoma ( F i g u r e 18 & 19) and t h i s hamster squamous c e l l carcinoma (Fig u r e 20) are s i m i l a r . However, i n c o n t r a s t t o these two c l a s s i c examples of squamous c e l l carcinoma, a s l i g h t l y d i f f e r e n t microscopic p a t t e r n was seen i n the m a j o r i t y of the hamster tumors. - 24 -S p i n d l e C e l l Tumors The majority" of the experimental hamsters t h a t developed squamous c e l l carcinomas a l s o developed s p i n d l e c e l l tumors i n the t r a c h e a l stroma. F i g u r e s 21, 22 and 23 are microscopic and gross photographs of an experimental hamster a f t e r exposure t o BP f o r 91 days. The low power l i g h t m i croscopic photomicrograph ( F i g u r e 21) shows the two tumor components: the s u p e r f i c i a l m i c r o i n v a s i v e squamous c e l l carcinoma (CA) and the stromal s p i n d l e c e l l tumor (SCT). In s e v e r a l i n s t a n c e s , the malignant s p i n d l e c e l l s appeared t o bud o f f from the invading tongues of squamous c e l l carcinoma. With a high power view ( F i g u r e 22), the c e l l u l a r f e a t u r e s of the a n a p l a s t i c s p i n d l e c e l l tumor are e v i d e n t . The c e l l s contained abnormal n u c l e i w i t h prominent n u c l e o l i , marked i r r e g u l a r i t y o f the nucleus and many m i t o t i c f i g u r e s . The c e l l s had an abundant cytoplasm. Although, the m a j o r i t y of the c e l l s were s p i n d l e or f u s i f o r m shaped, much v a r i a b i l i t y e x i s t e d i n c e l l u l a r shape (pleomorphism), and s i z e ( a n i s i o c y t o s i s ) . The only a d d i t i o n a l f e a t u r e t h a t e l e c t r o n microscopy revealed was t h a t the s p i n d l e c e l l s were f i l l e d w i t h markedly d i l a t e d rough endoplasmic r e t i c u l u m . The search f o r t r u e desmosomes between adjacent malignant s p i n d l e c e l l s was not s u c c e s s f u l , but pseudodesmosomes were found: the cytoplasmic plaques were d i s t i n c t i v e , but evidence of the intermediate l i n e and a s s o c i a t e d t o n o f i l a m e n t s was not seen. The a n a p l a s t i c s p i n d l e c e l l s comprised the m a j o r i t y of the tumor mass on the trachea of the experimental hamsters (Fig u r e 23). W i t h i n the primary t r a c h e a l tumor, the s p i n d l e c e l l s were o f t e n found invading the adjacent s k e l e t a l muscle. A m e t a s t a t i c s p i n d l e c e l l tumor was found as a subcutaneous lump i n the d o r s a l neck of one hamster (same hamster as i n F i g u r e s 21, 22 and 23). The m e t a s t a t i c tumor p l u s the l o c a l i n v a s i o n document the malignant - 25 -p o t e n t i a l of the s p i n d l e c e l l tumor. The t r a c h e a l c a r t i l a g e r i n g s encompassed by the thread underwent remodeling i n a l l of the animals. In the experimental hamsters, e c t o p i c f o c i of abnormal c a r t i l a g e (chondroid metaplasia i n the SCT) emerged o c c a s i o n a l l y and one presumed chondrosarcoma was i d e n t i f i e d . E v a l u a t i o n of the Histopathology of the Tracheal E p i t h e l i u m Tracheal s e c t i o n s were not a v a i l a b l e on a l l rodents. The thread c o u l d not be found at the time of autopsy i n 5 experimental and 3 c o n t r o l guinea p i g s , and thus, these tracheas were not evaluated; 3 experimental and 7 c o n t r o l guinea p i g s died w i t h i n 2 days of the o p e r a t i o n due t o p e n t o b a r b i t a l overdose or improperly i n s e r t e d threads, and these tracheas were not processed. In the hamsters, the thread could not be found i n 3 experimental t r a c h e a s ; one experimental trachea was l o s t f o r study because the animal d i e d , and the trachea was a u t o l y z e d ; no c o n t r o l tracheas were d e l e t e d . This means tha t the tracheas of 40/48 experimental and 36/46 c o n t r o l guinea p i g s , and 50/54 experimental and 16/16 c o n t r o l hamsters were a v a i l a b l e f o r h i s t o p a t h o l o g i c o b s e r v a t i o n . Table 1 shows the r e s u l t s o f the assessment of the o v e r a l l i n t e r o b s e r v e r e r r o r between two p a t h o l o g i s t s i n t h e i r h i s t o p a t h o l o g i c grading of the t r a c h e a l e p i t h e l i u m . This contingency t a b l e i n c l u d e s the morphologic data from c o n t r o l and experimental animals of both s p e c i e s , a t o t a l of 142 observations (76 guinea p i g s and 66 hamsters). The o v e r a l l c o r r e c t e d contingency c o e f f i c i e n t was 82%. The upper l e f t t o lower r i g h t d i a g o n a l c o n t a i n s 77/142 (52%) observations (dashed l i n e ) , where there was p e r f e c t c o r r e l a t i o n between the two observers. To be c o n s e r v a t i v e , i t was decided - 26 -t h a t a d i f f e r e n c e of one e p i t h e l i a l category between observers would be accepted, and t h a t the more benign l e s i o n of the two c a t e g o r i e s would be assigned. This range i n c l u d e d 120/142 (85%) of the data (data w i t h i n the s o l i d l i n e s ) . The remaining 15% (22/142) of the data was not i n c l u d e d i n the subsequent a n a l y s i s because the two p a t h o l o g i s t s d i f f e r e d i n t h e i r i n t e r p r e t a t i o n s of a given t r a c h e a l s l i d e by two or more e p i t h e l i a l c a t e g o r i e s . This l e f t 35 c o n t r o l and 35 experimental guinea p i g s and 15 c o n t r o l and 35 experimental hamsters f o r the f i n a l a n a l y s i s . F i g u r e s 24 & 25 show the h i s t o p a t h o l o g i c grade assigned t o the t r a c h e a l e p i t h e l i u m of each rodent when s a c r i f i c e d at the i n d i c a t e d days a f t e r surgery. Only mature e p i t h e l i u m or squamous metaplasia was seen i n the 35 c o n t r o l guinea p i g s . Two carcinomas were reported i n the 35 experimental guinea p i g s , one at 54 and one at 265 days po s t - o p e r a t i o n . On r e t r o s p e c t i v e a n a l y s i s , the squamous c e l l carcinoma at 54 days i n the experimental guinea p i g proved not to be a t r u e carcinoma (see d i s c u s s i o n ) . M i l d or moderate IEN was observed i n 17/35 (49%) of the experimental guinea p i g s , while ME and SM was reported i n the remaining 16/35 (46%) of the experimental guinea p i g s . In the c o n t r o l hamsters, the mature r e s p i r a t o r y e p i t h e l i u m was r e c o n s t i t u t e d by 80 days p o s t - o p e r a t i o n . The carcinoma reported i n the c o n t r o l hamster was spurious (see d i s c u s s i o n ) . On the other hand, 11/35 (31%) of the experimental hamsters demonstrated IEN. The f i r s t squamous c e l l carcinoma was seen at 55 days p o s t - o p e r a t i o n , another at 82 days and s e v e r a l t h e r e a f t e r . O v e r a l l , 15/35 (43%) of the experimental hamsters had developed squamous c e l l carcinoma by the end of the study. Spindle c e l l tumors (SCT) were a l s o p l o t t e d as a f u n c t i o n of time ( F i g u r e 25); 15/35 (43%) o f the experimental hamsters developed a s p i n d l e c e l l tumor. - 27 -E f f i c a c y of D i f f e r e n t Thread Options t o Induce Squamous C e l l Carcinoma Table 2 presents the data e v a l u a t i n g the e f f i c a c y of the d i f f e r e n t thread options t o induce squamous c e l l carcinomas i n the hamsters. The experimental hamsters t h a t had a thread w i t h a s i l i c o n e rubber sheath (SRS) developed carcinomas i n 13/25 (52%) animals; only 2/10 (20%) of the experimental hamsters w i t h threads l a c k i n g the SRS developed carcinomas. Furthermore, i t i s of i n t e r e s t that 1/23 (4%) of the experimental guinea p i g s c o n t a i n i n g a thread w i t h a SRS developed carcinoma; whereas, 0/12 of the experimental guinea p i g s t h a t had a thread l a c k i n g the SRS developed carcinoma. Inflammation and K e r a t i n i z a t i o n of the Tracheal E p i t h e l i u m In the guinea p i g s , the e p i t h e l i a l inflammation was comparable f o r both experimental and c o n t r o l animals. At the end of the study, 50% of the guinea p i g s showed m i l d e p i t h e l i a l inflammation, 30% moderate inflammation, 5% marked inflammation. In the hamsters, there was c o n s i d e r a b l y more e p i t h e l i a l inflammation i n the experimental than i n the c o n t r o l animals. In the experimental hamsters, 55% showed m i l d e p i t h e l i a l inflammation, 20% moderate inflammation and 25% no inflammation. Whereas i n the c o n t r o l hamsters, 15% show mil d e p i t h e l i a l inflammation and the remaining 85% showed no inflammation. The e p i t h e l i a l inflammation was not s p e c i f i c a l l y graded f o r the p r o p o r t i o n s of acute and c h r o n i c inflammatory c e l l s . But the o v e r a l l impression was t h a t the hamsters tended t o have more acute inflammatory c e l l s w i t h i n the e p i t h e l i u m than d i d the guinea p i g s . E a r l y i n the study, the stroma i n both.animals was i n f i l t r a t e d with.acute inflammatory c e l l s . But l a t e r on, c h r o n i c inflammatory c e l l s were more prominent, e s p e c i a l l y i n the form of a f o r e i g n body response t o the thread. - 28 -K e r a t i n i z a t i o n of the e p i t h e l i u m was a prominent f i n d i n g i n the experimental hamsters; 23/50 (46%) of the hamsters demonstrated k e r a t i n i z a t i o n . Conversely, only 3/48 (6%) of the experimental guinea p i g s showed evidence of k e r a t i n i z a t i o n . N e i t h e r the hamster nor the guinea p i g c o n t r o l s showed any k e r a t i n i z a t i o n . The k e r a t i n i z a t i o n was u s u a l l y found i n the s u p e r f i c i a l l a y e r s of e p i t h e l i u m (Figure 13) but some i n t r a e p i t h e l i a l k e r a t i n i z a t i o n (Figure 17) was noted, p a r t i c u l a r l y the k e r a t i n p e a r l s i n the nests of squamous c e l l carcinoma. H y p e r k e r a t i n i z a t i o n and parakeratoses was o f t e n found i n the t r a c h e a l e p i t h e l i u m of the experimental hamsters. Complications of the Model One of the main c o m p l i c a t i o n s of t h i s model i s t h a t i n a s m a l l percentage of animals, the threads were extruded i n t o the t r a c h e a l lumen. Ten percent (5/48) of the experimental guinea p i g s extruded t h e i r threads by the end of the study versus seven percent (3/46) of the c o n t r o l s . S i x percent (3/54) of the experimental hamsters extruded t h e i r threads while a l l (16) threads remained i n t a c t i n the c o n t r o l s . An abscess formed around the thread w i t h i n the t r a c h e a l w a l l i n one animal, mimicking a tumor g r o s s l y . Threads not t i e d f i r m l y around the c a r t i l a g e r i n g s hung l a x i n the lumen, causing o b s t r u c t i o n of the trachea and premature demise of the rodents. Lymph Nodes, L i v e r and Lung The v e n t r a l neck lymph nodes from 5/5 c o n t r o l hamsters were normal, as were the 8/21 lymph nodes from the experimental hamsters. The o t h e r 13/21 lymph nodes i n the experimental hamsters showed plasma c e l l h y p e r p l a s i a , but - 29 -there was no m e t a s t a t i c carcinoma present. A few of these lymph nodes contained d i s t i n c t f o r e i g n body granulomas; the nature o f the i n c i t i n g agent i s not known, but c a l c i f i e d d e b r i s , c o n s i s t e n t w i t h thread, was found i n some granulomas. In the c o n t r o l guinea p i g l i v e r s , A/10 l i v e r s were normal; 5/10 showed d i f f u s e f a t t y changes; 1/10 showed evidence of m i l d p e r i p o r t a l damage (hepatocyte dropout and f i b r o s i s around the p o r t a l t r i a d ) . S l i g h t l y more damage was noted i n the l i v e r s from the experimental guinea p i g s : A/12 l i v e r s appeared normal, while the remaining 8/12 l i v e r s demonstrated f a t t y changes and m i l d p e r i p o r t a l damage. I n t e r e s t i n g l y , 3/12 l i v e r s from the experimental animals showed i s l a n d s o f hematopoietic t i s s u e i n the l i v e r s i n u s o i d s ( e x t r a m e d u l l a r hematopoiesis). In the hamster c o n t r o l s , A/12 l i v e r s were normal w h i l e the remaining 8/12 showed f a t t y changes and m i l d p e r i p o r t a l damage. The same spectrum o f changes was observed i n 11 of the experimental hamster l i v e r s , but the p e r i p o r t a l damage was g r e a t e r than i n the c o n t r o l s . In the lungs from the c o n t r o l guinea p i g s , 3/1A showed h i s t o p a t h o l o g i c evidence of m i l d bronchopneumonia, compared t o 8/19 of the experimental guinea p i g s . The remaining lungs, 11/1A of the c o n t r o l and 11/19 of the experimental guinea p i g s , were normal. A l l 15 lungs from c o n t r o l hamsters were normal. One of 10 lungs from the experimental hamsters showed a f o c a l bronchopneumonia, l i k e l y due t o a s p i r a t i o n during the i n i t i a l o p e r a t i o n , as f o r e i g n body g i a n t c e l l s were a l s o present. C u r i o u s l y , one experimental hamster showed an intrapulmonary v a s c u l i t i s , without other evidence of parenchymal damage. - 30 -Animal S u r v i v a l T h e " o v e r a l l cumulative "morbidity and m o r t a l i t y i n the guinea p i g s and hamsters i s presented i n F i g u r e s 26 & 27. As would be expected, the c o n t r o l animals survived longer than the experimental animals. By the end of the study, 35% of the c o n t r o l guinea p i g s had e i t h e r d i e d spontaneously o r became s i c k enough that they were s a c r i f i c e d , as opposed t o 70% f o r the experimental guinea p i g s . The same o v e r a l l f i g u r e s were observed i n the hamsters. For both the guinea p i g and hamster c o n t r o l s , the mor b i d i t y and m o r t a l i t y does not inc r e a s e much a f t e r 150 t o 100 days, r e s p e c t i v e l y . However, both the experimental rodents undergo an increase i n morbidity and m o r t a l i t y w i t h time, t h a t l e v e l s o f f i n the l a t t e r t h i r d o f the study p e r i o d . - 31 -PLATE I F i g u r e 1 Impregnation of c o t t o n threads with benzo(a)pyrene (BP). Cotton . threads secured t o a p p l i c a t o r device (arrow) were dipped i n t o the aluminum block c o n t a i n i n g molten BP. Fi g u r e 2 P r e p a r a t i o n o f the s i l i c o n e rubber sheath (SRS). Cotton threads were slo w l y dragged through a puddle o f c a t a l y z i n g s i l i c o n e rubber (arrow). F i g u r e 3 Enlarged view o f apparatus used t o coat c o t t o n threads w i t h a SRS. Coated threads were allowed t o dry hanging f r e e l y (arrow). - 33 -PLATE I I F i g u r e 4 Diagram o f the s u r g i c a l procedure performed on the rodents. A f t e r i n t r a p e r i t o n e a l anesthesia, a m i d l i n e s k i n i n c i s i o n was made. The underlying subcutaneous t i s s u e and f a s c i a was teased apart, and the two strap muscles (arrow heads) were separated t o expose the trachea. In the m i d - t h i r d of the trachea, a thread (arrow) was sewn around 4 c a r t i l a g e r i n g s i n the v e n t r a l w a l l and t i e d on the e x t e r i o r s u r f a c e of the trachea. F i g u r e 5 Diagram of a tumor (arrowhead) forming around the thread (arrow) w i t h i n the v e n t r a l t r a c h e a l w a l l . The thread encompassed a l l l a y e r s o f the tr a c h e a : mucosa, submucosa, c a r t i l a g i n o u s l a y e r and a d v e n t i t i a . F i g u r e 6 Low power photomicrograph of a thread w i t h i n the t r a c h e a l w a l l o f an experimental hamster. Areas "B" and "C" are where the p r o l i f e r a t i n g e p i t h e l i u m was i n c l o s e contact w i t h the thread; the most advanced e p i t h e l i a l changes were seen i n t h i s area, and as such, t h i s i s the e p i t h e l i u m which was assessed i n the study. The subsequent photomicrographs are of p o r t i o n s o f areas "B" and "C". Area "A" served as an i n t e r n a l c o n t r o l ; f o r the most p a r t , i t d i d not undergo much change other than e p i t h e l i a l h y p e r p l a s i a and inflammation. The thread (TD) can be seen wrapped around the c a r t i l a g e r i n g s (arrows). The e p i t h e l i u m i s i n d i c a t e d by arrowheads, and an "S" marks the t r a c h e a l stroma (submucosa, c a r t i l a g e n o u s l a y e r and a d v e n t i t i a ) . The t r a c h e a l lumen (TL) separates the d o r s a l w a l l (area "A") from the v e n t r a l w a l l (area "B" and "C") x 25. ' • • " - 35 -PLATE I I I F i g u r e 7 E p i t h e l i u m adjacent to the s i t e where the thread p i e r c e s i n t o the t r a c h e a l lumen (TL), i n a guinea p i g c o n t r o l , A days p o s t - o p e r a t i o n . An arrow marks a c e l l undergoing m i t o s i s i n the p r o l i f e r a t i n g e p i t h e l i u m . The e p i t h e l i u m i s moderately inflamed, as i s the underlying stroma (S) x 172. F i g u r e 8 A high power view of c e l l s i n the aforementioned t r a c h e a l e p i t h e l i u m ( F i g u r e 7) undergoing m i t o s i s (arrows), documenting the rampant c e l l u l a r p r o l i f e r a t i o n . Acute inflammatory c e l l s (arrowheads) are found w i t h i n the e p i t h e l i u m x AAO. Figure 9 An attenuated l a y e r of regenerating squamous e p i t h e l i u m (arrow) covers the thread (TD) w i t h i n the t r a c h e a l lumen (TL), guinea p i g c o n t r o l , 20 days p o s t - o p e r a t i o n . Adjacent t o the thread i s r e l a t i v e l y mature r e s p i r a t o r y e p i t h e l i u m (arrowhead) which o v e r l i e s a moderately inflamed stroma (S) x 172. F i g u r e 10 Low power view of a trachea i n a guinea p i g c o n t r o l , 183 days p o s t - o p e r a t i o n . The mature r e s p i r a t o r y e p i t h e l i u m (arrowhead) i s completely r e c o n s t i t u t e d . Note the thread (TD) i n submucosa, the t r a c h e a l c a r t i l a g e r i n g (C) and the attendant f i b r o s i s (F) around the thread x 70. Figure 11 High power view of mature t r a c h e a l e p i t h e l i u m (arrowhead), corresponding to the aforementioned, low power photomicrograph ( F i g u r e 10). Chronic inflammatory c e l l s are seen i n the submucosa. F i b r o s i s (F) around a thread remnant i s present x 172. - 37 -PLATE IV F i g u r e 12 High power view of normal guinea p i g t r a c h e a l e p i t h e l i u m (hamster t r a c h e a l e p i t h e l i u m i s s i m i l a r ) . T h i s c i l i a t e d p s e u d o s t r a t i f i e d columnar e p i t h e l i u m c o n s i s t s of three c e l l t y p e s : b a s a l c e l l s (arrowheads), c i l i a t e d columnar c e l l s (curved arrows) and goblet c e l l s ( s t r a i g h t arrows) x 440. Fi g u r e 13 Squamous metaplasia (SM) with k e r a t i n i z a t i o n (K) i n an experimental hamster. The p o l a r i z e d b a s a l c e l l s (arrowhead) l i e beneath the s u p e r f i c i a l squamous c e l l s (arrow). A c a r t i l a g e r i n g (C) i s marked x 172. Fi g u r e 14 M i l d i n t r a e p i t h e l i a l n e o p l a s i a ( m i l d e p i t h e l i a l d y s p l a s i a ) i n an experimental hamster. Again the b a s a l c e l l s (arrowheads) are reasonably w e l l p o l a r i z e d , but there are a few immature c e l l s (arrows) i n the s u p e r f i c i a l e p i t h e l i u m x 172. Figure 15 Moderate i n t r a e p i t h e l i a l n e o p l a s i a (moderate e p i t h e l i a l d y s p l a s i a ) i n an experimental hamster. The b a s a l c e l l s (arrowheads) are now poo r l y p o l a r i z e d , and immature c e l l s (arrow) are found i n s u p e r f i c i a l l a y e r s of the e p i t h e l i u m . The u n d e r l y i n g c a r t i l a g e (C) i s i n d i c a t e d x 172. Fi g u r e 16 Marked i n t r a e p i t h e l i a l n e o p l a s i a (marked e p i t h e l i a l d y s p l a s i a ) i n an experimental hamster. The b a s a l c e l l s (arrowhead) are i n d i c a t e d . Immature c e l l s (arrows) can be found throughout the e p i t h e l i u m x 172. - 38 -F i g u r e 17 Marked i n t r a e p i t h e l i a l n e o p l a s i a (carcinoma i n s i t u ) i n an experimental hamster. "The basal aspect of the e p i t h e l i u m i s undulated (arrowhead), and the malignant e p i t h e l i a l c e l l s are ready to break the c o n f i n e s of the basement membrane. A t y p i c a l c e l l s (arrows) are present. K e r a t i n i z a t i o n i s e v i d e n t , both at the e p i t h e l i a l s u r f a c e (K) and i n t r a e p i t h e l i a l (IEK) x 172. - 40 -PLATE V F i g u r e 18 Low power view of nests of malignant squamous c e l l carcinoma (CA) invading the t r a c h e a l w a l l i n a guinea p i g exposed t o BP f o r 265 days x 70. F i g u r e 19 High power view of F i g u r e 18 i l l u s t r a t i n g the squamous c e l l carcinoma i n the guinea p i g trachea. The pleomorphism ( v a r i a t i o n i n c e l l u l a r shape), a n i s i o c y t o s i s ( v a r i a t i o n i n c e l l u l a r s i z e ) and m i t o t i c f i g u r e s (arrow) t y p i c a l o f malignant e p i t h e l i u m are evident i n t h i s photomicrograph x 440. Figure 20 Well d i f f e r e n t i a t e d squamous c e l l carcinoma (CA) i n a hamster exposed t o BP f o r 139 days x 172. F i g u r e 21 A photomicrograph of a s u p e r f i c i a l m i c r o i n v a s i v e squamous c e l l carcinoma (CA), and accompanying s p i n d l e c e l l tumor (SCT) i n the stroma. The two tumor components appear t o merge (arrows). F i g u r e s 21, 22 and 23 are of the same hamster, exposed t o BP f o r 91 days x 172. Figure 22 High power view of F i g u r e 21 showing the s p i n d l e c e l l tumor. S e v e r a l m i t o t i c f i g u r e s (arrows) are i n d i c a t e d , amongst the other a n a p l a s t i c s p i n d l e c e l l s x 440. Figure 23 Gross appearance of a t r a c h e a l tumor (TU) i n a hamster. - 42 -TABLE I ASSESSMENT OF THE OVERALL INTEROBSERVER ERROR IN THE HISTOPATHOLOGICAL GRADING OF THE TRACHEAL EPITHELIUM OBSERVER A ME SM IENi I E N 2 IEN3 CA 0 ME 32. 5 0 0 0 B S SM 5 7 0 0 E R I E N i 1 " 6 ^ 4 0 V E IEN2 1 7 5 ^ 1 R IEN3 0 2 2 1 ^ 3 B CA 0 2 1 4 ^ 2 " 18 STATISTICS: Corrected contingency c o e f f i c i e n t = 82%. (a p e r f e c t c o r r e l a t i o n i s 100%) These data represent the h i s t o p a t h o l o g i c o b s e r v a t i o n of the t r a c h e a l e p i t h e l i u m by two p a t h o l o g i s t s (observer A & B) on a t o t a l of 142 rodents (76 guinea p i g s and 66 hamsters). Each datum denotes the number of corresponding h i s t o p a t h o l o g i c o b s e r v a t i o n s ; the upper l e f t to lower r i g h t diagonal of data i s the number of p e r f e c t h i s t o p a t h o l o g i c c o r r e l a t i o n s between the observors. The data w i t h i n the s o l i d l i n e s are the acceptable morphologic o b s e r v a t i o n s . - 43 -PLATE VI Fi g u r e 24 H i s t o p a t h o l o g i c data of guinea p i g t r a c h e a l e p i t h e l i u m : c o n t r o l s (dashed l i n e ) and experimental ( s o l i d l i n e ) animals. Each p o i n t represents a rodent s a c r i f i c e d at the i n d i c a t e d time. Only those morphologic i n t e r p r e t a t i o n s w i t h an acceptable i n t e r o b s e r v e r e r r o r are i n c l u d e d . (35 c o n t r o l and 35 experimental guinea p i g s ) . The carcinoma (CA) reported i n the experimental guinea p i g at 54 days was not. a CA; i t was a pseudoepithelomatous h y p e r p l a s i a , w i t h m i l d i n t r a e p i t h e l i a l n e o p l a s i a . F i g u r e 25 H i s t o p a t h o l o g i c data o f hamster t r a c h e a l e p i t h e l i u m : c o n t r o l s (dashed l i n e ) and experimental ( s o l i d l i n e ) animals. Each p o i n t represents a rodent s a c r i f i c e d at the i n d i c a t e d time. Only those morphologic i n t e r p r e t a t i o n s w i t h an acceptable i n t e r o b s e r v e r e r r o r are i n c l u d e d . (15 c o n t r o l and 35 experimental hamsters). The hamsters t h a t developed a s p i n d l e c e l l tumor (SCT) i n the stroma are denoted above by an "x" at the i n d i c a t e d s a c r i f i c e times; only data p o i n t s w i t h p e r f e c t i n t e r o b s e r v e r c o r r e l a t i o n are i n c l u d e d (15 experimental hamsters). The carcinoma (CA) reported i n a c o n t r o l hamster at 20 days was not a CA; i t was a c o a l e s c i n g group o f a c t i v a t e d macrophages ( e p i t h e l o i d c e l l s ) i n the stroma which appeared to a r i s e from the o v e r l y i n g m e t a p l a s t i c squamous e p i t h e l i u m . I I 1 1 1 50 100 150 200 250 Days Post Operation - 45 -TABLE 2 EFFICACY OF THE DIFFERENT THREAD OPTIONS TO INDUCE SQUAMOUS CELL CARCINOMAS IN HAMSTERS CONTROL HAMSTERS EXPERIMENTAL HAMSTERS A B C . D non-impregnated non-impregnated BP-impregnated BP-impregnated thread thread & SRS thread thread & SRS # of hamsters - * not . 7 8 8 12 developing CA # of hamsters 0 1 2 13 developing CA Only the number o f squamous c e l l carcinomas (CA) which both observers independently agreed upon are i n c l u d e d i n t h i s data. The one carcinoma reported i n the c o n t r o l s was not a carcinoma (see legend f o r Fi g u r e 25). The presence of the s i l i c o n e rubber sheath (SRS) appears t o i n c r e a s e the e f f i c a c y of inducing squamous c e l l carcinomas i n hamsters; a chi-squared a n a l y s i s o f C versus D i s s i g n i f i c a n t at p £ 0.10. 46 Numbering error . Text f o r page 46 not a v a i l a b l e . - 47 -PLATE VII Fi g u r e 26 The o v e r a l l cumulative summed m o r t a l i t y and mor b i d i t y i n the guinea p i g s , expressed as a percentage of the i n i t i a l number of guinea p i g s r e c r u i t e d f o r surgery, i s shown i n s o l i d bars. The cumulative percentage of animals that were healthy but s a c r i f i c e d f o r morphology i s shown i n open bars. A t o t a l of 46 c o n t r o l (Con) and 48 experimental (Exp) guinea p i g s were used i n t h i s study. F i g u r e 27 The o v e r a l l cumulative summed m o r t a l i t y and mo r b i d i t y i n the hamsters, expressed as a percentage of the i n i t i a l number of hamsters r e c r u i t e d f o r surgery, i s shown i n s o l i d bars. The cumulative percentage of animals t h a t were healthy but s a c r i f i c e d f o r morphology i s shown i n open bars. A t o t a l o f 16 c o n t r o l (Con) and 54 experimental (Exp) hamsters were used i n t h i s study. - ¥ 8 -»-z U J o o a. < »— o t-X 100 •o 60 4 0 20 CUMULATIVE HAMSTER SURVIVAL L E G E N D C3 h e a l t h y H i s i c k & dead con con oxp con con oxp N*5 N » 7 N = « N*24 N-11 N-43 N = U N»51 N*16 N « 5 4 0-50 0-100 0-150 0 -200 DAYS POST-OPERATION 0 - 2 5 0 - 49 -DISCUSSION C o n s i d e r a t i o n of the H i s t o p a t h o l o g i c R e s u l t s Since we were i n t e r e s t e d i n developing a model of bronchogenic carcinoma which allowed us t o study the e p i t h e l i u m at v a r i o u s stages i n the development of the carcinoma, we purposely s a c r i f i c e d animals during the p r e n e o p l a s t i c phase, before carcinomas developed. Since grading IEN i s i n v a r i a b l y judgemental, i t was necessary t o c o n s t r u c t standards (Fi g u r e 12 t o 17) and t o a s c e r t a i n the degree of i n t e r o b s e r v e r c o r r e l a t i o n between the independent i n t e r p r e t a t i o n s of the two h i s t o p a t h o l o g i s t s . O v e r a l l , a good degree of i n t e r o b s e r v e r c o r r e l a t i o n was observed (see Table I ) , w i t h a c o r r e c t e d contingency c o e f f i c i e n t of 82%. E x c e l l e n t c o r r e l a t i o n was obtained when assess i n g mature e p i t h e l i u m , sguamous metaplasia and squamous c e l l carcinoma. And as t o be expected, there was some d i s p a r i t y between the two observers when reading the IEN. In order t o maintain the accuracy of the h i s t o p a t h o l o g i c data, readings which d i f f e r e d by more than one e p i t h e l i a l category were excluded (22 of 142) from the f i n a l r e s u l t s . Because the g r e a t e s t i n t e r o b s e r v e r e r r o r was seen when grading IEN, excluding these r e s u l t s t r a n s l a t e s t o excluding animals w i t h IEN. The i n t r a o b s e r v e r e r r o r was not assessed. The author spent c o n s i d e r a b l e time reviewing the same s l i d e s and agreed on a l l the d i a g n o s i s except 2 out of 142. On a r e t r o s p e c t i v e a n a l y s i s , the squamous c e l l carcinoma at 54 days i n the experimental guinea p i g proved not to be a t r u e carcinoma, but r a t h e r , pseudoepitheliomatous h y p e r p l a s i a mimicking a carcinoma. The other squamous c e l l carcinoma i n the guinea p i g was r e a l ( F i g u r e s 18 & 19). S i m i l a r l y , the carcinoma reported i n the c o n t r o l - 50 -hamster at 20 days p o s t - o p e r a t i o n was an exuberant stromal r e a c t i o n t o the thread, numerous a c t i v a t e d macrophages w i t h an e p i t h e l o i d appearance which appeared t o merge w i t h the o v e r l y i n g squamous m e t a p l a s t i c e p i t h e l i u m . The author's h i s t o p a t h o l o g i c assessment of the t r a c h e a l e p i t h e l i u m was excluded from the data because i t was not performed b l i n d . The IEN emerged e a r l y , w i t h i n 40 days, i n both experimental animals (Fi g u r e 24 & 25). Although i t was not s p e c i f i c a l l y t e s t e d , i t appeared.that the experimental animals f i t t e d with threads l a c k i n g the SRS developed IEN e a r l i e r than animals possessing threads w i t h a SRS. This might be expected, because without the SRS t o c o n t r o l the r e l e a s e r a t e , the BP would d i f f u s e out of the thread r e l a t i v e l y q u i c k l y t o induce IEN, but the l e s i o n s c o u l d then be r e p a i r e d ( r e c a l l t h a t IEN i s a p o t e n t i a l l y r e v e r s i b l e l e s i o n ) . The moderate degree of IEN i n the guinea p i g s s a c r i f i c e d at 30 t o 50 days p o s t - o p e r a t i o n , and the l a c k of p r o g r e s s i o n of the IEN w i t h time r e f l e c t s t h i s n o t i o n , most of the guinea p i g s i n t h i s e a r l y phase of the study were animals f i t t e d w i t h the l a r g e diameter (3.0) thread without a SRS. • IEN was seen i n the t r a c h e a l e p i t h e l i u m of s e v e r a l experimental hamsters, s a c r i f i c e d before 70 days p o s t - o p e r a t i o n . This i s not r e f l e c t e d i n the data presented i n f i g u r e 25, as there was r e l a t i v e l y more disagreement between the two observers when assessing these s l i d e s w i t h IEN, and many of these data p o i n t s were di s c a r d e d . A few of the experimental hamsters were found t o have only squamous metaplasia or mature r e s p i r a t o r y e p i t h e l i u m , d e s p i t e an adequate d u r a t i o n of exposure t o BP. This might be e x p l a i n e d by d i f f e r e n c e s amongst i n d i v i d u a l animals i n m e t a b o l i z i n g BP, c l e a r i n g the BP, or r e p a i r i n g the BP mediated c e l l damage. Since the amount of BP per thread v a r i e d by up t o 400 percent, another p o s s i b l e e x p l a n a t i o n i s t h a t the - 51 -experimental animals not developing IEN or CA r e c e i v e d threads with l e s s BP. When c o n s i d e r i n g the y i e l d of carcinoma i n t h i s animal model, r a t h e r than s t a t i n g the number of carcinomas that developed over the e n t i r e d u r a t i o n of the study as a f r a c t i o n of a l l the animals entered i n t o the study (a cumulative measure of cancer i n c i d e n c e ) , i t i s p r e f e r a b l e t o c a l c u l a t e the number of carcinomas that develop a f t e r a minimum time f o r i n d u c t i o n of cancer. In the experimental guinea p i g s , a reasonable minimal time f o r cancer development i s 200 days. There were 11 experimental guinea p i g s s a c r i f i c e d a f t e r 200 days and up t o 350 days, w i t h one squamous c e l l carcinoma - a carcinoma y i e l d of 1/11 ( 9 % ) . With the minimum cancer i n d u c t i o n time set at 120 days i n the hamsters, the carcinoma y i e l d i s 9/14 (65% ) . The data suggest t h a t the hamster i s the most u s e f u l animal t o study bronchogenic squamous c e l l carcinomas; and t o o b t a i n the g r e a t e s t y i e l d s , they should be s a c r i f i c e d between 120 t o 150 days a f t e r the ope r a t i o n . F u r t h e r t o the low y i e l d of carcinomas observed i n the guinea p i g s , the spontaneous tumor rat e i n guinea p i g s i s extremely low (Warren and Gates, 1941, and Rogers and Blumenthal, 1960). The pulmonary tumors t h a t form spontaneously are p e r i p h e r a l a l v e o l a r adenoma, hemangiosarcoma, lymphangioma, i n t r a b r o n c h i a l papilloma and adenomatosis ( H o c h - L i g e t i e t a l . , 1983). Guinea p i g s do not develop the common h i s t o l o g i c types o f pulmonary tumors seen i n humans. I t i s g e n e r a l l y considered t h a t guinea p i g s are r e l a t i v e l y r e s i s t a n t to e x p e r i m e n t a l l y induced r e s p i r a t o r y t r a c t tumors ( B l a c k l o c k , 1961, Russel and Ortega, 1952 and Hoc h - L i g e t i and Argus, 1970). However, some i n v e s t i g a t o r s have been able to induce pulmonary tumors w i t h o r a l • d i e t h y l n i t r o s a m i n e (Argus and H o c h - L i g e t i , 1963), gavage dimethylmorpholine ( L i j i n s k y and Reuben, 1981), and intravenous i n j e c t i o n o f - 52 -methylcholanthrene and dibenzanthracene (Heston and Deringer, 1952). Nevertheless, "the tumor y i e l d s of these guinea p i g models are low, and the tumors are p r i m a r i l y p e r i p h e r a l adenomas and adenomatous l e s i o n s . The one bona f i d e squamous c e l l carcinoma, which we observed i n the guinea p i g trachea i s the only t r a c h e o b r o n c h i a l squamous c e l l carcinoma and the only BP-induced carcinoma i n guinea p i g s found i n reviewing the l i t e r a t u r e . A l a r g e subset of bronchogenic carcinomas i n humans are squamous c e l l , as are the tumors i n these rodents. BP i s a major carcinogen i n c i g a r e t t e smoke, and i t i s the carcinogen used i n t h i s animal model. The human b r o n c h i a l e p i t h e l i u m can metabolize BP (Prough et a l . , 1979; Autrup et a l . , 1980; Grover et a l . , 1976; and H a r r i s et a l . , 1982), as can the t r a c h e a l e p i t h e l i u m of hamsters (Eastman et a l . , 1981, Moore and Cohen, 1978, and Mass and Kaufman, 1978). The human b r o n c h i a l versus the rodent t r a c h e a l s i t e of tumor formation i s an expected d i f f e r e n t and does not i n v a l i d a t e the comparison, e s p e c i a l l y when i t i s known t h a t repeated i n t r a t r a c h e a l i n s t i l l a t i o n of BP i n t o hamster lungs, s i m u l a t i n g i n h a l a t i o n o f c i g a r e t t e smoke i n t o human lungs, produces b a s i c a l l y the same spectrum and d i s t r i b u t i o n of pulmonary tumors as i n humans ( S a f f i o t t i et a l . , 1968). Although t h i s animal model may be not u s e f u l t o study human s m a l l or l a r g e c e l l carcinomas and adenocarcinomas, i t c e r t a i n l y w i l l be u s e f u l to study squamous c e l l bronchogenic carcinomas. E f f i c a c y of D i f f e r e n t Thread Options t o Induce Squamous C e l l Carcinoma I n i t i a l l y d uring the development of the model, the BP-impregnated threads were not coated w i t h a s i l i c o n e rubber sheath (SRS). L a t e r , i n an e f f o r t to i n c r e a s e the e f f i c a c y of i n d u c i n g carcinomas, SRS were added t o the - 53 -thread t o decrease the d i f f u s i o n r a t e of BP i n t o the t i s s u e s . The h a l f l i f e of r e l e a s e of BP from t h i s p a r t i c u l a r s i l i c o n e rubber matrix i s about 2 months (Shors et a l . , 1980). The r e l e a s e r a t e of BP from c o t t o n threads was not measured. But s i n c e there was l e s s impediment t o d i f f u s i o n , i t was assumed t h a t threads without the SRS would r e l e a s e the BP f a s t e r than threads w i t h a SRS. Work with tumors induced with 7,12-dimethylbenz(a)anthracene i n r a t tracheas have i n d i c a t e d t h a t lower doses of carcinogen d e l i v e r e d s l o w l y are more e f f e c t i v e i n producing d y s p l a s t i c and n e o p l a s t i c l e s i o n s than higher doses d e l i v e r e d r a p i d l y (Shiba et a l . , 1982). Presumably, the p r o t r a c t e d lower BP exposure i n c r e a s e s the i n c i d e n c e of c e l l i n i t i a t i o n and thereby i n c r e a s e s the p r o b a b i l i t y of inducing a carcinoma. Whereas, a s h o r t e r d u r a t i o n of concentrated BP exposure i s t o x i c t o the c e l l and thus i s l e s s e f f e c t i v e i n inducing carcinomas. The drawback of adding a SRS t o the thread i s t h a t i t takes s l i g h t l y longer to prepare the thread, but t h i s appears worthwhile as the data (Table 2) do suggest t h a t the SRS i n c r e a s e s the y i e l d of carcinomas. The c o n c e n t r a t i o n of BP i n the thread was an u n c o n t r o l l e d v a r i a b l e , as both 3.0 ( l a r g e diameter) and 5.0 ( s m a l l diameter) c o t t o n threads were used i n t h i s study. The 3.0 BP-impregnated thread exposes the animals t o three t o fo u r times the amount of BP as a 5.0 thread, 535 t o 800 ug and 150 t o 225 ug of BP r e s p e c t i v e l y . The d i f f e r e n t i a l response of the t r a c h e a l e p i t h e l i u m t o the d i f f e r e n t s i z e threads ( d i f f e r e n t p o t e n t i a l BP exposures) was not determined because the groups were s m a l l i n number i f a l s o subgrouped i n t o those w i t h and without a SRS. Nevertheless, the c o n c e n t r a t i o n of BP appeared to be l e s s important than the presence of the SRS i n the i n d u c t i o n of carcinomas, because no experimental animals w i t h a 3.0 thread (0/1 hamsters and 0/12 guinea p i g s ) developed CA. - 54 -Tissue O r i g i n of the S p i n d l e C e l l Tumor The"squamous c e l l " c a r c i n o m a s which were found i n the experimental animals are c l e a r l y d e r i v e d from the t r a c h e a l e p i t h e l i u m . However, the c e l l u l a r d e r i v a t i o n of the stromal s p i n d l e c e l l tumor (SCT) i n the hamsters i s not known. The term ' s p i n d l e c e l l tumor' i s merely d e s c r i p t i v e ; i t does not imply any p a r t i c u l a r c e l l u l a r o r i g i n . The SCT i s not a pseudosarcoma, a stromal r e a c t i o n t o the presence of squamous c e l l carcinoma, because the SCT e x h i b i t s malignant c e l l u l a r f e a t u r e s , l o c a l i n v a s i o n and i n one case, m e t a s t a t i c d i s e a s e . There are two p o s s i b i l i t i e s f o r the t i s s u e d e r i v a t i o n o f the SCT: the SCT may be a v a r i a n t of squamous c e l l carcinoma, a s p i n d l e c e l l carcinoma or i t may be d e r i v e d from t r a c h e a l mesenchymal elements, a fibrosarcoma f o r example. From l i g h t microscopic o b s e r v a t i o n s , i t appeared t h a t the SCT i n the hamsters d e r i v e s from the squamous c e l l carcinoma, and thus, i t c o n s t i t u t e s a s p i n d l e c e l l carcinoma. In s e v e r a l i n s t a n c e s ( r e f e r t o F i g u r e 21), the stromal SCT appeared t o be i n c o n t i n u i t y w i t h the s u p e r f i c i a l squamous c e l l carcinoma. This apparent i m p e r c e p t i b l e merging of the two tumor components i s i d e n t i c a l t o a s i m i l a r photomicrograph (Fi g u r e 4, page 23) of S t i n s o n and a s s o c i a t e s ( S t i n s o n et a l . , 1983) i n which they u n e q u i v o c a l l y demonstrated by TEM, t h a t a hamster t r a c h e a l SCT was a s p i n d l e c e l l carcinoma. The l i g h t microscopic observations suggest but do not prove t h a t the SCT i s a s p i n d l e c e l l carcinoma. U l t r a s t r u c t u r a l demonstration of desmosomes and t o n o f i l a m e n t s i s o f t e n reguarded as evidence f o r the e p i t h e l i a l d e r i v a t i o n o f a SCT. Yet there i s no u n i v e r s a l law i n b i o l o g y which s t a t e s t h a t s p i n d l e c e l l carcinomas must c o n t a i n these c e l l u l a r f e a t u r e s i n order t o be considered o f e p i t h e l i a l - 55 -o r i g i n . In f a c t , these c e l l u l a r elements become l e s s d i s t i n c t as the e p i t h e l i u m moves from h y p e r p l a s i a , t o squamous met a p l a s i a , t o i n t r a e p i t h e l i a l n e o p l a s i a , t o squamous c e l l carcinoma ( M u l l e r and Sutherland, 1971), and i t i s reasonable t o expect t o see a r e l a t i v e p a u city o f these elements when an even more a n a p l a s t i c form of a squamous, c e l l carcinoma forms, the s p i n d l e c e l l carcinoma. The TEM i n v e s t i g a t i o n of the hamster SCT showed pseudodesmosomes, which lacked the intermediate l i n e between the two opposing cytoplasmic plaques and the tonofilament a s s o c i a t i o n with the cytoplasmic plaques seen i n t r u e desmosomes. Poorly defined desmosomes are commonly found i n s p i n d l e c e l l carcinomas ( L i c h t i g e r e t a l . , 1970). Also i t i s u s u a l l y necessary t o cut s e v e r a l b l o c k s f o r TEM before t r u e desmosomes can be found ( B a t t i f o r a , 1976) and only one g r i d from each of four SCT was examined i n t h i s study. Therefore, our i n a b i l i t y to demonstrate desmosomes and t o n o f i l a m e n t s does not n e c e s s a r i l y preclude the p o s s i b i l i t y t h a t the hamster SCT i s a s p i n d l e c e l l carcinoma. A p r e l i m i n a r y study conducted i n an attempt t o type the intermediate f i l a m e n t s of the SCT was i n c o n c l u s i v e . The mouse-monoclonal a n t i b o d i e s used (Tissue O r i g i n I d e n t i f i c a t i o n K i t , #EAB-901, from Enzo Biochem Inc., New York, New York) were d i r e c t e d a g a i n s t human antigens (Gown and Vogel, 1982 and 1984). Because e p i t h e l i a l intermediate f i l a m e n t s ( t o n o f i l a m e n t s ) d i f f e r somewhat between sp e c i e s (Lane, 1982), i t was f e l t the mouse-monoclonal a n t i b o d i e s t o human antigens may not have been s u i t a b l e probes t o demonstrate hamster t o n o f i l a m e n t s . A n t i b o d i e s to hamster t o n o f i l a m e n t s would be i d e a l , f a i l i n g t h a t , a p o l y c l o n a l r a t h e r than a monoclonal s e r a may be of some value. TEM of the SCT revealed t h a t the tumor c e l l s were f i l l e d w i t h d i l a t e d rough endoplasmic r e t i c u l u m . This along w i t h the presence of c o l l a g e n i n the - 56 -i n t e r c e l l u l a r matrix i m p l i e d that the s p i n d l e c e l l s were a c t u a l l y producing c o l l a g e n . This o b s e r v a t i o n suggested that the SCT were o f f i b r o b l a s t i c o r i g i n , and could be considered fibrosarcomas. However, non-neoplastic e p i t h e l i u m i s known t o produce the type IV c o l l a g e n of the b a s a l lamina ( K e f a l i d e s , 1971), and c o r n e a l e p i t h e l i u m can sec r e t e c o l l a g e n (Hay and Dodson, 1973). Furthermore, i t has a l s o been c l e a r l y shown by B a t t i f o r a ( B a t t i f o r a , 1976) t h a t human s p i n d l e c e l l carcinomas, w i t h w e l l formed desmosomes, do demonstrate morphologic evidence o f c o l l a g e n production, as does the hamster SCT. The t r a c h e a l c a r t i l a g e r i n g s exposed t o BP underwent s e v e r a l changes tha t may provide c l u e s t o the o r i g i n of the SCT. There was at l e a s t one example o f an area o f chondroid metaplasia w i t h i n the SCT and one example of abnormal c a r t i l a g e which was presumed t o be a w e l l d i f f e r e n t i a t e d chondrosarcoma. The c a r t i l a g e was not evaluated i n a l l t r a c h e a l specimens, but abnormal c a r t i l a g e appeared i n many hamster tracheas. I f one mesenchymal element ( c a r t i l a g e ) can form a neoplasm, i t might be reasonable t o expect other mesenchymal elements ( f i b r o b l a s t s ) t o form neoplasms, perhaps suggesting t h a t the SCT_ was a sarcoma. In the f i n a l a n a l y s i s , i t i s Impossible t o s t a t e d e f i n i t i v e l y t h a t the hamster SCT i s a carcinoma o r a sarcoma. The l i g h t microscopic observations suggested that the SCT i s a s p i n d l e c e l l carcinoma, but the c o r r o b o r a t i v e TEM and intermediate f i l a m e n t t y p i n g r e s u l t s were e q u i v o c a l . I f the SCT i s i n f a c t a sarcoma, one may have to c a u t i o u s l y e x t r a p o l a t e the r e s u l t s of a p a r t i c u l a r a p p l i c a t i o n of the hamster model t o human bronchogenic carcinoma, because of the p o s s i b i l i t y t h a t the sarcoma may modify the response - 57 -of the squamous c e l l carcinoma t o the t e s t e d v a r i a b l e (eg. a t h e r a p e u t i c agent). On the other hand, i f the SCT i s a s p i n d l e c e l l carcinoma, the model i s p a r t i c u l a r l y v a l u a b l e , s i n c e i t a l l o w s the morphologist access t o a wide spectrum of p r e n e o p l a s t i c and n e o p l a s t i c e p i t h e l i u m . Future s t u d i e s may c l a r i f y t h i s i s s u e . The Role of Inflammation, Regenerative H y p e r p l a s i a and K e r a t i n i z a t i o n of the Tracheal E p i t h e l i u m i n Tumor Promotion The inflammation o f the t r a c h e a l e p i t h e l i u m was recorded because p e r s i s t e n t inflammation may c o n t r i b u t e t o the c a r c i n o g e n i c process. There were c o n s i d e r a b l y more acute inflammatory c e l l s (polymorphonuclear leukocytes) i n the e p i t h e l i u m of the experimental versus the c o n t r o l hamsters. Poly-morphonuclear leukocytes (PMN) i n inflammatory l e s i o n s are known t o produce oxygen f r e e r a d i c a l s (Babior, 1978). I t has a l s o been shown t h a t PMN s t i m u l a t e d w i t h phorbol e s t e r s generate oxygen f r e e r a d i c a l s ( G o l d s t e i n et a l . , 1981) which cause c y t o g e n e t i c changes i n c u l t u r e d mammalian c e l l s (Weitberg et a l . , 1983). A d d i t i o n a l l y , PMN can a c t i v a t e BP t o genotoxic m e t a b o l i t e s v i a a r e a c t i v e oxygen-dependent r e a c t i o n (Trush et a l . , 1985). So the PMN i n the experimental hamster tracheas may have generated s u f f i c i e n t oxygen f r e e r a d i c a l s t o c o n t r i b u t e t o the BP-mediated DNA damage and c a r c i n o g e n e s i s . The s u r g i c a l i n s u l t t o the trachea during the op e r a t i o n i n i t i a l l y i n c i t e d a regenerative b a s a l c e l l h y p e r p l a s i a : t i s s u e damage r e s u l t s i n n e c r o s i s , and c e l l death i s known t o act as a mitogenic stimulus (Columbano et a l . , 1981, and Farber, 1981). W i t h i n one month, the hamster t r a c h e a l - 58 -e p i t h e l i u m resembled a h y p e r p l a s t i c epidermis, w i t h a t h i c k l a y e r o f squamous e p i t h e l i u m and' attendant s u p e r f i c i a l " k e r a t i n i z a t i o n . In mouse models, repeated abrasion of ch e m i c a l l y i n i t i a t e d s k i n r e s u l t s i n epidermal h y p e r p l a s i a and s u f f i c i e n t tumor promotion f o r carcinoma formation ( A r g y r i s and Slaga, 1981 and A r g y r i s , 1982). Again based on models o f carcinoma i n mouse s k i n , wounding can act as a tumor promoter ( P e l l i n g and Slaga, 1985 and Marks et a l . , 1982). I t i s p o s s i b l e i n our model t h a t the s u r g i c a l i n s u l t , independent of the inflammation, may have induced a r e g e n e r a t i v e basal c e l l h y p e r p l a s i a and th a t t h i s c e l l p r o l i f e r a t i o n c o n t r i b u t e d t o the c a r c i n o g e n e s i s , p a r t i c u l a r l y t o tumor promotion. Phorbol e s t e r s , known tumor promoters, produce epidermal h y p e r k e r a t i n i z a t i o n i n mouse s k i n (Nelson and Slaga, 1982 and Slaga and Kl e i n - S z a n t o , 1983). I t i s of i n t e r e s t t h a t the experimental hamsters were the only group of animals which r e a d i l y developed carcinomas and that k e r a t i n i z a t i o n (as w e l l as inflammation) was a prominent f e a t u r e of t h i s e p i t h e l i u m . The r e l a t i v e l a c k of k e r a t i n i z a t i o n and carcinoma formation i n the guinea p i g s may suggest t h a t under these experimental c o n d i t i o n s there was i n s u f f i c i e n t tumor promotion t o r e s u l t i n carcinomas. Perhaps something was i n t e r f e r i n g w i t h the r o l e of tumor promotion i n the guinea p i g s ; t h i s p o s s i b i l i t y w i l l be elaborated on i n the d i s c u s s i o n o f a s c o r b i c a c i d and cancer. As c o r b i c A c i d and Cancer There was an obvious d i f f e r e n c e i n the h i s t o l o g i c response of hamsters and guinea p i g s t o the i n s e r t i o n of a BP-impregnated threads i n t o the tracheas. Carcinomas formed r e a d i l y i n the hamsters but r a r e l y i n the guinea - 59 -p i g s . The reason f o r t h i s d i f f e r e n c e i s not known, but s e v e r a l p o s s i b i l i t i e s e x i s t . F i r s t , carcinogens need to be h e l d i n contact w i t h the e p i t h e l i u m f o r s u f f i c i e n t p e r i o d s of time to be e f f e c t i v e (Kushner, 1968). Perhaps guinea p i g s are more e f f i c i e n t at c l e a r i n g the l i b e r a t e d BP, presumably a f u n c t i o n of macrophages. Another p o s s i b i l i t y i s t h a t the two animals possess d i f f e r e n t b a s a l l e v e l s or degrees of i n d u c i b i l i t y of the monooxygenase enzymes necessary t o convert BP t o the a c t i v e carcinogen (Mass and Kaufman, 1981). A l t e r n a t i v e l y , the guinea p i g s may produce a l e s s c a r c i n o g e n i c spectrum of BP m e t a b o l i t e s or b r i n g about a more e f f e c t i v e metabolic d e t o x i f i c a t i o n of BP (Autrup et a l . , 1980, and Moore and Cohen, 1978). The two animals may d i f f e r i n t h e i r c a p a c i t y t o r e p a i r the BP induced macromolecular damage, e s p e c i a l l y of the genome (Cohen and Ashurst, 1983, and Eastman et a l . , 1981). I t should be s t a t e d at t h i s p o i n t t h a t a l l these p o s s i b l e e x p l a n a t i o n s f o r the d i f f e r e n c e s i n the h i s t o l o g i c response of hamsters versus guinea p i g s are not mutually e x c l u s i v e and t h a t i n f a c t , the d i f f e r e n c e l i k e l y r e f l e c t s a complicated i n t e r r e l a t i o n of these and other c o n s i d e r a t i o n s . Another p l a u s i b l e e x p l a n a t i o n f o r the d i s p a r i t y of responses t o BP i n the two rodents, i s t h a t the d i e t of the guinea p i g s was supplemented with r e l a t i v e l y high doses of a s c o r b i c a c i d compared t o the hamsters which, l i k e most mammals, do not r e q u i r e exogenous a s c o r b i c a c i d . The proposed a n t i c a n c e r r o l e of a s c o r b i c a c i d has long been a source of c o n t r o v e r s y . In the 1970's, Cameron and P a u l i n g (Cameron and Campbell, 1974, Cameron and P a u l i n g , 1976 and 1978) suggested t h a t high dose a s c o r b i c a c i d (10 grams per day) was b e n e f i c i a l i n p a t i e n t s w i t h advanced cancer, based on a r e t r o s p e c t i v e comparison between s e l e c t e d study p a t i e n t s and h i s t o r i c a l c o n t r o l p a t i e n t s . L a t e r , a - 60 -p r o s p e c t i v e , randomized, double b l i n d c l i n i c a l t r i a l by Creagan and Moertel (Creagan et al.^"1979)" showed th a t high "dose a s c o r b i c a c i d therapy i n p a t i e n t s w i t h advanced c o l o r e c t a l carcinoma d i d not i n c r e a s e p a t i e n t s u r v i v a l . P a u l i n g argued ( P a u l i n g , 1980) t h a t Creagan's p a t i e n t s had r e c e i v e d chemotherapy which adversely i n t e r f e r e d w i t h the a b i l i t y of a s c o r b i c a c i d t o enhance the immune response against the tumor (Cameron and P a u l i n g , 1974). However, r e c e n t l y M o e r t e l and Creagan (Moertel et a l . , 1985) repeated the study on s i m i l a r p a t i e n t s without p r i o r chemotherapy and reported t h a t high dose a s c o r b i c a c i d showed no advantage-over placebo on disease p r o g r e s s i o n or p a t i e n t s u r v i v a l . Although the aforementioned c l i n i c a l s t u d i e s suggest that a s c o r b i c a c i d does not have an a n t i c a n c e r e f f e c t , a s c o r b i c a c i d ' s p o t e n t i a l a n t i c a n c e r r o l e should not be dismissed prematurely. These s t u d i e s assessed the e f f e c t of d i e t a r y a s c o r b i c a c i d i n t a k e on tumor p r o g r e s s i o n . I t i s p o s s i b l e t h a t a s c o r b i c a c i d e x e r t s an a n t i c a n c e r e f f e c t at other stages i n the n e o p l a s t i c process, such as tumor i n i t i a t i o n and tumor promotion. There e x i s t s i n v i v o (Cameron et a l . , 1975, P i e r s o n and Meadows, 1983, Sanders and Mahaffey, 1983, and Varga and A i r o l d , 1983) and i n v i t r o (Park et a l . , 1980; Prasad et a l . , 1980, and Bram et a l . , 1980) evidence a s c r i b i n g an a n t i c a n c e r r o l e t o a s c o r b i c a c i d . Although the mechanisms of a s c o r b i c a c i d ' s reputed a n t i c a n c e r e f f e c t are not c l e a r , some i n v e s t i g a t o r s ( C e r u t t i , 1985, and G o l d s t e i n et a l . , 1983) contend t h a t oxygen f r e e r a d i c a l s can mediate tumor promotion, and t h a t a s c o r b i c a c i d i s capable of scavenging oxygen f r e e r a d i c a l s . The t o t a l d i e t a r y i n t a k e of a s c o r b i c a c i d i n the guinea p i g was about 150 to 200 mg per kilogram body weight per day (mg/kg/day). Yet guinea p i g s r e q u i r e only 5 mg/kg/day to grow, reproduce and s u r v i v e normally (Veen-Baigent et a l . , 1975). Given t h a t oxygen f r e e r a d i c a l s c o u l d be produced i n our - 61 -rodent model of carcinoma by.the t r a c h e a l i n f i l t r a t e o f PMN; that oxygen f r e e r a d i c a l s may mediate c a r c i n o g e n e s i i s ; that a s c o r b i c a c i d can quench oxygen f r e e r a d i c a l s , i t i s p o s s i b l e t h a t the high supplemental d i e t a r y a s c o r b i c a c i d was r e s p o n s i b l e f o r the observed paucity of carcinomas i n the guinea p i g s . This hypothesis i s c u r r e n t l y being t e s t e d i n our l a b o r a t o r y by decreasing the d i e t a r y i n t a k e of a s c o r b i c a c i d i n guinea p i g s and n o t i n g the subsequent frequency and chronology of carcinoma development. Complications of the Model Th i s animal model of bronchogenic carcinoma i s s t r a i g h t forward and simple, but i t does have s e v e r a l drawbacks. The most obvious problem i s t r a c h e a l o b s t r u c t i o n , which leads t o bronchopneumonia and v e n t i l a t o r y f a i l u r e . The amount of BP d e l i v e r e d t o the t i s s u e s i s dependent on the l e n g t h and diameter of impregnated threads i n s e r t e d i n t o the t r a c h e a l w a l l . So i n c r e a s i n g the exposure of the trachea t o BP i s tantamount t o i n c r e a s i n g t r a c h e a l o b s t r u c t i o n . F o r t u n a t e l y , s u f f i c i e n t BP (150 t o 225 ug) can be d e l i v e r e d t o the hamster t i s s u e s ( w i t h the 5.0 c o t t o n threads) t o induce carcinomas without compromising r e s p i r a t i o n . One of the main c o m p l i c a t i o n s of t h i s model i s t h a t i n 5 t o 10 percentage of both groups of experimental animals, the thread appears to be extruded i n t o the t r a c h e a l lumen. In order t o p o s i t i o n the i n s e r t e d thread t a u t a g a i n s t the t r a c h e a l w a l l , the thread i s t i e d f i r m l y around the c a r t i l a g e r i n g s , o f t e n squeezing the c a r t i l a g e r i n g s t o g e t h e r . Presumably because of the pressure e f f e c t s of the thread and the inflammation, the c a r t i l a g e r i n g s remodel and resorb. I f t h i s process continues u n t i l the c a r t i l a g e r i n g s are completely resorbed, the thread i s no longer anchored i n the t r a c h e a l w a l l and - 62 -i s extruded i n t o the t r a c h e a l lumen. T h i s becomes more pronounced i n the l a t e phases"of"the"study; "aft'er~the c a r t i l a g e has had more time t o resorb. However as the hamsters develop tumours w i t h i n f o u r t o f i v e months, the magnitude of t h i s problem can be reduced by s a c r i f i c i n g the animals e a r l i e r . An obvious drawback t o the model i s t h a t the neoplasm can only undergo l i m i t e d growth before v e n t i l a t o r y f a i l u r e ensues. So by v i r t u e of design, t h i s model i s of most u t i l i t y i n s t u d y i n g the p r e n e o p l a s t i c l e s i o n s and e a r l y n e o p l a s t i c growth. Regardless of the p o t e n t i a l and r e a l problems w i t h the model, i t does produce squamous c e l l carcinoma i n a l a r g e percentage of the hamsters. Animal S u r v i v a l The s t a t e d s u r v i v a l r a t e s i n c l u d e a l l the animals used during the development of the model. W i t h i n t h i s s u r v i v a l data, many animals are i n c l u d e d which succumbed due t o c o m p l i c a t i o n s of the o p e r a t i o n such as p e n t o b a r b i t a l overdose, improperly placed threads, inadequate t e n s i o n on the thread t i e d around the c a r t i l a g e r i n g s , e x c e s s i v e t r a c h e a l damage due t o use of the wrong s u r g i c a l needles, and other minor problems which were appreciated l a t e r i n the study. These problems are r e f l e c t e d i n the i n i t i a l (0-50 days) mor b i d i t y and m o r t a l i t y of 10 t o 30% i n both animals ( F i g u r e s 26 & 27). Once the thread i n s e r t i o n technique i s mastered and the exact a n a e s t h e t i c doses are determined, the o v e r a l l morbidity and m o r t a l i t y could probably be reduced by 10 t o 20%. The guinea p i g s underwent an i n c r e a s e i n morbidity and m o r t a l i t y w i t h time. T h i s appeared t o be the r e s u l t of the development of bronchopneumonias, l i k e l y secondary t o p a r t i a l t r a c h e a l o c c l u s i o n . A r e l a t i v e l y l a r g e percentage - 63 -(approximately 40%) of the experimental guinea p i g s showed h i s t o p a t h o l o g i c evidence of bronchopneumonias. Only 20% of the c o n t r o l guinea p i g s developed bronchopneumonias. Despite the f a c t t h a t only one experimental guinea p i g developed a carcinoma, the t r a c h e a l lumen was u s u a l l y more d i s t o r t e d i n the experimental than i n the c o n t r o l guinea p i g s . These f i n d i n g s suggest t h a t guinea p i g s do not t o l e r a t e p a r t i a l t r a c h e a l o c c l u s i o n , as they are proned t o develope bronchopneumonias. Thus on t h i s b a s i s , they may be l e s s s u i t a b l e models of r e s p i r a t o r y t r a c t tumors than other rodents. In the experimental hamsters, the p r o g r e s s i v e i n c r e a s e of m o r b i d i t y and m o r t a l i t y , t h a t l e v e l l e d o f f towards the l a t t e r t h i r d o f the study p e r i o d c o i n c i d e d w i t h the development of the t r a c h e a l tumors. As they encroached on the t r a c h e a l lumen, v e n t i l a t o r y f a i l u r e ensued. I t i s l i k e l y the s p i n d l e c e l l tumor (SCT) r a t h e r than the squamous c e l l carcinoma (CA) component of the hamster t r a c h e a l tumor was r e s p o n s i b l e f o r the v e n t i l a t o r y f a i l u r e , s i n c e the SCT c o n s t i t u t e d the m a j o r i t y of the tumor mass. M e t a s t a t i c cancer probably d i d not c o n t r i b u t e appreciably t o the observed m o r b i d i t y and m o r t a l i t y i n the hamsters, because the sampled r e g i o n a l lymph nodes l i v e r and lung were negative f o r m e t a s t a t i c carcinoma, and only one example of tumor metastasis was found. In humans, metastasis of b r o n c h i a l carcinoma t o s k i n or subcutaneous t i s s u e i s r e l a t i v e l y r a r e i n comparison t o metastasis t o lymph nodes, l i v e r and lung. I t i s c u r i o u s t h a t a subcutaneous metastasis occured without evidence of metastasis t o other s i t e s i n any of the hamsters. Yet a d m i t t e d l y , a comprehensive search f o r tumor metas t a s i s was not conducted. Although i n h a l e d BP can enter the c i r c u l a t o r y system ( M i t c h e l l , 1982), and i t i s known that the l i v e r can metabolize BP t o mutagenic products - 64 -(Wood et a l . , 1976), we found no primary or secondary l i v e r tumors i n e i t h e r rodent. "Yet there was h i s t o p a t h o l o g i c evidence of l i v e r damage: d i f f u s e f a t t y changes and p e r i p o r t a l hepatocyte dropout and f i b r o s i s . T his may have c o n t r i b u t e d to the observed morbidity and m o r t a l i t y . The l i v e r damage was more prevalent and severe i n the experimental animals, but a few c o n t r o l animals a l s o showed m i l d l i v e r damage. I t was assumed t h a t the l i v e r damage was due t o a c y t o t o x i c a c t i o n of BP, but the m i l d l i v e r damage i n the c o n t r o l s may suggest t h a t a f a c t o r common to the two groups of animals, such as d i e t or the SRS, c o n t r i b u t e d t o the observed l i v e r damage. The s i g n i f i c a n c e of the extramedullary hematopoiesis i n the experimental guinea p i g s i s not known. I t c o u l d suggest t h a t BP i n s u l t e d the bone marrow, and t h a t the l i v e r was r e c r u i t e d f o r hematopoiesis; however, the hematologic s t a t u s of the guinea p i g s was not f o l l o w e d throughout the course of the study. CONCLUSION One of the major v i r t u e s of the model i s i t s p r a c t i c a l i t y : the threads are easy t o prepare; no e l a b o r a t e equipment or m a t e r i a l s are needed; the surgery i s simple and quick, and the carcinogen does.not need t o be r e p e t i t i v e l y administered. Therefore, l a r g e s c a l e animal s t u d i e s are f e a s i b l e . Besides being r e a d i l y a c c e s s i b l e , the i n c i p i e n t t r a c h e a l carcinomas are marked by the thread. This enables the i n v e s t i g a t o r t o l o c a t e and study the p r e n e o p l a s t i c e p i t h e l i a l l e s i o n s , simply by s a c r i f i c i n g the animal at the a p p r o p r i a t e time. Although the d e f i n i t i v e o r i g i n o f the hamster s p i n d l e c e l l - 65 -tumor i s unknown, i t i s presumed t o represent a s p i n d l e c e l l carcinoma. Therefore, morphologists can g a i n access t o d i f f e r e n t i a t e d mature r e s p i r a t o r y e p i t h e l i u m , squamous metaplasic e p i t h e l i u m , p r o g r e s s i v e degrees of i n t r a e p i t h e l i a l n e o p l a s i a , squamous c e l l carcinoma, and d e d i f f e r e n t i a t e d a n a p l a s t i c s p i n d l e c e l l carcinoma. Tumors can be r e a d i l y produced i n the hamsters. The m a j o r i t y of the hamsters l e f t t o s u r v i v e at l e a s t 120 days develop squamous c e l l carcinoma, a carcinoma incidence o f 65% (9/14). By a v o i d i n g the i n i t i a l c o m p l i c a t i o n s o f the o p e r a t i o n , by s a c r i f i c i n g the hamsters before thread e x t r u s i o n occurs, and by using only threads w i t h a s i l i c o n e rubber sheath, the carcinoma y i e l d could probably be increased another 20%. This model has innumerable a p p l i c a t i o n s . The u l t r a s t r u c t u r a l a l t e r -a t i o n s i n e p i t h e l i a l c e l l s which a l l o w s f o r l o s s of c e l l p o l a r i t y , c e l l u l a r a t y p i a , and l o c a l i n v a s i o n c o u l d be s t u d i e d i n t h i s model, as e p i t h e l i u m from v a r i o u s stages i n the n e o p l a s t i c process i s r e a d i l y a v a i l a b l e f o r e l e c t r o n microscopy and immunohistochemistry. A morphologic study of k e r a t i n i z a t i o n i n m e t a p l a s t i c , p r e n e o p l a s t i c and n e o p l a s t i c e p i t h e l i u m c o u l d be conducted. The r o l e o f inflammatory c e l l s i n p r e n e o p l a s i a and n e o p l a s i a might be c l a r i f i e d i n an i n v i v o model such as t h i s one. The model may be u s e f u l t o study the morphogenesis of c a r t i l a g e tumors or perhaps, the c e l l u l a r f e a t u r e s o f s p i n d l e c e l l carcinomas. The immunologic response to the development of bronchogenic carcinoma could be s t u d i e d i n t h i s model. I f s p e c i f i c immunologic markers of r e s p i r a t o r y p r e n e o p l a s ia or e a r l y n e o p l a s i a c o u l d be found, and i f a s i m i l a r immunologic response was mounted i n humans, a means of e a r l y d e t e c t i o n of bronchogenic carcinomas might be e s t a b l i s h e d . . - 66 -Because of the p r a c t i c a l i t y of the model, l a r g e s c a l e s t u d i e s could be i n s t i t u t e d " to study the e f f e c t s o f s p e c i f i c treatments or d i e t a r y manipula-t i o n on bronchogenic carcinoma. T h i s i s one avenue which we w i l l pursue i n the f u t u r e . This model i s a s e n s i t i v e i n viv o means of asses s i n g the e f f e c t of a s c o r b i c a c i d i n c a r c i n o g e n e s i s . I f a s c o r b i c a c i d can, i n f a c t , p a r t i a l l y negate oxygen f r e e r a d i c a l mediated tumor promotion or other stages i n c a r c i n o g e n e s i s , one might advocate t h a t i n d i v i d u a l s w i t h a high r i s k of developing bronchogenic carcinoma ( f o r example asbestos miners who smoke), in g e s t high d a i l y doses of a s c o r b i c a c i d , p a r t i c u l a r l y i n l i g h t of a s c o r b i c a c i d ' s non-toxic and inexpensive q u a l i t i e s . - 67 -REFERENCES Andervont, H.B. Pulmonary tumors i n mice. P u b l i c Health Reports U.S. 52:1584-1589, 1937. Argus, M.F., and H o c h - L i g e t i , C. 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