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Histochemical changes in colonic epithelial glycoproteins during the induction of cancer in rats Lazosky, Darien-Alexis 1985

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HISTOCHEMICAL CHANGES IN COLONIC EPITHELIAL GLYCOPROTEINS DURING THE INDUCTION OF CANCER IN RATS By DARIEN-ALEXIS LAZOSKY B.Sc, The University of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology, Faculty of Medicine) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1985 © Darien-Alexis Lazosky, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. PATHOLOGY Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e October 15, 1985 i i ABSTRACT Colonic e p i t h e l i a l glycoproteins were histochemically studied i n rats during the induction of c o l o r e c t a l cancer with 1,2,-dimethylhydrazine-diHCl (DMH). 310 male Wistar rats were separated into 3 groups: 10 control rats were s a c r i f i c e d without treatment, 150 rats were given weekly subcutaneous injections of the carcinogen and 150 rats were given sham Injections on the same schedule. Groups of rats (10 control and 10 treated) were s a c r i f i c e d at various time i n t e r v a l s i n an e f f o r t to obtain a large number of pre-neoplastic rat colons to be used to determine whether histochemical changes could be detected p r i o r to morphologic change. Three histochemically d i s t i n c t regions i n the Wistar rat d i s t a l colon have been described using recently developed techniques. Two major histochemical changes have been shown to occur prior to malignancy. Change 'A' represented a decrease or loss of sulphate from the glycoproteins; t h i s was the e a r l i e s t and predominant change and has been shown to occur p r i o r to any i d e n t i f i a b l e morphologic change. Change 'B' represented a decrease or loss of sulphate occurring i n the same c e l l s as a decrease or loss of side chain 0-acetylation of s i a l i c acid residues; t h i s change occured l a t e r and to a lesser extent than change 'A'. The data suggests that histochemical change may be a premalignant marker, however, further investigations are necessary to firmly establish t h i s conclusion. TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES vi LIST OF FIGURES v i i i LIST OF SLIDES ix ABBREVIATIONS x ACKNOWLEDGEMENTS xi INTRODUCTION 1 I. Objective 1 I I . Description of the colonic mucosa 1 I I I . Theories on the k i n e t i c s of colon carcinogenesis 2 IV. Colon cancer etiology: 4 ( i ) genetic factors 4 ( i i ) d iet and environmental factors 5 ( i i i ) premalignant lesions 6 (iv) bowel surgery 7 V. Early detection and survival 7 V I . Therapy 8 V I I . Colonic e p i t h e l i a l glycoproteins: 9 ( i ) nature 9 ( i i ) association with cancer and premalignant disease 9 states ( i i i ) i n normal and carcinogen treated rats 24 V I I I . The model 27 ( i ) rationale i n using an animal model to study 27 premalignant change ( i i ) current theories of the mechanism of DMH 28 carcinogenesis IX. Rationale i n the use of histochemistry as an investigative t o o l 32 X. Thesis rationale 34 iv MATERIALS AND METHODS PAGE I. MATERIALS 36 I I . METHODS 36 ( i ) Induction of tumours and i s o l a t i o n of colons 36 ( i i ) Gross observation and preparation of tissues for histology 37 ( i i i ) Staining procedures 40 a) preparation of stains 44 b) periodate oxidation 44 c) borohydride reduction 45 d) saponification 45 e) phenylhydrazine blockade 45 f) methods for the v i s u a l i z a t i o n of side chain O-acetylated s i a l i c acids 45 1) PBT/KOH/PAS 45 2) PAT/KOH/Bh/PAS 45 3) PAPT/KOH/Bh/PAS 46 g) methods for the simultaneous v i s u a l i z a t i o n of side chain O-acetylated s i a l i c acid and sulphate 46 1) AB 1.0/PAPS 46 2) PBT/KOH/AB l.O/PAS 46 h) methods for the simultaneous v i s u a l i z a t i o n of t o t a l s i a l i c acid and sulphate 46 1) KOH/AB 1.0/PAPS 46 2) HID/AB 2.5 47 (iv) Methods of assessment 47 a) h i s t o l o g i c c l a s s i f i c a t i o n 47 b) determination of the normal staining pattern 47 c) evaluation of abnormal crypts 48 d) h i s t o l o g i c c l a s s i f i c a t i o n of histochemically abnormal crypts 49 e) histochemistry of crypts with abnormal morphology 51 f) measuremment of mucosal length 52 RESULTS I. MACROSCOPIC OBSERVATION ( i ) General features of the colons ( i i ) D i s t r i b u t i o n of tumours I I . MICROSCOPIC OBSERVATIONS ( i ) Determination of the appropriate oxidation time for Wistar r a t c o l o n i c mucin ( i i ) Control sections ( i i i ) Histology a) normal histology b) abnormal histology c) tumours ( i v ) Normal histochemistry (v) Histochemical abnormalities i n the preneoplastic phase ( v i ) Morphological character of crypts e x h i b i t i n g abnormal histochemistry ( v i i ) H i s t o l o g i c abnormalities i n histochemically normal crypts ( v i i i ) Histochemical c h a r a c t e r i s t i c s of postneoplastic phase tumours ( i x ) Histochemical c h a r a c t e r i s t i c s of the non-neoplastic mucosa adjacent to tumours (x) Mucosa remote from tumours i n the post-neoplastic phase DISCUSSION BIBLIOGRAPHY APPENDIX SLIDES £tW/'-£ CjM*\y>>~^ C^jo vi LIST OF TABLES PAGE 1. The characteristics of human colonic epithelial 11 glycoproteins associated with: a) colorectal adenocarcinomas 12 b) the mucosa adjacent to tumours in the large bowel 13 c) adenomas 14 d) anastomotic sites 15 e) inflammatory diseases of the large bowel 16 f) other miscellaneous conditions of the large bowel 18 2. The characteristics of the Wistar rat distal colonic 25 epithelial mucin in normal rat and during the induction of colon cancer. 3. Characteristics of the histochemical stains used for the 41 identification of rat colonic mucosal epithelial glycoproteins. 3a. Predicted staining results for control sections. 43 A. The distribution of tumours in rat colons after 16-30 weeks 55 of treatment with DMH. 5. The staining characteristics of the three major regions of 60 the Wistar rat distal colon. 6. Characteristics of the colonic epithelial glycoproteins in 62 normal Wistar rat distal colon. 7. The time-related change in the character of the mucin present 65 in the lower halves of the crypts in the PE region of the Wistar rat distal colon. v i i PAGE 8. The mean number of histochemical changes .seen per decimetre 67 (dm) of colonic mucosa, classified according to the nature of the change. 9. The mean number of histochemical changes seen per decimetre 69 (dm) of colonic mucosa, classified according to morphologic cri teria . 10. The mean number of foci showing histologic abnormalities and 70 normal histochemistry. 11. Histochemical changes in the mucins of tumours of the Wistar 73 rat distal colon. 12. Histochemical changes in the non-neoplastic mucosa immediately 74 adjacent to tumours of Wistar rat distal colon. 13. The mean number of histochemical changes seen per decimetre of 76 colonic mucosa during the postneoplastic phase classified according to morphological criteria. 14. The mean number of foci showing histologic abnormalities and normal histochemistry in the postneoplastic phase. 77 v i i i LIST OF FIGURES PAGE 1. Quotation. xi i i 2. The N-acetyl-neuraminic acid molecule and its side chain 10 O-acetylated variants. 3. Current theories of DMH metabolism. 29 4. Quotation. 35 5. Experimental design. 38 6. Schematic diagram of a typical Wistar rat colon. 39 7. Quotation. 53 8. Quotation. 78 L I S T 0 F S L I D E S C o l l e t 1. Decreased mucin 2. Atypia 3. Dysplasia 4. Polypoid moderately w e l l d i f f e r e n t i a t e d adenocarcinoma 5. S e s s i l e moderately well d i f f e r e n t i a t e d adenocarcinoma 6. DE region stained with K0H/AB1.0/PAPS 7. DE region stained with PAPT/KOH/Bh/PAS 8. MID region stained with K0H/AB1.0/PAPS 9. MID region stained with PAPT/KOH/Bh/PAS 10. PE region stained with K0H/AB1.0/PAPS 11. PE region p r i o r to 8 weeks stained with PAPT/KOH/Bh/PAS 12. PE region a f t e r 8 weeks stained with PAPT/KOH/Bh/PAS 13. Change A stained with K0H/AB1.0/PAPS 14. Change B stained with PAPT/KOH/Bh/PAS X ABBREVIATIONS DMH - 1,2 - dimethylhydrazine - diHCl EDTA - ethylene diamine t e t r a a c e t i c a c i d C^ - s i a l i c a c i d with an 0-acyl su b s t i t u t e n t at p o s i t i o n C Cj - s i a l i c a c i d with an 0-acyl substitutent at p o s i t i o n C-- s i a l i c a c i d with an 0-acyl su b s t i t u t e n t at p o s i t i o n C, Cg - s i a l i c a c i d with an 0-acyl substitutent at p o s i t i o n C, or d i or t r i s u b s t i t u t e d s i a l i c a c i d C n - s i a l i c a c i d l a c k i n g side chain substituents NAT - non-neoplastic mucosa immediately adjacent to tumour DE - d i s t a l end of the r a t lower colon MID - middle region of the r a t lower colon PE - proximal end of the r a t lower colon x i ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to my supervisor Dr. PE Reid who taught me how to put 'old wine i n t o new wineskins', for the patience, guidance and nagging i t took to bring t h i s t h e s i s to f r u i t i o n ; to my supervisory committee Drs. HJ Freeman, WL Dunn, DA Owen and MG Clay fo r t h e i r constant encouragement and p a r t i c i p a t i o n i n many productive and l i v e l y committee meetings; and to Dr. Ed Jones for c o l l a b o r a t i n g on the h i s t o l o g i c assessment and for many valuable discussions on the histogenesis of cancer and the meaning of l i f e . I would l i k e to thank everyone who supported me throughout the course o f t h i s t h e s i s e s p e c i a l l y my family; P h i l and Jan Reid (and the WMS) for many memorable Sunday evenings; the Askews for providing a home away from home; Ken Pool for h i s beef stroganoff and h i s expert c a l l i g r a p h y ; and David f o r h i s f r i e n d s h i p and support, for keeping me sane and s i g n i f i c a n t l y decreasing my F.I.Fi The t e c h n i c a l expertise of the following people i s greatly appreciated; Dr. Freeman f or the execution of the i n i t i a l experiment; Betty Coret, Barbara Muelchen, and Linda Trueman for h i s t o l o g i c preparations; and Charl i e Ramey f o r trouble-shooting and general knowledge o f j u s t about everything. This study was supported by grants to Drs. Reid, Dunn and Clay from the Medical Research Council of Canada and to Dr. Freeman from the Canadian Foundation of I l e i t i s and C o l i t i s and the B r i t i s h Columbia x i i Health Care Research Foundation. I would also l i k e to thank the B r i t i s h Columbia Cancer Research Foundation for the award of two Summer Studentships, and a t r a v e l grant, and the University of B r i t i s h Columbia fo r the award of a Summer Graduate Fellowship, a t u i t i o n bursary, a t r a v e l grant and the Moses Foukes bursary. x i i i wider(udhum&tmccs is about aamfc scpfierb stone? - G^3L.3idms>. /Wv. Upkt R » . 7= i-62. O969) FIGURE 1. - 1 -INTRODUCTION I Objective The purpose of the i n v e s t i g a t i o n described i n t h i s t h e s i s was to determine whether changes i n c o l o n i c e p i t h e l i a l goblet c e l l mucin gl y c o p r o t e i n s as r e f l e c t e d i n a l t e r e d mucin histochemical reactions, preceded, p a r a l l e l e d , or followed the development of gross anatomic or h i s t o l o g i c evidence of malignancy during the induction of colon neoplasia i n r a t s with the carcinogen 1,2-dimethylhydrazine. II D e s c r i p t i o n of the c o l o n i c mucosa The c o l o n i c lumen i s l i n e d by a highly s p e c i a l i z e d s e l f renewing epithelium composed p r i m a r i l y of goblet c e l l s , which secrete mucin glycoproteins ( F i l i p e , 1979). Rather than forming a simple confluent l a y e r the epithelium invaginates at regular i n t e r v a l s forming glands commonly r e f e r r e d to as "crypts of Lieberkuhn". Pluripotent stem c e l l s , u n d i f f e r e n t i a t e d , immature c e l l s capable of DNA synthesis, self-renewal and p r o l i f e r a t i o n are found i n small numbers i n the bases of the crypts; these give r i s e to more f u n c t i o n a l l y mature col o n i c e p i t h e l i a l c e l l s ( F i l i p e , 1979). Newly formed c e l l s migrate from the lower portions o f the c r y p t s to the luminal surface. Migration takes from 3 to 8 days i n humans and from 2 to 3 days i n rodents ( L i p k i n , 1983). The l i f e expectancy of c e l l s that reach the surface epithelium approximates 2 days, i n humans, followed by desguamation i n t o the c o l o n i c lumen (Tutton & Barkla 1983). A strong c i r c a d i a n rhythm has been demonstrated i n - 2 -c o l o n i c crypt c e l l p r o l i f e r a t i o n (Bertalanffy, 1960; Chang, 1971; Hamilton, 1979, 1980). I l l Theories on the k i n e t i c s of colon carcinogenesis L i p k i n (1974, 1983) has proposed that there i s a two phase mechanism of p r o l i f e r a t i v e disturbances involved i n colon carcinogenesis. Phase I involves an a l t e r a t i o n i n the k i n e t i c properties of the c e l l s as they migrate from the bases of the crypts to the mucosal surface. Normally mature, non-dividing c e l l s continue to p r o l i f e r a t e even during migration. In phase II the impaired c e l l s accumulate at the surface of the i n t e s t i n a l mucosa thus leading to the formation of n e o p l a s t i c f o c i . In a study of c e l l k i n e t i c s during experimental carcinogenesis, P o z h a r i s s k i et a l (1982) state that the carcinogen 1,2-dimethylhydrazine (DMH) exerts i t s influence on the stem c e l l at the beginning of the m i t o t i c phase hence the c e l l continues to p r o l i f e r a t e but does not d i f f e r e n t i a t e r e s u l t i n g i n a preponderance of immature c e l l s which continue to r e p l i c a t e outside the normal p r o l i f e r a t i v e compartment. "Enterocyte d i f f e r e n t i a t i o n disturbances, i n turn, upset the steady s t a t e of the i n t e s t i n a l epithelium with time, because the rate of c e l l shedding i n t o the i n t e s t i n a l lumen i s decreased. As a r e s u l t , large numbers of a t y p i c a l c e l l s appear i n the s u p e r f i c i a l layers of the crypt, and the c e l l s form a carcinoma i n s i t u . " As shown by t r i t i a t e d thymidine l a b e l l i n g studies, DMH induces an extension of the p r o l i f e r a t i v e zone towards the surfaces of the c o l o n i c crypts (Thurnherr et a l , 1973; Chang, 1978). Tutton and Barkla (1983) have proposed two hypotheses that may account for these DMH-induced - 3 -p r o l i f e r a t i v e disturbances. F i r s t l y , i t has been suggested that r e p l i c a t i n g c e l l s appearing i n the middle and upper portions of the crypts are abnormal and are the precursors of c o l o n i c tumours. Secondly, i t may be that the d i v i d i n g c e l l s seen i n the upper portion of the crypts of DMH t r e a t e d animals are normal c e l l s that have been displaced upwards by abnormal c e l l s that have appeared i n the crypt bases, hence, the normal p r o l i f e r a t i v e compartment was s h i f t e d upwards. Current theory ( L i p k i n 1974, 1983; P o z h a r i s s k i et a l , 1982; Tutton & Barkla, 1983) suggests then that some unknown error occurs e a r l y i n the d i f f e r e n t i a t i v e pathway which, r e s u l t i n g i n c e l l s of a more immature phenotype, causes an imbalance i n the normal rate of c e l l turnover. The primary event causing that e a r l y f i r s t e r r o r remains the object of much speculation, and the impetus of numerous i n v e s t i g a t i o n s . It has been suggested that changes i n metabolic pathways and i n t r a c e l l u l a r s t r u c tures caused by free r a d i c a l damage may account for some of the properties of malignant c e l l s and may even play a r o l e i n malignant transformation (Hoffman et a l , 1985). The increased a c t i v i t y of Cu/Zn SOD (superoxide dismutase, an enzyme which functions i n the i n t r a c e l l u l a r d e t o x i f i c a t i o n of free r a d i c a l s ) i n DMH-induced r a t colon tumours i s thought to represent c e l l u l a r adaptation to free r a d i c a l s generated by the carcinogen. Further, Hoffman et a l (1985) suggest that damage to enzyme systems may preceed malignant changes and that free r a d i c a l s may be responsible for DNA damage leading to transformation. - A -IV Colon cancer e t i o l o g y Colon cancer i s thought to have a m u l t i f a c t o r i a l e t i o l o g y . There i s evidence for genetic (Lynch et a l , 1973; Knudson and K e l l y , 1983), di e t a r y and environmental f a c t o r s (Zaridze, 1983; M i l l e r , 1982; Barker and Godfrey, 198A; Nair, 198A), as well as increased r i s k associated with premalignant l e s i o n s (Morson, 197A; Fenoglio and Pascal, 1982), p r e - e x i s t i n g inflammatory bowel disease (Earnshaw et a l , 1982; Bulow, 198A; Boland et a l , 198A), and bowel surgery (Rubio et a l , 198A; C a l d e r i s i and Freeman, 198A; Habib et a l , 198A; Marcheggiano, 198A). ( i ) Genetic f a c t o r s In terms of genetics, d i f f e r e n t genes appear to be involved i n d i f f e r e n t cancers. Judging by the number of h e r e d i t a r i l y transmitted cancers i d e n t i f i e d to date, the number of such genes i s surely greater than 100 (Knudson & K e l l y , 1983). Most of these genes are i n h e r i t e d i n a dominant manner. The i n h e r i t e d polyposis syndromes ( f a m i l i a l polyposis c o l i , generalized j u v e n i l e polyposis,- Gardner, Peutz-Jegher, and Turcott syndromes) account f o r a very small percentage of colon cancers. Inherited non-polyposis syndromes (hereditary colon cancer, hereditary gastrocolonic cancer, hereditary adenocarcinomatosis, and Muir's syndrome) account for up to 25%. These syndromes are i n h e r i t e d as dominants, have onset i n the mid AO's and are associated with increased numbers of multiple primary colon tumours. Inherited carcinomas occur most freauently i n the cecum, ascending, and transverse colons whereas the unselected types more often appear i n the descending and rectosigmoid colon. Thus, cancers i n the r i g h t colon are more l i k e l y to have a strong - 5 -genetic component than those i n the l e f t colon (Knudson & K e l l y , 1983). ( i i ) Diet and environmental f a c t o r s Epidemiological geographic studies of high and low r i s k populations suggest that many environmental f a c t o r s f i g u r e prominently i n cancer e t i o l o g y . Members of low r i s k populations migrating to high r i s k areas generally assume the incidence of the host population. This suggests that e t i o l o g y i n most cases i s more environmental than genetic, and d i e t i s the most l i k e l y environmental f a c t o r that could account for such d i f f e r e n c e s (Nair, 1984). Dietary f a c t o r s associated with an increased r i s k f or colon cancer include obesity, f a t s (mainly saturated), meat, and c h o l e s t e r o l (Nair, 1984). Fiber i n d i e t i s e i t h e r associated with decreased r i s k or no e f f e c t and p r o t e c t i v e f a c t o r s include b e t a - s i t o s t e r o l , s y n t hetic antioxidants, ascorbic a c i d , alpha-tocopherol, selenium, vitamin A, and beta-carotene. A recent study i n B r i t a i n (Barker and Godfrey, 1984) revealed that, i n men, colon cancer incidence was highest i n towns with better socioeconomic conditions, whereas i n women the s i t u a t i o n was the reverse. The authors concluded that these r e s u l t s support the hypothesis that the dominant e t i o l o g i c a l influences causing c o l o r e c t a l cancer d i f f e r i n the two sexes. I t i s generally believed that more than 80% of human cancers are caused by environmental f a c t o r s . Much e f f o r t has gone into the i d e n t i f i c a t i o n of procarcinogens, ultimate carcinogens and anticarcinogens i n an attempt to discover i n i t i a t o r s and promoters of genetic damage that lead to malignant transformation (Bresnick, 1980; Ames, 1983). Glo b a l l y the highest rates for c o l o r e c t a l cancer occur i n - 6 -North America, Scotland and New Zealand, intermediate rates i n England and Scandinavia, and lowest rates i n Asia and the Caribbean (deJong et a l , 1972). In B r i t i s h Columbia i n 1981 (Cancer Control Agency of B r i t i s h Columbia, 1982-83) i n both males and females over 15 years of age, colon cancer was the t h i r d major cause of death due to cancer. In men i t was t h i r d to lung cancer (1st) and cancer of other digestive organs (2nd) and i n women i t was t h i r d to breast cancer (1st) and lung cancer (2nd), ( i i i ) Premalignant l e s i o n s Although i t has not been c o n c l u s i v e l y proven, i t i s generally believed that most col o n i c adenocarcinomas a r i s e i n premalignant l e s i o n s such as polyps or adenomas ( E n t e r l i n e , 1976). Most of the evidence that supports the hypothesis i s c i r c u m s t a n t i a l , derived from patient data and h i s t o l o g i c sections of biopsies and r e s e c t i o n s . Patients with adenomas, whether i n h e r i t e d or not, have an increased r i s k of developing cancer, (Morson, 1974) and d i f f e r e n t types of adenomas ( i . e . , tubular, t u b u l o - v i l l o u s , and v i l l o u s ) are associated with increasing degrees of malignant p o t e n t i a l , r e s p e c t i v e l y . H i s t o l o g i c a l l y , adenomas are benign n e o p l a s t i c growths of c o l o n i c glandular e p i t h e l i a l c e l l s which r e s u l t from normal or increased p r o l i f e r a t i o n concommittant with a decrease i n the a b i l i t y of these c e l l s to be shed i n t o the lumen (Pozharisski, et a l 1982). Adenomas have been shown to have areas of malignant change within them and adenocarcinomas have been shown to contain areas of benign n e o p l a s t i c t i s s u e ( E n t e r l i n e , 1976). Patients with inflammatory bowel diseases such as Crohn's disease and U l c e r a t i v e C o l i t i s have an increased r i s k of developing bowel cancer compared to the general population - 7 -(Earnshaw et a l , 1982; Shorter, 1983; Yardley et a l , 1983). (i v ) Bowel surgery The rate of tumour recurrence i n patients having undergone cur a t i v e bowel r e s e c t i o n i s highest at the anastomotic s i t e . Recurrence a f t e r r e s e c t i o n i s thought to be caused by micrometastases not v i s i b l e during surgery or by tumour c e l l dissemination through surgery i t s e l f (Metzger et a l , 1985). In experimental animals increased numbers of tumours occurred at anastomotic s i t e s i n both functioning colon and i n de f u n c t i o n a l i z e d s u r g i c a l l y occluded loops i n the same anatomic region (Rubio et a l , 1984). S p e c i f i c types of suture materials have been shown to promote tumourigenesis i n DMH r a t cecum, an area of the g a s t r o i n t e s t i n a l t r a c t which i s usually free of tumours i n t h i s experimental model, and s t a i n l e s s s t e e l increased the numbers of tumours downstream from the cecum ( C a l d e r i s i & Freeman, 1984). Pa t i e n t s having undergone ureterosigmoidostomy have a 500 f o l d r i s k of developing colon cancer. The promoting f a c t o r has not been i d e n t i f i e d but the e f f e c t of urine has been ruled out and fur t h e r the tumours mostly occur at s i t e s of ureterocoloanastomosis (Marcheggiano 1984). V E a r l y detection and s u r v i v a l Currently the most widely used t o o l s being evaluated f o r screening for large bowel cancer are f e c a l o c c u l t blood t e s t i n g and sigmoidoscopic examination; barium-contrast-enema x-ray and colonscopy may a l s o be required ( L e f a l l , 1981; G i l b e r t s e n et a l , 1980; Winawer, 1980; Fazio, 1979; Winawer & Sherlock, 1977). - 8 -Five-year s u r v i v a l rates depend on whether or not symptoms are present, the h i s t o l o g i c grade and stage of the neoplasm, gross c h a r a c t e r i s t i c s of the tumour and the extent of vascular or lymphatic invasion. Five year s u r v i v a l rates (Beahrs, 1982) i n asymptomatic pat i e n t s was 88% compared to 40% i f the symptoms were present for three months and 25% when present for seven months. The s u r v i v a l rate for p a t i e n t s having c o l o n i c polyps with no evidence of malignant change was 100%. VI Therapy Prognosis i s poor for those patients presenting with extensive and s u r g i c a l l y incurable disease. Unfortunately these tumours, by v i r t u e of t h e i r k i n e t i c properties are not very s e n s i t i v e to r a d i a t i o n therapy (Johnson, 1977) and are highly r e s i s t a n t to the c u r r e n t l y a v a i l a b l e chemotherapeutic agents (Davis, 1982). Confounding the problems of early diagnosis and resistance to therapy i s the f a c t that p a t i e n t s presenting with s i m i l a r tumours may follow widely d i f f e r i n g courses of progression and may react d i f f e r e n t l y to the same multimodal therapy (Carter 1976). Currently 5 - f l u o r o u r a c i l (5-FU) i s the therapeutic drug considered to be the most e f f e c t i v e i n man. A recent study i n DMH r a t s showed that razoxane produced a greater e f f e c t i n the reduction i n the number of malignant tumours than 5-FU ( G i l b e r t et a l , 1984). Razoxane has been used i n man for lymphoproliferative disorders and sarcomas and although not widely used at present i s one of the few drugs to have an e f f e c t i n the treatment of human c o l o r e c t a l cancer. - 9 -Evidence such as t h i s s t r e s s e s the possible importance of early diagnosis to prognosis and s u r v i v a l . The objective of t h i s study was to determine whether changes i n c o l o n i c mucin could be detected p r i o r to malignant transformation or even p r i o r to morphologic change. VII Colonic e p i t h e l i a l glycoproteins ( i ) Nature Glycoproteins are ubiauitous b i o l o g i c a l compounds composed of both protein and carbohydrate components. The carbohydrate p r o s t h e t i c groups of mucin glycoproteins are c o v a l e n t l y bound to the polypeptide backbone through an O - g l y c o s i d i c linkage between N-acetylgalactosamine and e i t h e r a serine or threonine residue (Sharon & L i s , 1982). Colonic e p i t h e l i a l glycoproteins contain carbohydrate p r o s t h e t i c groups composed of galactose, N-acetylgalactosamine, N-acetylglucosamine and terminal non-reducing s i a l i c a c i d and fucose residues (Forstner, 1978; Sharon & L i s , 1982). The penultimate sugar i s commonly galactose. In addition some sugars, excluding s i a l i c a c i d , may have O-sulphate e s t e r substituents ( F i l i p e , 1979; Dawson & F i l i p e , 1982). The s i a l i c acid residues ( f i g u r e 2) may be unsubstituted (C^) or have O-acetyl substituents at p o s i t i o n C^ and/or i n the polyhydroxy side chain (C-,, C Q, C Q ) , (Reid et a l , 1978, 1985b). o y ( i i ) A s sociation with cancer and premalignant disease s t a t e s The glycoproteins of human c o l o r e c t a l adenocarcinomas are s i g n i f i c a n t l y d i f f e r e n t i n many respects to those present i n normal t i s s u e (Table l a ) . In tumours, s i a l i c acid residues contain - 10 -FIGURE 2: THE N-ACETYL NEURAMINIC ACID MOLECULE AND ITS O-ACETYL VARIANTS H C H 3 C N / 3 f -OH \ C O O H h O H 9 ' C H 2 O H L - 0 O H H N-ACETYL NEURAMINIC ACID (SIALIC ACID) O H O H C H 2 O H 1-0 A c |-OH C H 2 O H t o H L o A c C H 2 O H O H O H C H 2 O A C DI SUBSTITUTED TRI SUBSTITUTED rOAC [okC [OH 1-0 AC [OH L O A C r o A c |-oAc C H 2 O A C C H 2 O H C H 2 O A C C H 2 O A C ( C ? + C 9 ) ( C 7 + C g ) ( C 8 + C g ) (C 7 +V C9 } - 11 -TABLE 1: THE CHARACTERISTICS OF HUMAN COLONIC EPITHELIAL  GLYCOPROTEINS ASSOCIATED WITH: A. COLORECTAL ADENCARCINOMAS; B. THE MUCOSA ADJACENT TO TUMOURS IN THE LARGE BOWEL; C. ADENOMAS; D. ANASTOMOTIC SITES; E. INFLAMMATORY DISEASES OF THE LARGE BOWEL; F. OTHER MISCELLANEOUS CONDITIONS OF THE LARGE BOWEL. SYMBOLS I ABBREVIATIONS : 1. p r e m a l i g n a n t c o n d i t i o n a s s o c i a t e d w i t h an i n c r e a s e d r i s k o f c o l o n c a n c e r . 2 . c o n s i d e r e d b e n i g n and does n o t c a r r y a h i g h r i s k o f m a l i g n a n c y . 3 . c o n s i s t e n t w i t h t h e p a t t e r n o b s e r v e d i n n o r m a l t i s s u e . 4 . a b n o r m a l p a t t e r n . PNA: p e a n u t a g g l u t i n i n ( s p e c i f i c f o r G a l - G a l N A c ) D E * : d o l i c h o s b i f l o r u s a g g l u t i n i n { s p e c i f i c f o r G a l N A c ) SBA: s o y b e a n a g g l u t i n i n ( s p e c i f i c f o r G a l N A c ) BPA: b a u h i n i a p u r p u r e a a g g l u t i n i n ( s p e c i f i c f o r G a l ) G a l : g a l a c t o s e G l c : g l u c o s e F u c : f u c o s e G a l N A c : N - a c e t y l g a l a c t o s a r n n e G l c H : g l u c o s a m i n e G l c N A c : N - a c e t y l g l u c o s a m i n e NANA: N - a c e t y l n e u r a m i n i c a c i d ( s i a l i c a c i d ) NGNA: N - g l y c o l y l n e u r a m i n i c a c i d S I A L O : a l l o r p r e d o m i n a n t l y s i a l o m u c i n SULPHO: a l l o r p r e d o i r i n a n t l y s u l p h o m u c i n ( n o r m a l p a t t e r n f o r human d e s c e n d i n g c o l o n ) MIXED: b o t h s i a l o m u c i n s and s u l p h o m u c i n s p r e s e n t C 4 : s i a l i c a c i d w i t h an O - a c e t y l s u b s t i t u e n t a t p o s i t i o n C 4 C 7 : s i a l i c a c i d w i t h an O - a c e t y l s u b s t i t u e n t a t p o s i t i o n C 8 : s i a l i c a c i d w i t h an O - a c e t y l s u b s t i t u e n t a t p o s i t i o n C8 OAc S A : O - a c e t y l a t e d s i a l i c a c i d REFERENCES: 1. B a r a i B u r t i n ( 1 9 8 0 ) 2 . B o l a n d e t a ^ ( 1 9 8 4 ) 3 . C u l l i n g e_t £L ( 1 9 " ) 4 . C u l l i n g e t a l . ( 1 9 7 9 ) 5 . Dawson & F i l i p e ( 1 9 7 6 ) 6 . Dawson & F i l i p e ( 1 9 8 2 ) 7 . Dawson e t a j ( 1 9 7 8 ) 8 . E h s a n u l l a h e t a l ( 1 9 8 2 a ) 9 . E h s a n u l l a h i i ^1 ( 1 9 8 2 b ) 1 0 . E h s a n u l l a h e t £ l ( 1 9 8 5 ) 11 . F e n g e r & F i l i p e ( 1 9 8 1 ) 1 2 . F i l i p e ( 1 9 6 9 ) 1 3 . F i l i p e ( 1 9 7 1 ) 1 4 . F i l i p e ( 1 9 7 2 ) 1 5 . F i l i p e S B r a n f o o t ( 1 9 7 4 ) 1 6 . F i l i p e & Cooke ( 1 9 7 4 ) 17. F r a n z i n e t i l ( 1 9 8 1 ) 1 8 . F r a n z i n e t a ^ ( 1 9 8 3 a ) 1 9 . F r a n z i n £ i a l ( 1 9 8 3 b ) 2 0 . F r a n z i n e t al_ ( 1 9 8 4 ) 2 1 . F r a s e r 4 Clamp ( 1 9 7 5 ) 2 2 . G o l d & M i l l e r ( 1 9 7 5 ) 2 3 . G o l d & M i l l e r ( 1 9 7 8 ) 2 4 . G r e a v e s e t a i ( 1 9 8 0 ) 2 5 . G r e a v e s e t a l ( 1 9 8 4 ) 2 6 . H a b i b e t a j . ( 1 9 8 4 ) 2 7 . I s a a c s o n i A t t w o o d ( 1 9 7 9 ) 2 8 . Katayanwi e t a l . ( 1 9 8 5 ) 2 9 . K i m & I s a a c s ( 1 9 7 5 ) 3 0 . K o r o h o n e n e t a j . ( 1 9 7 1 ) 3 1 . L a p e r t o s a e t a j . ( 1 9 8 4 ) 3 2 . L e v e t t± ( 1 9 8 5 ) 3 3 . U s t i n s k i & R i d d e l l ( 1 9 8 1 ) 3 4 . M a k e l a e t «1 ( 1 9 7 1 ) 3 5 . M a r c h e g g i a n o e t a l . ( 1 9 8 4 ) 3 6 . M o n t e r o » S e g u r a ( 1 9 8 0 ) 3 7 . R e i d e t a j . ( 1 9 8 0 ) 3 8 . R e i d e t a j . ( 1 9 8 4 a ) 3 9 . R e i d e t a j . ( 1 9 8 5 a ) 4 0 . R o g e r s e t a l ( 1 9 7 8 ) 4 1 . S u n t e r e t a l ( 1 9 8 5 ) 4 2 . Teague tt al ( 1 9 7 3 ) 4 3 . T e r h o t L a i t i o ( 1 9 7 7 ) 4 4 . U r b a n s k i e t a l ( 1 9 8 4 ) TABLE IA: The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h COLORECTAL ADENOCARCINOMAS. TISSUE CHARACTER OF MUCIN COMPARED TO NORMAL COMMENTS REFERENCE MIXED 4 ; 85% showed d e c r e a s e d OAc SA 81.5% i n l e f t c o l o n ; 33% non mucin s e c r e t i n g 25 COLORECTAL p r e d o m i n a n t l y n e u t r a l 4 mucin and MIXED4 e i t h e r SULPHO 3 , S I A L O 4 , o r MIXED 4 ; d e c r e a s e d C4 and s i d e c h a i n OAc SA v e r y l i t t l e mucin s e c r e t i o n 12 ADENO- m o s t l y w e l l d i f f e r e n t i a t e d tumours 38 CARCINOMA d e c r e a s e d F u c , NANA, G a l N A c , G l c N A c ; d e c r e a s e d a c t i v i t y o f g a l a c t o s y l t r a n s f e r a s e I I , f u c o s y l t r a n s f e r a s e 1 , 1 1 , p o l y p e p t i d y l : N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e , and s i a l y l t r a n s f e r a s e i n c r e a s e d c o n t e n t o f s i a l i c a c i d 29 23 d e c r e a s e d c a r b o h y d r a t e (g p e r l O O g ) ; d e c r e a s e d m o l a r r a t i o s o f Fuc and Gal p r i m a r y c o l o n i c a d e n o c a r c i n o m a 22 i n c r e a s e d c a r b o h y d r a t e (g p e r lOOg); i n c r e a s e d m o l a r r a t i o s o f F u c , G a l , G a l N A c , and NGNA l i v e r m e t a s t a s i s 22 d e c r e a s e d s i d e c h a i n OAc SA 3 100% d e c r e a s e d s i d e c h a i n OAc SA; 36% SULPHO 3 , 32% S I A L O 4 ? 2 8 % MIXED 4 , 4% no m u c i n 4 39 • - - d e c r e a s e d C4 and s i d e c h a i n OAc SA - 37 2 f o l d i n c r e a s e i n t o t a l hexosamine c o n t e n t 43 64% S I A L O 4 31% d e c r e a s e d C7 + C8 OAc SA 50% d e c r e a s e d C7 + C8 OAc SA 100% d e c r e a s e d C7 + C8 OAc SA c o l o n i c tumours w e l l d i f f e r e n t i a t e d tumours m o d e r a t e l y d i f f e r e n t i a t e d tumours p o o r l y d i f f e r e n t i a t e d tumours 36 52% SIALO 4 30% d e c r e a s e d C7 + C8 OAc SA 66% d e c r e a s e d C7 + C8 OAc SA 100% d e c r e a s e d C7 + C8 OAc SA r e c t a l turr.ours w e l l d i f f e r e n t i a t e d tumours m o d e r a t e l y d i f f e r e n t i a t e d tumours p o o r l y d i f f e r e n t i a t e d 36 MIXED 4 ; d e c r e a s e d OAc SA 32 no uptake o f 3 5 S u l p h u r 13 i n c r e a s e d n e u t r a l mucin and s u l p h a t e ; d e c r e a s e d C4 SA 30 COLONIC MUCOSA REMOTE FROM TUMOURS i n c r e a s e d S I A L O 4 ; d e c r e a s e d SULPHO4 normal c o l o n i c mucosa 15 RECTAL BIOPSIES 12/22 SULPHO3 - . 10/22 i n c r e a s e d SIALO ; d e c r e a s e d SULPHO t a k e n from p a t i e n t s w i t h a d e n o c a r c i n o m a o f t h e r e c t u m o r l o w e r s i g m o i d 14 RECTAL MUCOSA i n c r e a s e d SIALO ; d e c r e a s e d OAc SA c o l o r e c t a l type g l a n d u l a r e p i t h e l i u m i n the a n a l c a n a l 11 HARBOURING . CARCINOMA i n c r e a s e d SULPHO ; s l i g h t l y i n c r e a s e d OAc SA a n a l g l a n d s w i t h ; s c a n t y mucous s e c r e t i o n 11 - 13"-TABLE I B : The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h t h e MUCOSA ADJACENT TO COLONIC TUMOURS TYPE OF [ ~ TUMOUR CHARACTER OF MUCIN COMPARED TO NORMAL COMMENTS REFERENCE COLORECTAL ADENO-CARCINOMA SIALO1" S I A L O 4 100% o f c a s e s SIALO 4 63% o f c a s e s SIALO 4 90% o f c a s e s SIALof 83%' o f cases SIAL07 66% o f c a s e s SIALO i n c r e a s e d S I A L O 4 , d e c r e a s e d SULPHO4 37% SULPHO 3 , 63% SIALO 4 76% d e c r e a s e d s i d e c h a i n OAc SA 4 4 i n c r e a s e d SIALO , d e c r e a s e d SULPHO , d e c r e a s e d uptake o f 3 5 S u l p h u r 4 p o o r l y d i f f e r e n t i a t e d m o d e r a t e l y d i f f e r e n t i a t e d w e l l d i f f e r e n t i a t e d i n c r e a s e d SIALO SIALO 4 SIALO 4 SIALO 4 d e c r e a s e d SULPHO i n c r e a s e d SIALO \ d e c r e a s e d SULPHO and s i d e c h a i n OAc SA d e c r e a s e d C4 i n c r e a s e d s i d e c h a i n d i and t r i OAc SA i n c r e a s e d t o t a l hexosamine and t o t a l s i a l i c a c i d d e c r e a s e d % C4 OAc SA 3 d e c r e a s e d i n c o r p o r a t i o n o f ( H)Gal m i x t u r e o f M3 3 and M l 4 mucosal a n t i g e n s 9 3 / 9 5 c a s e s SIALO 4 M l i s n o r m a l l y p r e s e n t i n g a s t r i c & f e t a l c o l o n mucosa 35 12 27 33 11 15 39 39 13 43 36 32 5 7 40 16 6 1 ADENOMATOUS1 A n y - C T A , A 4 POLYPS c a s e s SIALO 14 PAPILLOMATOUS1 , n „ . C T n i n 4 POLYPS 6 0 2 0 c a s e s SIALO 14 HYPERPLASTIC2 , n n e / ^ 3 POLYPS 1 0 0 % o f c a s e s S U L P H 0 14 ANAL MELANOMA2; ANAL SQUAMOUS ? CELL CARCINOMA^' MALIGNANT . LYMPHOMA2 8 9 . 9 % o f cases SIALO 27 1 ABLE I C : The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h COLONIC ADENOMAS TYPE OF ADENOMA CHARACTER OF MUCIN COMPARED TO NORMAL COMMENTS REFERENCE JUVENILE AND INFLAMMATORY POLYPS2 4 MIXED i n an abnormal p a t t e r n 4 MIXED i n an abnormal p a t t e r n 18 - 19 HYPERPLASTIC (METAPLASTIC) POLYPS2 5/5 p e d u n c u l a t e d & 12/45 s e s s i l e showed SIALO 4 SULPHO3 SULPHO3 i n t h e m a j o r i t y ; SIALO 4 i n a few i n p e d i c l e o r a d j a c e n t t o m e t a p l a s t i c mucosa i n m e t a p l a s t i c mucosa p o l y p s w i t h very few g o b l e t c e l l s 18 20 5 0 % - S I A L O 4 , 50% SULPHO3 i m m a t u r e - p o l y p s w i t h predominance o f g o b l e t c e l l s 20 100% SIALO 4 SULPHO3 e l o n g a t e d c r y p t s w i t h i n o r a d j a c e n t t o p o l y p 20 abundant c o b l e t c e l l s 12 MIXED HYPER-PLASTIC-ADENOMATOUS POLYP SIALO 4 p r o m i n e n t g o b l e t c e l l s ; showed one a r e a o f m a l i g n a n t t r a n s f o r m a t i o n 44 ADENOMAS1 28% S I A L O 4 , 72% SULPHO3 m o s t l y SULPHO3 c o n s i s t e n t w i t h normal (SULPHO j_ i n c r e a s e d n e u t r a l m u c i n s ; i n c r e a s e d degree o f s u l p h a t i o n 4 4 MIXED ; i n c r e a s e d SIALO i n l a r g e r o r d y s p l a s t i c p o l y p s 1/53 c a s e s had l o s s o f OAc SA 33 12 3 34 25 25 SULPHO3 a s s o c i a t e d w i t h a n a p l a s i a 12 ADENOMAS1 ASSOCIATED M I X E D 4 ; i n c r e a s e d SIALO 4 and n e u t r a l mucin 4 4 MIXED ; i n c r e a s e d SIALO ; 14/47 c a s e s d e c r e a s e d OAc SA 12 25 WITH MALIGNANCY d e c r e a s e d s i d e c h a i n OAc SA 3 STALKS OF 83% SIALO 4 33 ADENOMAS1 100% SIALO 4 19 CRONKHITE-CANADA ~ SYNDROME 3 c o n s i s t e n t w i t h normal (SULPHO ) 28 POLYPECTOMY SCARS 2/5 c a s e s SIALO 4 3/5 c a s e s SULPHO 18 - 15 -TABLE ID: The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h ANASTOMOTIC SITES  TYPE OF CHARACTER OF MUCIN COMMENT REFERENCE ANASTOMOSIS COMPARED TO NORMAL COLONIC RESECTION MARGINS 20/24 c a s e s SIALO . 04/24 c a s e s SULPHO* 02/24 c a s e s SIALO 4 -22/24 c a s e s SULPHO* p a t i e n t s had 26 l o c a l r e c u r r e n c e no l o c a l 26 r e c u r r e n c e COLONIC 07/28 c a s e s S I A L o l 41 ANASTOMOSIS 21/28 c a s e s MIXED i n an abnormal p a t t e r n SITES REMOTE SULPHO3 « 41 FROM COLONIC r a r e l y MIXED ANASTOMOSIS COLOSTOMY A SITE 100% o f c a s e s SIALO 27 BIOPSY URETER- MIXED4 w i t h i n c r e a s e d SIALO 4 SIGMOIDOSTOMY 35 - 16 -TABLE I E : The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h INFLAMMATORY DISEASE OF THE LARGE BOWEL TISSUE CHARACTER OF MUCIN COMPARED TO NORMAL COMMENTS REFERENCE 4 " ' i n c r e a s e d PNA b i n d i n g i n 11/18 c a s e s 5/11 d y s p l a s t i c ; 1/11 had a d e n o c a r c i n o m a 2 ULCERATIVE d e c r e a s e d 4 DBA &/or SBA b i n d i n g i n 9/18 c a s e s 4/9 d y s p l a s t i c ; 0/9 had a d e n o c a r c i n o m a 2 4 i n c r e a s e d BPA b i n d i n g i n 4/18 c a s e s 1/4 d y s p l a s t i c ; 1/4 had a d e n o c a r c i n o m a 2 C O L I T I S 1 18/81-SULPHO3 , 17/81 MIXED 4 , 46/81 S I A L O 4 a c t i v e phase 8 09/13 SULPHO 3 , 01/13 MIXED 4 , 03/13 S I A L O 4 r e s o l v i n g phase 8 20/26 SULPHO 3 , 01/26 MIXED 4 , 03/26 S I A L O 4 r e m i s s i o n phase 8 4 99% o f c a s e s SIALO r e c t a l b i o p s i e s w i t h d y s p l a s i a 10 89/488 (18%) c a s e s SIALO 4 r e c t a l b i o p s i e s w i t h o u t d y s p l a s i a 10 S I A L O 4 12 SIALO 4 a c t i v e phase 18 4 SIALO q u i e s c e n t phase 18 4 MIXED - abnormal i r r e g u l a r d i s t r i b u t i o n e n d o s c o p i c a l l y u n i n v o l v e d mucosa 18 d e c r e a s e d C4 and s i d e c h a i n OAc S A ; d e c r e a s e d m o l a r c h r o n i c a c t i v e - d e s c e n d i n g c o l o n 38 r a t i o s o f G a l - F u c , G l c N A c - G a l N A c , Fuc-NANA, Gal-NANA d e c r e a s e d C4 and s i d e c h a i n OAc SA c h r o n i c i n a c t i v e - d e s c e n d i n g c o l o n 38 d e c r e a s e d C4 and s i d e c h a i n OAc SA; d e c r e a s e d m o l a r c h r o n i c a c t i v e a s c e n d i n g c o l o n 38 r a t i o s o f Fuc-NANA and Gal-NANA no change i n a c t i v i t y o f any g l y c o s y l t r a n s f e r a s e 29 o r g l y c o s i d a s e d e c r e a s e d % (w/w) c a r b o h y d r a t e & i n c r e a s e d Man c o n t e n t 21 i n c r e a s e d Man c o n t e n t ; normal r e l a t i v e amounts o f 42 F u c , G a l , G a l N A c , and GlcNAc d e c r e a s e d s i d e c h a i n OAc SA c o l o n 4 - 17 -TABLE I E : The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h INFLAMMATORY DISEASE OF THE LARGE BOWEL . TISSUE CHARACTER OF MUCIN COMPARED TO NORMAL COMMENTS REFERENCE d e c r e a s e d s i d e c h a i n OAc SA ( n o t as marked as i n UC) c o l o n 4 CROHN'S 15/31 SULPHO 3 , 03/31 MIXED 4 , 13/31 SIALO 4 t e r m i n a l i l e u m and c o l o n 8 DISEASE 1 02/11 SULPHO 3 , 02/11 MIXED 4 , 07/11 SIALO 4 SULPHO3 c o l o n o n l y i n r e c t u m 8 12 MIXED 4 i n c o l o n 12 DIVERTICU- s t a i n i n g f o r OAc SA c o n s i s t e n t w i t h n o r m a l ; d e c r e a s e d m o l a r OAc SA - NANA r a t i o ; i n c r e a s e d m o l a r G a l - F u c r a t i o 38 L I T I S 2 S I A L O 4 SULPHO3 32 12 SOLITARY S I A L O 4 u l c e r a t i o n and i n f l a m m a t i o n p r e s e n t 9 ULCER S I A L O 4 17 SYNDROME 8 9 . 9 % o f c a s e s SIALO 4 12 NON-SPECIFIC PROCTITIS 2 01/78 s p e c i m e n s f r o m 65 c a s e s SIALO 4 . , 77/78 s p e c i m e n s from 65 c a s e s SULPHO 0 2 / 7 8 c a s e s SIALO 4 -. 76/78 c a s e s SULPHO h i s t o l o g i c f e a t u r e s s i m i l a r c o l i t i s to u l c e r a t i v e 8 9 ISCHEMIC 2 / 5 c a s e s SIALO 4 18 C O L I T I S 2 3/5 c a s e s SULPHO3 NON-SPECIFIC COLONIC S I A L O 4 12 INFLAMMATION TABLE I F : The c h a r a c t e r i s t i c s o f human c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h MISCELLANEOUS CONDITIONS OF THE LARGE BOwEL^ DISEASE CHARACTER OF MUCIN COMPARED TO NORMAL COMMENT REFERENCE MUCINOUS C O L I T I S , AMYLOID, MELANOSIS COLI , P I L E S , ANAL FISSURE, AMEBIC DYSENTRY, s i l l P H n 3 INFECTIVE DIARRHOEA, IRRADIATION PROCTITIS, HIRSCHPRUNG'S DISEASE, CHRONIC CONSTIPATION FIBRO-LIPOMA MIXED4 d e s c e n d i n g c o l o n 31 MALIGNANT MELANOMA, BASAL CELL CARCINOMA SIALO 4 a n o - r e c t u m 31 SQUAMOUS CELL CARCINOMA 1/3 c a s e s SULPHO3 1/3 c a s e s MIXED4 1/3 c a s e s MIX ED 4 , v a r i a b l e c o n t e n t i n an abnormal d i s t r i b u t i o n p r e d o m i n a n t l y SIALO and i n OAc SA a n o - r e c t u m 31 LIPOMA, LEIOMYOMA, ANGIOLEIOMYOMA, LEIOMYOSARCOMA 17% SIALO 4 83% SULPHO3 mucosa o v e r l y i n g o r a d j a c e n t t o mesenchymal tumours o f t h e d e s c e n d i n g c o l o n 33 METASTATIC TUMOURS IN THE DESCENDING COLON 73% SIALO 4 27% SULPHO3 mucosa o v e r l y i n g o r a d j a c e n t t o m e t a s t a t i c tumours o f t h e d e s c e n d i n g c o l o n 33 ENDOMETRIOSIS IN THE DESCENDING COLON • 50% S I A L O 4 50% SULPHO3 c o l o n i c mucosa o v e r l y i n g e n d o m e t r i o s i s 33 METASTATIC TUMOURS, EPIDERMOID CANCER, CHRONIC VOLVULUS SIALO 4 32 - 19 -significantly less O-acetyl substitution at position and/or in the polyhydroxy side chain (C7, CQ, C 0)(Culling et al, 1977; Greaves et al, 1984; Korohonen et al, 1971; Montero & Segura, 1980; Reid et al, 1980, 1984a, 1985a). There is also a proportionate decrease in sulphomucin (Filipe, 1969, 1971; Greaves et al, 1984; Montero & Segura, 1980; Reid et al, 1984a, 1985a), increased neutral mucin (Filipe, 1969; Korohonen et al, 1971; Terho & Laitio, 1977), alterations in carbohydrate content (Gold & Miller, 1975, 1977; Kim & Isaacs, 1975; Terho & Laitio, 1977; Bresalier, 1984; Boland et al, 1982b; Cooper, 1982; Yonezawa et al, 1982), and decreases in the activities of several glycosyltransferases (Kim & Isaacs, 1975). Relative increases of sialomucin, and proportionate decreases in sulphomucin and O-acetylated sialic acid residues have also been reported in normal colonic mucosa remote from tumours (Fenger & Filipe, 1981; Filipe, 1972; Filipe & Branfoot, 1974). The mucosa adjacent to colonic tumours ("transitional mucosa") exhibits changes in glycoproteins similar to those observed in tumours (Table lb). Changes resulting in a proportionate increase in or predominance of sialomucin and a relative decrease in sulphomucin (Dawson & Filipe, 1976; Dawson et al, 1978; Fenger & Filipe, 1981; Filipe, 1969, 1971; Filipe & Branfoot, 1974; Greaves et al, 1980; Isaacson & Attwood, 1979; Listinski & Riddell, 1981; Marcheggiano et al, 1984; Montero & Segura, 1980; Reid et al, 1985a) have been shown to occur with incidences ranging from 63% (Listinski & Riddell, 1981; Reid et al, 1985a) to 100% (Isaacson & Attwood, 1979) of cases examined. The incidence may be related to the degree of differentiation of the tumour as Fenger & Filipe - 20 -(1981) have shown that a predominance of sialomucin was observed adjacent to 66% of well, 83% of moderately and 90% of poorly differentiated colonic adenocarcinomas. Filipe (1972) has demonstrated the predominance of sialomucins adjacent to colonic adenomas that have a high malignant potential in 60% of cases whereas this did not occur in the mucosa adjacent to benign neoplasms with no or very low malignant potential (Table lb). Further, Isaacson & Attwood (1979) have shown the predominance of sialomucin in mucosa adjacent to anal melanomas, anal sauamous cell carcinomas, and malignant lymphomas in 90% of cases 1 examined. Other alterations in glycoproteins adjacent to colorectal adenocarcinomas include a decrease in sialic acid residues with C^  and/or side chain O-acetyl substituents (Dawson et al, 1978; Filipe & Cooke, 1974; Reid et al, 1985a), a relative increase in side chain di and t r i 0-acyl substituted sialic acids (Rogers et al, 1978), increased content of total hexosamine and total sialic acid (Filipe & Cooke, 1974), altered carbohydrate metabolism (Dawson & Filipe, 1982), abnormal mucin antigens (Bara & Burtis, 1980), and altered lectin binding affinities (Boland et al, 1982b; Cooper, 1982; Yonezawa, 1982). Polyps or adenomas have varying degrees of malignant potential (Enterline, 1976), however i t is generally assumed that most cases of colonic adenocarcinoma follow the adenoma-carcinoma sequence (Enterline, 1976). In general, juvenile, inflammatory, inflammatory-fibroid, Peutz-Jeghersand hyperplastic (metaplastic) polyps, as well as polyps associated with colitis cystica profunda and the Cronkhite-Canada syndrome have no or-very low malignant potential, whereas adenomas which - 21 -are large and/or exhibit a villous pattern have a very high malignant potential (Enterline, 1976). Glycoproteins in colonic polyps show changes similar to those described above and the frequency of these changes may be related to malignant potential (Table lc). Juvenile and inflammatory polyps contain both sialo- and sulphomucins mixed in an abnormal distribution (Franzin et al, 1983a, b) and hyperplastic (metaplastic) polyps have been shown to contain only sulphomucin (Filipe, 1969; Franzin, 1983a, 1984) or a predominance of sialomucin (Franzin et al, 1983a, 1984) depending on the location of the crypt with respect to the metaplastic mucosa itself and the degree of maturity of the polyp (Table lc). Urbanski et al (1984) have reported the occurrence of a mixed hyperplastic (metaplastic) - adenomatous polyp which appeared immature by the criteria of Franzin et al (1984) and contained an area of malignant transformation, and which produced only sialomucin. Polyps associated with the Cronkhite - Canada Syndrome contained only sulphomucin, no abnormal mucin could be detected (Katayama et al, 1985). Adenomas not associated with malignant change mostly have a normal staining pattern of predominantly (Culling et al, 1977; Filipe, 1969; Listinski & Riddell, 1981) or increased (Makela et al, 1971) sulphomucins, increased content of neutral mucins (Makela et al, 1971) and, very rarely, a loss of 0-acetyl sialic acid side chain substitution (Greaves et al, 1984). Larger or severely dysplastic polyps show an increase in sialomucin (Greaves et al, 1984). Adenomas associated with malignant transformation show an increase in neutral mucin (Filipe, 1969), increased sialomucin (Filipe, 1969; Culling et al, 1977; Greaves - 22 -et al, 1984), and a decrease in side chain O-acetyl substitution of sialic acid residues (Culling et al, 1977; Greaves et al, 1984). Stalks of adenomas also show a predominance of sialomucins (Franzin et al, 1983b; Listinski & Riddell, 1981) and polypectomy scar biopsies more often contain only sulphomucins but in two of five cases showed a predominance of sialomucins (Franzin et al, 1983a). Yonezawa et al (1983) have shown that in cases of polyposis coli abnormal lectin binding could be demonstrated in the adenomatous epithelium and in the nonneoplastic mucosa intervening between the polyps whereas, in cases of solitary polyps, while the polyps showed abnormal binding, the adjacent nonneoplastic mucosa did not. The mucosa taken from colonic anastomoses, colostomy, and ureterosigmoidostomy sites present either with a predominance of sialomucins or with increased sialomucins mixed with sulphomucins in an abnormal distribution (Isaacson & Attwood, 1979; Marcheggiano et al, 1984; Sunter et al, 1985) and Habib et al (1984) have shown that the mucosa of colonic resection margins which contain a predominance of sialomucins are associated with local recurrence of malignancy whereas those showing a predominance of sulphomucins had no local recurrence (Table ld). Habib et al (1984) further suggest that the presence of sialomucins at the resection margin may be used to predict the recurrence of malignancy. Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are associated with an increased risk for the development of colorectal cancer (Earnshaw et al, 1982; Greenstein et al, 1981). Table - 23 -le summarizes the changes in colonic epithelial glycoproteins occurring in diseased and uninvolved mucosa. In cases of ulcerative colitis differences in lectin binding (Boland et al, 1984) reflect changes in carbohydrate content. A decrease in sulphate resulting in the predominance of sialomucins has been demonstrated in the active phase of ulcerative colitis (Ehsanullah et al, 1982a; Filipe, 1969; Franzin et al, 1983a), the auiescent phase (Franzin et al, 1983a), and in rectal biopsies associated with dysplasia (Ehsanullah e_t al, 1985). The endoscopically uninvolved mucosa has shown both sialo- and sulphomucins mixed in an abnormal, irregular pattern (Franzin et al, 1983a) and the resolving and remission phases more often present with a predominance of sulphomucins (Ehsanullah et al, 1982a). Significant decreases in and side chain 0-acetyl sialic acid substitution have been demonstrated in chronic active and chronic inactive ulcerative colitis (Culling et al, 1979; Reid et al, 1984a), and in the active phase there were changes in carbohydrate content (Fraser & Clamp, 1975; Reid et al, 1984a; Teague et al, 1973) but no observed changes in the activities of any glycosyltransferases or glycosidases (Kim & Isaacs, 1975). Crohn's disease of the colon shows decreased side chain 0-acetyl substituted sialic acid (Culling et al, 1979) and increases in the relative proportion of sialomucin (Ehsanullah et al, 1982a; Filipe, 1969) although these were not as marked as in ulcerative colitis. Colonic mucin in diverticular disease is consistent with normal mucin (Filipe, 1969; Reid et al_, 1984a) except for a decrease in the molar ratios of some carbohydrates (Reid et al, 1984a) (see Table le). Other inflammatory bowel diseases not associated with an increased cancer risk have been studied. Solitary ulcer syndrome presents with a predominance of sialomucin (Ehsanullah et al, 1982b; Franzin et al, 1981; Isaacson & Attwood, 1979) as does non-specific colonic inflammation (Filipe 1969) and ischemic colitis (Franzin e_t al, 1983a) whereas non-specific proctitis, which has histologic features similar to ulcerative colitis (Ehsanullah et al, 1982a), is usually associated with a predominance of sulphomucins (Ehsanullah et al, 1982a,b). Miscellaneous other diseases of the large bowel, not associated with increased cancer risk, presented in Table If are not usually associated with abnormal glycoprotein however recently increases in sialomucin have been observed in fibro-lipoma, malignant melanoma, basal cell carcinoma, sauamous cell carcinoma (Lapertosa et al, 1984), endometriosis and metastatic tumours from non-colonic primary sites (Listinski & Riddell, 1981). ( i i i ) In normal and carcinogen-treated rats Previous investigations have shown that the normal distal Wistar rat colonic mucin is predominantly composed of sulphomucin (Filipe, 1975; Reid et al, 1975; Decaens et al, 1983; Tsuyama et al, 1983) and contains O-acetyl substituted sialic acid residues (Tsuyama et al, 1983; Reid et al, 1975), as outlined in Table 2. Studies with lectins have shown distinct lectin binding affinities (Freeman et al, 1980; Tsuyama et al, 1983). Mucin changes in rats involving a decrease in sulphomucin and an increase in sialomucin have been observed in normal mucosa remote from tumours (Shamsuddin & Trump, 1981; Decaens et al, 1983), in dysplastic foci (Filipe, 1975; Decaens et al, 1983), transitional mucosa (Decaens et - 25 -TABLE 2: THE CHARACTERISTICS OF THE WISTAR RAT DISTAL COLONIC  EPITHELIAL MUCIN IN NORMAL RAT AND DURING THE  INDUCTION OF COLON CANCER. LEGEND: 3 ( H)Gal : t r i t i u m l a b e l l e d g a l a c t o s e ( H)Fuc : t r i t i u m l a b e l l e d f u c o s e M l Ag : mucin a n t i g e n s n o r m a l l y p r e s e n t i n human f e t a l and human a d u l t stomach mucin G l c : g l u c o s e GlcNAc : N - a c e t y l - g l u c o s a m i n e Gal : g a l a c t o s e GalNAc : N - a c e t y l - g a l a c t o s a m i n e Fuc ; f u c o s e Man : mannose NeuNAc ; N - a c e t y l - n e u a m i n i c a c i d ( s i a l i c a c i d ) LECTIN NAME (BINDS TO) DBA D o l i c h o s b i f l o r u s a g g l u t i n i n (GalNAc) PNA peanut a g g l u t i n i n ( G a l - G a l N A c ; G a l ) RCA R i c i n i s communis a g g l u t i n i n ( G a l ) SBA soybean a g g l u t i n i n (GalNAc ; G a l ) WGA wheat germ a g g l u t i n i n (GlcNAc ; NeuNAc) UEA U l e x europeus a g g l u t i n i n (Fuc) BPA B a u h i n i a p u r p u r e a a g g l u t i n i n (GalNAc) LPA L i m u l u s polyphemus a g g l u t i n i n (NeuNAc) Con A c o n c a n a v a l i n a g g l u t i n i n (Man ; G l c ) LCA Lens c u l i n a r i s a g g l u t i n i n (Man ; G l c ) - 26 -TISSUE MUCIN CHARACTER normal p r e d o m i n a n t l y s u l p h o m u c i n d i s t a l p r e d o m i n a n t l y s u l p h o m u c i n ; c o l o n no M l Ag s u l p h a t e d g l y c o p r o t e i n (1.91% w/w s u l p h a t e ) ; 65% o f s i a l i c a c i d r e s i d u e s have 0 - a c e t y l s u b s t i t u e n t s p r e d o m i n a n t l y s u l p h o m u c i n and O - a c e t y l a t e d s i a l i c a c i d s ; l e c t i n s : p o s i t i v e w i t h DBA.PNA, RCA,SBA,WGA; n e g a t i v e w i t h UEA l e c t i n s : p o s i t i v e w i t h PNA.SBA, WGA,UEA,BPA,LPA; n e g a t i v e w i t h Con A.'LCA.RCA normal i n c r e a s e d s i a l o m u c i n ; remote d e c r e a s e d s u l p h o m u c i n from tumours d y s - i n c r e a s e d s i a l o m u c i n ; p l a s i a d e c r e a s e d s u l p h o m u c i n i n c r e a s e d s i a l o m u c i n ; d e c r e a s e d s u l p h o m u c i n ; p r o d u c t i o n o f M l Ag d e c r e a s e d ( H)Gal u p t a k e ; no change i n ( 3 H ) F u c uptake t r a n s i - i n c r e a s e d s i a l o m u c i n ; d e c r e a s e d t i o n a l s u l p h o m u c i n ; p r o d u c t i o n o f Ml Ag mucosa c o l o n i c i n c r e a s e d s i a l o m u c i n ; adeno- d e c r e a s e d s u l p h o m u c i n c a r c i -noma p r o d u c t i o n o f M l Ag REFERENCE  F i l i p e (1975) Decaens e t al_ (1983) R e i d e t aj_ (1975) Tsuyama e t aj_ (1983) Freeman ejt aj_ (1980) Decaens e t al_ ( 1 9 8 3 ) ; Shamsuddin & Trump (1981) F i l i p e (1975) Deceans e_t al_ (1983) Dawson & F i l i p e (1980) Decaens et_ al_ (1983) F i l i p e (1975) Decaens e t a l (1983) - 27 -a l , 1983) and c o l o n i c adenocarcinomas ( F i l i p e , 1975). Decaens et a l (1983) have also shown the production of Ml mucin antigens i n dysplasia, t r a n s i t i o n a l mucosa, and tumours and Dawson & F i l i p e (1980) have observed decreased [^H] galactose uptake i n d y s p l a s t i c areas. This evidence suggests that there are mucin a l t e r a t i o n s i n rats associated with the development of cancer. Furthermore, the changes resemble those described i n human colon cancer. VIII The model ( i ) Rationale for using an animal model to study premalignant change Premalignant changes are inherently d i f f i c u l t to study i n man because a v a i l a b l e t i s s u e s are associated with e i t h e r established or suspected disease. Therefore, the 1,2-dimethylhydrazine (DMH)-induced rat c o l o n i c neoplasia model (Druckery, 1967, 1970) was chosen for study. DMH-induced tumours are a good model of human c o l o r e c t a l cancer (Deschner, 1974; Ward, 1974; P o z h a r i s s k i , 1975; F i l i p e , 1975; Maskens, 1976; Lamont & O'Gorman, 1978; Martins, 1982), and spontaneous carcinoma of the colon i n r a t s i s very rare (Crain, 1958; Burn et a l , 1966; Mitra, 1966; Sass et a l , 1975; Miwa, 1976). When appropriate doses are used (repeated subcutaneous i n j e c t i o n s at 7-25 mg/kg body weight or single i n j e c t i o n s of 40 or 200 mg/kg; F i a l a , 1975), DMH has a low t o x i c i t y , tumourigenesis i s almost e x c l u s i v e l y r e s t r i c t e d to the colon, and the anatomic d i s t r i b u t i o n of the tumours produced within the colon c l o s e l y resembles that found i n man. Further, epidemiologic studies have shown that r i g h t sided c o l o n i c tumours i n man predominate i n areas associated - 28 -with a low r i s k of large bowel cancer whereas i n high r i s k areas a higher percentage of tumours occur i n the l e f t colon. S i m i l a r l y , i n the rat model, when low doses of DMH are used tumours are more common i n the upper colon and when high doses are used tumours predominate i n the lower colon (Lament & O'Gorman, 1978). I t has been shown that even one dose of DMH i s "capable of d i s t o r t i n g the pre-programmed movement of d i f f e r e n t i a t e d c e l l s along the crypts of Lieberkuhn" and further i t i s postulated that t h i s may i n d i c a t e that "a small number of promutagenic l e s i o n s have the a b i l i t y to induce a t y p i c a l preneoplastic c e l l s . " (Herron & Shank, 1982). This evidence supports the use of the DMH model to study premalignant change. ( i i ) Current theories on the mechanism of DMH carcinogenesis DMH i s a procarcinogen which must be converted to the unstable metabolite methyldiazonium (MD) to exert i t s carcinogenic e f f e c t . MD undergoes r a p i d degeneration to a carbonium ion capable of a l k y l a t i n g DNA. Figure 3 i l l u s t r a t e s current theories of DMH metabolism. Following the subcutaneous i n j e c t i o n of DMH i n r a t s , a l k y l a t e d DNA bases, O^-methylguanine and 7-methylguanine can be demonstrated i n colon and l i v e r c e l l s (Herron & Shank, 1981). These bases are produced at a high rate i n c o l o n i c epithelium because of the high rate of c e l l d i v i s i o n normally present i n t h i s t i s s u e and because of the i n i t i a l DMH-induced 24 hour increase i n colon DNA synthesis (Hawks et a l 1974). In colon, a l k y l a t e d DNA bases have a h a l f l i f e of approximately 30 hours and removal of these bases i n c o l o n i c c e l l s i s determined by c e l l sloughing and c e l l d i v i s i o n whereas i n l i v e r the a l k y l a t e d bases are removed by DNA - 29 -FIGURE 3: THE METABOLISM OF 1,2-DIMETHYL,HYDRAZINE (DMH) (REFERENCES: F i a l a , 1975; H e r r o n & S h a n k , 1 9 8 1 , 1 9 8 2 ; M a r t i n , 1982 Newaz e t a l , 1983) (DMH) H 3 C-NH|NH-CH 3 ( s u b c u t a n e o u s i n j e c t i o n ) 11 v e r 1 (AM) H 3 C-N=N-CH 3 y may be e x p i r e d as a gas (AOM) H 3 C-N=N-CH 3 —V-» 1) may r e a c h c o l o n v i a b l o o d s t r e a m ; 0^ / 2) may be c o n j u g a t e d t o g l u c u r o n i c a c i d and s e c r e t e d i n t h e b i l e (MAM) H_C-N-N-CH OH (OH)" + H2C=0 H3C-N*=N ( a l k y l a t i n g a g e n t ) C P (RAPID) H 3 C* + N 2 - 30 -r e p a i r and spontaneous depurination (Herron & Shank, 1982). In a study comparing the e f f e c t s of DMH on micronucleus (an i n d i c a t i o n of chromosomal abberation) incidence i n bone marrow and colon i n v i t r o , Goldberg et a l (1983) showed that while both t i s s u e s are l a b i l e and have a high rate of c e l l p r o l i f e r a t i o n and turnover, only the c o l o n i c c e l l s developed micronuclei. The incidence of DMH induced nuclear aberration i n colon c e l l s was dose-related over a range of concentrations while DMH, regardless of the concentration used, had l i t t l e e f f e c t on the bone marrow c e l l s . DMH may be metabolized by the drug-metabolism systems of the l i v e r or of human colon microsomes. The highest a c t i v i t y rate of the c o l o n i c microsome system i s found i n the l e f t or descending colon, intermediate rates i n the transverse colon and lowest rates i n the r i g h t or ascending colon (Newaz et a l , 1983). In the l i v e r , DMH i s f i r s t oxidized to azomethane (AM). AM i s a poisonous gas, soluble i n both aqueous and organic solvents and i s expired by experimental animals. AM i s further ox i d i z e d , probably by a microsomal oxygenase, to azoxymethane (AOM) which i s a lso h i g h l y t o x i c and carcinogenic, hence the need to house the experimental animals i n a carcinogen containment f a c i l i t y . AOM may be hydroxylated, by the microsomal mono-oxygenase system i n the l i v e r , to methylazoxymethanol (MAM). MAM i s the aglycone of cycasin a n a t u r a l l y o c c u r r i n g carcinogen. In f a c t t h i s model of colon carcinogenesis was a c c i d e n t a l l y discovered by Laqueur (1965) who was a c t u a l l y looking for a d i e t a r y cause for the unusually high incidence of amylotrophic l a t e r a l s c l e r o s i s i n Guam since the Guamian d i e t i s high i n cycad meal. Feeding - 31 -cycad meal to r a t s , however, f a i l e d to produce neurologic disease and instead i n t e s t i n a l tumours were found i n some animals. They found that the carcinogen responsible was cycasin, a water soluble glucoside of MAM. AM, AOM, or MAM may be c a r r i e d to be colon v i a the blood stream and further metabolized there or may be conjugated to glucuronic a c i d and secreted i n the b i l e . In the colon, mucosal and b a c t e r i a l Q-glucuronidases hydrolyze the conjugated OAcetyl-methylazoxymethane to free MAM, thereby l i b e r a t i n g the carcinogenic a l k y l a t i n g agent methyldiazonium (MD) ( F i a l a , 1975; Martin, 1982). Evidence that b a c t e r i a l as well as host enzymes are involved i n the metabolism of the carcinogen r e s u l t s from studies with germ-free r a t s . Some such animals have decreased s e n s i t i v i t y to DMH ca r c i n o g e n i c i t y (Reddy et a l , 1974), others may never develop colon tumours, and s t i l l other s t r a i n s show a 100% incidence of colon tumours a f t e r 10 weekly doses of 15 mg/kg DMH (Asano & P o l l a r d , 1978). Carcinogenesis i n t h i s model involves two steps (Maskens, 1981). The f i r s t , i n i t i a t i o n , i s ( i ) caused by the carcinogen, ( i i ) a d d i t i v e , and ( i i i ) t r a n s m i s s i b l e within the renewing mucosa for prolonged periods. The second, promotion, ( i ) does not require the presence of the carcinogen, and ( i i ) randomly a f f e c t s c e l l s having undergone i n i t i a t i o n . The hypothesis that DMH acts s p e c i f i c a l l y on inherently susceptible c e l l s i s supported by the high degree of organotropism f o r the colon demonstrated by t h i s carcinogen even when the colon i s experimentally transposed to the small i n t e s t i n e (Gennaro et a l , 1973; C e l i k et a l , 1981), when the f e c a l stream i s div e r t e d (Senior et a l , 1981) or when a - 32 -segment of the colon is transplanted to the abdominal wall as an isolated loop (Rublo et al, 1980). These studies suggest that the carcinogen reaches the colon via the bloodstream and hence the organotropism is not due to preferential distribution but rather to some other factor such as selective metabolism by colonic cells (Grab & Zedeck, 1977) or a differential rate of repair of methylated DNA (Swenberg et al, 1979). As well, in rat different areas of colon show different susceptibilities to DMH carcinogenesis (Sunter et al, 1978a,b). IX Rationale for the use of histochemistry as an investigative tool Mucins may be studied either chemically or histochemically. Histochemical methods are more favourable for a study of premalignant changes in rat colon for numerous reasons. Rat colons are very small and the amount of mucosa required to produce enough mucin to analyse chemically would cover a large area and a large number of crypts. Hence, i f premalignant change involves a small area of mucosa the change would not be seen in chemical analysis. The whole colon may be analysed at once histochemically and directly related to morphology and anatomic location within the cell and throughout the colon. In a histochemical section the mucin within the goblet cell is a material uncontaminated with components of other cell types as may be the case when mucin is chemically extracted from tissue homogenates. In my experience, the purification of such extracts is tedious, time consuming and yields relatively small amounts of glycoprotein. Histochemical methods are more sensitive than chemical methods. Simple arithmetic emphasizes how small - 33 -an amount of material can be v i s u a l i z e d histochemically (Reid 1984, personal communication). The detection of c o l o n i c e p i t h e l i a l glycoproteins can be done using histochemical methods that detect s i a l i c a c i d residues. Assuming that the colon of the 150g Wistar r a t measures approximately 15 cm, 30,000 5 micron sections can be cut from one colon. Following the i s o l a t i o n of c o l o n i c e p i t h e l i a l c e l l s approximately 4 mg of p u r i f i e d g lycoprotein can be obtained by chemical procedures. Such glycoproteins contain 15% by weight of s i a l i c acid and therefore one colon would contain 0.6 mg s i a l i c a c i d . Each 5 micron sectionwould contain [(0.6)(1000)(1000)/(30,000)] = 20 nanograms (ng) s i a l i c a c i d . Assuming, i n a conservative estimate that there are 100 e p i t h e l i a l c e l l s per transverse section then each c e l l would contain 0.2 ng s i a l i c a c i d . Currently the most s e n s i t i v e chemical procedures for such s i a l i c acids w i l l j u s t detect 0.2 micrograms per tube, a d i f f e r e n c e i n s e n s i t i v i t y of 1000 times. The increased s e n s i t i v i t y of histochemical methods i s presumably a function of the large e x t i n c t i o n c o e f f i c i e n t of the "substrate/reaction" product and the, " i n e f f e c t " , high concentration of substrate (weight/volume). A problem freauently encountered i n histochemistry i s a poor understanding of the mechanism of the reactions involved i n the s t a i n i n g procedure. Without such knowledge i t i s d i f f i c u l t to p r e d i c t with any c e r t a i n t y the chemical nature of the material being stained. Previously there were very few procedures a v a i l a b l e for s t a i n i n g mucin glycoproteins. Recently however a s e r i e s of new s t a i n s has been developed (Reid et a l , 1985d) and most of them were used i n t h i s - 34 -investigation. Using these new methods i t was possible to determine, to a degree hitherto impossible, the character of the colonic epithelial glycoproteins and the identification of abnormalities associated with neoplasia. X Thesis rationale Colonic epithelial glycoproteins contain terminal sialic acid residues with 0-acetyl substituents at position C^  and/or in the polyhydroxy side chain (C^, CQ, C g), and O-sulphate esters (Filipe 1979). Colonic tumours and their immediately adjacent mucosae contain decreases in sulphate and 0-acetyl substituted sialic acid (Table la,b). Similar glycoprotein abnormalities have been observed in premalignant lesions such as adenomas (Table lc), anastomotic sites (Table Id), and inflammatory bowel diseases (Table le). Colon cancer is an increasingly common neoplastic disease in man, most often occurring in the rectum and descending colon and with greatest freauency in industrialized nations (Ames, 1983; Bresnick, 1980; de Jong et al, 1972; Zaridze, 1983). If the cancer is detected before metastasis has occurred then appropriate surgical intervention can, in most cases, effect a cure (Beahrs, 1982). Early diagnosis is of obvious fundamental importance to the patient's prognosis (Winawer, 1980). The objective of this study was to determine whether changes in colonic epithelial glycoproteins could be detected prior to any morphologic evidence of neoplasia during the induction of colon cancer in rats using the carcinogen 1,2-dimethylhydrazine. - 35 -otto ru^ifum^arui bofeeQuW dwL>icr6e& con&Ll tdk, ak-manu oon, 3b gfyenwint, Vafcriari, cwd>(knawes, Zbik oikcrcsQidac, i(1ki£fncfeUtkrier> Ourfan^famw^^ Ho alooutt ouronft, if tfiatiOcnum. Our fcnririap cdcof' cflkinacwurv, 5 F R O M - a l c ^ ^ S n a i i i , -Me.' IN ; FIGURE 4. - 36 -MATERIALS AND METHODS I MATERIALS Young male inbred Wistar r a t s weighing approximately 200 grams were purchased from the Amimal Care Unit of the University of B r i t i s h Columbia. The animals were housed i n a carcinogen containment f a c i l i t y at the B r i t i s h Columbia Cancer Research Centre with r e l a t i v e humidity and environmental a i r temperature constant and a twelve hour l i g h t / d a r k c y c l e . The r a t s were fed a standard laboratory d i e t (Purina Rat Chow P e l l e t s , Ralston Purina Co. Ltd., St. Louis, MO.) with water ad li b i t u m . At each i n j e c t i o n period and at each s a c r i f i c e the r a t s were weighed. Basic fuchsin, pararosanalin and hematoxylin were purchased from Fisher S c i e n t i f i c Co., t h i o n i n from JT Baker Chemical Co., eosin Y from B r i t i s h Drug House (BDH) and A l c i a n blue 8Gx from Matheson Coleman B e l l (MCB). Symmetrical 1,2-dimethylhydrazine d i HC1 was obtained from A l d r i c h Chemical Co., Milwaukee, WI. A l l other chemicals used were purchased from Fisher, MCB or Eastman - Kodak and were of reagent grade or b e t t e r . II METHODS ( i ) Induction of tumours This portion of the study was c a r r i e d out by Dr. Hugh J . Freeman, Head of the D i v i s i o n of Gastroenterology, Department of Medicine, Acute Care Unit, U n i v e r s i t y of B.C. Figure 5 illustrates the experimental design. Three hundred male Wistar rats were divided into 15 groups of 20 rats consisting of 10 control and 10 treated rats per group. Treated rats were given weekly subcutaneous injections of 0.083 M DMH (1,2-dimethylhydrazine-dihydrochloride) in 1 mM EDTA (ethylenediaminetetra-acetic acid) at pH 6.5 (25.0 mg/kg body weight). One group of rats was sacrificed with either nembutal or ether at each of the following time periods: 1 day (1 injection), 1 week (1 injection), 2 weeks (2 injections), 4 weeks (4 injections), 6 weeks (6 injections), 8 weeks (8 injections), 11 weeks (11 injections), 16 weeks (16 injections), 20 weeks (17 injections), 22 weeks (17 injections), 24 weeks (17 injections), 26 weeks (17 injections), 28 weeks (17 injections), 30 weeks (17 injections), 32 weeks (17 injections). Colons were resected, opened longitudinally, washed with saline, placed in 15 ml polypropylene test tubes and fixed in 10% formol calcium for periods in excess of seven days. (ii) Gross observation and preparation of tissues for histology Upon receipt, the fixed colons were pinned out onto mats and the number and location of tumours was recorded. Colons were cut into upper and lower halves with a scalpel at the juncture of the distinctly different muscular banding patterns (figure 6) and labelled at the proximal edges with India ink (slide 11). Specimens were assigned a five digit code consisting of letters and integers. The first letter, either D or E or C signified either DMH treated or EDTA treated or control rats receiving no treatment, respectively. The second letter denoted the time - 38 -FIGURE 5 : EXPERIMENTAL DESIGN 300 MALE WISTAR RATS 200 GMS 15 GROUPS; 20 RATS/GROUP; 10 CONTROL, 10 TREATED WEEKLY SUBCUTANEOUS INJECTIONS: EDTA OR DMH i n EDTA 1 GP SACRIFICED @ 1 DAY, 1 , 2 , 4 , 6 , 8 , 1 1 , 1 6 , 2 0 , 2 2 , 2 4 , 2 6 , 2 8 , 3 0 , 3 2 WEEKS COLONS RESECTED AND FIXED IN 10% FORMALIN FIXED COLONS I GROSS ANATOMICAL OBSERVATION I SWISS ROLL ROUTINE PROCESSING AND SECTIONING 1 HISTOLOGY AND HISTOCHEMISTRY - 39 -FIGURE 6: SCHEMATIC DIAGRAM OF THE WISTAR RAT COLON l y m p h o i d . a g g r e g a t e l y m p h o i d _ a g g r e g a t e l y m p h o i d . a g g r e g a t e UPPER (PROXIMAL) COLON ( 5 - 7 cm) j u n c t u r e o f upper & l o w e r c o l o n s LOWER (DISTAL) COLON (9 - 1 2 . 5 cm) - AO -at which the r a t was s a c r i f i c e d ; a = 1 day, b = 1 week, c = 2 weeks, d = A weeks, and so on. The number following the second l e t t e r r e ferred to the number of the r a t within the group s a c r i f i c e d at any one time period. The l a s t two l e t t e r s denoted the region of the colon. AB r e f e r r e d to upper colon and CD lower colon. For example, the code DalCD meant that the specimen was a lower colon (CD) from r a t no. 1 which had been treated with the carcinogen DMH (D) and was s a c r i f i c e d at one day a f t e r treatment (a). S i m i l a r l y the code Eh3AB denoted an upper colon from r a t no. 3 who received only sham i n j e c t i o n s of EDTA and was s a c r i f i c e d at sixteen weeks. ( i i i ) S t a i n i n g procedures S t r i p s of colon were r o l l e d i n t o "Swiss r o l l s " with the proximal end i n the centre, embedded i n paraplast and processed r o u t i n e l y ( C u l l i n g , 197A). No l e s s than twelve and not more than twenty-four s e r i a l s e c t i o n s were cut at a thickness of f i v e microns. Sections were mounted on clean glass s l i d e s using 0.1% chromealum i n 1.0% g e l a t i n ( C u l l i n g , 197A) as an adhesive and post fixed i n formalin vapour at 60°C for 2A hours. The s t a i n i n g procedures (table 3) were performed i n large batches so that a l l specimens were stained with one procedure at the same time with the same batch of s t a i n s and reagents. Control sections both untreated and pretreated with the PBT/KOH method (periodate oxidation, borohydride reduction, s a p o n i f i c a t i o n ) (table 3a), of normal human colon ( a c i d i c mucin, sulphated), normal r a t colon (taken from a region which contained a c i d i c mucin, non-sulphated), and normal human stomach (neutral - 41 -TABLE 3. CHARACTERISTICS OF THE HISTOCHEMICAL STAINS USED  FOR THE IDENTIFICATION OF RAT COLONIC MUCOSAL EPITHELIAL GLYCOPROTEINS * The i n i t i a l o x i d a t i o n t i m e u s e d was 2 h o u r s *' I n t e r p r e t a t i o n o f t h e s e h i s t o c h e m i c a l p r o c e d u r e s depends on t h e f o l l o w i n g a s s u m p t i o n s : ( i ) 0 - a c e t y l g r o u p s e x i s t o n l y on s i a l i c a c i d r e s i d u e s ( n o t on n e u t r a l h e x o s e r e s i d u e s ) ; ( i i ) C9 0 - a c e t y l s i a l i c a c i d r e s i d u e s a r e o x i d i z e d u n d e r t h e c o n d i t i o n s u s e d ; ( i i i ) P h e n y l h y d r a z i n e b l o c k a d e i s o n l y r e v e r s i b l e on s i a l i c a c i d m o n o a l d e h y d e s ; ( i v ) HID a t pH 1 . 0 s t a i n s o n l y s u ; p h a t e g r o u p s and s u b s e q u e n t s t a i n i n g w i t h AB a t pH 2 . 5 s t a i n s o n l y CI c a r b o x y l g r o u p s o f s i a l i c a c i d r e s i d u e s . 1 . C o l o u r s s e e n a r e e i t h e r a q u a m a r i n e b l u e (A) o r b l a c k ( B k ) . 2 . M i x t u r e s o f t h e v a r i o u s s t a i n i n g c o m p o n e n t s s t a i n i n d i f f e r e n t s h a d e s r a n g i n g f r o m a q u a m a r i n e b l u e ( A ) , t h r o u g h d a r k aqua ( D A ) , r o y a l b l u e ( R B ) , p u r p l e ( P ) , r e d p u r p l e ( R P ) , t o magenta ( M ) . 3 . M i x t u r e s o f t h e v a r i o u s s t a i n i n g c o m p o n e n t s s t a i n i n d i f f e r e n t s h a d e s r a n g i n g f r o m t h i o n i n e b l u e ( B ) , t h r o u g h b l u e p u r p l e ( B P ) ; p u r p l e ( P ) , and r e d p u r p l e ( R P ) , t o magenta ( M ) . 4 . When magenta (M) s t a i n i n g was p r e s e n t i t was g r a d e d on a n u m e r i c a l s c a l e o f i n t e n s i t y f r o m 1+ t o 4 + . HID/AB 2 . 5 = H i g h Iron D i a m i n e / A l c i a n B l u e pH 2 . 5 K 0 H / A B 1 . 0 / P A P S = K O H / A l c i a n B l u e pH 1 . 0 / P e r i o d i c A c i d ; P h e n y l h y d r a z i n e ; S c h i f f P B T / K 0 H / A B 1 . 0 / P A S = P e r i o d a t e ; B o r o h y d r i d e T r e a t m e n t / K O H / A l c i a n B l u e pH 1 , 0 / P e r i o d i c A c i d j S c h i f f AB 1 . 0 / P A P S = A l c i a n B l u e pH 1 . 0 / P e r i o d i c A c i d ; P h e n y l h y d r a z i n e ; S _ c h i f f PAPT/KOH/Bh/PAS = P e r i o d i c A c i d ; P h e n y l h y d r a z i n e ; T h i o n i n e / K O H / B o r o h y d r i d e / P e r i o d i c A c i d ; S c h i f f PAT/KOH/Bh/PAS = P e r i o d i c A c i d ; T h i o n i n e / K O H / B o r o h y d r i d e / P e r i o d i c A c i d ; S c h i f f PBT/KOH/PAS = P e r i o d a t e ; B o r o h y d r i d e T r e a t m e n t / K O H / P e r i o d i c A c i d ; S c h i f f B k = b l a c k ; A = a q u a m a r i n e b l u e ; M=magenta; B = t h i o n i n e b l u e ; 0 = n o s t a i n i n g ; - 0 S 0 . j H = 0 - s u l p h a t e e s t e r s C p = s i d e c h a i n u n s u b s t i t u t e d s i a l i c a c i d ; , Cg , and C g = s i a l i c a c i d w i t h 0 - a c e t y l s u b s t i t u e n t s a t p o s i t i o n s C 7 . C 8 , and C 9 , r e s p e c t i v e l y , (C8 may a l s o be d i o r t r i s u b s t i t u t e d ) . T i s s u e d i o l = o x i d i z a b l e d i o l on n e u t r a l h e x o s e o r h e x o s a m i n e r e s i d u e s . - 42 -HISTOCHEMICAL STAIN SIALIC ACID TISSUE -OS03H C Q C ? C g C g DIOL REFERENCE HID/AB 2 . 5 1 Bk A A A A S p i c e r 1965 KOH/ABl.O/PAPS A M M M M R e i d e t a l 1985c PBT/KOH/ABl.O/PAS A 0 M M 0 R e i d e t a l 1985c A B l . O / P A P S A M M 0 M R e i d e t a l 1985c PAPT/KOH/Bh/PAS 0 B B M B R e i d e t a l 1984b PAT/KOH/Bh/PAS 0 B B M B B R e i d e t a l 1984b PBT/KOH/PAS 0 0 M M 0 0 R e i d e t a l 1973; C u l l i n g e t a l 1974 - 43 -TABLE 3a: PREDICTED S T A I N I N G RESULTS FOR CONTROL SECTIONS f <? # # f X # CONTROL .<? -J" # v ^ * T I S S U E # # £ * <p> £ NORMAL HUMAN  STOMACH ( n e u t r a l mucin) UNTREATED 0 0 0 0 0 B 0 PBT/KOH PRETREATED O O O O O O O NORMAL HUMAN  COLON ( a c i d i c mucin s u l p h a t e d ) UNTREATED Bk P P P M M M PBT/KOH PRETREATED Bk P A P B B 0 NORMAL RAT  COLON ( a c i d i c mucin n o n - s u l p h a t e d ) UNTREATED A M M M M M M PBT/KOH PRETREATED A M 0 M B B 0 - 44 -mucin), were used with each batch of specimens where appropriate. This pretreatment f i r s t oxidized a l l v i c i n a l d i o l s to aldehydes, then reduced them to primary alcohols, and subsequent saponification, removing any O-acyl substituents present, created new v i c i n a l d i o l s on s i a l i c acid residues which were i n i t i a l l y substituted at position C 7 or Cg or (C ? + C 8 ) , or (C 8 + C 9 ) , or (C ? + C 9 ) f or (C ? + CQ + C^). a) Preparation of stains Thionine S c h i f f (Van Dujin, 1956), basic fuchsin or pararosanalin S c h i f f (Barger and De Lamater, 1948), and high iron diamine (HID) (Spicer, 1965) were prepared immediately before use. Alcian blue (AB) pH 2.5 and 1.0 (Cu l l i n g , 1974) were prepared as stock solutions and f i l t e r e d before each use. After staining with HID or AB the s l i d e s were drained thoroughly by inverting the st a i n i n g j a r onto papertowelling, then rinsed once or twice with a solu t i o n at the same pH as the s t a i n , either 3% acetic acid for pH 2.5 or 0.1N HC1 for pH 1.0, and drained again. b) Periodate oxidation Sections were oxidized i n 1.0% (w/v) aqueous periodic acid at room temperature. The appropriate i n i t i a l oxidation time for the tissues used i n t h i s study was determined to be 2 hours. When the staining procedure involved only one oxidation step, i t was performed for 2 hours. When 2 oxidation steps were used, the f i r s t was performed for 2 hours and the second for 30 minutes. Oxidative cleavage of v i c i n a l d i o l s r e s u l t s i n Schi f f - r e a c t i v e aldehydes. - 45 -Borohydride reduction Periodate engendered aldehydes were reduced to Schiff-unreactive primary alcohols using the sodium borohydride method of L i l l i e and Pizzolato (1972). Saponification Saponification with 0.5% (w/v) potassium hydroxide (KOH) in 70% ethanol for 15 minutes at room temperature resulted in the removal of 0-acyl substituents. Phenylhydrazine blockade Phenylhydrazine blockade was carried'out with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 hours at room temperature, followed by treatment with water for 16 hours at room temperature. Methods for the visualization of side chain O-acetylated sialic acids 1) PBT/KOH/PAS (Reid et al, 1973; Culling et al, 1974) Two hours of periodate oxidation was followed by reduction in sodium borohydride, saponification with potassium hydroxide, a second periodate oxidation for 30 minutes and finally staining with pararosanalin Schiff, (Table 3). 2) PAT/KOH/Bh/PAS (Reid et al, 1984b) Periodate oxidation, was followed by staining for 4 hours in Thionine Schiff reagent. Subseauent saponification, reduction, and second oxidation was followed by staining for 30 minutes with pararosanalin Schiff, (Table 3). - 46 -3) PAPT/KOH/Bh/PAS (Reid et a l , 1984b) This procedure was c a r r i e d out p a r a l l e l to the PAT/KOH/Bh/PAS with the addi t i o n of the phenylhydrazine blockade a f t e r the f i r s t periodate o x i d a t i o n (Table 3). Methods for the simultaneous v i s u a l i z a t i o n of side chain  O-acetylated s i a l i c a c i d and sulphate 1) AB1.0/PAPS (Reid et a l , 1985c) Sections were stained with a l c i a n blue at pH 1.0 for 30 minutes followed by 2 hours of periodate oxidation, phenylhydrazine blockade, and s t a i n i n g i n pararosanalin S c h i f f reagent for 4 hours, (Table 3). 2) PBT/K0H/AB1.0/PAS (Reid et a l , 1985c) Two hours of periodate oxidation was followed by reduction, s a p o n i f i c a t i o n , and s t a i n i n g with a l c i a n blue at pH 1.0 for 30 minutes. Sections were then oxidized again, for 30 minutes and stained with pararosanalin S c h i f f f o r 30 minutes, (Table 3). Methods for the simultaneous v i s u a l i z a t i o n of t o t a l s i a l i c acid and  sulphate 1) K0H/AB1.0/PAPS (Reid et a l , 1985c) Following s a p o n i f i c a t i o n , the sections were stained with a l c i a n blue at pH 1.0, oxid i z e d with p e r i o d i c a c i d for 2 hours, blocked with phenylhydrazine and f i n a l l y stained with pararosanalin S c h i f f f o r 30 minutes, (Table 3). - 47 -2) HID/AB2.5 (Spicer, 1965) Sections were stained with high iron diamine, drained, rinsed with 0.1N HC1, drained, blotted, stained with alcian blue at pH 2.5, drained, and rinsed with 3% acetic acid, (Table 3). Table 3 illustrates the characteristics of the histochemical stains described above. In addition to these procedures serial sections were stained with Hematoxylin and Eosin (H & E) (Culling, 1974) for morphological assessment. (iv) Methods of assessment All of the control sections from each time period were examined first to determine the normal staining pattern of rat colonic epithelial glycoproteins in each procedure used. Secondly the sections from DMH treated animals were observed and any changes from the established normal staining pattern were recorded. Histochemically abnormal areas were then examined in serial H & E sections to determine their morphological character. Finally, a l l H & E sections were scanned for histologic abnormalities and these were assessed histochemically. a) Determination of the normal staining pattern Each case was examined with a l l histochemical procedures used before another case was examined. The staining pattern of each colon was recorded beginning at the rectal end on the outer arm of the "swiss r o l l " and proceeding towards the proximal end of the distal colon in the centre of the r o l l , which had previously been - 48 -marked with India ink (slide 11). this procedure was performed by two observers using a double-headed microscope, and proved to be an accurate and time-efficient method of evaluating the 120 cases examined. After a substantial period of familiarization with the stains used and the colours produced, a reproducible agreement was reached between the two observers as to the scoring of each procedure. All of the control sections from each time period were evaluated first in order to firmly establish the normal staining pattern. Further at the start of each session of observation control sections were reviewed. In the same manner the DMH treated colons were observed and the general staining pattern for the whole colon in a l l stains was recorded. Evaluation of abnormal crypts The assessment of DMH treated colons for histochemical abnormalities was performed immediately after the general staining pattern for that particular colon had been recorded. Each abnormal crypt was evaluated in each stain used, crypts were numbered in the order they were detected, and the number assigned was written directly onto the glass slide with an arrow pointing to i t using a ballpoint pen. This method of labelling was performed under a Wild M5A dissecting microscope at a magnification of 25X. The crypt was then identified in the serial section closest to the one in which i t was first identified, labelled, and so on until the mucin in the - 49 -crypt had been observed i n each s t a i n used or u n t i l the crypt could not be detected i n any further sections. In most cases the crypts could be i d e n t i f i e d with a l l the st a i n s used. A crypt was only considered abnormal i f i t could be discerned i n more than two c e l l s i n any p a r t i c u l a r t i s s u e s i t e and was i d e n t i f i a b l e with no l e s s than two histochemical procedures. (c) H i s t o l o g i c c l a s s i f i c a t i o n of histochemically abnormal crypts H i s t o l o g i c c l a s s i f i c a t i o n of the histochemically abnormal crypts was done i n cooperation with Dr. E.A. Jones, Shaughnessy Hospital, Vancouver, B.C. The H & E s l i d e was taped to the AB 1.0/PAPS s l i d e . These sections were s e r i a l to each other or separated by no more than f i v e microns. In the AB 1.0/PAPS a change i n sulphate as well as a change i n s i a l i c a c i d could e a s i l y be detected. The two s l i d e s , taped together, were put onto the microscope stage and cl i p p e d i n t o place. Then, a histochemically abnormal crypt was located i n the AB 1.0/PAPS section and the corresponding H & E f i e l d was brought i n t o focus, and the crypt was assessed morphologically. The sections were double coded so that i t was not possible to know which colon was being observed at the time. The following morphologic c r i t e r i a were used i n the h i s t o l o g i c c l a s s i f i c a t i o n of crypts. NORMAL: No detectable h i s t o l o g i c abnormality as compared to the corresponding c o n t r o l group. - 50 -DECREASED MUCIN: No detectable h i s t o l o g i c abnormalities. Marked decrease i n goblet c e l l number, and/or smaller goblet s i z e ( s l i d e 1). ATYPIA: S l i g h t nuclear enlargement and hyperchromaticity; early p s e u d o s t r a t i f i c a t i o n may or may not be present. Atypia was always associated with decreased mucin presenting as a reduction i n goblet c e l l number and/or goblet c e l l s with l e s s mucin than normal ( s l i d e 2). DYSPLASIA: Nuclear abnormalities i n c l u d i n g enlargement, hyperchromaticity, crowding, and moderate p s e u d o s t r a t i f i c a t i o n with l o s s of nuclear p o l a r i t y . Crypts i n t h i s c l a s s i f i c a t i o n always had decreased mucin presenting as i n atypia ( s l i d e 3). TUMOURS: 1) Well d i f f e r e n t i a t e d : well-formed glands containing a varying amount of mucus; s t r a t i f i c a t i o n of c e l l s and n u c l e i , increased nuclear-cytoplasmic r a t i o , b i z a r r e n u c l e i and enlarged n u c l e o l i , and l o s s of nuclear and c e l l u l a r p o l a r i t y . 2) Moderately well d i f f e r e n t i a t e d : d i s t o r t i o n of crypt a r c h i t e c t u r e , a desmoplastic stromal response and an i n f i l t r a t i v e pattern to the glands, with or without invasion beyond the muscularis mucosa ( s l i d e s 4,5). - 51 -3) Poorly d i f f e r e n t i a t e d : small round u n d i f f e r e n t i a t e d c e l l s (carcinoma simplex), l o s s of glandular s t r u c t u r e , and marked anaplasia. The non-neoplastic mucosa d i r e c t l y adjacent to tumour equivalent to t r a n s i t i o n a l mucosa ( F i l i p e & Branfoot, 1976; F i l i p e , 1979). Histochemistry of morphologically abnormal crypts Following the h i s t o l o g i c c l a s s i f i c a t i o n of histochemically abnormal crypts, with the s l i d e s s t i l l taped together, the H & E was examined. Any abnormalities not previously detected were recorded and assessed h i s t o c h e m i c a l l y . This method permitted the i d e n t i f i c a t i o n of crypts with normal histochemistry and abnormal histology. Assessment of abnormal crypts i n the mucosa remote from tumours i n  the postneoplastic phase To determine the frequency of histochemical abnormalities a f t e r tumour formation the histochemical section s e r i a l to the H & E, e i t h e r the AB1.0/PAPS or the K0H/AB1.0/PAPS, was assessed. An examination of hi s t o c h e m i c a l l y abnormal f o c i revealed that of those detected i n the AB1.0/PAPS, 98% could also be i d e n t i f i e d i n the K0H/AB1.0/PAPS. S i m i l a r l y o f those f i r s t observed i n the K0H/AB1.0/PAPS, 99% could a l s o be detected i n the AB1.0/PAPS. - 52 -f) Measurement of mucosal length The amount of s t a i n a b l e mucosa present on each s l i d e was measured as follows. The s l i d e was placed on top of a l i g h t box (the type r o u t i n e l y used for viewing x-ray films) and the recorded with a colour t e l e v i s i o n camera which fed i n t o an APPLE II computer equipped with an image analyser, graphics t a b l e t , e l e c t r o n i c pen, and a morphometry package.* A f t e r c a l i b r a t i o n and c o r r e c t i n g f o r the magnification factor i n the t e l e v i s i o n camera, the image of the c o l o n i c mucosa on the computer screen was traced with the e l e c t r o n i c pen on the graphics t a b l e t and the length of mucosa traced was c a l c u l a t e d i n centimetres. The histochemical s t a i n which provided the optimum contrast for viewing on the computer screen was the HID/AB2.5. The histochemical changes observed were then expressed as the number of changes seen per decimetre of mucosa scanned. •This equipment was used courtesy of Dr. W.M. Thurlbeck, Dept. of Pathology, UBC and the morphometry program was set up by Dr. Larry Paul, Dept. of Pathology, UBC. - 53 -'p lOnwsfo Inifrbu fu^piopcr cause: 'VimjtHC fxmcmcrcVcr mote tvuxlq, -%tmasJ(cf-ttn. eftejisfi Akriembt C 1^50 F I G U R E 7. RESULTS I MACROSCOPIC OBSERVATION - 54 -( i ) General features of colons The Wistar r a t colon i s composed of two anatomically d i s t i n c t halves, the lower colon terminating at the rectum and the upper colon adjoining the cecum. The colon was separated from the cecum at an anatomical s t r i c t u r e . As i l l u s t r a t e d i n f i g u r e 6 the muscular banding pattern of the upper colon forms a "herring-bone" configuration while that of the lower colon e x h i b i t s l o n g i t u d i n a l s t r i a t i o n s . The Wistar r a t d i s t a l f i x e d colon ranged i n length from 14.0 to 19.5 cm; the lower colon was always longer ranging from 9.0 to 12.5 cm whereas the upper colon measured i n the range of 5.0 to 7.0 cm. Raised patches, l a t e r i d e n t i f i e d by H & E as lymphoid aggregates, appeared prominently i n a l l colons. In the upper colon one or two of these patches (2-5 mm i n length) appeared as thickenings of the muscular f o l d at approximately two centimetres from the proximal edge. In the lower colon a small patch (3-7 mm i n length) occured at approximately four centimetres from the juncture with the upper colon and a l a r g e r patch (5-15 mm) appeared at approximately one h a l f to two centimetres from the d i s t a l end of the specimen. In my experience these patches are d i f f i c u l t to see i n fresh t i s s u e but become more evident a f t e r f i x a t i o n . ( i i ) D i s t r i b u t i o n of tumours Tumours were v i s i b l e , g r o s s l y , a f t e r 16 weeks of treatment with - 55 -16 - 30 WEEKS OF TREATMENT WITH DMH GROUP CODE h i j k 1 m n TIME (WEEKS) 16 20 22 24 26 28 30 # OF DMH INJECTIONS 16 17 17 17 17 17 17 TOTAL # OF TUMOURS 3 6 17 30 35 54 69 # OF RATS WITH TUMOURS 2 (20) 4 (40) 9 (100) 10 (100) 10 (100) 10 (100) 8 (100) # OF RATS IN GROUP 10 10 9 10 10 10 8 AVE # OF TUMOURS PER RAT 0 . 3 0 . 6 1.7 3 . 0 3 . 5 5 . 4 8 . 6 LOCATION OF TUMOURS TOTALS Uu 0 0 0 0 0 0 0 0 U l . 0 2 2 2 1 4 3 14 (33) (12) (6) (3) (7) (4) ( 6 . 5 ) Lu 1 3 7 12 14 18 27 82 (33) (50) (41) (40) (40) (33) (39) ( 3 8 . 1 ) LI 2 1 8 16 20 32 40 119 (67) (17) (47) (54) (57) (60) (57) ( 5 5 . 4 ) *Numbers i n p a r e n t h e s e s are p e r c e n t a g e s . Uu = upper h a l f o f upper c o l o n Lu = upper h a l f o f l o w e r c o l o n Ul = l o w e r h a l f o f l o w e r c o l o n LI = l o w e r h a l f o f l o w e r c o l o n - 56 -DMH. Table 4 shows the number and anatomic l o c a t i o n of tumours observed i n a l l r a t s at each s a c r i f i c e period, the number of ra t s bearing tumours in each group, and the average number of tumours per r a t throughout the time course of the experiment. The number of ra t s bearing tumours and the number o f tumours per r a t increased with the period of treatment u n t i l at 22 weeks a l l the treated r a t s had tumours. Sixty-seven rats taken from the time periods 16-30 weeks were examined i n d e t a i l . A t o t a l of 215 tumours were observed i n the 53 animals that bore tumours. Of these 93.5% occurred i n the lower colon and 6.5% i n the upper colon. Because of t h i s d i s t r i b u t i o n only the lower colons were examined with histochemical methods. No tumours were observed i n the c o n t r o l r a t s . II MICROSCOPIC OBSERVATIONS ( i ) Determination of the appropriate oxidation time for Wistar rat  c o l o n i c mucin This was accomplished with the PBT/PAS (periodate oxidation, borohydride treatment, periodate oxidation, S c h i f f ) . F a i l u r e to s t a i n a f t e r a second period of oxidation i n d i c a t e d that the i n i t i a l period of oxidation was s u f f i c i e n t to cleave a l l a v a i l a b l e v i c i n a l d i o l s present i n the t i s s u e . For Wistar r a t c o l o n i c mucin the oxidation time chosen was 2 hours, a f t e r which time f a i n t s t a i n i n g was observed i n the upper portions of some erypts compared to darker s t a i n i n g i n the lower halves of the crypts a f t e r only 1 hour of i n i t i a l o xidation. - 57 -( i i ) Control sections Staining was controlled by the use of control sections. Of normal human stomach (neutral mucin), normal human colon (acidic mucin, sulphated) and normal rat colon (taken from a region which contained acidic mucin, non-sulphated). In control sections of stomach were a l l cut from the same block as were the other controls. A l l the control sections stained as expected (Table 3a) which indicated that the staining procedures were done correctly. There was no difference in the staining intensity of the control sections in different batches allowing the comparison of the test sections to one another regardless of the batch of stain or reagents used. ( i i i ) Histology (a) Normal histology The mucosa of the Wistar rat lower (also called d i s t a l or l e f t ) colon consisted of short straight crypts lined with goblet and absorptive c e l l s . In general, the bases of the crypts contained fewer goblet ce l l s than the remainder of the crypt. The mucosal surface was lined by a layer of surface mucin, which was continuous within the lumen of the crypts. The lymphoid aggregates varied greatly in size from small microscopic foci present in the lamina propria and submucosa to large macroscopically visible raised patches spanning millimetres of mucosa. Abnormal histology Abnormal h i s t o l o g y was only observed i n the DMH treated r a t s . Determination of normal histology was accomplished with the assessment of H & E sections of the sham c o n t r o l EDTA treated rats and comparing them to the c o n t r o l rats which had received no treatment and were s a c r i f i c e d at the beginning of the experiment. As o u t l i n e d i n M a t e r i a l and Methods se c t i o n I I ( i v ) ( a ) , three types of h i s t o l o g i c abnormalities were observed. Crypts c l a s s i f i e d as showing decreased mucin ( s l i d e 1) appeared, i n the H & E, to contain no goblet c e l l s or smaller than normal goblet c e l l s . The c e l l s present which lacked goblets were h i s t o l o g i c a l l y normal and i n d i s t i n g u i s h a b l e from absorptive c e l l s . Therefore, although i t s con s t i t u e n t c e l l s appeared normal, the gland was considered to be h i s t o l o g i c a l l y abnormal as i t contained l i t t l e mucin. I t was not p o s s i b l e to d i s t i n g u i s h whether the c e l l s i n question were a l l absorptive c e l l s or whether there were goblet c e l l s present which contained no mucin. Crypts e x h i b i t i n g atypia ( s l i d e 2) or d y s p l a s i a ( s l i d e 3) always showed a l o s s of mucin e i t h e r as a r e s u l t of a decrease i n goblet c e l l number, or because the c e l l s contained smaller goblets than normal, or both. Tumours A l l tumours examined i n DMH treated lower colons were c l a s s i f i e d as moderately well d i f f e r e n t i a t e d adenocarcinomas the majority of which ex h i b i t e d exophytic growth ( s l i d e 5) and some - 59 -appeared polypoid ( s l i d e 4). There were no benign adenomatous l e s i o n s observed and none of the polypoid carcinomas contained any regions which were not i n v a s i v e to some degree ( s l i d e 4). On occasion some tumours had endophytic growth but these were rare. There was no c o r r e l a t i o n between tumour l o c a t i o n and lymphoid aggregates. ( i i i ) Normal histochemistry As shown i n tables 5 and 6, the normal Wistar rat d i s t a l colon contained three histochemically d i s t i n c t regions. On the average the most d i s t a l end of the colon (DE) represented 43% of the t o t a l length while the middle region (MID) comprised approximately the next 30%. At the most proximal end of MID a patchy area was observed composed of a l t e r n a t i n g patches (3-8 crypts) of mucin e x h i b i t i n g a s t a i n i n g pattern c h a r a c t e r i s t i c e i t h e r of MID or of the proximal end of the d i s t a l colon (PE). The number of times the a l t e r n a t i n g pattern repeated varied from ra t to rat but i t was present i n a l l the colons examined and could be d i s t i n g u i s h e d i n each s t a i n used. The goblet c e l l mucin of the DE region contained both s i a l i c a c i d residues and O-sulphate e s t e r s (sialosulphomucin). Scattered throughout the DE were c e l l s containing a sialosulphomucin with a smaller r e l a t i v e quantity of sulphation than that present i n the vast majority of c e l l s . This was c l e a r l y i l l u s t r a t e d by the s t a i n i n g pattern observed i n the K0H/AB1.0/PAPS (table 5, s l i d e 6). The majority of c e l l s stained r o y a l blue, a combination of the magenta of s i a l i c a c i d and aqua of O-sulphate - 60 -TABLE 5 : THE STAINING CHARACTERISTICS OF THE THREE MAJOR  REGIONS OF THE WISTAR RAT DISTAL COLON DE = d i s t a l end o f d i s t a l c o l o n (43% o f t o t a l l e n g t h ) ; MID = m i d d l e r e g i o n o f d i s t a l c o l o n (30% o f t o t a l l e n g t h ) ; PE = p r o x i m a l end o f d i s t a l c o l o n (27% o f t o t a l l e n g t h ) ; SA = s i a l i c a c i d ; OS03H = O - s u l p h a t e e s t e r ; SPC = s c a t t e r e d p u r p l e c e l l s ; HEX = hexose o r hexosamine r e s i d u e s w i t h o x i d i z a b l e v i c i n a l d i o l s . HID/AB 2 . 5 AQUA = a l l o r p r e d o m i n a n t l y SA BLACK = a l l o r p r e d o m i n a n t l y 0S03H KOH/AB 1.0/PAPS AQUA = a l l 0S03H DARK AQUA c o l o u r s r a n g i n g f r o m dark aqua t o r e d p u r p l e r e p r e s e n t ROYAL BLUE d i f f e r e n t r e l a t i v e p r o p o r t i o n s o f SA and 0S03H p r e s e n t PURPLE w i t h i n t h e same g o b l e t c e l l ; i n g e n e r a l , t h e more aqua RED PURPLE the shade t h e g r e a t e r the r e l a t i v e p r o p o r t i o n o f 0S03H MAGENTA = a l l SA PBT/KOH/AB 1.0/PAS AQUA = a l l 0S03H DARK AQUA c o l o u r s r a n g i n g f r o m dark aqua t o r e d p u r p l e r e p r e s e n t ROYAL BLUE d i f f e r e n t p r o p o r t i o n s o f C7 + C8 SA and 0S03H p r e s e n t PURPLE w i t h i n t h e same g o b l e t c e l l ; i n g e n e r a l , t h e more aqua RED PURPLE t h e shade the g r e a t e r t h e r e l a t i v e p r o p o r t i o n o f 0S03H MAGENTA = a l l C7 + C8 SA AB 1 .0/PAPS AQUA = a l l 0S03H DARK AQUA c o l o u r s r a n g i n g f r o m dark aqua t o r e d p u r p l e r e p r e s e n t ROYAL BLUE d i f f e r e n t p r o p o r t i o n s o f C0+C7+C9 SA and 0S03H p r e s e n t PURPLE w i t h i n t h e same g o b l e t c e l l ; i n g e n e r a l , t h e more aqua RED PURPLE t h e shade t h e g r e a t e r the r e l a t i v e p r o p o r t i o n o f 0S03H MAGENTA = a l l CO, C 7 , and C9 SA PAPT/KOH/Bh/PAS BLUE = CO + C7+ C9 SA BLUE PURPLE c o l o u r s r a n g i n g from b l u e p u r p l e t o r e d p u r p l e r e p r e s e n t PURPLE d i f f e r e n t p r o p o r t i o n s o f s i d e c h a i n s u b s t i t u t e d S A ; i n RED PURPLE g e n e r a l t h e r e d d e r t h e shade the more C8 SA MAGENTA = C8 SA PAT/KOH/Bh/PAS BLUE = CO + C7 + C9 SA + HEX BLUE PURPLE c o l o u r s r a n g i n g f r o m b l u e p u r p l e t o r e d p u r p l e r e p r e s e n t PURPLE d i f f e r e n t p r o p o r t i o n s o f s i d e c h a i n s u b s t i t u t e d SA and RED PURPLE HEX; i n g e n e r a l , t h e r e d d e r t h e shade t h e more C8 SA MAGENTA = C8 SA PBT/KOH/PAS MAGENTA = C7 + C8 SA ( t h e g r e a t e r t h e i n t e n s i t y t h e more C7 + C8 SA) A = a q u a ; DA = dark a q u a ; RB = r o y a l b l u e ; P = p u r p l e ; RP = r e d p u r p l e ; BP = b l u e p u r p l e ; B = b l u e ; M = m a g e n t a ; Bk = b l a c k - 61 -HISTOCHEMICAL STAIN HID/AB 2 . 5 KOH/AB 1 . 0 / PAPS PBT/KOH/ AB 1 .0/PAS AB 1.0/PAPS DE Bk Bk RB + SPC RB + SPC DA + SPC DA + SPC RB + SPC RB + SPC MID Bk Bk RB + SPC RB t o P DA + SPC p a l e A t o DA RB + SPC P + some RB c e l l s UPPER OR LOWER PE CRYPT A + s c a t t e r e d Bk c e l l s M + s c a t t e r e d D A . R B . P . R P c e l l s M M + s c a t t e r e d D A . R B . P . R P c e l l s M M + s c a t t e r e d n D A , R B , P , R P c e l l s M L PAPT/KOH/ Bh/PAS PAT/KOH/ Bh/PAS PBT/KOH/ PAS BP BP 0 t o 1+ M 0 t o 1+ M BP t o P BP B t o BP 1 t o 2+ M 0 t o 1+ M P t o RP BP P t o RP 1 t o 2+ M 2 t o 4+ M - 62 -TABLE 6 : CHARACTERISTICS* OF THE COLONIC EPITHELIAL GLYCOPROTEINS IN NORMAL WISTAR RAT DISTAL COLON • D e t e r m i n e d u s i n g the h i s t o c h e m i c a l s t a i n s d e s c r i b e d i n T a b l e 1 . LEGEND: ++++ = p r e d o m i n a n t s p e c i e s +++ = p r e s e n t t o some e x t e n t ++ = p r e s e n t i n t r a c e amounts + = p r e s e n t i n s c a t t e r e d c e l l s SA = S i a l o m u c i n ( p r e d o m i n a n t l y c o n t a i n i n g C n , C 7 , o r C q c l a s s t e r m i n a l s i a l i c a c i d r e s i d u e s and not c o n t a i n -i n g O - s u l p h a t e e s t e r s ) OAcSA = O - a c e t y l a t e d s i a l o m u c i n ( c o n t a i n i n g p r e d o m i n a n t l y Cy o r Cn c l a s s s i a l i c a c i d r e s i d u e s and n o t c o n t a i n i n g O-suTphate e s t e r s ) SAS04 = S i a l o s u l p h o m u c i n ( c o n t a i n i n g p r e d o m i n a n t l y C n , C y , o r Cg c l a s s s i a l i c a c i d r e s i d u e s and O - s u l p h a t e e s t e r s ) S A ( s o * ) = S i a l o s u l p h o m u c i n ( p r e d o m i n a n t l y c o n t a i n i n g C , C , o r C „ c l a s s s i a l i c a c i d r e s i d u e s and a s m a l l e r p r o p o r t i o n o f O - s u l p h a t e e s t e r than SAS04) 0AcSAS04 = O - a c e t y l a t e d s i a l o s u l p h o m u c i n ( p r e d o m i n a n t l y composed o f C 7 o r C c c l a s s s i a l i c a c i d r e s i d u e s and O - s u l p h a t e e s t e r s ) a O A c S A ( s o - ) = O - a c e t y l a t e d s i a l o s u l p h o m u c i n ( c o n t a i n i n g p r e d o m i n a n t l y C ? o r L c l a s s s i a l i c a c i d r e s i d u e s and a s m a l l e r p r o -p o r t i o n o f O - s u l p h a t e e s t e r s t h a n 0AcSAS04) - 63 -REGION OF DISTAL COLON AVE. % OF TOTAL DIS-TAL COLON LENGTH TYPE OF MUCIN PRESENT WITHIN GOBLET CELLS LOCATION WITHIN CRYPT WEEKS 1-11 WEEKS 16-24 RECTAL END DISTAL END 43% (DE) ++++ SAS04 +++ 0AcSAS04 + S A ( s o - ) + 0 A c S A ? s o 4 ) ++++ SAS04 +++ 0AcSAS04 + SA(so ) + O A c S A t s o J SAME AS WEEKS 1-11 SAME AS WEEKS 1-11 UPPER HALF LOWER HALF MIDDLE (MID) 30% ++++ SAS04 +++ 0AcSAS04 + S A ( s o 4 ) + 0 A c S A ( s o 4 ) ++++ S A ( s o 4 ) ++ 0 A c S A ( s o 4 ) SAME AS WEEKS 1-11 SAME AS WEEKS 1-11 UPPER HALF LOWER HALF PATCHY v a r i e s PROXIMAL END EDGE OF PROXIMAL COLON 27% (PE) ++++ OAcSA ++++ SA + SAS04 + 0 A c S A ( s o 4 ) ++++ SA ++++ OAcSA ++++ SA + SAS04 + 0 A c S A ( s o 4 ) ++++ OAcSA UPPER HALF LOWER HALF - 64 -ester, while scattered c e l l s stained purple. The s i a l i c a c i d present i n t h i s area contained both O-acetylated and non-O-acetylated variants the former being predominant and more common i n the upper p o r t i o n of the crypts ( s l i d e 7). The surface mucin present d i f f e r e d from the mucin within the goblet c e l l s i n that i t was devoid of sulphate. The mucin i n the upper portion of the MID region was s i m i l a r in composition to that of the upper crypts of the DE region. However, the lower halves of the crypts of the MID region contained a sialosulphomucin with a much smaller proportion of sulphate and a smaller proportion of O-acetylated s i a l i c a c i d than found i n the DE region. The l o s s of sulphate was i l l u s t r a t e d by a proportional l o s s of the aqua s t a i n i n g component i n the K0H/AB1.0/PAPS ( s l i d e 8), the PBT/K0H/AB1.0/PAS, and the AB1.0/PAPS. The r e l a t i v e decrease i n O-acetylated s i a l i c a c i d was demonstrated by the increase i n the thionine blue s t a i n i n g together with a decrease o f the magenta s t a i n i n g component i n the PAPT/KOH/Bh/PAS ( s l i d e 9) and the PAT/KOH/Bh/PAS procedures, the very pale s t a i n i n g observed with the PBT/KOH/PAS method, and by the presence of intense magenta s t a i n i n g i n the AB1.0/PAPS together with the absence of magenta s t a i n i n g i n the PBT/K0H/AB1.0/PAS. The PE region contained predominantly sialomucin ( s l i d e s 10,11). Sulphate, when present, was confined to scattered c e l l s i n the upper halves of the crypts. In t h i s region Cg-O-acetyl s i a l i c acids occurred i n the mucins throughout the crypts. In the lower halves of the crypts of both c o n t r o l s and DMH treated animals, there was a time r e l a t e d change i n the character of the mucin (table 7, s l i d e s 11,12). S t a i n i n g with the - 65 -TABLE 7: THE TIME RELATED CHANGE IN THE CHARACTER OF THE  MUCIN PRESENT IN THE LOWER HALVES OF THE CRYPTS  IN THE PE REGION OF THE WISTAR RAT DISTAL COLON The c o l o u r s e e n i n each s t a i n was a s s i g n e d a n u m e r i c a l s c o r e from 0 t o 4 . In t h e PBT/KOH/PAS, the PBT/KOH/AB 1 . 0 / P A S , and the AB 1.0/PAPS the s c o r e was b a s e d on the i n t e n s i t y o f the magenta c o l o u r . In the r e m a i n i n g two s t a i n s the c o l o u r s were a s s i g n e d v a l u e s as f o l l o w s : b l u e = 0 ; b l u e p u r p l e = 1 ; p u r p l e = 2 ; r e d p u r p l e = 3 ; and magenta = 4 . The numbers i n the t a b l e are a v e r a g e v a l u e s o f the s c o r e s o f the i n d i -v i d u a l r a t s a t each time p e r i o d . AVERAGE HISTOCHEMICAL SCORE TIME PBT/KOH/ AB 1 . 0 / PBT/KOH/ PAPT/KOH/ PAT/KOH/ (WEEKS) AB 1.0/PAS PAPS PAS Bh/PAS Bh/PAS 0 (1 day) 1 - 2 3 - 4 1 1 - 2 1 1 1 - 2 3 - 4 2 1 - 2 1 2 1 - 2 3 - 4 2 1 - 2 1 4 1 - 2 3 - 4 2 1 - 2 1 6 4 2 4 1 2 1 8 4 2 . 4 1 2 2 11 4 2 4 3 - 4 3 16 4 2 4 4 3 20 4 2 4 4 3 22 4 2 4 4 3 24 4 2 4 4 3 - 66 -PBT/KOH/PAS and PBT/KOH/AB1.0/PA5 procedures increased markedly between 4 weeks (2+) and 6 weeks (4+) and concomittantly magenta s t a i n i n g i n the AB1.0/PAPS procedure decreased markedly from (4+) to (2+). The magenta component of the PAPT/KOH/Bh/PAS ( s l i d e 12) and PAT/KOH/Bh/PAS s t a i n s , however, di d not increase u n t i l 11 weeks. This implied that the change i n the character of the mucin involved the production of Cy-O-acetyl s i a l i c a c i d between week 4 and 6 followed by the production of C o - 0 - a c e t y l (or d i or t r i substituted) s i a l i c acids a f t e r 8 weeks. In o the upper halves of the PE c r y p t s s t a i n i n g for side chain O-acetyl s i a l i c acids was v a r i a b l e and no increase was observed throughout the course of the experiment. No d i f f e r e n c e s were noted between the EDTA treated sham c o n t r o l animals and the untreated c o n t r o l animals that were s a c r i f i c e d at the commencement of the i n v e s t i g a t i o n . (v) Histochemical abnormalities i n the preneoplastic phase Table 8 i l l u s t r a t e s the mean number of histochemical changes observed per decimetre of r a t colon p r i o r to neoplasia. These changes were f o c a l , occurring i n from one to no more than f i v e crypts per focus. Where c l u s t e r s of f o c a l changes were v i s i b l e each focus was a r c h i t e c t u r a l l y d i s t i n c t , separated from other f o c i by crypts e x h i b i t i n g the normal s t a i n i n g pattern. Two types of histochemical change were observed. Change A ( s l i d e 13) consisted of a r e l a t i v e decrease i n or absence of sulphate. This was characterized by the appearance i n the HID/AB2.5 of aaua s t a i n i n g i n a normally black f i e l d and the appearance - 67 -TABLE 8 : THE MEAN NUMBER OF HISTOCHEMICAL CHANGES SEEN PER  DECIMETRE OF COLONIC MUCOSA CLASSIFIED ACCORDING TO THE NATURE OF THE CHANGE* A t o t a l o f 211 f o c i o f h i s t o c h e m i c a l change were i d e n t i f i e d f r o m weeks 1 - 1 1 . # OF WEEK RATS PER CHANGE A CHANGE B TOTAL GROUP 1 4 0 0 0 2 5 1 . 6 + 2 . 3 0 1 . 6 + 2 . 3 4 3 8 . 8 + 0 . 1 0 8 . 8 + 0 . 1 6 5 9 . 9 + 3 . 7 2 . 6 + 2. .0 1 2 . 5 + 4 . 7 8 5 1 8 . 8 + 2 . 9 2 . 3 + 2, .2 2 1 . 1 + 3 . 7 11 5 16.7 + 1 . 5 4 . 2 + 1 . 6 2 0 . 9 + 2 . 6 * CHANGE A = a d e c r e a s e o r l o s s o f CHANGE B = a d e c r e a s e o r l o s s o f o r l o s s o f s i d e c h a i n s u l p h a t e o n l y s u l p h a t e accompanied by a d e c r e a s e O - a c e t y l a t e d s i a l i c a c i d r e s i d u e s - 68 -of magenta s t a i n i n g ( i . e . a l o s s of aqua) i n the K0H/AB1.0/PAPS, AB1.0/PAPS, and PBT/KOH/AB1.0/PAS procedures. This change was f i r s t seen at two weeks i n 2 out of 5 r a t s (40%) and i t s frequency increased with time. Change B ( s l i d e 14) c o n s i s t e d of a l o s s of sulphate together with a decrease i n the proportion of O-acetylated s i a l i c a c i d occurring i n the same c e l l . This was detected by the appearance of thionine blue s t a i n i n g i n the PAPT/KOH/Bh/PAS and PAT/KOH/Bh/PAS. In s e r i a l sections such s i t e s stained aoua i n the HID/AB2.5 and magenta i n the K0H/AB1.0/PAPS and AB1.0/PAPS procedures. They f a i l e d to s t a i n with the PBT/K0H/AB1.0/PAS and PBT/KOH/PAS s t a i n s . Change B was not observed u n t i l 6 weeks and occurred with a much lower frequency than change A. Because of t h e i r nature, the histochemical abnormalities could only be recognized i n the d i s t a l 73% of the lower colon, i . e . , the DE and MID regions. ( v i ) Morphological character of c r y p t s e x h i b i t i n g histochemical  abnormalities Of the 211 f o c i which showed histochemical change, 187 could be p o s i t i v e l y i d e n t i f i e d i n s e r i a l H & E sections. The r a t i o of changes A and B i n t h i s l a t t e r group of c r y p t s did not d i f f e r from that found, i n a l l 211 crypts showing histochemical change. Table 9 i l l u s t r a t e s the proportion of f o c i with histochemical change which were morphologically normal or which exhibited a reduction i n mucin, atypia, or d y s p l a s i a . No h i s t o l o g i c changes were detected at 1 day, 1 week, or 2 weeks. Further, the f o c i with histochemical change A observed at 2 weeks were h i s t o l o g i c a l l y normal. The number of morphologically normal crypts - 69 -TABLE 9 : THE MEAN NUMBER OF HISTOCHEMICAL CHANGES SEEN PER  DECIMETRE OF COLONIC MUCOSA CLASSIFIED ACCORDING TO MORPHOLOGICAL CRITERIA. A t o t a l o f 187 f o c i o f h i s t o c h e m i c a l change c o u l d be a s s e s s e d h i s t o l o g i c a l l y from 1 - 1 1 w e e k s . HISTOLOGICAL CLASSIFICATION DECREASED NORMAL MUCIN ATYPIA DYSPLASIA 1 4 0 0 0 0 2 5 1 . 6 ± 2 . 3 0 0 0 4 3 4.8 + 3 . 7 2 . 0 + 1 . 9 1 . 2 + 1 .1 0 . 8 + 1 . 3 6 5 5 . 1 + 1 . 6 1 . 2 + 1.1 2 . 9 + 2 . 8 2 . 0 + 1 . 8 8 4 3 . 0 + 1.7 3 . 9 + 1 . 5 4.3 + 4.2 11 .2 + 4.7 11 5 2 . 1 + 1 . 9 5 . 8 + 2 . 2 4.9 + 3 . 7 7 . 1 + 3 . 5 # OF RATS PER (WEEKS) GROUP TIME - 70 -TABLE 10: THE MEAN NUMBER OF FOCI SHOWING HISTOLOGIC  ABNORMALITIES AND NORMAL HISTOCHEMISTRY. A t o t a l o f 39 such f o c i were i d e n t i f i e d from weeks 1 - 1 1 . # OF HISTOLOGICAL CLASSIFICATION RATS TIME PER DECREASED (WEEKS) GROUP MUCIN ATYPIA DYSPLASIA 1 4 . 0 0 0 2 5 0 0 0 4 3 0 . 8 + 1 . 4 1 . 2 + 1.1 0 6 5 0 . 4 + 0 . 8 0 . 6 + 0 . 8 1 .1 + 1 . 6 8 4 1 . 6 + 1.1 2 . 6 + 2 . 1 0 . 7 + 0 . 8 11 5 2 . 1 + 2 . 3 0 . 9 + 1 . 3 0 . 6 + 0 . 8 - 71 -showing abnormal histochemistry increased up to 6 weeks and then decreased. Decreased mucin, atypia and a small number of d y s p l a s t i c f o c i were f i r s t observed at 4 weeks, and t h e i r incidence increased with time. ( v i i ) H i s t o l o g i c a l abnormalities i n histochemically normal crypts The incidence of h i s t o l o g i c abnormalities associated with normal histochemistry i s shown i n t a b l e 10. The phenomenon was most freauently associated with f o c i expressing e i t h e r a decrease i n the quantity of mucin as the only abnormality or a t y p i a . The number of crypts e x h i b i t i n g atypia together with normal histochemistry f l u c t u a t e d throughout the 11 weeks of the preneoplastic phase but, with the exception of 4 weeks, was always smaller than the number of such crypts with abnormal histochemistry (table 9). A small proportion of the d y s p l a s t i c crypts presented with normal histochemistry. Such crypts appeared l a t e r i n the time course of the experiment than d y s p l a s t i c crypts showing histochemical change. As the number of histochemically abnormal d y s p l a s t i c f o c i increased (table 9) the incidence of histochemically normal d y s p l a s t i c crypts decreased (table 10). ( v i i i ) Histochemical c h a r a c t e r i s t i c s of postneoplastic phase tumours The r e s u l t s of the histochemical study of 45 of the tumours presenting between 16 and 24 weeks are shown i n Table 11. F i f t y - t h r e e percent of these tumours had no mucin or i n s u f f i c i e n t mucin to make a r e l i a b l e histochemical assessment, 25% showed change A, and the remaining 22% showed change B. - 72 -(ix) Histochemical c h a r a c t e r i s t i c s of the non-neoplastic mucosa adjacent  to tumours Table 12 shows the histochemical c h a r a c t e r i s t i c s of the mucins i n the non-neoplastic mucosa (NAT) d i r e c t l y adjacent to the tumours shown i n Table 11. Of the NAT 'S showing no change from the normal histochemical pattern (21% 17/80), 82% (14/17) were associated with tumours having no mucin, while only 12% (2/17) were adjacent to tumours with change A, and 6% (1/17) with tumours presenting with change B. NAT 'S with change A (51%, 41/80) were marginally more common adjacent to tumours with no mucin (41%, 17/41) than adjacent to tumours with e i t h e r change A (32%, 13/41) or change B (28%, 11/41). NAT 'S presenting with change B were associated with tumours with no mucin (59%, 13/22), change A (14%, 3/22) or change B (27%, 6/22). (x) Mucosa remote from tumours i n the postneoplastic phase Histochemical change i n areas remote from neoplasms a f t e r the i n i t i a l tumours appeared was defined with the AB1.0/PAPS or, on occasion, with the KOH/AB1.0/PAPS methods (see table 3) as being a decrease i n the aaua component of the s t a i n s ( i n d i c a t i n g a r e l a t i v e l o s s of sulphate) since both change A and change B showed such a pattern. As shown i n Table 13, histochemical change occurred frequently i n s i t e s remote from the tumours and the incidence at 16, 20, 22 and 24 weeks d i d not d i f f e r s i g n i f i c a n t l y from that at 8 or 11 weeks (table 9). However, as compared with the preneoplastic phase, the incidence of crypts with normal histology or showing e i t h e r l o s s of mucin or atypia decreased while the - 73 -TABLE 11: HISTOCHEMICAL CHANGES1 IN THE MUCINS OF TUMOURS2 OF THE WISTAR RAT DISTAL COLON3. TUMOUR CHARACTERISTIC NUMBER OBSERVED % OF TOTAL NO MUCIN 24 53 CHANGE A 11 25 CHANGE B 10 22 1 . CHANGE A = a d e c r e a s e o r l o s s o f s u l p h a t e o n l y CHANGE B = a d e c r e a s e o r l o s s o f s u l p h a t e accompanied by a d e c r e a s e o r l o s s o f s i d e c h a i n O - a c e t y l a t e d s i a l i c a c i d r e s i d u e s 2. A l l tumours were m o d e r a t e l y w e l l d i f f e r e n t i a t e d . 3 . Two o t h e r tumours n o t i n c l u d e d i n t h i s t a b l e had h i s t o c h e m i c a l change t h a t d i f f e r e d from the tumours l i s t e d . One c o n t a i n e d o n l y n e u t r a l hexose and the o t h e r showed an i n c r e a s e i n n e u t r a l hexose p l u s a d e c r e a s e i n s u l p h a t e . - 74 -TABLE 12: HISTOCHEMICAL CHANGES1 IN THE NON-NEOPLASTIC MUCOSA2 ,  IMMEDIATELY ADJACENT TO TUMOURS OF WISTAR RAT  DISTAL COLON. TUMOURS NO CHANGE CHANGE MUCIN A B TOTAL NAT (23) (11) (10) NO CHANGE 14 2 1 17 CHANGE A 17 13 11 41 CHANGE B 13 3 6 22 TOTAL 44 18 18 80 1 . CHANGE A = a d e c r e a s e o r l o s s o f s u l p h a t e o n l y CHANGE B = a d e c r e a s e o r l o s s o f s u l p h a t e accompanied by a d e c r e a s e o r l o s s o f s i d e c h a i n O - a c e t y l a t e d s i a l i c a c i d r e s i d u e s 2 . NAT f e l l i n t o t h r e e g r o u p s : ( i ) where b o t h (on e i t h e r s i d e o f the tumour) had t h e same c h a r a c t e r i s r i c s ; ( i i ) where o n l y one was p r e s e n t ; ( i i i ) where the two NAT's had d i f f e r e n t c h a r a c t e r i s t i c s . NAT's from a l l t h r e e groups are i n c l u d e d i n t h i s t a b l e . 3 . The NAT o f one tumour had h i s t o c h e m i c a l change t h a t d i f f e r e d from t h o s e l i s t e d . On one s i d e o f t h e tumour t h e r e was an i n c r e a s e i n s i a l i c a c i d w i t h no change i n s u l p h a t e and on t h e o t h e r t h e r e was an i n c r e a s e i n s i a l i c a c i d p l u s a d e c r e a s e i n s u l p h a t e . - 75 -proportion o f d y s p l a s t i c crypts with abnormal histochemistry increased. In general, as shown i n Table 14, the number of histochemically normal crypts which showed l o s s of mucin only or e i t h e r atypia or dysplasia decreased between 16 and 24 weeks; at 22 and 24 weeks, no histochemically normal d y s p l a s t i c crypts were observed. - 76 -TABLE 13: THE MEAN NUMBER OF HISTOCHEMICAL CHANGES SEEN PER  DECIMETRE OF COLONIC MUCOSA DURING THE POST  NEOPLASTIC PHASE CLASSIFIED ACCORDING TO MORPHOLOGICAL CRITERIA. HISTOLOGICAL CLASSIFICATION / i i ! J ! ? c \ NORMAL D E u n r ? M E D ATYPIA DYSPLASIA ^WEEKS) MUCIN ; 16 1.1 + 1 . 2 6 . 3 + 3 . 5 4 . 2 + 2 . 3 8 . 0 + 2 . 8 20 0 . 9 + 1 .1 4 . 0 + 2 . 2 4 . 5 + 3 . 5 8 . 5 + 4 . 5 22 0 . 5 + 1 . 0 1 . 9 + 2 . 2 3 . 1 + 1 . 4 1 2 . 3 + 2 . 8 24 0 . 3 + 0 . 6 0 . 8 + 1 . 3 3 . 0 + 2 . 4 11 .5 + 8 . 7 - 77 -TABLE 14: THE MEAN NUMBER OF FOCI SHOWING HISTOLOGIC ABNORMALITIES AND NORMAL HISTOCHEMISTRY IN THE POST NEOPLASTIC PHASE. HISTOLOGICAL CLASSIFICATION  DECREASED (WEEKS) MUCIN ATYPIA DYSPLASIA 16 2 . 5 + 2 . 1 1 . 4 + 1 . 4 0 . 2 + 0 . 5 20 1.8 + 2 . 2 1.1 + 1 .2 0 . 1 + 0 . 4 22 0 . 9 + 1 .1 1 .6 + 1 .9 0 24 0 . 4 + 0 . 6 0 . 2 + 0 . 5 0 - 78 -~iZ.<$cott £ L o n d b r i , England. I970 FIGURE 8 . - 79 -DISCUSSION Human c o l o n i c e p i t h e l i a l mucin i s composed predominantly of a c i d i c sulphated g l y c o p r o t e i n ( C u l l i n g et a l , 1979, 1981; F i l i p e , 1969, 1979, F i l i p e & Branfoot, 1976; Franzin et a l , 1981; Isaacson & Attwood, 1979; L i s t i n s k i & R i d d e l l , 1981; Marcheggiano et a l , 1984; Reid et a l , 1978, 1980, 1984a, 1985d) with a large percentage of 0-acetyl substituted s i a l i c a c i d residues ( F i l i p e & Cooke, 1974; C u l l i n g et a l , 1979, 1981; Rogers et a l , 1978; Reid et a l , 1978, 1980, 1984a, 1985d). In man, the mucin of c o l o n i c tumours and the non-neoplastic mucosa (NAT) adjacent to tumours (also c a l l e d t r a n s i t i o n a l mucosa) may d i f f e r from normal i n that i t may contain s i a l i c a c i d residues with s i g n i f i c a n t l y l e s s 0-acetyl s u b s t i t u t i o n and/or have a proportionate decrease i n O-sulphate ester (table l a , b ) . Based on the c a t i o n i c dye s t a i n i n g technique, HID/AB2.5 (Spicer, 1965), NAT, or t r a n s i t i o n a l mucosa, has been characterized by an increase i n sialomucin and a decrease i n sulphomucin (Table l b ) . This reported a l t e r a t i o n i n the mucin has also been shown to occur at s i t e s remote from tumours ( F i l i p e & Branfoot, 1974). Although the s i g n i f i c a n c e of t h i s f i n d i n g i s unknown, Greaves et a l , (1980) have suggested that t h i s mucin a l t e r a t i o n may be of value i n prognosis and Habib (1984) proposes that i t may be u s e f u l i n monitoring patients for recurrence a f t e r c u r a t i v e bowel r e s e c t i o n . Further, a number of i n v e s t i g a t o r s have suggested that these mucin changes are premalignant (Bara & Burtin, 1980; C u l l i n g et a l , 1977, 1979; Dawson & F i l i p e , 1976, 1982; Dawson et a l , 1978; Ehsanullah et a l , 1982a,b,1985; Fenger & F i l i p e , 1981; F i l i p e , 1969, 1971, 1972, 1984; F i l i p e & Branfoot, 1974; F i l i p e & Cooke, 1974; - 80 -Greaves et a l , 1980, 1984; Habib et al, 1984; Lapertosa et al, 1984; Marcheggiano et al, 1984; Montero & Segura, 1980; Reid et al, 1984a, 1985a) however, others consider them to be non-specific secondary phenomena (Franzin et al, 1981, 1983a,b,1984; Isaacson & Attwood, 1979; Listinski & Riddell, 1981; Rhatigan & Saffos, 1979; Sunter et al, 1985). Chemical and histochemical studies indicate that, as compared to normal, mucins in colonic tumours, NAT, and the mucosa within five centimetres of tumours contain a proportionate decrease in C^ and/or side chain substituted sialic acid (Filipe & Cooke, 1974; Culling et al, 1977; Dawson et al, 1978; Rogers et al, 1978; Reid et al, 1980, 1984a, 1985a). Further, mucin changes have been observed in rats treated with colon carcinogens (Filipe, 1975, Dawson & Filipe, 1980; Shamsuddin & Trump, 1981; Decaens et al, 1983) as shown in Table 2. The suggestion that such changes in mucin glycoproteins may be premalignant lead to the Question of whether such changes preceed, parallel or follow the development of neoplasia. This thesis reports a detailed histochemical investigation of colonic epithelial glycoproteins during and after the induction of cancer using an animal model system. The DMH-induced Wistar rat colonic neoplasia model was selected because (a) this type of study would be impossible to do in humans, (b) the Wistar rat is inexpensive, easily maintained, and has the virtue of genetic homogeneity, (c) previous studies with this model have used Wistar strain rats and are therefore comparable, (d) this system is considered to be a good model of human colorectal cancer, and (e) a number of new histochemical techniques have recently become available - 81 -that permit, to a degree h i t e r t o impossible, the c h a r a c t e r i z a t i o n of e p i t h e l i a l mucin glycoproteins. The histochemical procedures which were used i n t h i s i n v e s t i g a t i o n are o u t l i n e d i n Table 3. The HID/AB2.5 has been used i n the majority of histochemical studies of c o l o n i c e p i t h e l i a l glycoproteins i n normal, diseased, and cancerous t i s s u e s (Dawson & F i l i p e , 1976; Dawson et a l , 1978; Ehsanullah et a l , 1982a,b,1985; Fenger & F i l i p e , 1981; F i l i p e , 1969, 1971, 1972, 1975; F i l i p e & Branfoot, 1974; Franzin et a l , 1981, 1983a,b,1984, Greaves et a l , 1980, 1984; Habib et a l , 1984; Isaacson & Attwood, 1979; Katayama et a l , 1985; Lapertosa et a l , 1984; L i s t i n s k i & R i d d e l l , 1981; Mareheggiano et a l , 1984; Reid et a l , 1984a, 1985a; Sunter et a l , 1985; Terho & L a i t i o , 1977; Urbanski et a l , 1984). However, for the simultaneous d i f f e r e n t i a l v i s u a l i z a t i o n of s i a l i c acid and sulphate, the r e c e n t l y developed K0H/AB1.0/PAPS (Reid et a l , 1985c) has been shown to be more s e n s i t i v e than the HID/AB2.5 (McFadden et a l , 1985) because i n the l a t t e r procedure the black colour of the HID covers up the aaua s t a i n i n g of A l c i a n Blue and therefore i f both species were present within the same c e l l only the black of the sulphate would be seen. In the KOH/AB 1.0/PAPS however, mixtures of O-sulphate ester and s i a l i c a c i d s t a i n i n various shades of purple; a c e l l which contained a preponderance of sulphate and p r o p o r t i o n a l l y l e s s s i a l i c a c i d would appear dark aqua to ro y a l blue, whereas those c e l l s containing predominantly s i a l i c a c i d with p r o p o r t i o n a l l y l e s s sulphate would s t a i n red purple. Further, while the K0H/AB1.0/PAPS sta i n s sulphate plus t o t a l s i a l i c a cid, the AB1.0/PAPS (Reid et a l , 1985c) d i s t i n g u i s h e s sulphate plus unsubstituted s i a l i c - 82 -a c i d , or those substituted at C ?, whereas the PBT/K0H/AB1.0/PAS sta i n s sulphate and side chain s u b s t i t u t e d (C 7, Cg, C-, + Cg, Cg + C^, C-, + C g, C 7 + Cg + C g) s i a l i c a c i d . Therefore using these three new s t a i n s i n conjunction with each other not only was i t po s s i b l e to observe s i a l i c a c i d plus sulphate on the same t i s s u e section, but i t was a l s o possible to s u b c l a s s i f y the s i a l i c a c i d residues i n terms of degree o f side chain O-acetylation. In any histochemical method s p e c i f i c i t y i s d i c t a t e d by the chemical re a c t i o n s involved i n the procedure. Therefore, a number of factors were considered before the s t a i n s were used. F i r s t l y , an appropriate i n i t i a l o x i dation time had to be chosen which t o t a l l y oxidized a l l a v a i l a b l e v i c i n a l d i o l s . In t h i s study a period of 2 hours was shown to be most e f f e c t i v e and was used c o n s i s t e n t l y i n a l l s t a i n s i n v o l v i n g periodate oxidation. I t should be noted that even though i t i s po s s i b l e that not a l l a v a i l a b l e d i o l s were oxidized i n 2 hours, the r e s u l t s of t h i s study would not be a f f e c t e d . This i s because the oxidation time chosen was used c o n s i s t e n t l y f o r a l l sections (controls and treated) and the histochemical abnormalities observed were recorded as changes from normal. Secondly, the i n t e r p r e t a t i o n of the PBT/KOH/AB1.0/PAS, PAPT/KOH/Bh/PAS, PAT/KOH/Bh/PAS, and the PBT/KOH/PAS procedures depends on the assumption that there are no carbohydrate residues, other than s i a l i c a c i d s , with 0-acyl e s t e r s s i t u a t e d on p o t e n t i a l v i c i n a l d i o l s . Such residues would add to the magenta s t a i n i n g a t t r i b u t e d to side chain s u b s t i t u t e d s i a l i c a c i d residues. Such residues have not yet been demonstrated, however, i f they d i d occur to such a degree that they might - 83 -i n t e r f e r e with the i n t e r p r e t a t i o n of the methods then the incorporation of a phenylhydrazine blockade a f t e r the f i n a l periodate oxidation would eliminate the problem (Reid et a l , 1984c). T h i r d l y , Haverkamp et a l (1975) have shown chemically that with l i m i t i n g a u a n t i t i e s of periodate, ( C g 0-acyl s i a l i c acids were o x i d i z e d more slowly than the corresponding s i a l i c a c i d without side-chain substituents. I f C n s i a l i c acids survived the i n i t i a l 2 hour oxidation period then magenta s t a i n i n g i n the PBT/K0H/AB1.0/PAS, PAPT/KOH/Bh/PAS, PAT/KOH/Bh/PAS and PBT/KOH/PAS procedures could be due to C g as well as C 7,C Q and d i or t r i s u b s t i t u t e d s i a l i c a c i d residues. However, Reid et a l , (1977, 1978) were unable to demonstrate C g s i a l i c acids i n g a s t r o i n t e s t i n a l mucins but obtained evidence f o r them i n bovine submandibular gland (Reid et a l , 1978). In a d d i t i o n , i n the concentration used, periodate would have been present i n excess and not i n l i m i t i n g q u a n t i t i e s . Fourthly, f a i l u r e to reverse the phenylhydrazine blockade of s i a l i c a c i d monoaldehydes w i l l lead to an underestimate of the quantity of s i a l i c a c i d while an overestimate w i l l r e s u l t from the r e v e r s a l of the blockage of sugar dialdehydes i n any procedures which incorporate the phenylhydrazine step (Reid et a l , 1985c). This would not a f f e c t the r e s u l t s of the present study as the standard procedure f o r phenylhydrazine blockade and r e v e r s a l (Reid e_t a l , 1984b,c) was used throughout on both c o n t r o l and treated t i s s u e s and abnormalities were expressed as changes as compared to normal. F i n a l l y , any method i n v o l v i n g the use of a c a t i o n i c dye depended on the s p e c i f i c i t y of the s t a i n i n g with the dye at a favourable pH. For example, i t was assumed - 84 -that e i t h e r HID or AB at pH 1.0 would s t a i n a l l a v a i l a b l e sulphate groups only and that AB at pH 2.5, applied a f t e r HID at pH 1.0, would have stained only those i o n i z e d carboxyl groups at p o s i t i o n of s i a l i c a c i d residues. Further, i t was assumed and has been shown previously (Reid et a l , 1985c) that none of the s t a i n i n g sequences used, e i t h e r p r i o r to or a f t e r the a p p l i c a t i o n of the c a t i o n i c dye, a f f e c t e d the i n t e n s i t y o f s t a i n i n g . With the scheme used, 4 of 7 stains detected sulphate 2, of 7 detected t o t a l s i a l i c a c i d , and 5 of 7 d i s t i n g u i s h e d between substituted and unsubstituted s i a l i c a c i d residues. The redundancy b u i l t into the s t a i n i n g scheme permitted the i d e n t i f i c a t i o n of the mucin with reasonable c e r t a i n t y even i n cases where i t was not p o s s i b l e to i d e n t i f y a focus i n a l l s e r i a l s due to mechanical a r t e f a c t s such as t i s s u e f o l d s , l i f t i n g s ections, or s e c t i o n i n g through the gland. The number of abnormalities seen per r a t colon was standardized by measuring the amount o f s t a i n a b l e mucosa present on the s l i d e . This was necessary because the amount of mucosa present for each colon examined varied g r e a t l y between cases due to colon length, t w i s t i n g of t i s s u e during embedding, and l o s s of t i s s u e during processing or s t a i n i n g . Previous studies i n r a t s have suggested that normal Wistar rat d i s t a l colon i s composed predominantly of sulphomucin ( F i l i p e , 1975; Reid et a l , 1975; Decaens et a l , 1983; Tsuyama et_ a l , 1983) and contain O-acetylated s i a l i c a c i d residues (Reid et a l , 1975; Tsuyama et a l , 1983), and also that normal Fischer F344 r a t d i s t a l colon i s composed of sulphomucin (Shamsuddin & Trump, 1981). In contrast, the data presented - 85 -i n t h i s t h e s i s i n d i c a t e s that the composition of the e p i t h e l i a l mucins of the d i s t a l colon i s complex varying with both the anatomic region of the colon and the l o c a t i o n of the glycoprotein within the crypts. These v a r i a t i o n s occurred c o n s i s t e n t l y and t h e i r pattern could e a s i l y be i d e n t i f i e d i n both normal and DMH treated r a t s . Furthermore, the pattern was so d i s t i n c t that i t was possible to determine the approximate l o c a t i o n of any tumour i n the d i s t a l colon by v i r t u e of the s t a i n i n g pattern i n the surrounding uninvolved mucosa regardless of the character of the mucin i n e i t h e r the tumour proper or the NAT. The changes i n s t a i n i n g pattern observed within a small l i n e a r distance i n d i c a t e that mucosal b i o p s i e s or random blocks are inadequate i n studies of colonic e p i t h e l i a l glycoproteins of the d i s t a l rat colon and therefore i t i s d e s i r a b l e to employ e i t h e r "swiss r o l l s " or blocks taken from an anatomically defined l o c a t i o n i n the colon. I f such defined blocks are used the l o c a t i o n must be selected with care as, the lengths of the regions DE, MID and PE vary between ra t s and one must be c e r t a i n that one i s w e ll within the boundaries of the desired region to ensure that none of the block i s taken from an area of t r a n s i t i o n from one region to another. The b i o l o g i c a l s i g n i f i c a n c e of the r e g i o n a l d i f f e r e n c e s i n mucin composition i s unknown. The data c l e a r l y shows that, moving d i s t a l l y from the proximal end of the lower colon towards the r e c t a l end, there i s a gradual increase i n the degree to which the mucin i s sulphated and a decrease i n the proportion of side chain O-acetylated s i a l i c acids. The c o l o n i c mucin i s assumed to serve a v a r i e t y of functions including - 86 -l u b r i c a t i o n of the f e c a l mass, pro t e c t i o n of the mucosa from abrasion by s o l i d f e c a l matter by s e a l i n g breaches i n the epithelium, and i n forming a b a r r i e r ( F i l i p e & Branfoot, 1976; F i l i p e , 1979, Forstner, 1978). As a b a r r i e r the mucin may r e i n f o r c e the c e l l membranes and t i g h t junctions by maintaining e l e c t r o l y t i c gradients across the mucosal surface (Winzler, 1970). Sialomucins are more viscous and are therefore thought to be p r i m a r i l y p r o t e c t i v e i n function whereas sulphomucins are l e s s v i s c i d and are thought to have a l u b r i c a t i v e e f f e c t (Smith and Butler, 1974). Since the f e c a l mass becomes mo r e 1 s o l i d and abrasive as i t passes through the i n t e s t i n e , the increased proportion i n sulphomucin towards the r e c t a l end of the colon would be required to keep the mucosa well l u b r i c a t e d . It has been shown that i t i s the s i a l i c acid residue which i s a c t u a l l y responsible for much of the v i s c o s i t y of ovine submaxillary mucin (a sialomucin). Gottschalk (1960) demonstrated that enzymatic removal of 80% of the terminal s i a l i c a c i d residues at pH 6.0 decreased the reduced v i s c o s i t y from 3.2 to 1.3. The s i a l i c a c i d present on the c e l l surface i s a lso thought to function i n the transport of potassium ions through the plasma membrane and the transport of p r o t e i n out of the c e l l (Rambourg, 1971). Since substituted s i a l i c acids are r e s i s t a n t t o ' d i g e s t i o n with b a c t e r i a l neuraminidases and s i a l i c acids have been shown to protect some proteins against p r o t e o l y s i s ( F a i l l a r d & Schauer, 1972) i t has been suggested that they serve to protect the glycoprotein against b a c t e r i a l degradation and would also therefore help to maintain the i n t e g r i t y of the mucin b a r r i e r . Goblet c e l l mucin appears to be a l t e r e d a f t e r i t s s e c r e t i o n from the c e l l ; f o r example, the i n t r a c e l l u l a r mucin - 87 -of the DE region consisted of sialosulphomucin but the surface mucin i n the same region was sialomucin. However, i n t r a c e l l u l a r glycoprotein synthesis i s a continuous process and Neutra & Leblond (1966) have estimated that i n the r a t colon a fresh droplet of mucin i s released from the Golgi membrane every 2-4 minutes. The cause of the increase i n side chain O-acetylated s i a l i c acids observed during the course of the experiment i s unknown. Presumably i t i s r e l a t e d to the age of the r a t s but the p o s s i b i l i t y that i t i s an e f f e c t of EDTA has not been eliminated. It was of i n t e r e s t , however, that histochemical reactions of normal c o l o n i c mucin of the older rats more c l o s e l y resembled that of adult human c o l o n i c mucin ( C u l l i n g et a l , 1981) than that of the younger r a t s . This suggests that i s may be more appropriate to s t a r t with older r a t s when using t h i s model of c o l o r e c t a l cancer. The DMH induced tumours of r a t colon are considered to be a good model of human c o l o r e c t a l cancer (Ward, 1974; Deschner, 1974; P o z h a r i s s k i , 1975; F i l i p e , 1975; Maskens, 1976; Lamont & 0'Gorman, 1978; Martin, 1982, Freeman, 1983), because the agent has a low t o x i c i t y , tumourigenesis i s almost e x c l u s i v e l y r e s t r i c t e d to the colon, and the anatomic d i s t r i b u t i o n of the tumours within the colon c l o s e l y resembles that found i n man. A number of i n v e s t i g a t o r s have reported evidence of an adenoma-carcinoma sequence i n the model (Lingeman & Garner, 1972; Sunter et a l , 1978b; Hagihara et a l , 1980; Madara et a l , 1983), however, other have presented evidence to the contrary (Pozharisski, 1975; Maskens & D u j a r d i n - L o i t s , 1981; S h i r a i et a l , 1983). A number of morphological - 88 -and biochemical changes have been reported i n the mucosa harbouring DMH induced tumours (Lamont et j d , 1974; F i l i p e , 1975; Barkla, 1977; Freeman et a l , 1978; DeRubertis & Craven, 1980; Dawson & F i l i p e , 1980; Maskens & D u j a r d i n - L o i t s , 1981; Shamsuddin & Trump, 1981; Barthold, 1981; Chang, 1981; Decaens et a l , 1983; Traynor et a l , 1983; Freeman, 1983), but there have been r e l a t i v e l y few s t u d i e s o f the pre-neoplastic phase of the disease and none which have employed the range of histochemical procedures used i n t h i s i n v e s t i g a t i o n . The incidence and pattern of neoplasia observed i n t h i s study was consistent with that reported by other i n v e s t i g a t o r s (Lingeman & Garner, 1972; P o z h a r i s s k i , 1975; F i l i p e , 1975; Maskens, 1976; Hagihara et a l , 1980; S h i r a i e t a l , 1983; Freeman, 1983; Scudamore et a l , 1983; C a l d e r i s i et a l , 1984). The e a r l i e s t and predominant pre-neoplastic histochemical change was a f o c a l l o s s of sulphate. Loss of 0-acetyl groups from s i a l i c a c i d residues i n addition to l o s s of sulphate occurred l a t e r and to a l e s s e r extent. The s i g n i f i c a n c e of t h i s apparent sequential a l t e r a t i o n in mucin biochemistry i s unknown. The r a r i t y of f o c a l changes i n 0-acetylation of s i a l i c a c i d residues without a change i n sulphate i s s u r p r i s i n g e s p e c i a l l y i n view of the f a c t that i t does occur i n humans (Reid et a l , 1985a). The observed f o c a l mucin changes may, i n theory, have res u l t e d from one or more of a v a r i e t y of f a c t o r s , for example, from a l t e r a t i o n s i n the biosynthetic pathway due to ( i ) the absence of a required enzyme such as a s u l f o t r a n s f e r a s e and/or an O-acetyltransferase; ( i i ) the i n a c t i v a t i o n of an a c t i v e form of the enzyme(s); ( i i i ) a mutation r e s u l t i n g i n the - 89 -production of an i n a c t i v e form of the enzyme(s); ( i v ) a mutation r e s u l t i n g i n the production of an enzyme with a l t e r e d a c t i v i t y ; or (v) a mutation a f f e c t i n g the r e g u l a t i o n of glycoprotein syntehsis such as the a c t i v a t i o n of genes normally a c t i v e only i n l e s s d i f f e r e n t i a t e d c e l l s , i . e . : f e t a l type goblet c e l l s , or the i n a c t i v a t i o n of genes normally a c t i v e i n the adult. V e l i c a n and V e l i c a n (1969) state that "^S-sulphate incorporation i s influenced by the degree of c e l l u l a r d i f f e r e n t i a t i o n . A l t e r n a t i v e l y , the change may involve a mutation r e s u l t i n g i n the production of a c a t a b o l i c enzyme(s) which i s capable of removal of sulphate or O-acetyl groups a f t e r they have been added such as a sulphatase or de-O-acetylase, r e s p e c t i v e l y . Another p o s s i b i l i t y may be the presence of a substance or substances capable of e f f e c t i v e l y "soaking up" a v a i l a b l e "stores" of sulphate or a c e t y l groups or a d e f i c i e n c y i n t h e i r sources. The observation that histochemical change occurred before any detectable morphologic change and, further, that i t occurred i n t i s s u e s i t e s with normal morphology at a l l time periods suggested that i t was a pre-malignant change. This hypothesis i s consistent with the observations that d y s p l a s i a when i t f i r s t presented was associated with l o s s of mucin and abnormal histochemistry while the presumably r e v e r s i b l e changes, reduction i n mucin and atypia, presented, at the same time period, with both normal and abnormal histochemistry. However, the hypothesis i s not consistent with the observation that, at l a t e r time periods, a small number of d y s p l a s t i c f o c i presented with a l o s s of mucin and normal histochemistry. This could be a consequence of the r e l a t i v e - 90 -i n s e n s i t i v i t y of v i s u a l observation when determining histochemical change; there may be an i n s u f f i c i e n t l o c a l concentration of dye to be v i s i b l e to an observer. Also, i n s t a i n s where two dyes are used, the i n t e n s i t y and shade of the f i n a l colour produced i s a function of the r e l a t i v e concentrations of the stained components and any di f f e r e n c e s that may e x i s t between the e x t i n c t i o n c o e f f i c i e n t s of the dyes employed. The l a t t e r could r e s u l t i n e i t h e r an overestimate or underestimate of one of the colours or, i n extreme cases, the f a i l u r e to observe one colour i n the presence of a large excess of the other. However, i n a comparative study of chemical and histochemical methods, Reid e_t al (1985b) determined that, despite the d i f f i c u l t i e s inherent i n the nature of histochemical studies, on average, v i s u a l histochemical observations provide a r e l i a b l e , comparative, semiquantitative method for the estimation o f side chain O-acylated s i a l i c a c ids. A l t e r n a t i v e l y , that d y s p l a s i a was r a r e l y associated with normal histochemistry, could be evidence of the existence of two pathways, one i n which mucin changes preceed morphologic change, the other i n which morphologic change occurs without change i n mucin composition. Possibly, two such pathways are r e f l e c t e d i n the observation that 82% of the histochemically normal NATS were observed adjacent to tumours with no mucin while only 18% of these were adjacent to tumours showing histochemical change. Although the tumours were a l l moderately d i f f e r e n t i a t e d , tumour heterogeneity i n morphologically s i m i l a r tumours i s well established (Drewinko et a l , 1984; Dexter, 1983). The data reported here d i f f e r s i n a number of respects from that - 91 -reported i n e a r l i e r s t u d i e s ( F i l i p e , 1975; Decaens et a l , 1983) where although f o c a l reductions i n sulphate associated with dysplasia were detected, histochemical change i n normal f o c i were apparently not observed. Further, the time course of the disease d i f f e r e d . These d i f f e r e n c e s could be due to a number of factors i n c l u d i n g our use of male instead of female r a t s (Martin, 1982), environmental conditions, a d i f f e r e n t dosage of carcinogen or our use of the KOH/AB 1.0/PAPS, a more s e n s i t i v e method for the evaluation of s i a l o and sulphomucins than HID/AB 2.5 (McFadden et a l , 1985). The s i m i l a r i t i e s between the histochemical changes observed i n the mucosa adjacent to human carcinoma and those seen i n d y s p l a s t i c f o c i i n the pre-neoplastic phase of the DMH induced tumours i n r a t l e d F i l i p e and her coworkers ( F i l i p e & Branfoot, 1976; F i l i p e , 1979; Greaves et a l , 1980; Ehsanullah et a l , 1982a,b) to suggest that the former changes are premalignant. The r e s u l t s of t h i s study i n d i c a t e , however, that mucin changes i n the DMH model d i f f e r i n some important respects to those observed i n man. F i r s t l y , although the los s of sulphate from the mucins of NAT ( t r a n s i t i o n a l mucosa) observed both i n t h i s study and that of Decaens et a l , (1983) (to our knowledge the only other study to date to a c t u a l l y examine the t r a n s i t i o n a l mucosa i n ra t s ) i s s i m i l a r to that i n man and a l s o occurs i n the preneoplastic phase, the proportion of NAT's showing normal histochemistry was much larger than that reported i n man ( F i l i p e , 1984). Secondly, the changes seen i n r a t NAT were f o c a l while the t r a n s i t i o n a l mucosa i n man generally shows f i e l d changes ( C u l l i n g et a l , 1981). T h i r d l y , i n r a t , l o s s of O-acetyl groups from s i a l i c acid - 92 -residues without a p a r a l l e l l o s s of sulphate was very rare but, i n man, t h i s type o f change i s r e l a t i v e l y common (Reid et a l , 1985a,b). 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J Histochem Cytochem 28:217 Reid PE, Dunn WL, Ramey CW, Corect E, Truman L and Clay MG (1984b). Histochemical i d e n t i f i c a t i o n of side chain O-acylated s i a l i c a c i d : the PAT/KOH/Bh/PAS and the PAPT/KOH/Bh/PAS procedures. Histochemical Journal 16:623 Reid PE, C u l l i n g CFA, Dunn WL, Clay MG, and Ramey CW (1978). A c o r r e l a t i v e chemical and histochemical study of the O-acetylated s i a l i c acids of human c o l o n i c e p i t h e l i a l glycoproteins i n formalin f i x e d p a r a f f i n embedded t i s s u e s . J. Histochem. Cytochem. 26:1033 Reid PE, C u l l i n g CFA, Dunn WL, Ramey CW and Clay MG (1984a). Chemical and histochemical studies of normal and diseased human g a s t r o i n t e s t i n a l t r a c t I: A comparison between h i s t o l o g i c a l l y normal colon, c o l o n i c tumours, u l c e r a t i v e c o l i t i s and d i v e r t i c u l a r disease of the colon. Histochem. 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An animal study. Dis. Colon. Rectum 27:468 Sass B, Rabstein LS, Madison R, Mims RM, Peters RL and K e l l o f GO (1975). Incidence of spontaneous neoplasms i n F344 rat s throughout the n a t u r a l l i f e span. J . N a t l . Cancer Inst. 54:1449 Scudamore CH and Freeman HJ (1983). E f f e c t s of small bowel transection, r e s e c t i o n of bypass i n 1,2-dimethylhydrazine-induced rat i n t e s t i n a l n eoplasia. Gastroenterology 84:725-731. Senior PV, Sunter JP, Appleton DR, Watson AJ (1984). Morphological studie s on the long-term organ c u l t u r e of c o l o n i c mucosa from normal and dimethylhydrazine treated r a t s . Br. J . Cancer 49:281-290 Shamsuddin AKM and Trump BF (1981). Colonic epithelium I I : i n vivo s t u d i e s of colon carcinogensis. Light microscopic, histochemical and u l t r a s t r u c t u r a l studies of histogenesis of azokymethane-induced colon carcinomas i n Fisher F344 r a t s . J . Natl. Inst. 66:389 Sharon N, and L i s H (1982). Glycoproteins: research booming on long-ignored ubiquitous compounds. Mol. C e l l Biochem. 42:167 S h i r a i T, Nakanowatari J, Kurata Y, Fukushima S and Ito N (1983). D i f f e r e n t dose-response r e l a t i o n s h i p s i n the induction of d i f f e r e n t types of c o l o n i c tumour i n Wistar r a t s by 1,2-dimethylhydrazine. Gann 74:21 Shorter RG (1983). Risks of i n t e s t i n a l cancer i n Crohn's disease. Dis. Colon. Rec. 26:686 Smith B and Butler M (1974). The autonomic c o n t r o l of co l o n i c mucous se c r e t i o n i n the mouse. B r i t J Exp Pathol 55 Spicer SS (1965). Diamine methods for d i f f e r e n t i a t i n g mucosubstances histochemically. Journal of Histochemistry and Cytochemistry 13:211 - 105 -Steplewski Z and Koprowski H (1983). Monoclonal antibody-defined antigens of human g a s t r o i n t e s t i n a l tumors. In: Oncodevelopmental  Markers: B i o l o g i c , Diagnostic and Monitoring Aspects. Fishman WH (ed), Academic Press, p 159 Sunter JP, Appleton DR, Wright NA, and Watson AJ (1978a). K i n e t i c s of changes i n the crypts of the j e j u n a l mucosa of dimethylhydrazine treated r a t s . Br. J . Cancer 37:662 Sunter JP, Appleton DR, Wright NA, and Watson AJ (1978b). Patho l o g i c a l features of the c o l o n i c tumours induced i n r a t s by the administration of 1,2-dimethylhydrazine. Virchows Archives 29:211 Sunter JP, Higgs MJ and Cowan WK (1985). Mucosal abnormalities at the anastomosis s i t e i n patients who have had i n t e s t i n a l r esection f or c o l o n i c cancer. J . C l i n . Path. 38:385 Swenberg JA, Cooper HK, Bucheler J and Kleihues P (1979) 1,2-dimethylhydrazine-induced methylation of DNA base i n various rat organs and the e f f e c t of pretreatment with d i s u l f i r a m . Cancer Res. 39:465 Tatematsu M , Katsuyama T, Nakanowatari J , Hasegawa R, Imaida K, Fukushima S, and Ho N, (1982) Region s p e c i f i c patterns of mucin reaction demonstrated by paradoxical concanavalin A-staining i n normal c o l o n i c epithelium and i n c o l o r e c t a l cancers induced i n r a t s by 1,2-dimethylhydrazine. Gann. 73:77 Teague RH, Fraser D and Clamp JR (1973). Changes i n monosaccharide content of mucosa glycoproteins i n u l c e r a t i v e c o l i t i s . Br. Med. J . 2:645 Terho, T and L a i t i o M (1977). Hexosamine containing macromolecules i n human colon carcinomas. Scand J . Gastroenterol. 12:7 Thurnherr N, Deschner EE, S t o n e h i l l E and L i p k i n M (1973). Induction of adenocarcinoma of the colon i n mice by weekly i n j e c t i o n s of 1,2-dimethylhydrazine. Cancer Res. 33:940 Traynor OJ, Costa NL and Wood CB (1983). A scanning e l e c t r o n microscopy study of changes i n the co l o n i c mucus layer during chemical carcinogenesis. Cancer 51:1847 Tsuyama S, Suzuki S, and Murata F (1983). The histochemical di f f e r e n c e s of i n t e s t i n a l gland e p i t h e l i a i n the r a t colon with s p e c i a l reference to t h e i r qlycoconjugates. Acta Histochemica Cytochemica 16:456 Tutton PJM and Barkla DH (1983). Regulation of c e l l k i n e t i c s and colon cancer. In: Experimental Colon Carcinogenesis. Autrup & Williams (Eds). CRC Press Inc., Boca Raton, F l o r i d a p 200 - 106 -Urbanski SJ, Marcon N, Rossakowska A and Bruce WR (1984). Mixed h y p e r p l a s t i c adenamatous polyps - an underdiagnosed e n t i t y . Am. J . Surg. Path. 8:551 Vel i c a n C and V e l i c a n D (1969). Histochimie des glucides en pathologies humaine. G a u t h i e r - V i l l a r s , P a r i s pp 60-62, 371 Ward JM (1974). Morphogenesis of chemically induced neoplasms of the colon and small i n t e s t i n e i n r a t s . Lab Invest. 30:505 Weisberger JH and F i a l a ES (1983). Experimental colon carcinogenesis and t h e i r mode of a c t i o n . In: Experimental Colon Carcinogenesis. Autrup & Williams (Eds), CRC Press, Inc. Boca Raton, F l o r i d a p 27 Winawer SO, and Sherlock P (1977). Detecting e a r l y colon cancer. H o s p i t a l P r a c t i c e 12:49 Winawer SJ (1980). Screening for c o l o r e c t a l cancer: An overview. Cancer 45:1093 Winzler RJ (1970). Carbohydrates i n c e l l surfaces. Int Rev Cytol 29:77 Yardley JH, Ransoff DF, Riddel RH and Goldman H (1983). Cancer i n inflammatory bowel disease: How serious i s the problem and what should be done about i t ? Gastroenterology 85:197 Yonezawa S, Nakamura T, Tanaka S and Sato E (1982). Glyconjugate with Ulex europacus agglutinin-1 binding s i t e s i n normal mucosa, adenoma and carcinoma of the human large bowel. J.N.C.I. 69:777 Yonezawa S, Nakamura T, Tanaka S, Maruta K, Nish i M and Sato E (1983). Binding of Ulex europeus agglutinin-1 i n polyposis c o l i : comparative study with s o l i t a r y adenoma i n the sigmoid colon and rectum. J.N.C.I. 71:19 Zaridze DG (1983). Environmental e t i o l o g y of large-bowel cancer. J. Nat'l Cancer Inst. 70:389 Zweibaum A, Hauri HP, S t e r c h i E, Chantret I, Haffen K, Bamat J, Sordat B (1984). Immunohistological evidence obtained with monoclonal antibodies of small i n t e s t i n a l brush border hydrolases i n human colon cancers and f o e t a l colons. Int. J . Cancer 34:591 - 107 -Descri p t i o n of S l i d e s S l i d e s 1-14 are l i g h t micrographs of t h i n sections of formalin f i x e d Wistar r a t d i s t a l colon. The mucosal surface i s at the tops of the s l i d e s and the crypts extend down through the lamina propria to the muscularis mucosa, the narrow band of muscle near the crypt bases which separates the mucosa from the sub mucosa. S l i d e 1 was stained with H & E and photographed at a magnification of 200x. The three c e n t r a l crypts e x h i b i t "decreased mucin" as there i s a d i s t i n c t absence of mucin goblets compared to the normal crypts on e i t h e r s i d e . The mucin-depleted c e l l s have s l i g h t l y enlarged n u c l e i and abundant e o s i n o p h i l i c cytoplasm. S l i d e 2 was stained with H & E and photographed at a magnification of 200x. The c e n t a l mucin depleted crypt e x h i b i t s "atypia". In comparison to the normal adjacent crypt the a t y p i c a l gland contains c e l l s with l e s s mucin, enlarged n u c l e i and some l e s s of nuclear p o l a r i t y . S l i d e 3 was stained with H & E and photographed at a magnification of 200x. The c e n t r a l mucin depleted crypt e x h i b i t s "dysplasia" and shows enlarged p s e u d o s t r a t i f i e d hyperchromatic n u c l e i crowded together. As nuclear p o l a r i t y i s l o s t , i t i s poss i b l e to see nu c l e i at d i f f e r e n t l e v e l s e s p e c i a l l y i n the middle to upper portion of the crypt. - 108 -Slide 4 was stained with H & E and photographed at a magnification of 31.25x. This moderately well differentiated polypoid adenocarconoma shows the formation of glandular elements with a distortion of the crypt architecture and budding, and a dismoplastic stromal response. The carcinoma has invaded beyond the muscularis mucosa and glandular structures have formed in the submucosa. Slide 5 was stained with H & E and photographed at a magnification of 31.5x. This moderately well differentiated adenocarconoma is exophytic but not polypoid in nature. Well defined glands are distorted in structure and form an infiltrative pattern with budding. The stromal response is dismoplastic with an increase in fibrous elements. The tumour has invaded beyond the muscularis mucosa and glandular elements can be seen in the sub mucosa. Slide 6 illustrates the DE region stained with K0H/AB1.0/PAPS and photographed at a magnification of 125x. The royal blue colour represents sialosulphomucin, a mixture of aaua (sulphate) and magenta (total sialic acid). The scattered purple cells represent sialosulphomucin containing a smaller relative proportion of sulphate than present in the royal blue cells. Slide 7 illustrates the DE region stained with PAPT/KOH/Bh/PAS and photographed at a magnification of 125x. The purple colour represents a mixture of O-acetylated (magenta) and non O-acetylated (blue) sialic acid. - 109 -S l i d e 8 i l l u s t r a t e s the MID region stained with K0H/AB1.0/PAPS and photographed at a magnification of 125x. The roy a l blue colour represents sialosulphomucin and the purple colours r e f l e c t a l o s s of the aaua component of the s t a i n hence a sialoculphomucin with a r e l a t i v e l y smaller proportion of sulphate than that present i n the royal blue c e l l s . S l i d e 9 i l l u s t r a t e s the MID region stained with PAPT/KOH/Bh/PAS and photographed at a magnification of 200x. The purple colour i n the tops of the crypts represents a mixture of O-acetylated (magenta) and non-O-acetylated (blue) s i a l i c a c i d . The blue colour i n the crypt bases represents a r e l a t i v e preponderance of C Q, C 7 or C g O-acetylated s i a l i c a c i d . S l i d e 10 i l l u s t r a t e s the PE region stained with K0H/AB1.0/PAPS and photographed at a magnification of 125x. The magenta colour repesents t o t a l s i a l i c acid and the purple and royal blue c e l l s i n the tops of the crypts r e f l e c t s the presence of varying r e l a t i v e amounts of sulphate as well as t o t a l s i c l i c a c i d . S l i d e 11 i l l u s t r a t e s the PE region before 8 weeks stained with PAPT/KOH/Bh/PAS and photographed at a magnification of 125x. The black colour i n the ti s s u e i s the India ink which was used to l a b e l the proximal end of the Wistar r a t d i s t a l colon. The purple colour represents a mixture of O-acetylated and non O-acetylated s i a l i c acid residues. - 110 -Sl i d e 12 i l l u s t r a t e s the PE region a f t e r 8 weeks stained with PAPT/Bh/PAS and photographed at a magnification of 125x. The magenta colour represents an overwhelming preponderance of O-acetylated s i a l i c a c i d residues. The purple colours i n the top halves of the crypts represent mixtures of O-acetylated and non O-acetylated s i a l i c a c i d residues. S l i d e 13 i l l u s t r a t e s CHANGE A occurring i n the DE region stained with K0H/AB1.0/PAPS and photographed at a magnification of 125x. The roy a l blue colour represents s i a l i c sulphomucin and the magenta colour represents t o t a l s i a l i c a c i d . The two magenta crypts have l o s t the aaua component of the s t a i n hence a loss of sulphate. S l i d e 14 i l l u s t r a t e s CHANGE B occurring i n the DE region stained with PABT/KOH/Bh/PAS and photographed at a magnification of 125x. The purple colour represents a mixture of O-acetylated s i a l i c a c i d residues. The two blue crypts have l o s t the magenta component of the s t a i n , hence a lo s s i n the C f t-0-acetylated s i a l i c a c i d . 

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