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THE EFFECT OF VOLATILE THIOL COMPOUNDS ON PERMEABILITY OF ORAL MUCOSA BY WILLIAM MAN FAI NG B.Sc., The University of British Columbia, 1980 D.M.D. , The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Oral Biology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1986 •William Man Fai Ng, 1986 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. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Department DE-6(3/81) ABSTRACT Cumulative clinical and experimental evidence indicates that volatile sulphur compounds (VSC) the principal components of oral malodour, may play an important role in the pathogenesis of periodontal disease. As their (H2S and CH3SH) concentrations in gingival sulci increase with the severity of periodontal involvement, the objective of this investigation is to ascertain if they exert an effect on the permeability of oral mucosa. Permeability determinations were performed on excised porcine sublingual mucosal specimens which consisted of non-keratinized epithelium, basal membrane and connective tissue layers mounted in a two compartment perfusion apparatus. Using radioactive and fluorescent-labelled penetrants, it was found that exposure of the epithelial surface to an atmosphere containing physiological concentrations of both thiols (15 ng H2S or CH3SH / ml of 95% air - 5% C02) increased the permeability of the mucosa to (35S)-S04~2, (3H)-prostaglandin E 2 (PGE2) and fluorescein isothiocyanate labelled JL. coli lipopolysaccharide (F-LPS). A three hour exposure of the mucosa to H2S and CH3SH resulted in a 75% and 103% increase respectively in permeability to .(35S)-labelled sulphate ion. ii Similarly, the mercaptan induced up to a 70% increase in permeability of the mucosa to (3H)-prostaglandin E 2 . The magnitude of changes in the permeability were found to depend on duration of exposure to the thiols and to their concentration. Studies using (3 5S)-H2S suggest that the observed changes in the tissue permeability are related to the reaction of the thiols with tissue components. In addition, the (35S)-H2S is capable of perfusing through all three layers of the mucosa at 12.3 ng / cm2. In contrast to H2S , the CH3SH effect was irreversible in control air / C02 environment. This infers that CH3SH is potentially a more deleterious agent . to the tissue barrier. However, its effect can also be reversed by treatment of tissues with 0.22% ZnCl2 either prior to or after exposure to mercaptan. This suggests that Zn + 2 ion may be useful in preventing the potentially harmful effects of VSC. Fluorescent studies with F-LPS indicate that thiols can also potentiate the penetration of endotoxin. Whereas the fluorescence of the F-LPS in control systems was confined to the superficial epithelial layer in contact with the endotoxin, the CH3SH-exposed mucosa exhibited fluorescence throughout the epithelial and connective tissue layers. Fluorescent staining of the mucosal iii specimens with fluorescein diacetate followed by counter staining with ethidium bromide provides evidence of membrane impairment to some cells by CH3SH. Collectively these observations provide strong experimental evidence that the VSC, products of putrefaction produced in the gingival sulcus by oral microflora, may adversely affect the integrity of the crevicular barrier to deleterious agents and thus contribute to the etiology of periodontal disease. iv Table of Contents Page Abstract ii List of Tables ix List of Figures x Acknowledgment xi General Introduction Chapter I A. Inflammatory Periodontal Diseases 1 B. Volatile Sulphur Compounds 3 C. Microorganisms Implicated in the Production of Volatile Sulphur Compounds 4 D. The Effect of VSC on Collagen, Procollagen, Total Protein and DNA Synthesis by Human Gingival Fibroblast Cultures 5 E. Clinical Manifestations Ascribed to VSC 7 F. Histological and Morphological Characterization of Sulcular Epithelium and Basal Lamina 8 G. Review of Studies on Permeability of Oral Mucosa 10 1. The Epithelial Intercellular Space Compartment 13 2. Basal Lamina Compartment 14 v Table of Contents (Continued) Page H. Membrane Coating Granules, an Integral Protein of the Matrix in the Intercellular Space Compartment 16 I. Properties of Penetrants 18 J. Permeability of Tissues to Endotoxin 19 K. Prostaglandins in Gingival Tissues 20 L. Role of Zinc Ion 23 M. Principal Objectives 23 Methods and Materials Chapter II A. Mucosal Tissue Specimens 25 B. In Vitro Permeability System Using Perfusion Apparatus 26 C. Calculation of Permeability Coefficients 29 D. Protocol for Permeability Studies 31 1. Experimental Protocol for Permeability of Tissue Exposed to VSC and Control Environments 32 2. The Effect of Zinc Chloride on Thiol-Induced Permeability of Mucosal Specimens 34 3. Perfusion Studies with [35S]-H2S 35 vi Table of Contents (Continued) Page E. Reaction of [35S]-H2S with Mucosa 35 F. Stability of the [35S]-H2S Tissue Complex 36 G. Penetration of Mucosa by ]L_ coli Endotoxin 37 H. Culture Conditions for Epithelial and Fibroblastic Cells 38 I. Vitality Staining of Mucosa with Fluorescein Diacetate (FDA) and Ethidium Bromide (EB) 38 Materials 40 RESULTS Chapter III A. Variations in Permeability 42 B. Histological Survey of Porcine Sublingual Mucosa 42 C. Development of in vitro System 43 D. Relationship Between Gas Pressure and Permeability of Tissues 45 E. Concentrations of Volatile Sulphur Compounds Used to Treat Mucosa. 45 F. Comparative Permeability Studies on Control, H2S and CH3SH-Treated Tissues 48 G. Permeability of [14C]-Ovalbumin 49 vii Table of Contents (Continued) Page H. Permeability of [3H]-Prostaglandin E 2 49 I. The Effect of ZnCl2 on Permeability of CH3SH-Treated Mucosa 51 J. Protective Effect of ZnCl2 Against PGE2 53 K. Reactivity of [35S]-H2S with Non-keratinized Mucosa 56 L. Permeation of [35S]-H2S Through Intact Mucosa 59 M. Stability of [35S]-H2S Binding to Mucosa 60 N. Penetration of Mucosa by Fluorescent Isothiocynate Labelled JL. coli Lipopolysaccharides (LPS) 62 0. Assessment of Cellular Viability after CH^ SH Treatment 67 DISCUSSION Chapter IV A. The Effect of VSC on Diffusion of Antigens through Non-keratinized Oral Mucosa. Factors Contributing to Periodontal Disease. 73 References viii 85 List of Tables Table Title Page Number I Percentage change in permeability of mucosal specimens subjected to various concentrations of h\S 47 II Percentage increase in permeability of oral mucosa subjected to various periods of time to H2S and CH.SH 50 III Percentage increase in permeability of methyl mercaptan-treated oral mucosa to PGE2 52 IV Protective effect of zinc chloride against CH3SH-induced increase in permeability of mucosa 54 V Reversal of CH3SH-induced increase in permeability of mucosa 55 VI Reversibility of CH3SH effect on permeability of mucosa to PGE2 57 VII Retention of H2S by oral mucosa 58 VIII Diffusion of H2S through oral mucosa 61 IX Treatment of [35S]-H2S-exposed mucosa with Air/C02and phosphate buffered saline 63 i x List of Figures Figure Title Page Number 1 Design of perfusion apparatus used for permeability-studies of mucosal specimens. 27 2 Components of the perfusion apparatus. 28 3 Assembly of the system used for the permeability studies. 30 4 Attainment of steady state of perfusion of penetrants before and after gaseous treatment. 44 5 Cytofluorography of control oral mucosa treated with FITC fluorescent-labelled endotoxin. 65 6 Cytofluorography of methyl mercaptan-induced penetration of FITC-labelled endotoxin. 66 7 Cytofluorography of fresh tissue differentially stained with fluorescein diacetate (FDA) and ethidium bromide (EB). 69 8 Differential staining with fluorescein diacetate (FDA) and ethidium bromide (EB) of previously frozen mucosa. 70 9 Differential staining of mucosa following 9 hour of experimental manipulation. 71 10 a,b CH3SH-treated mucosa stained with FDA and counter stained with EB. 72 x A C K N O W L E D G E M E N T I would like to express my deepest gratitude to my M.Sc. supervisor Dr. Joseph Tonzetich for his guidance and supervision throughout the course of the project without which the successful completion of this project would not have been possible. Besides his dedication to research, he is also a superior teacher and a friend. I am very fortunate to have been supervised by the real expert and authority in the field. He has made my study in research very entertaining and exciting and he has opened the door for me into the field of research. Moreover, Dr, Tonzetich has devoted numerous precious hours proof-reading the initial drafts of this thesis in spite of his busy schedule. I am most appreciative for all his assistance. I also want to thank my other advisors: Drs. D. Brunette, A. Hannam, L. Kraintz and B. McBride for their valuable comments and constructive criticism and the staff in the Department of Oral Biology. Special thanks is extended to Mr. Anthony Ng for his immense help in the construction of the system employed for the volatile sulphur compound studies. Mr. Ng has also helped me a great deal in the use of the computer. Lastly, I am especially grateful to my parents, Ching Fong and Grace, and to my brothers, Peter and Stanley for their encouragement and support throughout the years of my study. I would also like to acknowledge the Medical Research Council of Canada for their financial support. CHAPTER I GENERAL INTRODUCTION A. Inflammatory Periodontal Diseases Inflammatory periodontal diseases are disorders associated with the destruction of soft and hard tissues that provide support to the dentition. The progression of the diseases ultimately results in the loss of teeth, impairment in optimal masticatory function, proper phonation and acceptable esthetics, and in social and psychological stigmata. Periodontal diseases are considered to be an episodal disorder. They may reside in a quiescent state where they exhibit no overt inflammatory manifestation or it may be in a virulent inflammatory stage. The latter active stage is characterized by marked disruption and loss of connective tissue of gingivae and periodontal ligament, conversion of junctional epithelium to pocket epithelium, ulceration, and variable amounts of loss in alveolar bone. These alterations lead to a loss of attachment and to pocket formation, tooth mobility, and finally exfoliation of teeth as manifested in advanced periodontitis. Although the progression of gingival inflammation is generally a prerequisite to 1 periodontitis, the etiology of the disease is not known and appears to be multifactorial. Microbial by-products, mechanical irritants, autoimmunity, occlusal traumatism, endocrine imbalance, nutritional status, age, and psychosomatic problems have all been implicated as modulators of the disease process. Among this group of putative factors, the presence of bacteria and their by-products in or near the gingival sulcus is almost universally accepted as the primary etiological factor responsible for the establishment of inflammatory periodontal disease. The intact epithelial-basal lamina barrier functions as the first line of defense and, therefore, comprises a significant component of the overall resistance of the host to the infection. Hence, the bacteria or their by-products that can induce changes in the structure of the tissue barrier and enhance the accessibility of microbial substances to the underlying connective tissue layer would be conducive to initiating tissue destruction. The maintenance of the tissue barrier is important, as toxic substances such as endotoxins and bacterial dextrans, per se, have been shown to be incapable of causing inflammation to healthy sulcular gingiva (1,2). 2 Bu. V o l a t i l e S u l p h u r C o m p o u n d s Hydrogen sulphide (H2S), methyl mercaptan (CH3SH), dimethyl disulphide (CH3S)2 and dimethyl sulphide (CH3)2S collectively are termed as volatile sulphur compounds (VSC). These malodorous intermediate products of putrefaction are formed by predominantly oral gram negative anaerobic microflora from proteinaceous substrates rich in thiol containing amino acids. The reduction of cystine to cysteine followed by dethiolation of cysteine and reductive demethiolation of methionine are the primary pathways for the production of hydrogen sulphide and methyl mercaptan (3). In human mouth air, H2S and CH3SH generally occur in 1:1 ratio and account for 90% of the physiological content of VSC. The concentration of these thiols in human mouth air is in the range of 10"8 to 10"10 g / 10 ml mouth air. Levels greater than 1.5 ng of H2S / 10 ml air (120 ppb) and 0.5 ng CH3SH / 10 ml air (40 ppb) are considered socially objectionable (4). Studies have shown that the production of these sulphur containing compounds is intensified in cases of chronic periodontitis, gingivitis, oral hemorrhage, acute ulcerative gingivo-stomatitis, and certain lung-borne systemic diseases (5). 3 * L _ M f c r p p r g f l n i s m s I m p l i c a t e d i n t h e P r o d u c t i o n o f V o l a t i l e S u l p h u r C o m p o u n d s Gingival crevice provides a favorable environment for optimal growth of facultative and strict anaerobes. It is a stagnant environment enriched with proteinaceous serum exudates, erythrocytes, leukocytes, lymphocytes and desquamated epithelial cells. A large number of strains of bacteria have been isolated from the crevice. Among the isolated and identified anaerobes, pathogenic strains of Bacteriodes melaninogenicus were demonstrated to produce copious amounts of volatile sulphur compounds. In comparison to the non-pathogenic strains, Bacteriodes gingivalis produced ten-fold more total VSC, fifteen-fold more CH3SH and two hundred and fifty fold more (CH3S)2 (6). The production of VSC is optimal at pH > 6.5 and is inhibited by glucose concentrations greater than 0.02M (3). Thus, the level of VSC emission is expected to be higher in deep sulcular pockets where the pH is basic and the exogenous availability of carbohydrates is limited. 4 P_ The Effect of VSC on Collagen. Procollagen. Total  Protein and DNA Synthesis by Human Gingival  Fibroblast Cultures Initially, inflammatory periodontal diseases involve the loss of gingival collagen followed by alteration of periodontal ligament. Since the half-life of collagen from gingiva and periodontal ligament is extremely short compared to other connective tissues, any imbalance in its metabolism would be expected to induce an adverse effect on both structures. This is attested by an observed 60% to 70% loss of collagen at the affected crevicular sites as early as four days after the onset of inflammation. Type III collagen which constitutes 20% to 30% of gingival and normal periodontal ligament collagen content has been reported to be as low as 4% at disease sites (113). In addition, the presence of a type V collagen and an aberrant form of type I collagen termed as Type I trimer have also been documented in disease states. The type I trimer has been recovered from biopsied diseased tissue and fibroblast cell cultures derived from affected tissues. It is believed that its origin is attributed to a deviant gene expression of the affected fibroblasts. Recently, Johnson and Tonzetich reported a significant depression of collagen, procollagen, total non-collagenous protein 5 and DNA synthesis in human fibroblasts cultures that were exposed to volatile sulphur compounds (7,8,9). In vitro reactions of [35S]-H2S with Type I acid soluble and insoluble fibrillar collagen have demonstrated that this agent can bind to collagen and in the process alter its structure and solubility. As this reaction results in an uptake of sulphur, exposure of free aldehyde groups and in conversion of the protein to a more soluble form, it is postulated that these thiols cleave certain aldehyde mediated linkages, possibly Schiff Base or aldol condensation cross linkages (7,8,9). Furthermore, CH3SH treatment was found to interfere with conversion of type I procollagen to collagen. This is in accord with reported analyses of diseased tissues which show a loss of insoluble and acid soluble collagen forms while the content of the neutral salt - soluble fraction remained unchanged (10). Although both H2S and CH3SH can suppress protein synthesis by fibroblast cultures, the effect of CH3SH is more pronounced and is not reversible up to 24 hours of incubation. Thus, thiols produced through putrefaction appear to be potential collagenolytic agents (10). 6 J L C l i n i c a l M a n i f e s t a t i o n s A s c r i b e d t o V S C An increase in mouth malodour has been clinically observed in many inflammatory periodontal disease conditions. There is convincing evidence that the production of volatile sulphur compounds increases in periodontal pockets. Rizzo (1967) using strips of filter paper impregnated with lead acetate, demonstrated a correlation between the amount of lead sulphide formed and the depth of periodontal pockets (l). More recently methods were developed to quantitatively measure sulphur compounds in parts per billion range by use of a gas-chromatograph equipped with a flame ionization detector. Using this method, Coil (62) reported active production of H2S, CH3SH and (CH3)2S in periodontal pockets with amounts corresponding to the severity of disease. Similarly, it has been reported that concentrations of VSC in mouth air correspond to the severity of periodontal disease. (13) Three weeks after corrective periodontal therapy involving curettage and surgery, the levels of VSC were reduced by 60 % to baseline levels (12,13). It has also been reported that systemic administration of metronidazole (Flagyl), which selectively reacts with thiols, significantly reduced both the pocket depth and black-pigmented Bacteroides counts (14). In 7 view of the considerable body of experimental evidence implicating the involvement of VSC in periodontal disease, it is reasonable to propose that VSC may play a significant role in the pathogenesis of the disease. £ L _ H i s t o l o g i c a l a n d M o r p h o l o g i c a l C h a r a c t e r i z a t i o n o f S u l c u l a r E p i t h e l i u m a n d B a s a l L a m i n a The junctional and the crevicular epithelia are generally considered to possess the weakest barrier to penetration of substances in the oral cavity. They are constantly exposed to inflammatory agents and mechanical stresses and ironically, both are devoid of a protective keratin layer (15). Morphologically, crevicular epithelium has fewer and randomly distributed hemidesmosomes, desmosomes, tight junctions and other interlocking processes which are required to form a continuous seal and thereby retard the passage of water, ions and small molecules through the intercellular space (16,17,18,19). In comparison to keratinized oral epithelium, the intercellular space of sulcular epithelium is wide (19). It is believed to serve as a principal pathway for transport of substances across the epithelial surface. The minimal width of 8 the intercellular space was reported to be in the range of 150-155 A (20,21). The junction between the epithelium and the underlying connective tissue is demarcated by a 300 to 900A thick layer of basal lamina consisting of lamina lucida adjacent to the epithelium and lamina densa in contact with the connective tissue (21,22,23,24). Basal lamina is comprised of three main components: poorly organized molecules resembling type IV collagen, glycoproteins, and proteoglycans (25). The composition of its collagen resembles interstitial collagen in that glycine makes up one third of the amino acid residues and proline and hydroxyproline account for 20%-22% of total amino acids. These molecules differ from interstitial collagen in three respects. (a) They have 50% more hydroxyproline and eight-fold more hydroxylysine. (b) 80% of the hydroxylysine is glycosidically substituted primarily with glucosylgalactosylhydroxylysine and galactosylhydroxylysine (26). (c) The presence of eight half-cystine per 1000 amino acid residues is a unique feature of basal lamina collagen. Both Spiro and Kefalides propose that the formation of disulphide linkages involving cystine residues may have an important role in protein adhesion in the matrix (26,27). 9 Several functions have been assigned to basal lamina. They include the attributes of structural integrity, flexibility, tensile strength, direction of cellular regeneration, and a barrier to antigens (28). £ L R e v i e w o f S t u d i e s o n P e r m e a b i l i t y o f O r a l M u c o s a It has been suggested that the onset of periodontal disease is elicited by damage to the tissue's barrier (29,30, l) . Previous studies have shown that penetration can occur through several mechanisms which include endocytosis and simple diffusion through intercellular spaces (31,32,33,34). Although basal and prickle cells are capable of endocytosis, this route does not appear to be a primary transport mechanism across the entire stratified epithelium (31,32,35). Similarly, active transport, which is the principal route for passage of ions through epithelial cell layers in other parts of the body, does not appear to play an important role in oral mucosa (36). Recent studies support simple diffusion as the mechanism for penetration of substances through non-keratinized epithelium (33,37). 10 Permeability through the barrier is considered a passive process because refrigeration, of epithelial tissues for several days has been shown to have negligible effect on the permeability of these specimens when compared to freshly biopsied tissues, or to the permeability of epithelium studied in vivo (33,38,39). The passive phenomenon is further supported by Fick's law of simple diffusion which states that the speed of penetration of a substance is proportional to the concentration gradient (33). Permeability of substances through the mucosal barrier is not limited to one direction. In chronic inflammation, an accumulation of gingival fluid, lysosomal enzymes, polymorphonuclear leucocytes and lymphoctyes can be found in the sulcus (40,41,43). The presence of an inflammatory exudate indicates that reverse permeation of macromolecules occurs through the crevicular and/or junctional epithelium. The outflow of serum protein exudate is probably a net result of hydrostatic-osmotic pressure phenomena (43). Other studies have demonstrated absorption of [3H]-H20 and drugs through the crevicular epithelium of animals (36,44,45,46). These and other morphological findings provide good evidence that reverse permeability occurs and that the crevicular epithelium can be highly permeable. 11 Various methods have been used to study the permeability of epithelium including, intravenous injections and in vivo studies of buccal absorption following rinsing and topical application of antigens. In vitro studies are based on use of diffusion cells (47,48,49,50,38,51,45,52,53). Of the methods studied, the in vitro approach using radioactive isotope as a tracer has been found most reliable and sensitive. Great interest has been devoted to the molecular size of penetrants that can transverse the epithelial barrier. Tolo reported absorption of labelled human albumin through healthy crevicular epithelium in vivo (43). Fine, Pechersky and Mckibben reported penetration of even carbon particles with diameter of 1 to 3 microns through crevicular epithelium (54). Tolo and Jonsen performed an in vitro study of the size-limiting barrier by exposing rabbit lingual frenum to radiolabelled dextran. Using preparations with molecular weight ranging from 16,000 to 250,000 daltons, they found that 90 per cent of the molecules that penetrated had molecular weights between 16,000 and 70,000 daltons (52). To appreciate and understand the function of tissue it is necessary to know its composition. The tight junctions, intercellular spaces, and the basal lamina all contribute to the 12 compartmentalization of non-keratinized stratified epithelium. Thus transport of substances across an epithelial membrane can be conceived as a stepwise process from one compartment to another. 1 . T h e E p i t h e l i a l I n t e r c e l l u l a r S p a c e C o m p a r t m e n t As previously cited, the intercellular space has a minimal width of 150A to 155A and has adequate width to permit the passage of ferritin molecules with 95A diameter (20). In addition, electron microscopic analyses have demonstrated that the perfusion of horseradish peroxidase follows the intercellular space of gingival epithelium (55,56). In spite of the presence of tight junctions, the intercellular space is considered to be continuous with the basal lamina (19). It is believed to be the principal communication channel between the epithelial surface and the connective tissue layer. During an inflammatory episode, the intercellular space is widened, which presumably allows the passage of serum protein exudate into the gingival crevice (57). The expansion and contraction of the intercellular space is a form of adaptation to the different physiological conditions in many parts of the body. For example, 13 the intercellular space of gall bladder epithelium is known to dilate during water transport across the epithelial membrane and to contract when the transport ceases (58,59). 2 ^ B a s a l L a m i n a C o m p a r t m e n t Since basal lamina constitutes the only continuous connection, Gavin proposes that the basal laminar compartment is the functional barrier of crevicular epithelium (19). This assumption is in agreement with the observations by Frithiof who found localized breaks in the continuity of the basal lamina concomitant with emigrating leucocytes observed in inflammatory cases (24). Ultrastructural studies of basal lamina of individuals with chronic periodontitis corroborate observations of marked disruption of basal lamina at the pathologically involved regions. Takarada characterized two regions of changes which he terms positive for the upper region of the pocket and negative corresponding to the lower part of the pocket (60). In the upper area of the pocket, positive change was characterized by thickening (more than 900A) detachment and subsequent duplication, fragmentation, multilayer formation, and dislocation of the basal lamina. The 14 negative change, which is conceivably more detrimental, was associated with the lower region of the pocket where bacterial growth and tissue insults were more pronounced. This was reflected by a decrease in thickness (less than 350A) and density, interruption, and disappearance and breakdown of the basal lamina. It is believed that the negative zone is the primary area of active inflammation. The importance of basal lamina as the rate limiting barrier is supported by two additional observations. Firstly, the basal laminar layer of human skin stripped of the keratin layer is impermeable to methylene blue and Evans blue. However, treatment with collagenase or mechanical damage to basal lamina readily permits the permeation of both dyes (39). Secondly, while exposure of blood brain barrier to hyaluronidase and neuramidase has negligible effect on its permeability, treatment with pepsin and pronase significantly increases the permeability (61). The actions of these proteolytic enzymes on the barrier suggests that the basal lamina collagens and glycoproteins may play a primary role in the maintenance of tissue barrier. The permeability of the basal lamina barrier is more complex than merely being dependent on the size of the penetrant molecule. For example, while rabbit non-keratinized mucosa is 15 permeable to dextran-70 with molecular weight of 70,000 daltons, it is impermeable to horseradish peroxidase which has a molecular weight of 40,000 (52,32). Additional support attesting to the intrinsic complexity of basal lamina barrier is that non-keratinized sublingual mucosa of guinea pigs is permeable to dextran-20 (molecular weight 20,000 dalton) but not to inulin (molecular, weight 5,000 dalton) (63). Based on these observations, Alfano hypothesized that the transport of molecules may be mediated by either carrier molecules, molecular sieving, energy dependent processes, or simple filtration (63). From these reports it appears that simple filtration is the most probable route. The impediment to penetration by smaller proteins may be due to an inherent reactivity of these molecules with basal lamina components. Conversely, the matrix of basal lamina may retard the passage of the (charged) molecules in a fashion analogous to ionic exchange chromatography or affinity chromatography. I L _ M e m b r a n e C o a t i n g G r a n u l e s , a n I n t e g r a l P r o t e i n o f t h e M a t r i x i n t h e I n t e r c e l l u l a r S p a c e C o m p a r t m e n t . Although the concept that basal lamina serves as the 16 functional barrier appears attractive, currrent findings attribute the effectiveness of the barrier, in part, to the presence of membrane coating granules found in the intercellular space compartment (32,64). Horseradish peroxidase introduced intradermally into epidermis was ultrastructurally demonstrated to permeate only to the level of stratum corneum where the membrane coating granules or Odland bodies are deposited (65). These granules are not restricted to keratinized epithelium. Although they may differ in composition, they have been found in non-keratinized oral epithelium, uterine cervix and oesophagus (66,67). The membrane coating granules are believed to be of epithelial origin. Morphological studies suggest that the granules are synthesized in the Golgi apparatus at the prickle cell layer and then are extruded into the intercellular space where they are modified and become components of intercellular matrix (64). Cytochemical analysis indicates that the granules contain an amorphous material consisting of hydrolytic enzymes and glycoproteins (68,69,70,71,72). It is noteworthy that both the junctional epithelium and pocket epithelium are devoid of the membrane coating granules (73). 17 L P r p p e x t j g s o f P e n e t r a n t s Important determinants of the permeability are the physical and chemical properties of the penetrants. While the principal factor governing the penetration of an ionic compound is its pK value, the absorption of a non-ionic compound is dependent on the solvent and its partition coefficient. Since the permeability barrier is predominantly composed of neutral and polar lipids, it is presumed that substances with partition coefficients close to unity diffuse more readily. According to Wills, substances that possess the following properties are considered to be more effective penetrants (14). (1) Smaller molecules (2) Molecules diffuse faster than ions (3) Volatile compounds faster than non-volatiles compounds (4) Solubility in both nonpolar and polar solvents. These observations imply that small volatile molecules such as H2S and CH3SH, which are soluble in polar and nonpolar solvents, may be effective penetrants of the oral epithelium. 18 J . P e r m e a b i l i t y o f T i s s u e s t o E n d o t o x i n It is generally accepted that the presence of microorganisms and their byproducts of metabolism in gingival pockets is a prerequisite to inflammatory periodontal disease. There is ample evidence that bacterial endotoxin is an extremely harmful antigen. Its presence and destructive potential is supported by the following reported findings. (1) There is a shift of gram positive to gram negative organisms as pocket depth increases (75,76). (2) Gram negative organisms are the primary source for endotoxin. (3) Most endotoxins contain a similar lipopolysaccharide moiety. (4) Topical application of endotoxin to clinically healthy gingiva can induce acute inflammatory response, increase of gingival exudate, and vascular leakage (77,78,79). (5) In vitro, bone resorption and inhibition of bone synthesis can be induced by endotoxin (80,81). (6) Exposure of crevicular epithelium to endotoxin for 24 hours reveals a widening of intercellular space (82). (7) Endotoxin can activate lymphokines in the immune system. 19 Whether or not endotoxin penetrates through intact healthy gingiva is highly controversial. The work by Rizzo indicated that healthy rabbit gingival pocket tissue is impermeable to Salmonella  enteriditis endotoxin (2). In contrast, in vivo and in vitro studies by other investigators demonstrated the penetration of tritiated JL_ eoii endotoxin through the crevicular epithelium (82,83,84). Their autoradiographic analysis indicated a dissemination of the labelled compound with the highest concentration delineated at the basal lamina layer. These conflicting findings may be attributed to the differences in method and tissue specimens employed. Thus, there is experimental evidence indicating the possibility of endotoxin involvement in the initial stage of pathogenesis of periodontal disease, 1 L _ P r o s t a g l a n d i n s i n G i n g i v a l T i s s u e s Several potent substances, some of which are believed to be mediators of inflammatory response, are released locally at the site of inflammation. One group of such mediators are prostaglandins whose levels are elevated in inflammatory reactions associated with thermal injury, allergic contact eczema, ultraviolet irradiation, primary irritant dermatitis and 20 inflammatory periodontal diseases (85,86,87,88,89). In inflamed human gingiva, the presence of PGE2 has been found twenty-fold greater than in healthy tissues (90). The PGE2 levels were positively correlated with the progression of the disease process. PGE2 was absent from non-inflamed tissue but detectable amounts were present in areas adjacent to the margins of periodontal pockets. Highest PGE2 levels occurred at the advancing periodontal disease front (91). Since prostaglandins, in particular PGE2, are released in the purulent exudate and since the levels correlate with severity of periodontal disease, it is reasonable to suggest that they assume a role in mediating the inflamatory reaction and affect the integrity of the supportive tissue. Prostaglandins are ubiquitous compounds produced by a number of different cell types. On stimulation, they can be synthesized by osteoblasts, macrophages, eosinophils, and monocytes (92,93,94). These cells readily migrate to the affected area and contribute to the primary response of local inflammation. It is known that prostaglandins E 2 can increase vascular permeablity during the early inflammatory stage causing pronounced erythema and edema (95). 21 As a mediator of immune response, PGE can cause suppression of some lymphocyte functions, including mitogen and antibody response, T-lymphocyte cytotoxicity, lymphokine secretion and antibody dependent cell-mediated cytotoxicity (96). Connective tissue alteration and an increase in osteoclastic activity can also be induced in vitro by prostaglandins. Furthermore, the interplay between monocytes and lymphocytes in the production of osteoclastic activating factor may be mediated by prostaglandins which along with lymphokines are known as potent local mediators of bone resorption (97). Clinically, a number of therapeutic agents are employed to reduce inflammation through suppression of prostaglandin levels. The most prominent and widely used agents are the nonsteroidal anti-inflammatory drugs such as indomethacin fenamates and salicylates which inhibit the synthesis of prostaglandins. More recently, flurbiprofen has been found to be a potentially effective agent in the treatment of inflammatory periodontal diseases in experimental animals. A study on Beagle dogs demonstrated that systemic administration of flurbiprofen inhibited resorption of alveolar bone in chronic marginal periodontitis (98). Thus, it is plausible that prostaglandin E may play a significant role in the pathogenesis of periodontitis. 22 L . R o l e o f Z i n c I o n Zinc is an essential element which possesses cell membrane stabilizing properties. The uptake and incorporation of zinc ion can change the structural and functional components of cell membrane. Change in cellular function is supported by findings that uptake of silica by macrophage decreases after zinc treatment (99). Furthermore, the presence of Zn + 2 results in increased yield of purified viable lymphocytes (99). Zinc has also been found to accelerate the rate of healing of thermal and excised wounds, and to be a useful agent for reducing organ transplant rejection (100,101). In addition, it is believed that zinc has an immunological effect on transformation and mitotic activity of lymphocytes and macrophages (102). As zinc is a thiol-binding agent that has the propensity to stabilize the cell membrane, it is imperative that a study be performed to determine the degree of protection that this ion may have on the mucosal barrier that has been exposed to VSC. M . P r i n c i p a l O b j e c t i v e s The objective of this thesis is to investigate: (l) The effect of volatile sulphur compounds on the 23 permeability of oral mucosa to an anion. Permeability of volatile sulphur compounds through the intact mucosa. Reactivity of volatile sulphur compounds with tissue components. The effect of volatile sulphur compounds on the permeability of tissue's barrier to prostaglandin E 2 and ovalbumin. The effect of volatile sulphur compounds on the penetration of tissue's barrier by fluorescein isothiocynate labelled EL coli lipopolysaccharide. Reversibility of methyl mercaptan and hydrogen sulphide effect on permeability. Effect of zinc chloride on mucosal tissue barrier that has been exposed to volatile sulphur compounds. 24 Chapter II  METHODS AND MATERIALS A. Mucosal Tissue Specimens Porcine sublingual oral mucosa is composed of non-keratinized stratified epithelium which exhibits characteristic rete ridge extensions histologically similar in appearance to human crevicular epithelium. As the area of crevicular mucosal specimens is inadequate in size to cover the aperture of the perfusion apparatus, easily obtainable porcine sublingual mucosa was selected for the study. Porcine mandibles were obtained from a local abatoir and tissue specimens were biopsied within 60 minutes after sacrifice. Biopsied preparations of mucosal tissue were taken from the floor of the mandible ventral to the tongue. These specimens consisted of intact layers of non-keratinized epithelium, basal lamina and connective tissue having a total thickness of approximately 1 mm. The presence of an intact epithelial layer was verified by its shiny surface observed under a dissecting 25 microscope at 5 X magnification. The biopsy samples were sectioned into 5 mm X 5 mm pieces then either immediately used for permeation studies or frozen up to a maximum of two months in 10 % glycerol / phosphate buffer saline (PBS) at -70°C. Prior to use, the frozen sections were equilibrated to room temperature then immersed in PBS at 37°C for 30 minutes. Evaluation of fresh and frozen specimens indicated that the freezing procedure had no discernable adverse effect on permeability of mucosa (53). B. In Vitro Permeability System Using Perfusion  Apparatus Permeability of substances were studied using a perfusion apparatus designed and constructed by the author from Teflon. The apparatus consists of two chambers each with a central aperture of 0.0491 cm 2 and inlet-outlet ports (Figs. 1 & 2). The design of the employed unit is a modification of the apparatus used by Ainsworth and Alfano (38,83). The modified unit has the advantage that it minimizes the dead space volume and facilitates rapid measurements of steady state perfusion. 26 MODIFIED PERFUSION APPARATUS A" UPPER CHAMBER WHERE ISOTOPE IS APPLIED Fig. 1. Design of perfusion apparatus used for permeability-studies of mucosal specimens. Perfusion apparatus units were constucted from teflon. The mucosal tissues were mounted over the central aperatures (0.0491 cm2) separating the two chambers held in place by three tightened screws. The labelled penetrants were placed in the upper chamber (A) and perfused through the tissue into the lower chamber. The inlet-outlet ports in the upper chamber provided the passage of gaseous components over the epithelial surface of the mounted specimens, while the inlet-outlet ports in the lower chamber provided a constant flow of PBS bathing the underlying connective tissue surface. 2 7 Fig. 2. Components of the perfusion apparatus. Mucosal specimens consisting of intact epithelial, basal lamina and connective tissue layers (laminar propria), were biopsied to approximately 1 mm thickness then mounted with the epithelial surface facing upwards between the two chambers. Teflon tubes were then inserted into the inlet-outlet ports of the apparatus. 28 For each test system, a biopsied mucosal section was mounted over the 0.0491 cm 2 aperture between the two chambers with the epithelial surface facing upwards. The specimens were clamped firmly between the chambers by means of three equally tightened screws (Fig. l ) . In the lower chamber, a continuous flow of PBS (4.0 ml/hr), regulated by a Gilson minipuls peristalit pump, was in contact with the underlying connective tissue layer. Aliquots of 1,3 ml PBS effluent were collected at 20 minute interval using an LKB 7000 fraction collector, A device was incorporated in the PBS line to eliminate air bubbles and thus prevent any gaseous interface from forming between the tissue and the liquid in the lower chamber, The entire experimental operation was conducted at 37°C in a constant temperature incubator (Fig. 3). C . C a l c u l a t i o n o f P e r m e a b i l i t y C o e f f i c i e n t s Permeability changes were determined by means of penetration coefficients 'p', calculated on the basis of diffusion rate, exposed surface area, time, and concentration of the applied penetrant according to procedure described by Tregear (33). The determined 'p' values were then used to compare the permeability of control and test systems. For calculation of the 29 Fig. 3. Assembly of the system used for the permeability studies. Permeability experiments were conducted at 37°C in an incubator. The VSC permeation tube standards were equilibrated to 30°C, calibrated to yield 6.8 m l / m i n of 1.5, 15, and 150ng of V S C / m l 95% air/5% C 0 2 , then channelled into the upper chambers inlet port of the perfusion apparatus. A constant flow 4 ml /h r of PBS (37°C) was delivered v i a a peristaltic pump to the inlet port of the lower chamber of the perfusion apparatus. Effluent fractions of PBS were collected at 20 m i n intervals for LSC analyses. 30 'p' values, the activity of the perfusate is divided by the exposed tissue area and plotted against time. The slope at steady state is then divided by the concentration of the applied labelled substance. Penetration rate at steady state (cpm/cm 2 min) 'p' (cm/min) = Concentration of penetrant applied (cpm/cm 3) The initial permeability value was determined on all tested specimens and used as a control for assessing the effect of exposure to tested thiols. Thus each specimen served as its own control and the calculated 'p' value differences between the control and test values yielded reliable results attributed .to the tested volatile compounds. Change in permeability of the tested specimens were compared to control specimens exposed to 95% air / 5% C0 2 . D. Protocol for Permeability Studies To assess the effect of VSC on the permeability of the tissue barrier, Na 2 S0 4 , ovalbumin, prostaglandin E 2 and EL coli endotoxin were selected as penetrants for the following reasons. 31 (1) [35S]-Na2S04 was chosen as an initial marker for permeation studies as it is representative of a simple ion that has been shown to have minimal reactivity with proteins and cellular elements of saliva (104), (2) Since the introduction of PGE2 can induce inflammatory reaction similar to periodontal disease, the study of permeability of this inflammatory agent is of great interest. It also represents diffusion of a small physiologically active molecule. (3) Permeability studies were conducted on [14C]-ovalbumin preparation because its molecular weight corresponds to the limiting molecule size that can pass through the tissue barrier. (4) As endotoxin is an extremely potent inflammatory agent, the ability of tissue to function as a barrier to this antigen is of paramount importance. In order to ascertain the distribution pattern of endotoxin, fluorescein isothiocyanate labelled EL coli lipopolysaccharide was used as a tracer for this study. 1 . E x p e r i m e n t a l P r o t o c o l f o r P e r m e a b i l i t y o f T i s s u e  E x p o s e d t o V S C a n d C o n t r o l E n v i r o n m e n t s For the penetration phase of the study, mucosa was firmly mounted between the two chambers and 50 ul volumes of 32 labelled penetrant solution of isotopes were dispensed over the epithelial layer in the upper chamber. Aliquots of 1.3 ml of PBS (pH 7.4) perfusate were collected from the lower chamber until a steady state of perfusion was achieved. Then 100 ul of each collected perfusate fraction was admixed with 3 ml of aqeuous scintillation counting solution and assayed for radioactivity by liquid scintillation counting (LSC). A 10 ul sample of radioisotope containing solution was withdrawn from the upper chamber and analyzed for total radio-activity remaining in the top chamber. Subsequently, the epithelial surface facing the upper chamber was washed thrice with PBS and subjected for specific periods of time (5 to 180 min.) to an atmosphere of constant flow (6 ml/min) of H2S or CH3SH (x ng/ml) admixed with 95% air/5% C02. In the control system, the tissues were exposed for similar periods of time to only 95% air/5% C02. Then 50 ul of the isotope solution was reapplied onto the epithelial surface and the effluent was collected from the lower chamber until a steady state of perfusion was once again established. The desirable concentrations of hydrogen sulphide and methyl mercaptan were obtained by diluting the VSC emitted at 30°C from a permeation tube standard with x ml/min of 95% air/5% C02. Specific concentrations were determined from the calibrated 33 flow rate using a flow meter. A constant flow of 6 ml/min of x ng/ml VSC was channelled over the tissue surface in the upper perfusion chamber in a closed system. 2 . T h e E f f e c t o f Z i n c C h l o r i d e o n T h i o l - I n d u c e d  P e r m e a b i l i t y o f M u c o s a l S p e c i m e n s As Zn + 2 ion has been reported to have a protective effect on biological membranes, its ability to stabilize the integrity of the tissue barrier was investigated. The concentration of Zn + 2 (0.1%) employed in the study was in accordance with the level present in a commercially available mouth" wash (Lavoris). The effectiveness of Zn + 2 ion at the concentration present in Lavoris in suppressing oral malodour is well documented (105). For these studies, labelled penetrants ([ 3 5S]-S0 4 - 2, [3H]-PGE2) were initially applied over the epithelial surface of the tissue until a steady state of perfusion was obtained. Then the surface was subjected to an aqueous solution of 0.22% ZnCl2 (0.1 % Zn+ 2) for a period of 15 min., either immediately before or after exposure to 15 ng of CH3SH/ml air/C02 (flow rate of 6 ml/min). This was followed by reapplication of the labelled penetrant until a steady state of perfusion was again achieved. 3 4 3 L _ P e r f u s i o n S t u d i e s w i t h [ 3 5 S ] - H 2 S The penetration of [35S]-H-,S per se through intact tissue was studied by exposing the epithelial surface to 15 ng H2S, with specific activity of 46 mCi/mmole of [35S]-H2S/ml 95% air/5% C02 at a flow rate of 6.8 ml/min for 180 min. In this series of experiments, continuous flow af [35S]-H2S was regulated by displacement with room air of a standard [35S]-H"2S / H2S/air mixture prepared in a 1000 ml dilution flask. Aliquots of 1.3 ml PBS / 20 min of the effluent were collected from the lower chamber and analyzed for [35S]-activity by LSC. To ensure complete retention of H9S, 0.22% ZnCl2 was incorporated into the PBS. The entire experiment was performed at 37°C in an incubator housed in a fumehood. E . R e a c t i o n o f [ 5 5 S ] - H 2 S w i t h M u c o s a As thiols have been found to react with certain salivary proteins and cellular elements, a study was undertaken to find if 35 they also react with mucosal tissue components (106). For these [35S]-H2S experiments, mucosae were exposed to 15 ng [35S]-H2S at a flow rate of 6.8 ml/min for a period of 30 min. After the tissues were washed thrice with PBS, the areas peripheral to the exposure were trimmed away under a dissecting microscope. The exposed specimens were then digested in 5 ml of Soluene™ 100 for 2 hours at 60°C and assayed (LSC) for 35S-activity. F \ S t a b i l i t y o f t h e [ 5 5 S ] - H 2 S T i s s u e C o m p l e x Following 30 min. exposure of mucosa to [35S]-H2S, the epithelial surface was washed three times, for 20 min. each, with 1.0 ml volumes of PBS to remove unreacted or weakly bound [35S]-H2S. The tissues were then trimmed, digested in 5 ml of soluene™ 100, and assayed for radioactivity (LSC). A combined effect of aeration and washing with PBS on stability of [35S]-H2S tissue reaction was also investigated. In this series, immediately after exposure to [35S]-H2S the epithelial surface was aerated for 180 min. with 95% air/5% CO2 at a flow 36 rate of 6.8 ml/min. This was followed by washing the tisssues under constant agitation with 10 ml of PBS for 30 min. The specimens were then trimmed, digested, and assayed for radioactivity. G . P e n e t r a t i o n o f M u c o s a b y E . c o l i E n d o t o x i n The effect of CH3SH on the permeability of mucosa to endotoxin was determined using a highly purified commercially available fluorescent labelled E. coli preparation. As the analysis of the eluted fraction failed to detect fluorescence in either control or test systems, the tissues were retained for cytofluorometric analysis. The failure to demonstrate fluorescence in eluates may be due to either the low sensitivity of the method or to inability of the endotoxin to penetrate mucosa in detectable amounts. The micoscopic examination was intended to show if and how deep the endotoxin penetrated the tissues. Following the permeability experiment protocol previously described under section D, fluorescent labelled endotoxin was applied for three hours to the epithelial surface. The tissues were then washed with PBS, areas peripheral to the test site trimmed, and frozen in liquid nitrogen. They were then 37 mounted in 'tissue-TEK II OCT compound' embedding medium and sectioned into 8 um thick slices using a cryostat. Both control and test sections were examined under a FITC filter at optimum fluoresence emission (4400A to 5 0 0 0 A ) using a Zeiss photo-microscope. Photographs were taken using a Ektachrome (ASA 400) film with automatic exposure control. H . C u l t u r e C o n d i t i o n s f o r E p i t h e l i a l a n d F i b r o b l a s t i c C e l l s Epithelial cells and fibroblasts were obtained from areas of periodontal ligament of freshly extracted porcine mandibular teeth. The cells were grown to confluency over coverslips that were fastened to the bottom of Falcon-3002 culture dishes by silicone grease; these cells were supplied by Dr. Brunette's laboratory. L _ V i t a l i t y S t a i n i n g o f M u c o s a w i t h F l u o r e s c e i n D i a c e t a t e  ( F D A ) a n d E t h i d i u m B r o m i d e ( E B ) A differential staining procedure was employed to establish whether the cells throughout the tissue remain vital under the 38 conditions and duration (9 hours) of the experiment (107, 108). In order to conform to the described experimental conditions, the epithelial surface of the mounted specimens was first overlaid with 100 ul of PBS for 3 hours then exposed to either 95% air/ 5% C02 or to CH3SH 15 ng/ml air/C02 at 6.8 ml/min. for 3 hours. Then 100 ul of PBS was reapplied over the epithelium for 3 more hours to prevent tissue dehydration. The 0-3 hours and 6-9 hours intervals in this study correspond to periods that the mucosa was subjected to labelled penetrants in permeation experiments. The tissue was removed from the perfusion apparatus and unexposed areas were trimmed away and discarded. The remaining tissue was bathed in 10 ml of Dulbecco's modified Eagle Medium (DMEM) supplemented with 50 ul of FDA (5 mg/ml acetone) and 50 ul of freshly prepared EB (0.2 mg/ml PBS) then incubated for 30 min. at 37°C. After the reaction, the specimens were washed thrice for 5 minutes with 10 ml portions of DMEM to remove the excess stains. Following the last wash, the specimens were counter stained with 10 ul of EB. Again, the tissues were frozen in liquid nitrogen and sectioned into 8 um thick slices for cytofluorometry. The visualization of FDA-EB staining under blue or ultraviolet light permits a clear distinction between cells with intact and impaired membrane. By this method the EB is rapidly taken up 39 by DNA of only the damaged cells while FDA is hydrolyzed intracellular^ by an esterase in intact cells (107). Under a FITC filter, at wavelength (4400A - 5000A), EB incorporated in 'damaged' cells emits a red fluorescence which is in contrast to a green background of intact cells stained with FDA. Materials Phosphate Buffered Saline (PBS) pH 7.4: Grand Island Biological Company (GIBCO), New York. Dulbecco's Modified Eagle Medium supplemented with NaHC03: (GIBCO), New York. Zinc Chloride dissolved in distilled water to 0.22% w/v: Fisher Scientific Company 97.7% purity. Fluorescein Diacetate made to 5 mg/ml acetone and stored at -20°C until just before use: Sigma Chemical Company No. F-7378. Freshly prepared Ethidium Bromide made to 0.2 mg/ml PBS: Sigma Chemical Co. Soluene™ 100: Packard Instrument Co., Downers Grove, 111. Hydrogen Sulphide: Analytical Instrument Development Incorp., Batch calibrated at 30°C, Avondale, USA. 40 Methyl Mercaptan; Analytical Instrument Development Incorp,, Batch calibrated at 30°C, Avondale, USA. [35S]-Sodium Sulfate in water: New England Nuclear sp. act. 529.44 mCi/mmole, 5 mCi/ml, MW 131. [3H]-Prostaglandin E 2 [5,6,8,11,12,14,15,-3H (N)] ; N e w E n g i a n d Nuclear, 165 Ci/mmole in 0.5 ml ethanol/water (7:3), MW. 352.5, concentrate of isotope used for study 12.5 piCi/ml, 76 x IO - 9 M. Methyl- 1 4 C Ovalbumin: New England Nuclear, stock solution 5 liCi, sp. act. 0.01 mCi/mg, MW 45,000 concentration of isotope used for study 1.25 |iCi/ml distilled H20. Fluorescein isothiocyanate labelled EL coli lipopolysaccharide: Sigma Chemical Co., St. Louis USA, 055:B5 phenol extract, chromatographically purified, stock solution 0.5 mg/ml. Hydrogen [35S]-Sulphide: Amersham International Ltd., 5 mCi (185 MBQ), 24.2 mCi/mmole, 5.1 ml at STP. Aqeuous Scintillation Counting Solution: Amersham, 111, USA. 41 C H A P T E R III R E S U L T S V a r i a t i o n s i n P e r m e a b i l i t y An inherent disadvantage of the present in vitro system is the difficulty of attaining uniformity of thickness in all tissue specimens studied and the morphological variance of tissues among the animals. Consequently, the amount of labelled substances perfused through the different tissues were found to vary. To overcome this difficulty, it was necessary to establish steady state of perfusion on each tissue before and after treatment. Since permeability changes were determined by changes of rate of steady state of perfusion on the same specimen, each tissue served as its own internal control. E L Histological S u r v e y of Porc ine Sub l ingua l M u c o s a The tissue of interest in this investigation is the human crevicular and junctional epithelia. However, because of the difficulty in obtaining human crevicular and junctional tissues of 4 2 sufficient size for these studies, it was imperative to adapt an animal model system. Hence porcine sublingual mucosa obtained from the floor of the mandible was selected for the study, as it is readily available. Hematoxylin and eosin staining demonstrates that this tissue is composed of non-keratinized stratified squamous epithelium (6 to 10 cells thick) with rete ridge extensions and intact basal lamina. Lamina propria showed no sign of infiltration by polymorphonuclear leucocyte or lymphocyte. Histologically, the mucosa is almost analogous to healthy human gingival sulcular tissue. £ L Development of in vitro system As the employed system was leak proof, all of the tested penetrants permeated through the tissue and not through any perforations or improper seals. Tissues with leaks or perforations were readily identified at the onset of the experiment. It was found that isotopes diffused totally from the upper chamber into lower chamber within 15 min. in these situations. Another indication of 'leakiness' was the presence of reverse diffusion of PBS from the lower to the upper chamber. The steady state of diffusion was considered attained when four successive fractions yielded a constant rate of perfusion. (Fig. 4) This was 43 PERMEABILITY OF MUCOSA TO Na 2S0 4 BEFORE AND AFTER I HOUR EXPOSURE TO I50ng/ml H2S Fig, 4. Attainment of steady state of perfusion of penetrants before and after gaseous treatment. A steady state of perfusion of a penetrant was deemed established when four consecutive effluent fractions yielded a constant radioactive counts. The slope of cpm/cm2 to time represented a constant rate of permeation of a penetrant. The 'p' value was determined by dividing the permeation rate by the total amount of penetrant applied, The difference in 'p' values before and after gaseous treatment was attributed to the exposure of the mucosa to the VSC. As the control and test 'p' values were determined on the same tissue, each study served as its own control. 44 consistently reached within initial 180 min. of perfusion. It is noteworthy that the steady state was almost instantaneously reached in the majority of experiments (Fig. 4) following the exposure of mucosa to the H2S and CH3SH. E L . R e l a t i o n s h i p B e t w e e n G a s P r e s s u r e a n d P e r m e a b i l i t y o f  T i s s u e s Since aeration of tissue specimens with volatiles was carried out under positive pressure, it was conjectured that the pressure itself may induce affects on the tissue barrier. To examine this possibility, comparative studies were performed on open and closed systems. It was found that permeability was identical when experiments were conducted under an open and closed system in the upper chamber. To induce optimal saturation of tissues with thiols, volatile sulphur compounds were passed through the upper chamber at a rate of 6.8 ml/min. E L _ C o n c e n t r a t i o n s o f V o l a t i l e S u l p h u r C o m p o u n d s U s e d t o  T r e a t M u c o s a Although early morning human mouth air contains on 45 average 0.5 ng/10 ml CH3SH and 1.5 ng/10 ml H2S, these concentrations are not considered representative of the levels present in the gingival crevice, as volatile sulphur compounds are subjected to dilution in mouth air. The crevice is an enriched environment for thiol production and an area of active tissue destruction. Thus, it is postulated that the level of VSC in the crevice would be greater than that in mouth air. For example, the head space of incubated saliva exhibits levels of VSC concentration up to 15,500 ng/ml (110). Another important parameter that was found to affect permeability of the tissue was the duration of exposure of mucosa to thiols. Since crevicular tissue is exposed to constant presence of VSC produced in the sulcus, the effect of duration of exposure and concentration of thiols on mucosal barrier were concomitantly studied. The results in Table I show that the increase in permeability to sulphate ion was dependant on the duration of exposure of tissue to the tested 1,5, 15, and 150 ng/ml concentrations of H 2S, An increase in rate of permeability was observed during the initial 5 to 60 min. of exposure to 15 ng H 2S/ml air/C0 2 atmosphere. No apparent further increase in the rate of perfusion of the penetrant was observed between the 60 and 180 min. periods of exposure. The change in permeability was also influenced by the level of FLS present. (Table I) It is evident that, with the exception of 46 Tabic I P e r c e n t a g e c h a n g e i n p e r m e a b i l i t y o f m u c o s a l s p e c i m e n s s u b j e c t e d t o v a r i o u s c o n c e n t r a t i o n s o f H 2 S Time (min) C o n c e n t r a * ' o n °f H 2 S *n9'm'* 1.5 15.0 150.0 5 -12.3 10.6 — 30 16.8 25i 3 — 60 25.1 59.1 74.8 120 25.8 — — 180 74.9 61.7 15a o 240 — — 91.5 To determine the significance of duration and concentration of exposure of mucosal specimens to H2S, 1 mCi [55S]-Na2S04/ml H20 (529.44 mCi/mmol) was applied to the epithelial surface of the tissue to obtain control rate of diffusion. Then the epithelial surface were exposed to 1.5, 15 or 150 ng/ml H2S for periods of 5 to 240 min following which 100 ml of [35S]-Na2S04 was reapplied to the upper chamber until steady states were again achieved. Each value in the table represents a percentage change in permeability obtained on separate specimens. 47 1.5 ng H 2S/ml at 180 min., higher concentrations of H2S induced greater changes in permeability. A 60% to 75% increase was obtained with 1,5, 15, and 150 ng H 2S/ml air -within 3 hr, 1-2 hr, and 1 hr, respectively. I L _ C o m p a r a t i v e P e r m e a b i l i t y S t u d i e s o n C o n t r o l , H 2 S a n d C H 3 S H T r e a t e d T i s s u e s As methyl mercaptan has been implicated as a more cytotoxic agent than H2S (8), a study was undertaken to ascertain whether mercaptan also has a more pronounced effect on tissue permeability. Hence, parallel studies were performed on control tissues and tissues treated with 15 ng/ml CH3SH or 15 ng/ml H 2S. At 15 ng/ml air concentration both CH3SH and H2S exposed tissues exhibited a similar increase in permeability up to 60 min. of exposure. On longer exposure, the increase in permeability appears to plateau out. Comparative studies demonstrate that methyl mercaptan is a more potent agent, capable of increasing the permeability of mucosa to Na 2S0 4 up to 103%. The values 48 for control systems indicate an insignificant change in permeability following aeration with air/C0 2. (Table II) £ L _ P e r m e a b i l i t y o f [ 1 4 C ] - O v a l b u m i n As other investigators have reported the passage of dextran-70 (molecular weight 70,000 daltons) in in vitro studies, the effect of thiols on the permeation of [14C]-labelled ovalbumin was investigated. The results indicate that both control and methyl mercaptan-treated tissues are impermeable to [ 1 4C]-ovalbumin. Although ovalbumin has a smaller molecular weight than dextran-70, a difference in structural configuration and surface ionization of the protein may account for its lower penetrability. This finding illustrates the importance of selective mechanism regulating permeation of mucosal barrier. £ L Permeability p f [ 5 H ] - P r o s t a g l a n d i n E 2 The presence of prostaglandin E 2 in in vivo and in in vitro systems induces tissue reactions similar to those manifested in periodontal disease. As the concentration of PGE2 is markedly elevated in the crevicular fluid at inflammatory periodontal sites, 49 Table II Percentage increase in permeability of oral mucosa subjected various periods of time to H2S or CH3SH+ Exposure % Increase in permeability time (min) C0 2 /air? H2S CH3SH 5 10.6 30 - 25.3 19.0 60 -6.6 59.1 34.6 120 -6.9 - 103.0 180 9.3 61.7 73.0 + 15.0 ng H2S or CH3SH/ml 95% air/5% CO ¥ 95% air/5% C02 represents control atmosphere Individual mucosal specimens were exposed for 5, 30, 60, 120, and 180 min to either 15.0 ng H2S or 15.0 ng CH3SH/ml 95% air /5% C02. In comparison, the control specimens were exposed to the same periods of time to 95% air/5% C02. Changes in steady-state of perfusion were determined by applying 1 mCi [35S]-Na2S04/ml H20 to epithelial surface of the mucosa before and after exposure to the volatiles. The values represent percentage change in permeability to [35S]-Na2S04of the same specimen subjected to the tested gas. Negative values indicate a percentage decrease in permeability of control systems to [35S]-Na2S04 after exposure to air/C02 atmosphere. 50 the purpose of this study is to determine whether methyl mercaptan at physiological concentration has the capability to increase the permeability of oral mucosa to PGE2. It was found that exposure of the tissue to 15 ng CH3SH/ml air for 30 to 120 min. significantly increased the permeability of mucosa to pH]-PGE2. Whereas the change in the control systems were minimal, the increases in permeability induced by CHjSH relative to the controls were 47%, 73% and 56% at 30, 60 and 120 min. respectively. These results are similar to the changes in permeability of thiol-treated mucosa to [35S]-S04~2 ion. The effect appears to plateau on prolonged period of exposure. (Table III) L u T h e E f f e c t o f Z n C l 2 o n P e r m e a b i l i t y o f C H 3 S H - T r e a t e d  M u c o s a It is known that zinc ion stabilizes cell membranes and increases the rate of wound healing. Furthermore, an increase in penetration of protein through the sulcular epithelium lining has been demonstrated in zinc deficient rabbits. These observations suggest that the integrity of intercellular space and basal lamina is zinc dependent ( i l l ) . 51 Table III I i. PERCENTAGE INCREASE IN PERMEABILITY OF METHYL MERCAPTAN-TREATED ORAL MUCOSA TO PGE Exposure % Change in Permeability Time (min) Control" 1" CH 3SH-treated + 30 6.9 ' 54 60 -7.7 66 120 2 0 76 Control atmosphere, 95% a i r / 5 % CO ?; t e s t atmosphere, 15ng CH^SH per ml 95% a i r / 5 % CO Initially, lOOul of 12.5u.Ci (76 x IO"9 M) [3H]-prostaglandin E 2 solution [5,6,8,ll,12,14,15-3H(N)]/ml was applied to the epithelial surface of the tested specimens and perfusate fractions were collected until steady states of perfusion of [3H]-PGE2 were established. The surface of the tissues were then washed with PBS, exposed to 15.0 ng CH3SH/ml 95% air/5% C02 for either 30, 60, or 120 min., and re-exposed to [3H]-PGE2. The control specimens were exposed to only 95% air/5% C02. 52 Mouth air analyses have demonstrated that Zn + 2 is highly effective in suppression of oral malodour. Since oral malodour is primarily attributed to the presence of volatile sulphur compounds, namely CH3SH and H2S, and since zinc is a thiol reacting agent, it was deemed imperative to investigate the effect of this ion on the thiol-treated tissues. It is evident that application of 0.22% ZnCl2 to mucosal surface for 15 min. exerted a protective effect on the tissue barrier against methyl mercaptan. Treatment of the tissues with ZnCl2 prior to exposure to CHjSH nullified the expected increase of 103% in permeability to [35S]-Na2S04. (Table IV) Tissues exposed to methyl mercaptan prior to ZnCl2 treatment yielded similar results. Again zinc ion totally nullified thiol-induced permeation to [35S]-Na2S04, from expected 103% to essentially a control state. (Table V) s L . P r o t e c t i v e E f f e c t o f Z n C l 2 A g a i n s t P G E 2 To ensure that the reversal by ZnCl2 of thiol-induced permeability by ZnCL was not merely a result of reaction 53 Table IV P r o t e c t i v e e f f e c t o f z i n c c h l o r i d e a g a i n s t C H 3 S H -i n d u c e d i n c r e a s e i n p e r m e a b i l i t y o f mucosa Exposure time % Change i n (min) t o CH3SH p e r m e a b i l i t y 3 0 + -4.4 6 0 + -0.8 120 + +17.0 UQj +103.0 + The t i s s u e was t r e a t e d f o r 15 min w i t h 0.22% Z n C l 2 » then exposed t o 15ng CH 3SH/ml 95% a i r - 5% CO2 atmosphere f o r 30 t o 120 min. i The CH3SH-exposed t i s s u e t h a t d i d not r e c e i v e Z n C l 2 p r e t r e a t m e n t . After steady states of perfusion of [35S]-Na2S04 were established the mucosae samples were exposed to 0.22% ZnCl2 (w/v H20) for 15 min. then to 15 ng CH3SH/ml 95% air/5% C02 for 30, 60, or 120 min. The difference in percentage change in permeability following treatment with 15.0 ng CH3SH/ml air for 120 min demonstrates the ability of ZnCl2 to maintain the permeability of the mucosa. 54 Table V Reversal of Ch^SH-induced increase in permeability of mucosa by zinc chloride Treatment time , % Change in CH3SH (min) permeability 5 30 60 120 +13.0 + 0.8 - 9.3 + 5.0 120 (control) 120 (test) - 6.9 +103.0 Mucosa was treated for indicated periods of time with a i r /C0 2 atmosphere containing 15ng Ch^SH/ml, then subjected to 0.22% ZnCl 2 for 15 min. The values are compared to control and test systems not treated with ZnCl2-[35S]-Na2S04 was used as penetrant in this study. Following exposure of mucosa to 15.0 ng CH3SH/ml air for 120 min, 0.22% w/v ZnCl2 was applied to the epithelial surface for 15 min. From comparison of values obtained in the control and test systems, it is evident that 0.22% ZnCl2 nullified the CH3SH-induced increase in permeability. 55 between zinc and sulphate ions, ZnCl2 studies were also performed with [3H]-PGE2. As in the case with [35S]-S04, exposure of mucosa to 15 ng of CH3SH/ml air for 60 min. increased its permeability to [3H]-PGE2 by 66%. Again it was found that the treatment of the tissues with 0.22% ZnCl2 for 15 min. immediately after CH3SH exposure completely nullified the thiol induced changes in permeability. These findings indicate that the reversal of permeability by Zn + 2 ion is not a result of a direct reaction between Zn + 2 and the penetrant. (Table VI) JL. Reactivity of [ 3 5S]-H 2S with Non-keratinized Mucosa It is plausible to propose that the disruption of the mucosal barrier is a result of a reaction between thiols and intercellular and/or extracellular components which regulate the permeability of mucosa, This contention is supported by LSC analysis of mucosal specimens that were exposed for 30 min. to radioactive labelled H2S. The results showed a substantial retention of H2S by the mucosa. The amount ranged from 53 ng to 86 ng of H2S/cm2. (Table VII) While these results demonstrate reactivity between H2S and mucosa, they do not provide information as to 56 Table VI i i REVERSIBILITY OF CH^SH EFFECT ON PERMEABILITY OF MUCOSA TO PGE„ System % Change in Permeability Control -+ -7.7 CH3 SH-treated§ +66.0 CH 3SH/ZnCl 2-treated * -2.5 £ 95% a i r / 5 % CO? f o r 60 min. I § 15ng CH3SH per ml 95% a i r / 5 % C0 2 f o r 60 min * Following 60 min exposure to 15ng CH3SH , per ml 95% a i r / 5 % CO? atmosphere, mucosa was treated f o r 15 min with 0.22% ZnCl 2« I The depicted increase in permeability to [3H]-PGE2 was established on mucosal specimens exposed to CH3SH. Immediately following 60 minutes of CH3SH treatment, the specimens were exposed to 0.22% ZnCl2 for 15 min., washed thrice with PBS, then again exposed to [3H]-PGE2. The anticipated methyl mercaptan-induced increase in permeability of PGE2 was reversed by ZnCl2 treatment. These values are similar to those obtained with [35S]-Na2S04 in Table V. 57 Table VII Retention of H2s by Oral Mucosa [ 3 5 S] -H 2 S/H 2 S retained/cm 2 I Experiment dpm[ 3 5S]-H 2S ngH2s 1 79,185 53.1 2 113,419 76.1 3 127,964 85.9 4 85,043 57.1 Average 101,403 68.1 Specif ic Act ivi ty of [ 3 5 S] -H 2 S was 46m Ci/m mole i Following the exposure of mucosa to 15 ng (46 mCi/mmole) [35S]-H2S/H2S at a flow rate of 6.8 ml/min for 30 min., the surface of the specimens was washed thrice with PBS at 2 min. intervals and the areas of specimens not exposed to [35S]-H2S were trimmed and discarded. The remaining tissues were digested in 5 ml portions of Soluene™ 100 for 2 hours at 60°C. One ml of each digest was dissolved in 3 ml of aquous scintillating counting solution and analyzed by LSC. 58 whether the H2S reacted with the cellular membrane, the intracellular organelles and/or extracellular matrix components. This phase of the study was limited to reaction with [35S]-H2S as gas chromatographic analyses showed that all commercially available [35S]-CH3SH preparations were grossly contaminated with [35S]-H2S. Permeat ion of [ 5 5 S j - f l 2 S Through Intact Mucosa Studies by Tonzetich and Johnson (1983) have demonstrated that hydrogen sulphide and methyl mercaptan possess collagenolytic properties and adversely affect protein metabolism by human gingival fibroblasts. It follows that in order to exert a direct influence on collagen and metabolism of fibroblasts, the thiols must be able to transverse both epithelial and basal laminar layers of the mucosa to gain access to the underlying connective tissue. Using [35S]-labelled H2S, it was found that in addition to _ being retained, [35S]-H2S penetrated through all three tissue layers of mucosa, LSC analysis of effluent fractions that diffused 59 through tissues that were treated with 15 ng [35S]-H2S/ml air for 3 hr contained 12.3 ng H2S/cm2 tissue (table VIII) into the lower chamber of the perfusion apparatus, showing that the amount of H2S that perfused through the tissues is of sufficiently high concentration to be considered deleterious to fibroblasts, as attested by previous in vitro studies (8,9). hL. S t a b i l i t y o f [ 3 5 S ] - H 2 S B i n d i n g t o M u c o s a The stability of [35S]-H2S tissue complex was investigated to ascertain whether the H2S was covalently bonded with tissue components. It is of importance to find whether the bonding is stable or it is of transient nature under physiological conditions. A stable state would maintain the thiol-induced permeation effect during the periods of low VSC production and as periodontal infections are believed to cycle through very active and quiescent periods of disease. These experiments were conducted on mucosa that were exposed for 30 min to 15 ng of [35S]-H2S, then washed for 60 min with PBS to remove any unreacted or loosely bound H2S. It is evident from the results that elution with PBS had no effect on displacing tissue bound ~SH while the washed specimens 60 Table VIII D i f f u s i o n of H 2S through o r a l mucosa [ 3 5 S ] - H 2 s / H 2 s d i f f u s e d / c m 2 Experiment :  dpm[ 3 5S]-H 2S ngH 2 s i 1 18,670 12.5 2 18,358 12.3 3 18,045 12.1 Average 18,358 12.3 S p e c i f i c a c t i v i t y o f [ 3 5 S ] - H 2 s was 46mCi/m mole. The tissues were exposed to 15 ng of 46 mCi / mmole P5S]-H2S at a flow rate of 6.8 ml / min for 180 min, Aliquots of 1.3 ml / 20 min of PBS supplemented with 0.22% ZnCl2 were collected using a fraction collector. One ml of each aliquot was dissolved in 3 ml of aqueous scintillating counting solution for [35S]-analysis. The tabulated values represent the total amount of [35S]-activity and ng H2S / cm 2 tissue that permeated through all three tissue layers during the 180 min duration of the experiments, 61 retained 80.5 ng H2S/cm2 tissue, the rinsed control specimens retained 68.1 ng H2S / cm2. (Table IX) The difference between the two values is attributed to the use of two tissue preparations with different diffusion properties. In contrast, displacement of [35S]-H2S that had reacted with tissue components was observed when the mucosa was aerated with a constant stream of 95% air/5% C02 for 3 hours followed by 30 min elution with PBS. Under these conditions, approximately half of the bound SH was displaced. (Table IX) Partial displacement of [35S]-H2S implies that hydrogen sulphide induced change in permeability is reversible. £ L . P e n e t r a t i o n o f Mucosa b y F l u o r e s c e n I s o t h i o c y n a t e L a b e l l e d E . C o l i L i p o p o l y s a c c h a r i d e s ( L P S ) It is well established that bacterial endotoxins are primary etiologic factors initiating tissue destruction. However, it is not established whether endotoxins can penetrate through the intact healthy mucosal barrier. Hence one of the aims of this investigation is to determine whether CH3SH can potentiate the diffusion of LPS. 62 Table IX , , i \ i Treatment of [ 3 5S]-H 2S-exposed mucosa with a i r / C 0 2 and phosphate buffered s a l i n e [ 3 5S]-H 2S/H 2S retained/cm 2 I Treatment dpm [ 3 5 s]-H 2S ngH2S [ 3 5S]-H 2S t r e a t e d 101,403 68.1 PBS + 120,000 80.5 95% a i r / 5 % C02/PBS?< 52,496 35.2 — — I + Tissue was exposed f o r 30 min to 15ng [ 3 5S]-H 2S followed by l h wash i n phosphate buffered s a l i n e (PBS). f Tissue was trea t e d f o r 30 min with 15ng [ 3 5 S ] - i H 2S followed by 3h exposure t o 95% a i r / 5 % C0 2 and ! 30 min wash with PBS. This table compares the effect of washing the mucosa with three 1.0 ml volume PBS and aeration for 180 min with 95% air / 5% C0 2 of [ 3 5S]-H 2S treated mucosa. Specimens that were exposed to 15 ng P5S]-H2S for 30 min, were trimmed and digested in Soluene™ 100 (60°C) for 2 hr. The solublized specimens were then analysized for retained [3 5S]-activity by LSC. 63 Fluorescent analysis of perfusates of FITC-treated tissues indicated that the amount of fluorescence in the perfusate that had permeated through the tissue was insufficient to permit detection by spectrof luorgraphy. However, cytofluorescent analysis of the control specimens revealed intense fluorescent emission at the superficial surface of the epithelium limited to the exposed 1 to 2 cell layers. (Fig. 5) These results are in agreement with results of other investigators who found that healthy oral tissue is impermeable to endotoxin. Mucosal specimens that were exposed to 15 ng/ml CH3SH for 3 hr also yielded intense fluorescent labelling at the superficial epithelial layer and, in addition, showed evidence of distribution of fluorescence throughout the underlying tissue layers. (Fig. 6) No clear demarcation of fluorescence was observed at the basal laminar level as claimed by other workers. Therefore, it is unlikely that the observed uniform distribution of fluorescence throughout the mucosa is a result of perforation in the tissue as damaged sites would have shown discrete localized areas of fluorescence. The comparison of control and test tissues readily demonstrates that treatment with CH3SH had altered the tissue barrier and thereby potentiated the penetration of ]L_ coli endotoxin. 64 Fig. 5. Cytofluorography of control oral mucosa treated with FITC fluorescent-labelled endotoxin. The epithelial surface of the mucosa was exposed to (0.5 mg / ml) of fluorescein isothiocynate labelled £^ coli lipopolysaccharide for 180 min. The exposed surface of the tissue was washed thrice with PBS, immediately frozen in liquid nitrogen, then sectioned into 8 um thick slices and mounted in 'tissue-TEK OCT compund'. The fluorescent labelled epithelial surface areas emitted a strong green colour under a FITC filter at 4400°A to 5000°A. Basal lamina region, and the underlying connective tissue layer (lamina propria), (arrow), are devoid of fluorescence indicating that the endotoxin did not penetrate beyond the surface epithelium. 65 Fig. 6, Cytofluorography of methyl mercaptan-induced penetration of FITC-labelled endotoxin. Fluorescein isothiocynate labelled ELopJi lipopolysaccharide (0.5 mg / ml) was applied on to the CH3SH-treated epithelial surface for 180 min. Detection of penetrability of the fluorescein-labelled endotoxin throughout the 3 tissue layers of mucosa was analyszed as described under Fig. 5. The epithelial surface of the mucosa is shown on the top portion of the picture. A cross section of a vessel at the connective tissue layer is identified by the arrow. 66 A s s e s s m e n t o f C e l l u l a r V i a b i l i t y a f t e r C H j S H T r e a t m e n t Differential staining with fluorescein diacetate and ethidium bromide of fresh biopsied tissues (Fig. 7) and frozen tissues (Fig. 8) demonstrated similar staining characteristics. The absence of ethidium bromide fluorescence from tissues of both samples indicated freezing had no discernible effect on tissue viability. The basal lamina layer was delineated by absence of uptake of either stain. Tissues that were exposed for 3 hr to 95% air/5% C02 out of a total of 9 hr of experimentation (Fig. 9) exhibited cellular intactness analogous to fresh biopsied samples. The indicated isolated areas of ethidium bromide uptake at the periphery of the sample were a result of damage to the cells by the biopsy procedure. Microscopic examination of unstained preparations of basal lamina and lamina propria (Fig. 9) showed that the integrity of the control tissues remained intact at the end of the 9 hr experimentation period. The intense uptake of ethidium bromide by the superficial epithelium of CHjSH-exposed mucosae implied that mercaptan had induced a significant change in membrane permeability, (Fig. 10 a,b) Although the implied cytotoxic effect was most intense at 67 the superficial layer, evidence of ethidium bromide uptake by cells in the lamina propria suggests that the damage to the cells was not limited only to the surface layer. 68 Fig. 7. Cytofluorography of fresh tissue differentially stained with fluorescein diacetate (FDA) and ethidium bromide (EB). Freshly biopsied tissue samples were stained with 10 ml of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 50 ul of 5 mg FDA / ml acetone and 50 ul of freshly prepared 0.2 mg EB / ml PBS for 30 min at 37°C. The specimens were frozen in liquid nitrogen and sectioned to 8 um thick slices. Under the FITC filter (4400°C to 5000°C), FDA coated cell membrane emitted a green fluorescence while impaired cells emitted a red fluorescence due to counter staining with EB. The epithelial layer of the mucosa is located at the top right corner of the picture. Epithelial layer (E), basal lamina (arrow) which follows the rete ridges of the epithelial layer, cross section of capillary and lymphatic vessels (V), and connective tissue (CT) are identified as indicated. 69 Fig. 8, Differential staining with fluorescein diacetate (FDA) and ethidium bromide (EB) of previously frozen mucosa. Tissues that have been frozen in 10% glycerol / PBS at -70°C for three months were equilibrated to room temperature, bathed in 10 ml PBS at 37°C for 30 min, then stained with FDA and EB under the procedure described under Fig. 7. FDA stained cells appeared through out all three tissue layers with no apparent uptake of EB. 70 Fig. 9. Differential staining of mucosa following 9 hrs of experimental manipulation. The epithelial surface of the mucosa was exposed to 100 ul of PBS for 3 hr followed by aeration of the same surface with 6.8 ml / min of 95% air / 5% C02 for 3 hr. Then 10 ml of PBS was reapplied over the epithelium for another 3 hr. This was followed by differential staining with fluorescein diacetate and ethidium bromide. 71 10-a 10-b Fig. 10a,b. CH3SH-treated mucosa stained with FDA and counter stained with EB. The specimens were overlaid with 100 ul of PBS for 3 hr. The epithelial surface was then exposed to 15 ng CH3SH / ml air for 3 hr and PBS was reapplied over the epithelium for another 3 hr prior to differential staining according to the procedure described for Fig. 6. While Fig. 10a demonstrates marked uptake of EB by the superficial epithelial cells (at the top of the picture), Fig. 10b taken from another region of the same tissue also shows areas of intense uptake of EB by the connective tissue layer. 72 C H A P T E R IV D I S C U S S I O N A . T h e E f f e c t o f V S C o n D i f f u s i o n o f A n t i g e n s t h r o u g h N o n - k e r a t i n i z e d O r a l M u c o s a . F a c t o r s C o n t r i b u t i n g t o P e r i o d o n t a l D i s e a s e It is universally accepted that bacteria and their by-products of metabolism are involved in the etiology of periodontal diseases. To cause inflammatory destruction at the supportive tissue level, it is neccessary for the toxic agents to penetrate the surface epithelium. Lining the crevice are several cell layers of non-keratinized epithelium which act as primary deterrents against the penetration of deleterious substances. Although the pathogenesis of inflammatory periodontal diseases is believed to be multifactorial, the present thesis specifically investigates the potential pathogenic role of volatile sulphur compounds produced primarily by gram negative anaerobic oral microflora. These compounds have been demonstrated to have the ability to react with collagen and in the process alter its structure and solubility. In addition, physiological concentrations of volatile sulphur compounds have been shown to suppress total protein, collagen, and DNA synthesis of cultured gingival fibroblasts (19). 73 The ability of volatile thiols to permeate through all three intact mucosal tissue layers at cytotoxic levels implies that the physical barrier per 5e is inadequate protection against these products of putrefaction. The deleterious effect of thiols to both epithelial cells and fibroblasts is further substantiated by results obtained from differential staining studies using fluorescein diacetate and ethidium bromide. Differential staining results provide positive evidence that membrane integrity of some fibroblasts is severely affected by the presence of volatile sulphur compounds. Since the permeability of membranes of cells is regulated by sulphydryl groups, the observed membrane impairment to cells and induced change in permeability may well be a result of interaction between the free sulphydryl groups of membranes and volatile sulphur compounds. For example, critical changes in membrane permeability and cell vitality were demonstrated in erythrocytes when the sulphydryl groups were reacted with reducing agents (113). As fluorescent staining results showed that only certain cells in the superficial and underlying tissues were affected by CH3SH, this implies that the thiol inflicted impairment in cell membranes was a selective phenomenon. Structural cellular changes may only in part be responsible for the alterations in permeability since the primary tissue barrier is also composed of intercellular matrix proteins produced 7 4 and secreted by epithelial cells and fibroblasts. Therefore, changes in the viability and metabolic activity of these cells would invariablly affect the protein matrix which is a component of the mucosal barrier. Some bacterial species, indigenous to the gingival crevice, are known to possess protease and collagenolytic activity. Some of these proteolytic enzymes can be activated by reducing agents, such as SH compounds, to contribute to tissue degradation (114). As volatile thiol compounds are strong reducing agents, they are suspected to exert similar stimulatory affects on these enzymes. It is also plausible to conjecture that volatile sulphur compounds, per se, are immunogens. In cell harvesting, 2-mercaptoethanol is routinely added to cultures to enhance macrophage proliferation. Hence H2S and CH3SH thiols may be presumed as potent mitogens since the thiol functional groups of 2-mercaptoethanol and methyl mercaptan is common to both compounds. As putative mitogens, the volatile thiols could stimulate macrophages to release a number of mediators initiating the inflammatory process. At the initial stage of inflammation, markedly increased amounts of prostaglandins, particularly prostaglandin E 2 , are produced by numerous inflammatory cells. Prostaglandin E 2 induces bone resorption and is believed to be an important mediator of inflammatory periodontal disease, rheumatoid 75 arthritis and malignant osteolysis. The fact that PGE2 levels are increased by 10 to 20 fold in periodontally involved tissues and that volatile sulphur compounds can increase the penetration of prostaglandin E 2 by 76% strongly indicate that disruption of the permeability barrier by the thiols will enhance the antigenic and immunogenic challenge of this inflammagen to the connective tissue. Exogenous prostaglandins are potent inhibitors of several lymphocytic functions. Elevated levels of PGE are immunosuppressive to lymphoid mitogenesis, cytolysis, antibody production and secretion of lymphokines (interleukin-2) (115, 116). These local immunosuppressive responses can hinder the host's second line of defense. As prostaglandins are believed to have an immunological effect, the volatile sulphur compound induced increase in permeability of PGE2 through intact mucosal tissue is of the paramount importance. Furthermore, it has been shown that prostaglandins can directly suppress synthesis and turnover of collagen. In addition, prostaglandin PGE2 has been reported to augment the production of endotoxin-induced collagenase which could contribute to the increased destruction of connective tissue collagen observed at the inset of inflammatory stage of periodontal disease. Several other sources of evidence provide further support for a relationship 76 between prostaglandin E 2 and inflammatory periodontal disease.Prostaglandins are believed to be important in regulating bone deposition and resorption. The production of osteoclastic activating factors by monocytes and lymphocytes is regulated by the relative amount of prostaglandin E 2 present ( 6 ) . Therefore, the ability of zinc ion to nullify the C H 3 S H - induced increase in access of exogenous prostaglandin E2 to the underlying tissue is of paramount significance. In vivo, prostaglandins are considered to have a short half-life. It is noteworthy in this context that recently a sulfur-bound analog of prostaglandin, thiaprostacyclin, was found to be stable and to possess enhanced bone resorption activity (122). Thus, it is plausible to suggest that a similar stable configuration of sulfur-bound prostaglandin E analoque might be formed with VSC in the present study. Similar to prostaglandins, extracts of bacterial lipopolysaccharides are known to act as inflammatory agents and as potent immunogens. "LPS components of numerous strains of microorganisms associated with periodontitis stimulate the production of antibodies and osteolytic activating factors by non-specific B lymphocytes and polyclonal B cells. They also function as chemotatic stimulants of lymphocytes in the local release of lysosomal enyzmes. 77 In order to evoke an immune response, the endotoxin must first gain access through the epithelial barrier of the mucosa. In non-treated tissues, the mucosal barrier served as an effective primary deterrent against endotoxin penetration. In contrast the tissues previously exposed to methyl mercaptan demonstrated a marked distribution of labelled endotoxin in both the superficial epithelial and the underlying connective tissue layers. However, as the presence of bacteria has been demonstrated in the connective tissue layer of gingival biopsies obtained from patients with periodontal disease, this suggests that both the endotoxins and VSC could also be actively produced within the affected tissues. Nevertheless, once the endotoxin has gained access to connective tissue layer, it is known to be capable of activating the complement system through the properidin pathway. Activation of complement system ultimately leads to the release of hydrolytic enzymes by polymorphonuclear leukocytes and secretion of lymphokines by T-lymphocytes (118). The presence of interleukin II can cause several local inflammatory responses including activation of osteoclasts and macrophages, inhibition of macrophage migration, and induced fibroblast production of collagenase and prostaglandin (124). The resulting stimulation of macrophages and leukocytes would lead to an increase in the secretion of proteolytic enzymes. Endotoxin can also directly stimulate macrophages and neutrophils to produce hydrolytic 78 enzymes and collagenase (117). Ultimately these enzymes contribute to further destruction of both the tissue's protein matrix and cells. For example, human gingival fibroblasts exposed to endotoxin have been shown to exhibit impaired metabolic activity (119). Bacterial plaque appears to have an adjuvant effect on cell mediated immunity. In some generalized gingivitis and periodontitis cases, it is possible that the enhanced permeation of endotoxin into deeper tissue layers induces hypersensitive reaction to the host's immunological system. It is not known how the toxic substances penetrate into the underlying tissue without directly disrupting the superficial epithelium to initiate the inflammatory and immunological responses. It is believed that both the epithelial cells and fibroblasts synthesize and secrete non-fibrous proteoglycans and glycoproteins into the extracellular matrix. They are the major components of the extracellular matrix and are of the paramount importance in maintaining and regulating tissue function and structural integrity. In destructive inflammatory periodontal diseases, it is plausible that the integrity of proteoglycans and glycoproteins which regulate the rate of diffusion through epithelium and basal lamina layers of extrinsic antigens from the sulcus to the underlying connective tissue is disrupted. Furthermore the access of irritants to the connective tissue layer 79 may also affect the maintenance of the cellular and fibrillar components. Hence the susceptibility of the sulcular tissues degradation and their capacity to regulate the perfusion of molecules are important considerations in the initial stage of periodontal disease. Heparan sulphate is the predominant proteoglycan synthesized by gingival epithelium. Once excreted into the intercellular space, the molecules of this glycosaminoglycan, varying in molecular weights from 105 to several million dalton aggregates. Such putative molecular interactions among the proteoglycans are presumed to act as 'first-line' barrier to the penetration of potential irritants through the epithelial-basal lamina layers. It has also been reported that alterations in proteoglycans of inflamed gingiva are associated with a loss of integrity of molecular structure (120). Thus, de-aggregation of intercellular substances may be responsible for eventual loss of epithelial integrity as seen in the latter stage of periodontal destruction. Proteoglycans in their aggregated state act as a molecular sieve for the passage of both interstitual water and solutes. Aggregation of proteoglycans has been shown to depend on disulfide linkages. Similarly the glycoproteins in the extracellular compartment are also believed to interact with collagen to form stable matrices via disulfide bonds. It is noteworthy that membrane coating granules in the intercellular space are 80 composed predominantly of sulfur-rich proteins and glycoproteins. The membrane coating granules have been assigned an important function in regulation of the permeability barrier. The importance of disulfide linkages of proteoglycans and glycoproteins is clearly demonstrated in systems treated with dithiothreitol, a disulphide cleaving agent which causes deaggregation of proteoglycans and a concomitant increase in permeability. Blockage of aggregation can also be achieved by alkylation of thiol residues which prevents formation of disulphide linkages (123). It is conjectured that H2S and CH3SH enhance the permeation of toxic substances through disulphide cleavage mechanism. This is a reasonable supposition as the results of this demonstrated that mucosal specimens readily interact with H2S. Although mucosal interaction with H2S appears to be relatively stable, the fact that permeation change can be partially reversed following aeration suggests that re-aggregation of proteoglycans is also possible, Restoration of integrity of the tissue barrier's is presumed to be less likely with methyl mercaptan as methiolation of free thiol groups would prevent reoxidation of free SH groups. By analogy, the structure and function of both intercellular and extracellular matrices of the proposed system can be considered as the stationary phase of a chromatographic column. 81 The charged groups of acid mucopolysaccharides can exhibit properties of ion-exchange chromatography and the intricate aggregated proteoglycan matrix as molecular sieve similar to polymers in gel filtration. By virtue of simple filtration and selective ionic interactions, the passage of many metabolites through the tissue is thus regulated. Therefore, an alteration of the charged groups or the aggregative state of the mucopolysaccharides' proteoglycans and glycopoteins by the volatile sulphur compounds would invariably affect the rate of permeation and penetration of substances across the mucosal barrier. Zinc ion was found to be a protective agent for maintenance of the primary barrier of the tissue. Exposing the epithelial surface of mucosa to ZnCl2 either prior to or after thiol treatment restored tissue permeability to control values. From these observations, it is apparent that zinc ion can nullify the effect of the sulphur compounds. It is proposed that zinc protects extracellular matrices by the following mechanisms. When mucosal tissue is exposed to zinc, the ion readily reacts with cellular and tissue components. A reservoir of zinc ions is provided by ionic interaction between zinc and free anionic groups such as free carboxyls. In the presence of volatile sulphur compounds, Zn + 2 ion readily reacts with the mercaptides to form inactive zinc mercaptides before the reactive thiols can oxidize 82 the SH groups in the matrices. In addition, zinc ion can induce re-aggregation of the affected proteoglycans by functioning as anionic bridge between two sulphur atoms. At the cellular level, zinc has been used extensively by cytologists to stabilize cell membranes. Zinc ion prevents distortion to the membranes by interfering with the formation of disulfide linkages from free radical oxidation of SH groups. The ability of this ion to stablize labile membranes infers that the release of destructive enzymes and other mediators responsible for inflammation can thus be modulated. Karls et al's finding that zinc ion is incorporated by the intact cells within 15 minutes of exposure corroborates our findings that thiol-induced increase in permeability can be nullified by a short treatment with Zn + 2 (121). Although a portion of the observed permeability change could be related to cell death and impairment to cell membranes, it is not likely that this is a major factor for increased permeability of mucosa. This is supported by the observation that zinc ions prossess the inherent ability to restore the permeability of the mucosa to control state. Hence it is proposed that the principal change in tissue barrier is attributed to alterations in the intercellular and extracellular matrices. The implications of these findings are of major significance. The results suggest a mechanism how the products of putrefaction 83 can affect the periodontal tissue and in process assist toxic substances to gain access to connective tissue and how they initiate a series of reactions resulting in a marked destruction of the tissue. 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