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 degree this at the thesis in partial fulfilment of University of British Columbia, I agree freely available for reference copying of department publication this or of thesis by this his for or and study. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) that the representatives. may be It thesis for financial gain shall not permission. requirements I further agree scholarly purposes her the is an advanced Library shall make it that permission for extensive granted by the head understood be for that allowed without of my copying or my written 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. gingival sulci As their (H S and CH SH) concentrations 2 increase with 3 the severity of in periodontal involvement, the objective of this investigation is to ascertain if they exert an effect on the permeability of oral mucosa. Permeability porcine sublingual non-keratinized tissue determinations layers apparatus. mucosal epithelium, mounted were specimens basal in performed a which membrane two on excised consisted and compartment of connective perfusion 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 H S or CH3SH / ml of 95% air - 5% C0 ) increased the 2 2 permeability of the mucosa to ( S)-S0 ~ , ( H)-prostaglandin E 35 2 3 4 (PGE ) 2 and fluorescein lipopolysaccharide (F-LPS). to H S and 2 respectively CH3SH isothiocyanate labelled JL. 2 coli A three hour exposure of the mucosa resulted in a 75% and in permeability to .( S)-labelled 35 ii 103% increase sulphate ion. Similarly, the mercaptan induced up to a 70% increase in permeability of the mucosa to ( H)-prostaglandin E . The 3 2 magnitude of changes in the permeability were found to depend on duration of exposure to the thiols and to their concentration. Studies using ( S)-H S suggest that the observed changes in the 35 2 tissue permeability are related to the reaction of the thiols with tissue components. perfusing cm . 2 In addition, the ( S)-H S is capable of 35 2 through all three layers of the mucosa at 12.3 ng / In contrast to H S , the CH SH effect was 2 control air / potentially C0 environment. This infers 2 a more irreversible in 3 deleterious agent . to the that CH SH is 3 tissue barrier. However, its effect can also be reversed by treatment of tissues with 0.22% ZnCl either prior to or after exposure to mercaptan. 2 This suggests that Z n +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. of the F-LPS epithelial Whereas the fluorescence in control systems was confined to the superficial layer in contact with the endotoxin, the CH SH3 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 impairment bromide to some provides cells by evidence CH SH. 3 of membrane 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 D. 4 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 G. 8 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 3. Perfusion Studies with [ S]-H S 35 2 vi 34 35 Table of Contents (Continued) Page E. Reaction of [ S]-H S with Mucosa F. Stability of the [ S]-H S Tissue 35 2 35 35 2 Complex 36 G. Penetration of Mucosa by ]L_ coli Endotoxin 37 H. Culture Conditions for Epithelial and Fibroblastic Cells I. 38 Vitality Staining of Mucosa with Fluorescein Diacetate (FDA) and Ethidium Bromide (EB) Materials 38 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 E. Concentrations of Volatile Sulphur Compounds Used to Treat Mucosa. F. 45 Comparative Permeability Studies on Control, H S and CH SH-Treated Tissues 48 Permeability of [ C]-Ovalbumin 49 2 G. 45 3 14 vii Table of Contents (Continued) Page H. Permeability of [ H]-Prostaglandin E 49 I. The Effect of ZnCl on Permeability of CH SH- 3 2 2 3 Treated Mucosa 51 J. Protective Effect of ZnCl K. Reactivity of [ S]-H S with Non-keratinized 2 Against PGE 2 53 35 2 Mucosa 56 L. Permeation of [ S]-H S Through Intact Mucosa 59 M. Stability of [ S]-H S Binding to Mucosa 60 N. Penetration of Mucosa by Fluorescent Isothiocynate 35 2 35 2 Labelled JL. coli Lipopolysaccharides (LPS) 0. 62 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 85 References viii List of Tables Table Number I II Title Page Percentage change in permeability of mucosal specimens subjected to various concentrations of h\S 47 Percentage increase in permeability of oral mucosa subjected to various periods of time to H S and 2 CH.SH III 50 Percentage increase in permeability of methyl mercaptan-treated oral mucosa to PGE 52 Protective effect of zinc chloride against CH SH-induced increase in permeability of mucosa 54 2 IV 3 V Reversal of CH SH-induced increase in permeability 3 of mucosa VI 55 Reversibility of CH SH effect on permeability of 3 mucosa to PGE 57 VII Retention of H S by oral mucosa 58 VIII Diffusion of H S through oral mucosa 61 IX Treatment of [ S]-H S-exposed mucosa with Air/C0 and phosphate buffered saline 63 2 2 2 35 2 2 ix List of Figures Figure Number 1 Title Page Design of perfusion apparatus used for permeabilitystudies of mucosal specimens. 27 2 Components of the perfusion apparatus. 28 3 Assembly of the system used for the permeability studies. 30 Attainment of steady state of perfusion of penetrants before and after gaseous treatment. 44 Cytofluorography of control oral mucosa treated with FITC fluorescent-labelled endotoxin. 65 Cytofluorography of methyl mercaptan-induced penetration of FITC-labelled endotoxin. 66 Cytofluorography of fresh tissue differentially stained with fluorescein diacetate (FDA) and ethidium bromide (EB). 69 Differential staining with fluorescein diacetate (FDA) and ethidium bromide (EB) of previously frozen mucosa. 70 Differential staining of mucosa following 9 hour of experimental manipulation. 71 4 5 6 7 8 9 10 a,b CH SH-treated mucosa stained with FDA and 3 counter stained with EB. x 72 ACKNOWLEDGEMENT 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 A. Hannam, comments my other advisors: Drs. D. Brunette, L. Kraintz and B. McBride for their valuable 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 encouragement and support for their throughout the years of my study. I would also like to acknowledge of Canada for their financial support. the Medical Research Council 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 inflammatory stage. or it may be in a virulent 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 teeth as manifested in advanced periodontitis. progression of gingival inflammation is generally 1 exfoliation of Although the a prerequisite to periodontitis, the etiology of the disease is not known and appears to be multifactorial. Microbial by-products, mechanical irritants, autoimmunity, occlusal traumatism, age, endocrine imbalance, nutritional status, 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 dimethyl sulphide disulphide (H S), methyl 2 (CH S) 3 and 2 mercaptan dimethyl sulphide (CH SH), 3 (CH ) S 3 2 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, H S and CH SH generally occur in 1:1 2 3 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" to 10" 8 10 g / 10 ml mouth air. Levels greater than 1.5 ng of H S / 10 ml air (120 ppb) and 0.5 ng CH SH / 10 ml 2 air 3 (40 ppb) are considered socially objectionable have shown that compounds gingivitis, is (4). Studies the production of these sulphur containing intensified in cases of chronic oral hemorrhage, acute ulcerative and certain lung-borne systemic diseases (5). 3 periodontitis, gingivo-stomatitis, *L_ Mfcrpprgflnisms V o l a t i l eS u l p h u r Implicated Compounds in the Production of Gingival crevice provides a favorable environment for optimal growth of facultative and strict anaerobes. environment enriched with proteinaceous It is a stagnant serum exudates, erythrocytes, leukocytes, lymphocytes and desquamated epithelial cells. A large number of strains of bacteria have been isolated from the crevice. pathogenic Among the isolated and identified anaerobes, strains of Bacteriodes melaninogenicus were demonstrated to produce copious amounts of volatile sulphur compounds. In comparison to Bacteriodes fifteen-fold gingivalis non-pathogenic ten-fold more strains, total VSC, more CH SH and two hundred and fifty fold more (CH S) (6). 3 produced the 2 inhibited by 3 The production of VSC is optimal at pH > 6.5 and is 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 availability of carbohydrates is limited. 4 basic and the exogenous P_ The Effect Protein of and V S C on DNA Collagen. Synthesis Procollagen. by Human Total 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 observed 60% to 70% sites as early as four structures. This is attested by an loss of collagen at the affected crevicular 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 [ S]-H S with Type I acid soluble and insoluble fibrillar 35 2 collagen have demonstrated that this agent can bind to collagen and in the process alter its structure and solubility. As this reaction results of free in an uptake of sulphur, exposure aldehyde groups and in conversion of the protein to a more soluble form, aldehyde it is postulated that these thiols cleave certain mediated linkages, possibly Schiff Base or aldol condensation cross linkages (7,8,9). Furthermore, CH SH treatment was found to interfere with 3 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 H S and CH SH can suppress protein synthesis 2 3 by fibroblast cultures, the effect of CH SH is more pronounced 3 and is not reversible up to 24 hours of incubation. Thus, thiols produced through putrefaction appear to be potential collagenolytic agents (10). 6 JL Clinical Manifestations Ascribed to VSC An increase in mouth malodour has been clinically observed in many inflammatory periodontal disease conditions. convincing evidence that the There is production of volatile compounds increases in periodontal pockets. sulphur 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. reported active production of Using this method, Coil (62) H S, CH SH 2 and 3 (CH ) S 3 2 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 disease. (13) severity of periodontal 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 selectively reacts with thiols, significantly (Flagyl), which reduced both the pocket depth and black-pigmented Bacteroides counts (14). 7 In 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_ Histologicaland Morphological Characterization of Sulcular Epithelium and Basal Lamina 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). crevicular epithelium hemidesmosomes, has fewer desmosomes, and tight Morphologically, randomly junctions distributed 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 intercellular space of believed to serve as substances to sulcular keratinized epithelium oral is wide (19). a principal pathway across the epithelial surface. 8 epithelium, for transport the It is of The minimal width of 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 epithelium and lamina lucida adjacent to the lamina densa in contact with the connective tissue (21,22,23,24). components: of Basal lamina is comprised of three main 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 Review of Studies on Permeability of Oral Mucosa 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 through intercellular spaces (31,32,33,34). simple diffusion 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 process because refrigeration, of epithelial tissues for a passive 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). passive phenomenon is further supported by Fick's law The 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 accumulation direction. of In gingival chronic fluid, inflammation, lysosomal an enzymes, polymorphonuclear leucocytes and lymphoctyes can be found in the sulcus (40,41,43). indicates that The presence of an inflammatory exudate reverse permeation of macromolecules through the crevicular and/or junctional epithelium. of serum protein exudate is probably a occurs The outflow net result of hydrostatic-osmotic pressure phenomena (43). Other studies have demonstrated absorption of [ H]-H 0 and drugs through 3 2 crevicular epithelium of animals (36,44,45,46). morphological permeability findings provide good evidence the These and other that reverse 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 of buccal absorption following rinsing and antigens. in vivo studies topical application of 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. preparations with Using 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 12 to the 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. The Epithelial Intercellular Space Compartment 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 perfusion of horseradish peroxidase demonstrated follows that the 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. inflammatory During an 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 physiological conditions in many parts of the body. 13 different For example, 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^ Basal Lamina Since basal Compartment 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 900A) detachment and subsequent (more than duplication, fragmentation, multilayer formation, and dislocation of the basal lamina. 14 The 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, lamina. and disappearance and breakdown of the basal 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 treatment lamina blue and Evans blue. with collagenase or readily permits the However, mechanical damage to basal 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 barrier The actions of these proteolytic enzymes on 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 molecule. on the size of the penetrant 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). to the intrinsic complexity of Additional support attesting 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 sieving, mediated by either carrier molecules, molecular energy dependent processes, or simple filtration From these reports it appears that probable route. (63). simple filtration is the most 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. IL_ M e m b r a ne Coating Granules, an Integral Protein of the Matrix in the Intercellular Space Compartment. Although the concept that basal 16 lamina serves as the functional barrier appears attractive, the effectiveness of the membrane coating compartment barrier, granules (32,64). currrent findings attribute in part, found in Horseradish to the the presence of intercellular peroxidase space introduced intradermally into epidermis was ultrastructurally demonstrated to permeate only to the level of stratum corneum where membrane coating granules or Odland bodies are deposited These granules are not restricted to the (65). 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 epithelial origin. coating granules are believed to be of 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 Cytochemical amorphous analysis material of intercellular matrix indicates that consisting of glycoproteins (68,69,70,71,72). the granules hydrolytic (64). contain enzymes an and It is noteworthy that both the junctional epithelium and pocket epithelium are devoid of the membrane coating granules (73). 17 L Prppextjgs of Penetrants 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 H S and CH SH, which are soluble in polar and nonpolar solvents, 2 3 may be effective penetrants of the oral epithelium. 18 J. Permeability of Tissues to Endotoxin It is generally accepted that the presence of microorganisms and their byproducts of metabolism in gingival pockets is a prerequisite to inflammatory periodontal disease. evidence that antigen. bacterial endotoxin is an There is ample extremely harmful 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 gingiva can increase of induce gingival acute to clinically healthy inflammatory exudate, response, 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 system. 19 lymphokines in the immune 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. possibility Thus, there is experimental evidence indicating the of endotoxin involvement in the initial stage of pathogenesis of periodontal disease, 1L_ Prostaglandins in Gingival Tissues Several potent substances, some of which are believed to be mediators of inflammatory response, are released locally at the site of inflammation. prostaglandins whose One group of levels are such elevated mediators in are inflammatory reactions associated with thermal injury, allergic contact eczema, ultraviolet irradiation, primary 20 irritant dermatitis and inflammatory periodontal diseases (85,86,87,88,89). In inflamed human gingiva, the presence of PGE has been found twenty-fold 2 The PGE2 levels were greater than in healthy tissues (90). positively correlated with the progression of the disease process. PGE was 2 absent from amounts were present periodontal pockets. non-inflamed tissue but detectable in areas adjacent to the margins of Highest PGE 2 levels occurred at the advancing periodontal disease front (91). Since prostaglandins, in particular PGE , are released in the purulent exudate and since 2 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 number of different cell types. synthesized by osteoblasts, compounds produced by a On stimulation, they can be 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 permeablity during the early can increase vascular inflammatory pronounced erythema and edema (95). 21 2 stage causing 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 activity tissue alteration can also be and induced an in increase vitro by in osteoclastic 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. systemic A study on Beagle dogs demonstrated that administration of flurbiprofen inhibited resorption of alveolar bone in chronic marginal periodontitis (98). plausible Thus, it is that prostaglandin E may play a significant role in the pathogenesis of periodontitis. 22 L. Role of Zinc Ion 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. that Change in cellular function is supported by findings uptake treatment of (99). silica by macrophage decreases Furthermore, the presence of Z n after +2 zinc 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. Principal Objectives 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 and 2 ovalbumin. The effect penetration of of volatile sulphur tissue's compounds barrier by on the 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 extensions crevicular histologically epithelium. specimens is inadequate perfusion apparatus, exhibits characteristic similar in As the area appearance of rete to ridge human crevicular mucosal in size to cover the aperture of the 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, lamina and connective approximately 1 m m . tissue having a total basal thickness of The presence of an intact epithelial layer was verified by its shiny surface 25 observed under a dissecting 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 -70°C. Prior to use, the frozen sections were (PBS) at equilibrated to room temperature then immersed in PBS at 37°C for 30 minutes. Evaluation of fresh and frozen specimens indicated that the freezing discernable adverse on System Using procedure had no effect permeability of mucosa (53). B. In Vitro Permeability Perfusion Apparatus Permeability of substances were studied using a perfusion apparatus designed and The constructed by the author from Teflon. apparatus consists of two chambers each with a central aperture of 0.0491 cm The design of the 2 and employed inlet-outlet ports (Figs. 1 & 2). unit is a modification of apparatus used by Ainsworth and Alfano (38,83). the 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" Fig. 1. UPPER CHAMBER WHERE ISOTOPE IS APPLIED Design of perfusion apparatus used for permeabilitystudies of mucosal specimens. Perfusion apparatus units were constucted from teflon. The mucosal tissues were mounted over the central aperatures (0.0491 cm ) 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 27 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 m m 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 mounted test system, the 0.0491 over chambers with a biopsied mucosal cm aperture 2 the epithelial surface section between facing the upwards. was two 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 underlying connective tissue layer. in contact with Aliquots of 1,3 effluent were collected at 20 minute interval using an fraction collector, the m l PBS LKB 7000 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. Calculation of Permeability Coefficients Permeability determined changes penetration coefficients 'p', rate, exposed surface area, were by means calculated on the basis of diffusion time, and concentration of the applied penetrant according to procedure described by (33). of Tregear The determined 'p' values were then used to compare the permeability of control and test systems. 29 For calculation of the Fig. 3. Assembly of the system used for the permeability studies. Permeability experiments were conducted at 37°C i n 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 , then channelled into the upper chambers 2 inlet port of the perfusion apparatus. A constant flow 4 m l / 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. then divided by the The slope at steady state is concentration of the applied labelled substance. Penetration rate at steady state (cpm/cm min) 2 '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 exposure to tested thiols. effect of Thus each specimen served as its own control and the calculated 'p' value differences control and test values yielded reliable results tested volatile compounds. the between the attributed .to the Change in permeability of the tested specimens were compared to control specimens exposed to 95% air / 5% C 0 . 2 D. Protocol for Permeability Studies To assess the effect of VSC on the permeability of the tissue barrier, Na S0 , 2 4 ovalbumin, prostaglandin endotoxin were selected as penetrants 31 E 2 and EL coli for the following reasons. (1) [ S]-Na S0 35 2 4 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 PGE can induce inflammatory 2 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 [ C]-ovalbumin 14 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. Experimental Protocol for Permeability of Tissue Exposed to VSC and Control Environments For the penetration phase of the study, mounted between the two mucosa was firmly 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 H S or CH SH (x ng/ml) admixed with 95% air/5% 2 C0 . 2 3 In the control system, the tissues were exposed for similar periods of time to only 95% air/5% C0 . 2 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% C0 . 2 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. The Effect of Zinc Permeability of Mucosal As Z n +2 Chloride on Thiol-Induced Specimens 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 Z n +2 ion at the concentration present in Lavoris in suppressing oral malodour is well documented (105). For these studies, labelled penetrants ([ S]-S0 , [ H]-PGE ) 35 -2 3 4 2 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% ZnCl (0.1 % Zn ) for +2 2 a period of 15 min., either immediately before or after exposure to 15 ng of CH SH/ml air/C0 (flow rate of 6 ml/min). 3 2 This was followed by reapplication of the labelled penetrant until a steady state of perfusion was again achieved. 34 3L_ Perfusion Studies with [ S]-H S 3 5 2 The penetration of [ S]-H-,S per se through intact tissue was 35 studied by exposing the epithelial surface to 15 ng H S, with 2 specific activity of 46 mCi/mmole of [ S]-H S/ml 95% air/5% C0 35 2 at a flow rate of 6.8 ml/min for 180 min. experiments, continuous flow af 2 In this series of [ S]-H S was 35 2 regulated by displacement with room air of a standard [ S]-H" S / H S/air 35 2 mixture prepared in a 1000 ml dilution flask. 2 Aliquots of 1.3 ml PBS / 20 min of the effluent were collected from the lower chamber and analyzed for [ S]-activity 35 by LSC. To ensure complete retention of H S, 0.22% ZnCl was incorporated into the 9 PBS. 2 The entire experiment was performed at 37°C in an incubator housed in a fumehood. E. Reaction of [ S]-H S with 5 5 2 Mucosa 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 [ S]-H S experiments, mucosae were exposed to 15 ng 35 2 [ S]-H S at a flow rate of 6.8 ml/min for a period of 30 min. 35 2 After the tissues were washed thrice with PBS, the areas peripheral to the exposure were trimmed away under a dissecting microscope. Soluene™ 35 The exposed specimens were then digested in 5 ml of 100 for 2 hours at 60°C and assayed (LSC) for S-activity. F\ Stability of the [ S]-H S 5 5 2 Tissue Complex Following 30 min. exposure of mucosa to [ S]-H S, the 35 2 epithelial surface was washed three times, for 20 min. each, with 1.0 ml volumes of PBS bound [ S]-H S. 35 2 to remove unreacted or weakly 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 [ S]-H S tissue reaction was also investigated. 35 2 In this series, immediately after exposure to [ S]-H S the epithelial 35 2 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. specimens were then trimmed, digested, and assayed The for radioactivity. G. Penetration of Mucosa The effect of by E. coli Endotoxin CH SH on the 3 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 described under section D, fluorescent applied for three hours to the protocol previously labelled endotoxin was epithelial surface. The tissues were then washed with PBS, areas peripheral to the test site trimmed, and frozen in liquid nitrogen. 37 They were then 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 5000A) using a Zeiss photo-microscope. Photographs were taken using a Ektachrome (ASA 400) film with automatic exposure control. H. Culture Conditions for Epithelial and Fibroblastic Cells Epithelial cells and fibroblasts were obtained from areas of periodontal ligament teeth. of freshly extracted porcine mandibular 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_ Vitality Staining of Mucosa with Fluorescein (FDA) and Ethidium Bromide (EB) Diacetate A differential staining procedure was employed to establish whether the cells throughout the tissue remain vital 38 under the 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% C0 or to CH SH 15 ng/ml air/C0 at 6.8 ml/min. for 3 hours. 2 3 2 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 experiments. apparatus discarded. to labelled penetrants The tissue was and unexposed areas removed were The remaining tissue was in permeation from the trimmed perfusion away and 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). FITC filter, at wavelength (4400A - 5000A), Under a 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 NaHC0 : 3 (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. [ S]-Sodium Sulfate in water: New England Nuclear sp. act. 35 529.44 mCi/mmole, 5 mCi/ml, MW 131. [ H]-Prostaglandin E 3 2 [5,6,8,11,12,14,15,-3 H ( )] 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 76 x I O -9 piCi/ml, M. Methyl- C Ovalbumin: 14 liCi, 12.5 sp. act. New England Nuclear, stock solution 5 0.01 mCi/mg, MW 45,000 concentration of isotope used for study 1.25 |iCi/ml distilled H 0. 2 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 [ S]-Sulphide: 35 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 RESULTS Variations in Permeability An inherent disadvantage of the present in vitro system is the difficulty of attaining uniformity of specimens among studied the and animals. the thickness morphological Consequently, variance the substances perfused through the different in all tissue amount of of tissues labelled tissues were found to vary. To overcome this difficulty, it was necessary to establish steady state of treatment. changes of perfusion Since rate of on each permeability steady tissue before changes were state of perfusion and after determined on the by same specimen, each tissue served as its own internal control. EL H i s t o l o g i c a l S u r v e y of P o r c i n e S u b l i n g u a l M u c o s a The tissue of interest in this investigation crevicular and junctional epithelia. difficulty in obtaining However, is the human because of the human crevicular and junctional tissues of 42 sufficient size for these studies, animal model system. it was imperative to adapt an 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) lamina. Lamina with rete ridge extensions and intact propria showed no sign of polymorphonuclear leucocyte or lymphocyte. basal infiltration Histologically, by the mucosa is almost analogous to healthy human gingival sulcular tissue. £L Development of in vitro system As the employed system was leak proof, penetrants all of the tested 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. 43 (Fig. 4) This was PERMEABILITY OF MUCOSA TO Na S0 BEFORE AND AFTER I HOUR EXPOSURE TO I50ng/ml H S 2 4 2 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/cm 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. 2 44 consistently reached within initial 180 min. of perfusion. noteworthy that the steady state was almost reached in the majority of experiments It is instantaneously (Fig. 4) following the exposure of mucosa to the H S and CH SH. 2 3 EL. Relationship Between Gas Pressure and Permeability of Tissues 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. possibility, comparative studies were To examine this 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. thiols, volatile To induce optimal saturation of tissues with sulphur compounds were upper chamber at a rate of 6.8 ml/min. EL_ Concentrations Treat Mucosa of Volatile Sulphur Although early morning human 45 passed through Compounds mouth Used the to air contains on average 0.5 ng/10 ml concentrations not are CH SH and 3 considered present in the gingival crevice, are subjected 1.5 ng/10 ml representative as H S, these the levels 2 of volatile sulphur compounds 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, and concentration of the effect of duration of exposure 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, H S, 2 the 15, and 150 ng/ml concentrations of An increase in rate of permeability was observed during initial 5 to 60 min. of exposure to 15 ng H S/ml 2 atmosphere. No apparent further increase in the air/C0 rate 2 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 Percentage change in permeability of mucosal specimens subjected to various concentrations of H S 2 Time (min) 5 30 60 120 180 240 C o n c e n t r a *' o n °f 2 * 9' '* H S n m 1.5 15.0 150.0 -12.3 16.8 25.1 25.8 74.9 10.6 25i 3 59.1 — — 74.8 61.7 15a o — — — — 91.5 To determine the significance of duration and concentration of exposure of mucosal specimens to H S, 1 mCi [ S]-Na S0 /ml H 0 55 2 2 4 2 (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 H S for periods of 5 2 to 240 min following which 100 ml of [ S]-Na S0 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. 35 2 47 4 1.5 ng H S/ml at 180 m i n . , higher concentrations of H S induced 2 2 greater changes in permeability. obtained with 1,5, A 60% to 75% increase was 15, and 150 ng H S/ml air -within 3 hr, 1-2 2 hr, and 1 hr, respectively. IL_ Comparative Permeability Studies on Control, H S and 2 CH SH Treated Tissues 3 As methyl cytotoxic agent ascertain whether mercaptan than HS 2 has been (8), a implicated study was as a more undertaken to 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 CH SH or 15 3 ng/ml H S. 2 At 15 ng/ml air concentration both CH SH and H S exposed 3 2 tissues exhibited a similar increase in permeability up to 60 min. of exposure. On longer exposure, appears to plateau out. the increase in permeability Comparative studies demonstrate that methyl mercaptan is a more potent agent, capable of increasing the permeability of mucosa to Na S0 2 48 4 up to 103%. The values for control systems indicate an insignificant change in passage of permeability following aeration with air/C0 . (Table II) 2 £L_ Permeability of [ As other 1 4 investigators C]-Ovalbumin have reported the dextran-70 (molecular weight 70,000 daltons) in in vitro studies, the effect of thiols on the permeation of [ C]-labelled ovalbumin 14 was investigated. methyl The results mercaptan-treated [ C]-ovalbumin. 14 tissues are both control and impermeable to Although ovalbumin has a smaller molecular weight than dextran-70, and indicate that a difference in structural configuration 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 [ H ] - P r o s t a g l a n d i n E 5 2 The presence of prostaglandin E in in vivo and in in vitro 2 systems induces tissue reactions similar to those manifested in periodontal disease. As the concentration of PGE 2 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 H S or CH SH+ 2 3 Exposure % Increase in permeability time (min) HS - 10.6 25.3 59.1 2 5 30 60 120 180 + ¥ C0 /air? CH3SH 2 -6.6 -6.9 9.3 19.0 34.6 103.0 73.0 - 61.7 15.0 ng H S or CH SH/ml 95% air/5% CO 95% air/5% C0 represents control atmosphere 2 3 2 Individual mucosal specimens were exposed for 5, 30, 60, 120, and 180 min to either 15.0 ng H S or 15.0 ng CH SH/ml 95% air 2 3 /5% C0 . In comparison, the control specimens were exposed to the same periods of time to 95% air/5% C0 . Changes in steadystate of perfusion were determined by applying 1 mCi [ S]-Na S0 /ml H 0 to epithelial surface of the mucosa before and after exposure to the volatiles. The values represent percentage change in permeability to [ S]-Na S0 of the same specimen subjected to the tested gas. Negative values indicate a percentage decrease in permeability of control systems to [ S]-Na S0 after exposure to air/C0 atmosphere. 2 2 35 2 4 2 35 2 35 2 4 2 50 4 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 PGE . 2 It was found that exposure of the tissue to 15 ng CH SH/ml air 3 for 30 to 120 min. significantly increased the permeability of mucosa to pH]-PGE . Whereas the change 2 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 [ S]-S0 ~ 35 2 4 ion. The effect appears to plateau on prolonged period of exposure. (Table III) Lu The Effect of ZnCl 2 on Permeability of CH SH-Treated 3 Mucosa 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 Time (min) % Change i n P e r m e a b i l i t y Control" " CH SH-treated 1 + 3 6.9 ' -7.7 2 0 30 60 120 54 66 76 C o n t r o l atmosphere, 95% a i r / 5 % CO ; t e s t atmosphere, 15ng CH^SH p e r ml 95% a i r / 5 % CO ? Initially, lOOul of 12.5u.Ci (76 x IO" 9 M) [ H]-prostaglandin E 3 solution [5,6,8,ll,12,14,15- H(N)]/ml was applied to the epithelial surface of the tested specimens and perfusate fractions were collected until steady states of perfusion of [ H]-PGE were 3 3 2 established. The surface of the tissues were then washed with PBS, exposed to 15.0 ng CH SH/ml 95% air/5% C0 for either 30, 3 2 60, or 120 min., and re-exposed to [ H]-PGE . The control specimens were exposed to only 95% air/5% C0 . 3 2 2 52 2 Mouth air analyses have demonstrated that Z n effective in suppression of oral malodour. primarily attributed to the compounds, namely CH SH presence +2 is highly Since oral malodour is of volatile sulphur and H S, and since zinc is a thiol 3 2 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% ZnCl 2 to mucosal surface for 15 min. exerted a protective effect on the tissue barrier against methyl mercaptan. Treatment of the tissues with ZnCl prior to exposure to CHjSH nullified the expected increase of 2 103% in permeability to [ S]-Na S0 . (Table IV) Tissues exposed 35 2 4 to methyl mercaptan prior to ZnCl results. 2 treatment yielded similar Again zinc ion totally nullified thiol-induced permeation to [ S]-Na S0 , from expected 103% to essentially a control 35 2 4 state. (Table V) sL. Protective Effect of ZnCl To ensure that the Against 2 reversal permeability by ZnCL was not 53 by PGE 2 ZnCl 2 of merely a result thiol-induced of reaction 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 CH3SHi 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 (min) t o CH3SH 30 60 120 % Change i n permeability -4.4 + -0.8 +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 SH/ml 9 5 % a i r - 5% CO2 atmosphere f o r 30 t o 120 m i n . i The CH3SH-exposed t i s s u e t h a t d i d n o t r e c e i v e ZnCl2 pretreatment. 3 After steady states of perfusion of [ S]-Na S0 were established the mucosae samples were exposed to 0.22% ZnCl (w/v H 0) for 15 min. then to 15 ng CH SH/ml 95% air/5% C0 for 30, 60, or 35 2 4 2 3 2 2 120 min. The difference in percentage change in permeability following treatment with 15.0 ng CH SH/ml air for 120 min 3 demonstrates the ability of ZnCl to maintain the permeability of the mucosa. 2 54 Table V Reversal of Ch^SH-induced increase i n permeability of mucosa by zinc chloride Treatment time , CH SH (min) % Change i n permeability 3 +13.0 + 0.8 - 9.3 + 5.0 5 30 60 120 - 6.9 +103.0 120 (control) 120 (test) Mucosa was treated for indicated periods of time with a i r / C 0 atmosphere containing 15ng Ch^SH/ml, then subjected to 0.22% ZnCl for 15 min. The values are compared to control and test systems not treated with ZnCl22 2 [ S]-Na S0 was used as penetrant in this study. Following exposure of mucosa to 15.0 ng CH SH/ml air for 120 min, 0.22% w/v ZnCl 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% ZnCl nullified the CH SH-induced increase in permeability. 35 2 4 3 2 2 3 55 between zinc and sulphate ions, ZnCl studies were also performed 2 with [ H]-PGE . As in the case with [ S]-S0 , exposure of 3 35 2 4 mucosa to 15 ng of CH SH/ml air for 60 min. increased its 3 permeability to [ H]-PGE by 66%. 3 2 treatment of the tissues with Again it was found that the 0.22% ZnCl 2 for 15 min. immediately after CH SH exposure completely nullified the thiol 3 induced changes in permeability. These findings indicate that the reversal of permeability by Zn ion is not a result of a direct reaction between Z n +2 +2 and the penetrant. (Table VI) JL. Reactivity of [ S]-H S with Non-keratinized Mucosa 35 2 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 of mucosa, permeability This contention is supported by LSC analysis of mucosal specimens that were exposed for 30 min. to radioactive labelled H S. 2 The results showed a substantial retention of H S by the mucosa. 2 The amount ranged from 53 ng to 86 ng of H S/cm . (Table VII) 2 2 While these results demonstrate reactivity between H S and mucosa, they do not provide information as to 2 56 Table VI i i REVERSIBILITY OF CH^SH EFFECT ON PERMEABILITY OF MUCOSA TO PGE„ System % Change i n C o n t r o l -+ CH SH-treated§ CH SH/ZnCl -treated * -7.7 +66.0 -2.5 3 3 Permeability 2 £ 95% a i r / 5 % CO f o r 60 min. § 15ng CH SH per ml 95% a i r / 5 % C 0 f o r 60 min * F o l l o w i n g 60 min exposure t o 15ng CH SH p e r ml 95% a i r / 5 % CO atmosphere, mucosa was t r e a t e d f o r 15 min w i t h 0.22% Z n C l « ? I 3 2 3 , ? 2 I The depicted increase in permeability to [ H]-PGE was established 3 2 on mucosal specimens exposed to CH SH. Immediately following 60 3 minutes of CH SH treatment, 3 the specimens were exposed to 0.22% ZnCl for 15 min., washed thrice with PBS, then again 2 exposed to [ H]-PGE . The anticipated methyl mercaptan-induced increase in permeability of PGE was reversed by ZnCl 3 2 2 treatment. These values [ S]-Na S0 in Table V. are similar to those obtained with 35 2 2 4 57 Table VII Retention of H s by Oral Mucosa 2 [ S ] - H S / H S retained/cm 3 5 2 Experiment 1 2 3 4 Average I 2 2 dpm[ S]-H S ngH s 35 2 2 79,185 113,419 127,964 85,043 53.1 76.1 85.9 57.1 101,403 68.1 S p e c i f i c A c t i v i t y of [ S ] - H S was 46m Ci/m mole 3 5 2 i Following the exposure of mucosa to 15 ng (46 mCi/mmole) [ S]-H S/H S at a flow rate of 6.8 ml/min for 30 min., the 35 2 2 surface of the specimens was washed thrice with PBS at 2 min. intervals and the areas of specimens not exposed to [ S]-H S 35 2 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 H S reacted with the 2 cellular membrane, the intracellular organelles and/or extracellular matrix components. This phase of the study was limited to reaction with [ S]-H S as 35 2 gas chromatographic analyses showed available that all commercially [ S]-CH SH preparations were grossly 35 3 contaminated with [ S]-H S. 35 2 P e r m e a t i o n of [ S j - f l S Through I n t a c t Mucosa 5 5 2 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 thiols must be able to transverse fibroblasts, the both epithelial and basal laminar layers of the mucosa to gain access to the underlying connective tissue. Using [ S]-labelled H S, it was found that in addition to _ 35 2 being retained, layers of mucosa, [ S]-H S penetrated through all three tissue 35 2 LSC analysis of effluent fractions that diffused 59 through tissues that were treated with 15 ng [ S]-H S/ml air for 35 2 3 hr contained 12.3 ng H S/cm tissue (table VIII) into the lower 2 2 chamber of the perfusion apparatus, showing that the amount of H S that perfused through the tissues is of sufficiently high 2 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 SBinding to Mucosa 2 The stability of [ S]-H S tissue complex was investigated to 35 2 ascertain whether the H S was covalently bonded with tissue 2 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 [ S]-H S, then washed 35 2 for 60 min with PBS to remove any unreacted or loosely bound H S. 2 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 Diffusion of H S through o r a l 2 [ 3 5 mucosa S]-H s/H s diffused/cm 2 2 2 Experiment : ngH s dpm[ S]-H S 3 5 2 2 i 1 18,670 12.5 2 18,358 3 18,045 12.3 12.1 18,358 12.3 Average Specific mole. activity of [ 3 5 S ] - H s was 46mCi/m 2 The tissues were exposed to 15 ng of 46 mCi / mmole P S]-H S 5 2 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% ZnCl were collected 2 using a fraction collector. One ml of each aliquot was dissolved in 3 ml of aqueous scintillating counting solution for [ S]-analysis. The tabulated values represent the total amount of [ S]-activity and ng H S / cm tissue that permeated through all three tissue layers during the 180 min duration of the experiments, 35 35 2 2 61 retained 80.5 ng H S/cm 2 2 tissue, the rinsed control specimens retained 68.1 ng H S / cm . (Table IX) The difference between 2 2 the two values is attributed to the use of two tissue preparations with different diffusion properties. In contrast, displacement of [ S]-H S that had reacted with 35 2 tissue components was observed when the mucosa was aerated with a constant stream of 95% air/5% C0 for 3 hours followed by 2 30 min elution with PBS. half of the bound Under these conditions, approximately SH was displaced. (Table IX) Partial displacement of [ S]-H S implies that hydrogen sulphide induced 35 2 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 Labelled E. Coli Lipopolysaccharides (LPS) It is well established that bacterial endotoxins are primary etiologic factors initiating tissue destruction. established healthy However, it is not whether endotoxins can penetrate through the intact mucosal barrier. Hence one of the aims of this investigation is to determine whether CH SH can potentiate the 3 diffusion of LPS. 62 Table IX , , i \ i Treatment o f [ S ] - H S - e x p o s e d mucosa w i t h a i r / C 0 and phosphate b u f f e r e d s a l i n e 35 2 [ S]-H S/H S retained/cm 35 2 2 101,403 120,000 52,496 3 5 2 + — — ngH S 35 [ S]-H S treated PBS 95% a i r / 5 % C0 /PBS?< 2 I 2 2 dpm[ s]-H S Treatment 2 2 68.1 80.5 35.2 I + T i s s u e was exposed f o r 30 min t o 15ng [ S ] - H S f o l l o w e d by l h wash i n phosphate b u f f e r e d s a l i n e (PBS). f T i s s u e was t r e a t e d f o r 30 min w i t h 15ng [ S ] H S f o l l o w e d by 3h exposure t o 95% a i r / 5 % C 0 and 30 min wash w i t h PBS. 3 5 2 3 5 2 i ! 2 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 of [ S]-H S treated mucosa. Specimens that were exposed 35 2 2 to 15 ng P S]-H S for 30 min, were trimmed and digested in Soluene™ 100 (60°C) for 2 hr. The solublized specimens were then analysized for retained [ S]-activity by LSC. 5 2 35 63 Fluorescent analysis of perfusates of FITC-treated tissues indicated that the amount permeated through detection analysis of by of fluorescence the in the tissue was spectrof luorgraphy. the perfusate insufficient However, control specimens that revealed had to permit cytofluorescent 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 CH SH for 3 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. unlikely that the observed Therefore, uniform distribution of it is 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 CH SH had altered the tissue barrier thereby and 3 potentiated endotoxin. 64 the penetration of ]L_ coli 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 CH SH-treated epithelial 3 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 Assessment of Cellular Viability after CHjSH Treatment Differential staining with fluorescein diacetate and ethidium bromide of (Fig. 8) fresh biopsied tissues (Fig. 7) demonstrated similar staining and frozen tissues 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% C0 out of a 2 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. basal Microscopic examination of unstained preparations of 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% C0 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. 2 71 10-a 10-b Fig. 10a,b. CH SH-treated mucosa stained with FDA and counter 3 stained with EB. The specimens were overlaid with 100 ul of PBS for 3 hr. The epithelial surface was then exposed to 15 ng CH SH / ml air for 3 hr and 3 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 CHAPTER IV DISCUSSION A. The Effect of VSC on Diffusion of Antigens through Non-keratinized Oral Mucosa. Factors Contributing to Periodontal Disease 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 non-keratinized the crevice are epithelium which act several as cell layers of primary deterrents against the penetration of deleterious substances. Although the diseases is believed pathogenesis to of inflammatory periodontal be multifactorial, the present thesis specifically investigates the potential pathogenic role of volatile sulphur anaerobic compounds oral produced microflora. primarily These by gram compounds negative 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 diacetate and ethidium bromide. provide positive differential evidence staining that studies using fluorescein Differential staining results membrane integrity of some fibroblasts is severely affected by the presence of volatile sulphur compounds. regulated Since the permeability of membranes of cells is 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 CH SH, this implies that the 3 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 74 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 compounds, per se, 2-mercaptoethanol to are conjecture that immunogens. volatile In cell harvesting, is routinely added to cultures to macrophage proliferation. sulphur enhance Hence H S and CH SH thiols may be 2 3 presumed as potent mitogens since the thiol functional groups of 2-mercaptoethanol compounds. and methyl mercaptan is common to both 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, amounts of prostaglandins, produced by numerous markedly increased particularly prostaglandin E , 2 inflammatory cells. are Prostaglandin E 2 induces bone resorption and is believed to be an important mediator of inflammatory periodontal 75 disease, rheumatoid arthritis and malignant osteolysis. The fact that PGE levels are 2 increased by 10 to 20 fold in periodontally involved tissues and that volatile sulphur compounds can increase the penetration of prostaglandin E by 76% strongly indicate that disruption of the 2 permeability barrier by the thiols will enhance the antigenic and immunogenic challenge of this inflammagen to the connective tissue. Exogenous prostaglandins lymphocytic functions. are potent Elevated inhibitors of several levels of PGE are immunosuppressive to lymphoid mitogenesis, cytolysis, antibody production and secretion of lymphokines (interleukin-2) 116). (115, These local immunosuppressive responses can hinder the host's second line of defense. have an immunological effect, As prostaglandins are believed to the volatile sulphur compound induced increase in permeability of PGE through intact mucosal 2 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. sources of evidence provide further support 76 for Several other a relationship 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 present ( 6 ) . 2 the ability of zinc ion to nullify the C H 3 S H Therefore, - induced increase in access of exogenous prostaglandin E to the underlying tissue is of 2 paramount significance. In vivo, half-life. prostaglandins are considered to have a short 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. as the presence of bacteria has been However, 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 tissues. Nevertheless, affected 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 secretion of lymphokines by T-lymphocytes (118). and The presence of interleukin II can cause several local inflammatory responses including activation of osteoclasts and macrophages, inhibition of macrophage migration, and induced collagenase and prostaglandin (124). fibroblast production of 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 78 produce hydrolytic enzymes and collagenase (117). Ultimately these enzymes contribute to further destruction of both the tissue's protein matrix and cells. exposed to For example, endotoxin have human gingival been shown fibroblasts 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 epithelium to responses. without initiate directly the It is believed disrupting inflammatory that and the superficial immunological 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. degradation Hence the susceptibility and their capacity of the sulcular tissues to regulate molecules are important considerations the perfusion of in the initial stage of periodontal disease. Heparan sulphate is the predominant proteoglycan synthesized by gingival epithelium. space, the Once excreted molecules of this into the intercellular glycosaminoglycan, varying in molecular weights from 10 to several million dalton aggregates. 5 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 molecular structure (120). a loss of integrity of 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 Aggregation of proteoglycans disulfide linkages. interstitual has water been shown and solutes. to depend on Similarly the glycoproteins in the extracellular compartment are also believed to interact with collagen to form stable matrices via disulfide bonds. It is noteworthy membrane intercellular coating granules in the 80 that space are 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, deaggregation permeability. a disulphide cleaving agent which of proteoglycans and a concomitant causes increase in Blockage of aggregation can also be achieved by alkylation of thiol residues which prevents formation of disulphide linkages (123). It is conjectured that H S and CH SH enhance the permeation 2 3 of toxic substances through disulphide cleavage mechanism. This is a reasonable supposition as the results of this demonstrated that mucosal specimens readily interact with H S. 2 Although relatively mucosal stable, the interaction fact that with H S appears 2 permeation change to be can be partially reversed following aeration suggests that re-aggregation of proteoglycans is also possible, tissue barrier's is Restoration of integrity of the 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 properties of ion-exchange aggregated can exhibit chromatography and the intricate proteoglycan matrix as molecular sieve similar to polymers in gel filtration. By virtue of simple filtration and selective ionic interactions, the passage of many through the tissue is thus regulated. the charged groups mucopolysaccharides' or the Therefore, an alteration of aggregative proteoglycans metabolites and state of the 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 ZnCl either prior to or after thiol treatment 2 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. compounds, Z n +2 In the presence of volatile sulphur ion readily reacts with the mercaptides to form inactive zinc mercaptides before the 82 reactive thiols can oxidize 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, cytologists to stabilize zinc has been used extensively by 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 Z n +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. change Hence it is proposed that the principal 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 substances to gain access to connective assist toxic tissue and how they initiate a series of reactions resulting in a marked destruction of the tissue. Moreover, the ability of zinc ion to prevent an increase in permeability of the mucosa implies that zinc ion may be an useful therapeutic agent for treatment and prevention of periodontal diseases. 84 REFERENCES 1. Gaffar, A . , Coleman, E . J . , and Marcussen, H.W. 1981. 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Membrane coating granules in non-keratinizing oral epithelium. J. Ultrastruct. Res. 60, 212. 65. Squier, C.A., and Johnson, N.W. 1975. oral mucosa. Br. Med. Bull. 31, 169. 66. Squier, C A . and Hall, B.K. 1984. The permeability of mammalian non-keratinized oral epithelia to horseradish peroxidase applied in vivo and in vitro. Archs. Oral Biol. 29, 45. 67. Hayward, A.F. 1979. Rev. Cytol. 59, 97. 68. Silverman, S. Jr. and Kearns, G. 1970. Ultrastructural localization of acid phosphatase in human buccal epithelium. Archs. Oral Biol. 15, 169. 69. Silverman, S. Jr. 1971. Current concepts of histology of oral mucosa (Squier, C A . and Meyer J . eds.) Thomas Spring field 111. 70. Innes, P.B. 1973. The nature of the granules within sulcular epithelial cells. J. Periodont. Res., 8, 252. 71. Hayward, A.F. 1973. Electron microscopic observation on cell coat and membrane coating granules of the epithelium of the hard and soft palate in the rat. Archs. Oral Biol. 18, 67. 72. 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The effect of volatile thiol compounds on permeability of oral mucosa Ng, William Man Fai 1986
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Title | The effect of volatile thiol compounds on permeability of oral mucosa |
Creator |
Ng, William Man Fai |
Publisher | University of British Columbia |
Date Issued | 1986 |
Description | 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 E₂ (PGE₂) and fluorescein isothiocyanate labelled E. 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. Similarly, the mercaptan induced up to a 70% increase in permeability of the mucosa to (³H)-prostaglandin E₂. The magnitude of changes in the permeability were found to depend on duration of exposure to the thiols and to their concentration. Studies using (³⁵S)-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 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. |
Subject |
Cell Membrane Permeability Thiols Sulfhydryl Compounds Oral mucosa Cells -- Permeability Mouth Mucosa |
Genre |
Thesis/Dissertation |
Type |
Text |
Language | eng |
Date Available | 2010-07-16 |
Provider | Vancouver : University of British Columbia Library |
Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
DOI | 10.14288/1.0097012 |
URI | http://hdl.handle.net/2429/26508 |
Degree |
Master of Science - MSc |
Program |
Dental Science |
Affiliation |
Dentistry, Faculty of |
Degree Grantor | University of British Columbia |
Campus |
UBCV |
Scholarly Level | Graduate |
Aggregated Source Repository | DSpace |
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