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THE EFFECT OF VOLATILE THIOL COMPOUNDS ON PERMEABILITY OF ORAL MUCOSA  BY  WILLIAM MAN FAI NG B.Sc., The University of British Columbia, 1980 D . M . D . , The University of British Columbia, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Department of Oral Biology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November 1986 •William Man Fai Ng, 1986  In  presenting  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. 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