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Effect of zinc on the metabolism of thiol-treated human gingival fibroblasts Ouyang, Ying 1991

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EFFECT OF ZINC ON THE METABOLISM OF THIOL-TREATED HUMAN GINGIVAL FIBROBLASTS by  YING OUYANG B. M.Sc, Beijing Medical University, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF DENTAL SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Department of Oral Biology, School of Dentistry) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1991. © Y. Ouyang, 1991.  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by  his  or  her  representatives.  It is  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Oral Biology  The University of British Columbia Vancouver, Canada  D a t e  DE-6 (2/88)  October 1 0 , 1991  Abstract  The etiology of periodontal diseases is multifactorial. Cumulative in vivo and in vitro observations implicate volatile sulphur compounds (VSC), namely, hydrogen  sulphide (H S) and methyl mercaptan (CH SH), produced through putrefaction of 2  3  sulphur-containing proteinaceous substrates by gram negative bacteria, in the pathogenesis of the diseases. Their concentrations in the mouth air correlate with the severity of periodontal involvement. They significantly inhibit total protein, DNA and collagen synthesis, and proline transport in cultured human gingival fibroblast (HGF) systems. The inhibition on total protein synthesis is irreversible, with CH SH exerting a 3  more profound inhibitory effect than H S. In addition, they have been found to increase 2  the permeability of porcine sublingual mucosa to [ H]-PGE , [ S]-S0 and E. coli 3  35  2  4  endotoxin thus suggesting that VSC may be capable of altering the permeability of the epithelial barrier and thereby assist the pathogens to gain access to the underlying connective tissues. However, treatment of the mucosa with 0.22% ZnCI totally nullifies 2  the effect of VSC and restores the tissue permeability to control state. The objective of this thesis is to investigate whether zinc is also capable of reversing the inhibitory effect of CH SH on total protein, DNA and collagen synthesis, and proline transport by 3  cultured HGF. While ZnCI at concentration of 0.22% (1.6x10" M) is protective against VSC 2  2  and without any apparent harmful effects when applied to the intact tissue, HGF cells, when in direct contact with this agent, are more sensitive to it. In order to determine the maximal zinc concentration that is not deleterious to HGF, studies of total protein synthesis, cell attachment and proliferation were conducted on cultured HGF  ii  exposed to various concentrations of ZnCI ranging from 1'.0x10-M to 1.5x10-M. It 6  2  2  was found that zinc at concentrations higher than "I.OxKHM significantly decreased the total protein synthesis by HGF over a 24-hr labeling period in DMEM devoid of L-proline but supplemented with L-[ C]-proline. Fibroblast attachment was reduced by 14  47% and 21% when they were treated with 1.5x10-M and 0.5x10 M ZnCI , 2  3  2  respectively. Fibroblasts incubated in the presence of 1.5x10" M and 0.5x10" M ZnCL 2  3  2  failed to proliferate over a 11-day growth period and under the light microscope were rounded in appearance. Transmission electron microscopic studies of fibroblasts treated for 24 hours with different concentrations of ZnCI revealed that cells exposed 2  to higher than 1.0x10' M ZnCI underwent pathologic changes characterized by 4  2  clumping of nuclear chromatin, presence of large amount of myelin figures and overall disintegration of cytoplasmic organelles. In addition, it was found that exposure of HGF cultures for 24 hours to [ Zn] resulted in an accumulation of zinc by fibroblasts in a concentration-dependent manner. 65  To assess the ability of zinc to counteract CH SH, control HGF cultures were 3  incubated in an atmosphere of 95% air / 5% C0 while test cultures were subject to 2  95% air / 5% C0 admixed with 15ng / ml CH SH with or without the presence of ZnCI 2  3  2  in the culture media. It was found that during a 24-hr L-[ C]-proline pulsing period, 14  exposure of HGF to CH SH resulted in a 24% reduction in total protein synthesis which 3  could be totally reversed by the presence of 0.5x10-M or 1.0x10" M ZnCI in the 4  5  2  media. Furthermore, at these two concentrations, zinc was also found effective in  iii  reversing the CHSH-induced inhibition of proline transport. HPLC analysis of total 3  protein and collagen confirmed that ZnCI present at a concentration of 1.0x10 M in -5  2  the media totally nullified the inhibitory effect of CH SH and restored total protein 3  synthesis by HGF to control levels. However, at this concentration, ZnCI did not 2  reverse the effect of CH SH on collagen synthesis, and at non-inhibitory 3  concentrations (below 0.5x10 M), it was ineffective in reversing the adverse effect of -4  CH SH on DNA synthesis by cultured HGF. 3  In summary, zinc at non-inhibitory concentrations in HGF culture has been shown to be capable of counteracting the CHSH-induced suppression of total protein 3  synthesis and proline transport by HGF, which implies that zinc may have a therapeutic role in counteracting some of the adverse effects of volatile thiols and modify their effect on tissue destruction that occurs in periodontal disease.  iv  Table of Contents Page Abstract Table of contents List of Tables List of Figures Acknowledgment  ii v x xii xiv  General Introduction Section I A. Structure of Human Periodontal Tissues B. Inflammatory Periodontal Diseases C. Volatile Sulphur Compounds (VSC)  1 4 6  1.  Production  6  2.  Bacteria Responsible for VSC Production in Periodontal Disease  6  D. Implications Derived from in vivo and in vitro Studies on the Roles of VSC in Pathogenesis of Periodontal Disease  v  7  Table of Contents (Continued) Page 1. 2. 3.  4.  Comparison of Clinical Observations with VSC Levels Effect of VSC on Permeability of Non-Keratinized Porcine Sublingual Mucosa Effect of VSC on DNA, Total Protein and Collagen Synthesis, and Proline Transport by Human Gingival Fibroblasts (HGF) Effect of VSC on lnterleukin-1 and cAMP Production by HGF  E. Literature Review of the Actions of Zinc on Biological Systems  7 7  8 9 10  1.  General Information on the Metabolism and Functions of Zinc  2.  Transportation of Zinc Across the Cell Membrane  12  3.  Stabilization of Biomembrane and Protection by Zinc of Cells against Membrane Damage by Haemolytic Agents Role of Zinc in Gene Expression and Cell Division Effect of Zinc on Collagen Synthesis Role of Zinc in Immune Response In vivo and in vitro Adverse Effects of Zinc  13 15 16 18 19  4. 5. 6. 7.  F. Objectives of This Thesis  10  19  Materials and Methods  vi  Table of Contents (Continued) Page Section II A. Human Gingival Fibroblast Cultures  21  B. Zinc Chloride Stock Solutions  22  C. Assessment of the Effect of Zinc on HGF Cultures  23  1.  Analysis of the Total Protein Synthesis in the Presence  2.  of Zinc by Trichloric Acid (TCA) Precipitation Measurement of [ Zn] Uptake by HGF  23 25  Effect of Zinc on the Attachment of the Fibroblasts Effect of Zinc on the Proliferation of the Fibroblasts Light Microscopic (LM) and Transmission Electron Microscopic (TEM) Studies of HGF Treated with Zinc  26 26  3. 4. 5.  65  27  D. Measurement of the Reversal Effect of Zinc on the Metabolism of HGF Exposed to CH SH 3  1.  CH SH Gassing System  2.  Analysis of the Reversal Effect of Zinc on the Synthesis of Total TCA-Precipitable Proteins Reversal Effect of Zinc on Proline Transport HPLC Analysis of the Effect of Zinc on Collagen and Total Protein Synthesis  3. 4.  30 30  3  vii  31 33 34  Table of Contents (Continued) Page 5.  Effect of Zinc on DNA Synthesis by CHSH-Exposed HGF 3  E. Statistics  38 39  Results Section III A. Effect of Zinc on the Total TCA-Precipitable Protein Synthesis by HGF B. Quantitation of Zinc Transport by HGF C. Effect of Zinc on HGF Attachment D. Effect of Zinc on HGF Proliferation E. LM and TEM Observations of Morphological Changes in Fibroblasts Treated with Zinc  40 45 47 49 52  F. Reversal Effect of Zinc on Suppression of Protein Synthesis 57  by CH SH 3  G. Reversal Effect by Zinc on CHSH-lnduced Inhibition of 3  Proline Transport H. HPLC Analysis of the Effect of Zinc on the Total Protein and Collagen Synthesis by CHSH-Exposed HGF 3  viii  62 65  Table of Contents (Continued)  I. Effect of Zinc on DNA Synthesis by CHSH-Exposed HGF 3  Discussion Section IV A. Molecular Mechanisms of the Detrimental Effect of High Levels of Zinc on HGF Cultures B. Preventive and Therapeutic Aspects of Zinc in Periodontal Disease: Its Counteractive Effect against CH SH 3  References  ix  List of Tables Table Number  Title  Page  Distribution of [ C]-activity between medium and cellular fractions of 24-hr cultures in the presence of FCS and various concentrations of ZnCI  42  Distribution of [ C]-activity between medium and cellular fractions of 24-hr cultures in the absence of added FCS but in the presence of various concentrations of zinc.  44  14  2  14  The amount of zinc transported into the fibroblasts during a 24-hr labeling period with [ Zn]. 65  46  Reversal by zinc of CHSH-induced suppression of total protein synthesis.  63  Percent decrease of total TCA-precipitable protein synthesis by HGF in the presence of CH SH and ZnCI .  64  Reversal effect of zinc on CH SH-induced suppression of proline transport by HGF.  66  HPLC analysis of the medium fraction for total protein and collagen synthesis by fibroblasts cultured in the presence of CH SH and ZnCI . 2  69  HPLC analysis of the cellular fraction for total protein and collagen synthesis by fibroblasts cultured in the presence of CH SH and ZnCI .  70  3  3  6  7  3  3  8  2  3  2  x  List of Tables (Continued)  DNA synthesis by HGF cultured in the presence of CH SH and ZnCI . 3  2  List of Figures Figure Number  Title  1  Some of the major steps in the biosynthesis of collagen.  2  Apparatus for incubating control and test cultures.  3  Total TCA-precipitable protein synthesized by HGF during a 24-hr labeling period in the presence of FCS and various concentrations of ZnCI . 2  4  Total TCA-precipitable protein synthesized by HGF during a 24-hr labeling period in the absence of FCS but in the presence of various concentrations of ZnCI . 2  5  The effect of different concentrations of ZnCI on HGF attachment. 2  6  HGF proliferation over a 11-day incubation period in the presence of different concentrations of ZnCI following a 48-hr quiescence. 2  7  HGF proliferation over a 11-day incubation period in the presence of different concentrations of ZnCI . 2  8  Light microscopic appearance of 24-hr incubated HGF in the presence of FCS and different concentrations of ZnCI. 2  XII  List of Figures (Continued)  Light microscopic examinations of HGF incubated for 24 hrs in the absence of FCS but in the presence of different concentrations of ZnCI . 2  Lower magnification TEM appearance of HGF incubated for 24 hrs in the presence of different concentrations of ZnCI . 2  Higher magnification TEM appearance of HGF incubated for 24 hrs in the presence of different concentrations of ZnCI . 2  Retention times for [ C]-labeled hydroxyproline and proline. 14  xiii  ACKNOWLEDGEMENT I would like to express my deepest gratitude to my supervisor, Dr. Joseph Tonzetich for his guidance, and patience throughout this project. I am especially grateful for the time he took to proof-read the initial draft of this thesis. I feel fortunate to have been supervised by someone I truly respect as both a mentor and an academic authority. I also wish to thank Drs. D. Brunette and J. Douglas Waterfield for their valuable comments and suggestions, especially Dr. Waterfield for the time he spent reading the first draft of this thesis. Special thanks are extended to Mr. Anthony Ng for his immense help in the lab. I appreciate all his generous assistance in everything from the experimental techniques to the use of the computer. Thanks are also expressed to Mr. Andre Wong for his help in TEM lab and to Ms. Leslie Weston for her assistance in the cell culture lab, and to all the staff in the Department of Oral Biology who have made my study here a memorable experience. I am especially grateful to my parents, Yi Ouyang and Zhichun Wu, and to my brother, Xiaolu Wu, for their unconditional encouragement and support throughout the years of my study. I would also like to acknowledge the Medical Research Council of Canada, the University of British Columbia and the Department of Oral Biology for their financial support.  xiv  Section I Introduction  A.  Structure of human periodontal tissues  Periodontal tissue is primarily composed of gingiva, crevicular and junctional epithelium, periodontal ligament and alveolar bone. Separating the epithelium from the underlying connective tissue, is a layer of basement membrane consisting mainly of type IV collagen, laminin, glycoproteins and proteoglycans. Sulcular epithelium, which is non-keratinized and characterized by wide intercellular space, together with basement membrane serves as a barrier that is essential for the integrity of underlying connective tissue. Collagen, an essential structural component of all connective tissues, provides primary support for teeth. The basic collagen molecule is made up of 3 polypeptide a-chains, each having approximately 1,000 amino acids coiled into a left handed triple helix. In each chain, every third amino acid residue is occupied by glycine in a X-Y-Gly. triplet structure with proline located in X position and hydroxyproline or hydroxylysine in Y position. The presence of an unusual amino acid, hydroxyproline, serves as an accepted indicator of the amount of collagen present since aside from collagen only small amount of hydroxyproline has so far been found in elastin, the C1q subcomponent of the complement system (67) and the tail structure of acetylcholinesterase (72). In the collagen molecule, hydroxyproline accounts for approximately 10% of total amino acids and only 1-2% in elastin. Beside the triple helical region, short non-helical telopeptide domains are also present at the amino and carboxyl terminal ends. After intracellular formation of the triple helix, the procollagens  1  are secreted across the cell membrane into the extracellurlar space where the two peptide extensions are removed so that the intramolecular and intermolecular cross-linking that designates the maturation of collagen can take place. The solubility of collagen depends on its degree of maturation. Newly synthesized collagen molecules are readily soluble in neutral salt solutions. As cross-linking takes place, it is changed into the acid soluble state which upon further crosslinking is converted to insoluble, mature or fibrillar form. Schiff base and aldol condensation are the two principle types of cross-linkages found in collagen. The aldol condensation involves a reaction of two aldehydes formed through deamination of hydroxylysine and lysine while Schiff base is formed by reaction of one aldehyde and one NH-group on the 2  adjacent molecules. Both types of cross-linking are catalyzed by an enzyme named lysyloxidase, which oxidizes the NH groups to aldehydes. Figure 1 depicts some of 2  the major steps in collagen synthesis (95). Up to now, at least fifteen genetically different types of collagen have been identified in the human body. In the periodontal ligament, collagen accounts for 50-60% of the organic matter, of which 85-90% and 10 -15% are type I [a (l) a (l)] and 1  2  2  type III [cx-, (111)3], respectively. The type I collagen molecule is composed of two identical a-] chains and one cc chain while type III tropocollagen consists of three identical a-| 2  chains. In gingiva, the collagen content is 50% of organic matter, of which 80-85% is type I and 15-20% is type III. The remainder of the organic matter of periodontal connective tissue is predominantly proteoglycans and glycoproteins. They appear to play a role in stabilizing the collagen molecule and are involved in cell-cell and cell-matrix interactions, which also contribute to the integrity of periodontal tissue. Gingival and periodontal ligament fibroblasts are responsible for the production and maturation of these extracellular matrix proteins.  2  DNA Transcription RNA Translation Synthesis of 3 polypeptide chains Hydroxylation  Synthesis of hypro and hylys  Glycosylation  Addition of sugars to hylys Formation of interchain S-S bonds  AL  Formation of triple helix  Procollagen  PLASMA Removal of peptide extensions Collagen molecule Polymerization  Formation of Schiff base and aldol condensation  Mature collagen  Figure 1.  Some of the major steps in the biosynthesis of collagen. hypro: hydroxyproline, hylys: hydroxylysine  3  Type IV collagen is a component of basement membrane. It is secreted by epithelial cells and consists of two identical a-j chains and one a chain. Unlike the 2  structure of type I and type III collagen, type IV collagen monomer consisting of three pro oc-chains possesses a distinctive non-collagenous globular domain (NC1) at its COOH-terminus and a 30 nm long triple helical 7S-domain at the NH-terminus. These 2  two terminal extensions are not proteolytically processed when deposited in the matrix before self-assembly in extracellular space. Four triple helical type IV collagen molecules form a tetramer by binding together at the NH-terminal 7S-domain region 2  while segment NC1 at the COOH-terminal ends serves to connect two molecules. This forms a network structure that is different from the cross-linked fibrillar structures of type I and type III collagen (85).  B.  Inflammatory periodontal diseases  Periodontal diseases are inflammatory disorders that are believed to cycle through quiescent and active phases. The active stage is characterized by a breakdown of the crevicular junctional epithelial barrier, periodontal pocket formation and destruction of tooth supporting tissues such as collagen, which eventually result in the loss of teeth. The etiology of periodontal disease is believed to be multifactorial. Bacteria, including their structural components, enzymes, and metabolic products, host enzymes and immune response reactions have all been assigned a role in its pathogenesis. It is generally accepted that bacteria and their by-products in dental plaque play a primary role in the initiation and establishment of periodontal disease. They may induce a change in the intact epithelial barrier and thereby increase the accessibility of various  4  pathogens to the underlying connective tissue where they activate the host immune response system and initiate a series of destructive inflammatory reactions that finally result in the loss of collagen. The breakdown of the epithelial barrier is an initial event in the establishment of periodontal disease since toxic substances such as bacterial dextrans and endotoxins, per se, do not appear to cause inflammation in healthy sulcular gingiva (29, 70). Collagen destruction is a major event during periodontal disease. Both quantitative and qualitative changes have been observed. Comparison between normal and diseased gingival tissues reveals that while there is no significant difference in neutral salt-soluble collagen content between the two groups, the acid-soluble fraction is markedly reduced in inflamed tissues indicating that more mature, more insoluble and more cross-linked forms of collagens are affected in inflamed gingiva. Type III collagen, which normally constitutes up to 30% of total gingival collagen, accounts for only 4% in diseased tissue. In addition, type I trimer [00-1(1)3]  ar,  d type  | V  collagen, which are normally absent, are found in inflamed tissues.  Type I trimer, which consists of three identical [ 0^ (I)] chains, is considered to be a product of a deviant gene expression and has been found in tissues under pathological conditions and in fibroblast cell cultures perpetuated through large number of passages (57). Noncollagenous proteins in periodontal tissues which consist mostly of proteoglycans and glycoproteins also undergo degeneration in periodontal diseases and their destruction usually proceeds that of collagen (24).  5  C.  Volatile sulphur compounds (VSC)  1.  Production Volatile sulphur compounds are derived through putrefaction of  sulphur-containing proteinaceous material by oral microorganisms. Three volatile sulphur compounds, namely hydrogen sulphide (H S), methyl mercaptan (CHSH) and 2  3  dimethyl sulphides [(CH ) S], are present in the mouth air of all individuals (87). In 3 2  humans, H S and CH SH occur in the range of 1CV to 10" g per 10 ml mouth air and 8  2  account for approximately 90% of its sulphur content. physiological and pathological oral malodor. 2.  10  3  They are the major cause of  Bacteria responsible for VCS production in periodontal disease  During the onset of periodontal disease, there is a shift in oral microflora in gingival crevice from gram-positive cocci to motile, gram-negative anaerobic rods and spirochetes which are believed to be the primary periodontal disease pathogens. The gram-negative organisms are proteolytic and are primarily responsible for VSC production. A comparison between pathogenic and non-pathogenic strains of Bacteroides gingivalis showed that pathogens produced 10-fold more VSC than the  non-pathogens. In addition, pathogens generated large amount of CH SH and 3  dimethyl disulfide while non-pathogenic strains emitted only H S and limited amount of 2  CH SH (89). The production of VSC occurs at pH>6.5 (54). The presence of blood, 3  which is required for growth by some species such as Porphyromonas gingivalis, provides a utilizable sulphur-containing substrate that enhances VSC production (8).  6  Hence in periodontal disease, the condition in deep periodontal pockets where the pH is basic, the rich proteinacous serum and tissue exudates resulting from bleeding and inflammation greatly facilitate the production of VSC.  D.  Implications derived from  in vivo and in vitro studies on the  roles of V S C in pathogenesis of periodontal disease  1.  Comparison of clinical observations with VSC levels Clinically, an increase in oral malodor has been observed in many  periodontal disease conditions. Using filter paper strips impregnated with lead acetate, Rizzo was able to demonstrate that the amount of H S detected was in direct 2  correlation with the degree of periodontal destruction (69). Additional evidence comes from studies by Tonzetich (87) and other investigators using gas chromatographic methods of analysis. These studies indicate that the concentration of VSC in putrefying saliva (49), mouth air (88) and gingival crevice (20) correlates with the severity of periodontal involvement. Furthermore, after corrective periodontal therapy, the level of VSC declines to baseline levels (88). 2.  Effect of VSC on permeability of non-keratinized porcine sublingual mucosa The exposure of  porcine sublingual mucosa, which consists of  non-keratinized epithelium, basement membrane and connective tissue, to VSC increases its permeability to [ H]-PGE , [ C]- and FITC-labeled E. Coli endotoxin, and 3  14  2  [ S]-S0 (59). Both PGE and endotoxin have been recognized as potential 35  4  2  7  mediators of inflammatory periodontal disease process (60,78). Furthermore it has been reported that [ S]-H S can react with the mucosa and is capable of penetrating 35  2  all three layers of tissues. However, treatment of the mucosa with 0.22% (1.6x10" M) 2  ZnCI for 15 minutes, either before or after the exposure, can totally nullify the effect of 2  VSC and restore the permeability to control state (59). 3.  Effect of VSC on DNA, total protein, and collagen synthesis, and proline transport by human gingival fibroblasts (HGF) Previous studies have demonstrated that VSC exerts an inhibitory effect on  the metabolism of the cultured human gingival fibroblasts. Exposure of HGF to VSC results in a reduction of total protein (40) , collagen (40) as well as DNA (93) synthesis and proline transport (92) by 32%, 39%, 25% and 47%, respectively. In these studies, CH SH has been found to be a stronger inhibitor thanH S(91). 3  2  Loss of collagen in periodontal disease can be a result of both decreased synthesis and / or increased degradation. The decrease in synthesis may largely be attributable to the reduction in proline transport by HGF. Proline uptake by fibroblasts involves a membrane associated active transport mechanism. Its inhibition by VSC suggests possible damage to cell membranes. Supporting evidence is derived from a fluorescent staining study of oral mucosal sections. When the tissues are exposed to CH SH, the uptake of Ethidium Bromide (EB) is intensified, which implies an 3  impairment to the membrane and / or the death of the cells (59). Other in vitro studies have shown that thiols are capable of binding with collagen. Reaction of H S and CH SH with rat tail type I tendon collagen results in the 2  3  8  transformation of some acid soluble collagen to a neutral salt soluble form (39). It has been suggested that -SH combines with the aldehyde groups of collagen to form a more soluble product. DEME-cellulose and SDS-page analyses show that while CH SH reduces both type I and type III collagen synthesis, the amount of type I 3  procollagen is increased (39). The effect of CH SH on HGF cultures was found to be 3  more profound than H S and is irreversible for at least 24 hours in control air / C0 2  2  atmosphere (94). 4.  Effect of VSC on interleukin-1 and cAMP production by HGF  The present information suggests that host immune response plays an important role in the destruction of periodontal tissue (60). The involvement of interleukin-1 (IL-1) has been intensively studied. A variety of cell lines including macrophages, lymphocytes, epithelial cells and fibroblasts have been shown in vitro to be capable of secreting IL-1 or IL-1-like molecules with similar biological activities (32). Some of the effects of IL-1 include inducing acute phase protein and neutrophilia. In vitro, IL-1 has been found to increase the production of lymphocyte derived lymphokines and the production of antibodies. It is mitogenic for cultured fibroblasts and chemotactic for neutrophils (32). Studies also suggest that bone loss in progressive periodontal disease is partially attributable to the involvement of IL-1, which is also known as osteoclast activating factor. Its activation of osteoclasts results in bone resorption (75). There are two genetically different types of IL-1, a and P, both forms have been identified in the gingival crevicular fluid of patients with moderate and advanced periodontitis (52). In addition, marked reduction in IL-1 level has been observed following effective periodontal treatment. In gingival and  9  periodontal ligament fibroblast cell cultures, treatment with IL-1 results in an increase in procollagenase production and an increase in the level of procollagenase mRNA. In gingival fibroblast cultures, increased collagenase activity is induced by IL-1 (68). IL-1 also stimulates in a dose and time-related manner human periodontal ligament fibroblasts to synthesize PGE , another factor or mediator that has been implicated in 2  the pathogenic process (74). The levels of PGE are raised during inflammatory 2  periodontitis and correlate with the level of cAMP, an intracellular molecule that serves as a "second messenger" in signal transduction (78). Recently, it has been demonstrated that CH SH is capable of inducing and 3  augmenting IL-1 production by  LPS activated mononuclear leukocytes (84).  Furthermore, in CHSH-exposed HGF cultures, the cAMP content is increased by 34%. 3  When HGF cultures were supplemented with cell extract containing IL-1, the amount of cAMP is increased by 58% (90). These results imply that CH SH is capable of 3  increasing proteolysis indirectly through immunological pathways.  E.  Literature review of the actions of zinc on biological systems  1.  General information on the metabolism and functions of zinc  Like Fe, Cu, and Mg, zinc is an essential trace element. Dietary zinc is absorbed in the intestine through both carrier-mediated and diffusion mechanisms, which are under homeostatic control. Newly absorbed zinc is bound by albumin and transported first to the liver and then redistributed to other tissues.  10  In humans, over 99% of zinc is located in tissues, of which 62% is in the muscle, 38% in bone and 0.3% in blood. Normal plasma zinc concentration is 15 uM with normal range of 10 to 18 uM. Approximately 65% of plasma zinc is loosely bound to albumin. The remainder of zinc is bound to oc-macroglobulin, amino acids such as 2  histidine and cysteine, as well as other small molecular weight ligands. Endogenous zinc is excreted either through the gastrointestinal tract or urine (1). Zinc was first found essential for the growth of Aspergillus niger more than a century ago (64). It was not until 1963 that zinc deficiency was discovered in man characterized by severe growth retardation and sexual hypofunction. This condition was greatly improved after administration of zinc (64). Since then, mounting evidence has been accumulated which demonstrates  that zinc deficiency in humans and  animals results in teratological, genetic and medical abnormalities (27, 50). Clinical manifestation include dermatological, gastrointestinal, endocrinological, reproductive, skeletal, developmental, immunological and neurological disorders (97). It is generally recognized now that zinc plays a significant role in many biological and biochemical processes. It has been identified as a component or a co-factor of more than 200 enzymes (104). They are zinc metalloenzymes that require zinc for the activation or at their active sites. These enzymes participate in various synthesis and degradation reactions of major metabolites such as carbohydrates, lipids, proteins and nucleic acids. In addition, zinc has been related to cell-mediated immunity and other aspects of host defense system (77, 82). It is also regarded as a stabilizer of macromolecules and biomembranes (17).  11  2.  Transportation of zinc across the cell membrane Zinc uptake across cell membranes has been studied using several cell lines  including human seminal spermatozoa (79), primary mouse hepatocytes, human skin fibroblasts (2) and rat tumor cells (11). The kinetics of zinc uptake in different types of cells investigated so far appears similar. Cells incorporate zinc from culture medium in a biphasic manner with an initial rapid phase which usually takes a few minutes of incubation, followed by a slower linear phase (80). It is suggested that the process is carrier-mediated, saturable and does not depend on ATP (80). However, it seems that different types of cells may transport zinc across the membrane by different mechanisms (11). Experiments on human seminal spermatozoa suggests that functional membrane sulfhydryl groups are not involved in the process while in human fibroblasts, use of sulfhydryl blocking agent such as mersalyl significantly reduces zinc uptake, indicating sulfhydryl groups may be important for the uptake process (2, 79). In addition, studies on human skin fibroblasts suggest that at 37°C, initial rapid zinc uptake is membrane bound which reaches a plateau within 10 minutes, then is followed by a linear increase of the internalized portion of zinc. The internalization of zinc is supported by the correspondence between the amount of zinc accumulated in the cells and the estimated zinc requirement by growing fibroblasts. The conversion from membrane bound to the internalized compartment requires sulfhydryl groups and does not seem to depend on metabolic energy (2). Electron microscopic analysis based on a rat macrophage ascetic tumor cell line revealed that upon incubation in the presence of 23 mM ZnCI , intracellular^/ 2  12  incorporated zinc is located as electron dense particles associated with the plasma membrane, endoplasmic reticulum, mitochondrial membrane, nuclear membrane and chromatin, and in the cytoplasm (66). Data is lacking on cellular location of zinc in normal, untransformed cells. It may differ from that of tumor cells since during malignant transformation, the plasma membrane acquires several new glycolipids or glycoproteins (66). In vivo, zinc is capable of binding to more than one ligand in plasma. It is  unclear which ligand is involved in the zinc uptake process in vitro and whether information obtained via in vitro experiments is representative of physiological zinc transport across the membrane. 3.  Stabilization of biomembrane and protection by zinc of cells against membrane damage by haemolytic agents  It has long been known that zinc stabilizes various biological membranes (19). Several experiments based on gastric ulcer models in rats indicating that zinc has a significant anti-ulcer effect that has been ascribed to its stabilizing effect on the gastric mast cells and lysosomal biomembranes (16,103). It has been suggested that zinc stabilizes membranes by 1) interaction with some functional groups of intrinsic macromolecular components of the membrane such as thiol groups of membrane proteins to form stable mercaptides. This theory is  13  supported by the finding that membrane stabilization can also be obtained by using thiol blocking agents and that -SH blocking agents decrease the uptake of zinc by cells. 2) interference with metal catalyzed lipid peroxidation and 3) interaction with enzymes controlling the integrity of the membranes (19). Both of the two latter mechanisms have been ascribed a role in lysosomal membrane stabilization (17). Recently, a novel role has been attributed to zinc protecting cells against membrane damage by pore-forming agents. Some cytotoxic agents such as certain paramyxoviruses, bacterial and animal toxins as well as activated complement and cytolysin isolated from cytotoxic lymphocytes damage cells by creating pores or channels on membrane through which ions and metabolites leak. This kind of direct damage to plasma membrane integrity can be prevented by zinc (5). It has been shown that one of the cytotoxic agents, S. aureus toxin, increases the permeability across the plasma membrane of mouse mammary gland cells both in vivo and in vitro, and that the increase can be inhibited by 0.6x1 CH to 0.2x10" M Zn (51). Other in vitro experiments using erythrocytes and Lettre cells also demonstrated that the increase in permeability can be prevented by increasing extracellular Zn concentrations 3  2+  2+  (<10-4 M) (6).  Zinc acts from extracellular sites by closing preformed pores (5, 62). Supporting evidence comes from the experiment using bilayer models of pure phospholipids, in which, when exposed to the pore forming agent, single channels were induced. In the presence of Zn , channels were still formed. However, the channels spent a longer time in the closed state as opposed to the open configuration. This implies that channels are "gated" or "guarded" by Zn+ (62). 2+  2  14  Other divalent cations such as Ca , Cu have been shown to possess similar effects (6). Since zinc, unlike Ca and Cu , is comparatively innocuous in experimental animals and humans and is more than 10 times as effective as Ca in inhibiting leakage (51), and it is impractical to raise plasma Ca levels except in case of hypocalcaemia while raising Zn level is feasible and moderate increase eg. 50% above normal is not detrimental (11, 28), the use of zinc to combat certain infectious and inflammatory diseases seems possible. 2+  2+  2+  2+  2+  2+  2+  4.  Role of zinc in gene expression and cell division  Zinc is involved in a variety of functions, among which its role in gene regulation is of primary importance. Evidence has been obtained which shows that eukaryotic DNA and RNA polymerases, thymidine kinase, and reverse transcriptase are zinc metalloenzymes that require tightly incorporated zinc at active sites (97). Several in vivo studies have demonstrated a general reduction of DNA synthesis, decreased incorporation of thymidine into nuclear DNA and reduction in activity of thymidine kinase in animals fed a zinc deficient diet (23).  In vitro  experiments showed that cell growth in zinc-deprived cultures is arrested in S and G  2  phases of cell cycles, suggesting that a critical zinc dependent step in cell cycle is blocked by zinc deficiency (25). A recent study on 3T3 fibroblasts revealed that zinc is required before the start of S and at the  IS transition phases (15).  It is reported that zinc at concentrations from 1x10' M to 5x10-4 M stimulate 4  DNA and RNA synthesis and proliferation of human peripheral lymphocytes (9, 46). In addition, zinc at concentration of 4x10' M also increase EGF-stimulated DNA 5  15  synthesis of primary mouse hepatocytes in vitro. However, in the presence of fetal calf serum, supplementation of zinc to the medium has no effect on DNA synthesis, possibly due to the binding of zinc by serum proteins (48). Zinc reacts with DNA nucleotide bases and stabilizes the double helix. The role of zinc for biological nucleotide synthesizing systems is considered to coordinate the 3'-hydroxyl group of the primer chain and accelerate deprotonation from the hydroxyl group. Theoretical studies suggests that zinc may have been selected inevitably instead of being a historical accident during the process of prebiotic evolution as zinc binds with 3'-hydroxyl group with higher affinity and accelerates the deprotonation more efficiently than other divalent cations (86). Zinc is also an important structural component in nucleic acid binding and gene regulatory proteins. The activity of these proteins include sequence-specific interactions with single and double stranded DNA. Structural studies of these proteins revealed that they are zinc-finger proteins. Basically, zinc finger designates a small structural domain in which a short amino acid sequence binds a zinc ion through the cysteine and histidine residues. Genetic and biochemical experiments have identified the zinc finger regions of these proteins as the sites of the specific binding of protein-DNA interactions ( 4, 7, 14). 5.  Effect of zinc on collagen synthesis The effect of zinc on collagen synthesis has received attention because of its  influence on wound healing, which was discovered by chance. In 1953, during studies of the effect of dietary amino acid on wound healing in rats, commercially bought beta-phenyllactic acid, an analog of phenylalanine, surprisingly was found to increase  16  the rate of healing over that in controls. However, purified beta-phenyllatic acid itself had no effect. This product was later found contaminated with zinc (64). Epithelialization and granulation tissue formation are two of the major steps in wound healing. In experimental zinc-deficient animals, delayed epithelialization has been observed along with a lower collagen content in granulation tissue and a proportionally larger amount of soluble collagen. Supplementation with zinc was found to normalize wound healing in these animals (99). A close relationship between zinc and wound healing, especially granulation tissue formation is clearly demonstrated by a study using [ Zn], which shows more zinc is accumulated in the wound than in the adjacent tissue and that its concentration reaches maximum at the time of granulation tissue formation. After the wound is completely healed, no [ Zn] is found in scar tissue (64). This migration of zinc into the wound and out of scar tissue suggests that zinc plays a role in the healing process. Macroscopically, less secretion and a quicker healing rate were observed in zinc supplemented subjects. Microscopically, a larger number of fibroblasts, collagen fibers and less inflammatory response were observed in granulation tissue of the experimental group supplemented with zinc (34). 65  65  It has also been claimed that zinc deficiency reduces the activity of lysyloxidase and alters the cross-linking pattern of the collagen. It was reported that in zinc-deficient rats, the solubility of skin collagen was about 20% higher than that of pair-fed controls, and that there was more than 45% reduction in aldehyde content of collagen, implicating a possible inhibition of lysyloxidase (53). In addition, zinc is involved in the metabolism of bone and cartilage matrix. At lO-stolO^M, zinc increases in vitro bone total protein synthesis by 30% (107). Type II collagen and proteoglycan are the prominent organic components of cartilage matrix. It is reported that the addition of zinc prevents cartilage swelling which is associated with increased degradation of proteoglycans but collagen seems to be unaffected (13).  17  It is clear now that chronic zinc deficiency impairs wound healing, which can be corrected by restoration of zinc to the normal level. However, it is unclear as to whether the administration of supplemental zinc can accelerate healing to an above-normal rate, and whether it is as effective in the zinc sufficient as in the zinc deficient group. 6.  Role of zinc in immune response Zinc has been shown to play a significant role in host immune response,  especially cell-mediated immune response (19). Patients with congenital defects in zinc homeostasis have shown improvement in neutrophil migration by oral supplementation of zinc (3, 47). Mouse peripheral macrophages from mice fed a diet deficient in zinc show a reduced capacity to take up and destroy parasites; both functions are restored by the addition of 7.5x10 M zinc (102). Treatment with 1x10 M zinc of aging spleen cells with diminished immune potency enhances antibody production to the level of young counterparts provided that zinc is added within 24 hrs of incubation, suggesting that early events in antibody formation which include macrophage processing and presenting of antigen as well as IL-1 production, may be affected by zinc (101). _5  _4  Zinc has been reported to stimulate the release of cytokines such as tumor necrosis factor and IL-1 B of cultured human peripheral blood mononuclear cells in a concentration-depended manner with secretion peaks occurring at 0.25 mM and 0.12 mM zinc, respectively (77). It has also been shown that zinc inhibits pokeweed mitogen-induced generation of immunoglobulin secreting cells through an non-specific augmentation of T-lymphocytes (82). Furthermore, zinc can inhibit both in vivo and  18  in vitro histamine release by mast cells (19). Generally it is conjectured that the action  of zinc in host defense systems is through its anti-inflammatory and anti-bacterial mechanisms. 7.  In vivo and in vitro adverse effects of zinc  Generally zinc is considered relatively innocuous compared with other divalent cations and humans exhibit tolerance to high intakes of zinc. The Recommended Dietary Allowance for zinc is 15 mg/day. Symptoms of acute toxicity due to accidental ingestion of large doses of zinc, though uncommon, are mainly gastrointestinal such as vomiting and diarrhea. Prolonged pharmacological use of zinc (100-300 mg / day) usually results in copper deficiency, characterized by classical symptoms of hypocupremia, anemia, leukopenia and neutropenia (28). Alterations in immune response in human subjects on pharmacological doses of zinc intake have also been reported. Depression in lymphocyte stimulation index, chemotactic migration and phagocytosis of bacteria have been observed (11). The in vitro detrimental effect of zinc has been studied on several cell lines including human prostate cells, lymphoid cells, fibroblasts, B16 melanoma cells and mammalian epithelial cells. The preponderance of in vitro data suggests that zinc becomes inhibitory at concentrations above 10' M. Reduction of cell proliferation and detachment of cells from the substrate have been observed (11). 4  F.  Objectives of this thesis  Both in vivo and in vitro studies strongly suggests that VSC may contribute to the pathogenesis of destructive periodontal disease. 19  Zinc is an essential trace element that participates in a variety of biological processes in humans. Zinc has been used in mouth rinse and tooth paste formulations which have been reported capable of inhibiting plaque accumulation and oral malodor, which is primarily due to the presence of VSC. Its ability to nullify the effect of VSC on permeability of mucosa implies that it may also be capable of reversing the adverse effect of VCS on the metabolism of cultured human gingival fibroblasts. The principal aims of this thesis are to employ in vitro human gingival fibroblast cell culture system, to establish the maximal zinc concentration that is non-inhibitory, then investigate whether this zinc can counteract the adverse effects of CH SH on the total protein, DNA, and collagen synthesis, and proline transport. 3  20  Section II Materials and Methods A.  Human gingival fibroblast cultures The fibroblast cell lines employed in this study were derived from healthy  gingival tissue explants previously frozen and stored in liquid nitrogen. Frozen fibroblasts (1x10 per vial) maintained in a suspension of 92% FCS 6  and 8% dimethyl sulphoxide were thawed in lukewarm water, then transferred to 25 cm Falcon flasks in 5 ml growth medium and incubated at 37°C in an 2  atmosphere of 95% air / 5% C0 . The employed growth medium consisted of mixtures 2  of 94 ml of Dulbecco's Modified Eagle Medium (DMEM) (glucose 4.5 mg / ml) supplemented with 1.0 ml penicillin / streptomycin (104 units / ml and 10 mg / ml), respectively, (Gibco Laboratory), 2.0 ml glutamine (0.2 M, Gibco) , 1.0 ml sodium pyruvate (11 mg /1, Gibco), 1.0 ml non-essential amino acids (L-alanine, 0.89 mg / ml, L-asparagine»H 0,1.5 mg / ml, L-aspartic acid, 1.33 mg/ml, L-glutamic acid, 1.47 2  mg/ml, L-glycine, 0.75 mg / ml, L-proline, 1.15 mg/ml and L-serine, 1.05 mg/ml, Gibco Laboratories), 1.0 ml ascorbic acid (5 mg / ml) and 15% FCS. After overnight attachment of cells, the medium was changed to normal growth medium or complete DMEM containing the same ingredients as previously described except for 10% FCS. The medium was changed twice a week until fibroblasts grew to confluence. To harvest cells, medium was discarded, cultures were rinsed twice with phosphate buffer saline (PBS). The cells were then detached by treatment with 0.25% trypsin (Gibco) for  21  three minutes. After trypsinization, normal growth medium was added to protect cells from damage by trypsin. The cells were then pelleted by centrifugation for five minutes at 1,500 rpm (Centaur 2, MSE). The supernatant was decanted, and the cells were resuspended in 75 cm Falcon flasks and incubated in normal growth medium. After 2  cultures reached confluence, cells were again trypsinized and subcultured. Since different culture conditions, such as medium composition and cell density greatly affect the amount and the types of collagen produced by cultured fibroblasts, control and test cultures in all experiments were carried out in media of similar composition and at similar cell densities. For each individual experiment, confluent cultures of the same passages were trypsinized, pooled and the cell number per unit volume was determined by an electronic cell counter (Coulter Electronics, Inc. Hialeah, Florida). Fibroblasts of equal number were attached in Petri dishes and subsequently subjected to control and test conditions. In addition, because of the fibroblast heterogeneity eg. the possible presence of trypsin-resistant and trypsin-sensitive subgenotypes, the fibroblasts used in all experiments were from cultures of less than ten passages. In all studies, cultures were routinely washed with PBS three times before the changing of the incubation media.  B.  Zinc chloride stock solutions Zinc chloride stock solutions (15x10" M, pH, 5.7) were made from 97.7% pure 2  ZnCI powder at a concentration approximately ten times higher than that of 2  22  Lavoris  mouth rinse (0.22%,1.6x10 M). -2  Direct  flame  atomic absorption  spectrophotometric analysis (CanTest) of ZnCI solution samples showed that the 2  prepared ZnCI solutions were within an accuracy of 10% and a precision of 2  (13.6 ±0.1 )x10- M. 2  For each experiment, fresh solutions of ZnCI were prepared. Serial dilutions 2  of ZnCI from stock solutions were made to obtain ZnCI concentrations as follows: 2  2  1.5x10- M, 1.0x10-M, 0.5x10- M, 1.0x10-M, 0.5x10"M, 1.0x10-M, 0.5x10 M, 1  1  1  2  2  3  -3  1.0x10-4 M, 0.5x10- M and 1.0x10" M (ten times the final concentrations present in 4  5  the cultures). The stock solution contained a ten-fold higher concentration of ZnCI than 2  the final concentration. For example, 0.2 ml of 1.5x10-M ZnCI could be added to 1  2  1.8 ml of DMEM resulting in the final concentration of 1.5x10- M. 2  C.  Assessment of the effect of zinc on HGF cultures  1.  Analysis of the total protein synthesis in the presence of zinc by trichloric acid (TCA) precipitation  This study was designed to establish the maximal zinc concentration that itself was not inhibitory to cultured HGF. For this study, confluent fibroblast cultures were trypsinized, pooled and cell number per unit volume was determined by the cell counter. Equal number of fibroblasts (3.5x10) were plated in 60 mm Petri dishes and incubated overnight in 2 ml 5  23  DMEM supplemented with 10% FCS. After the cells were attached, the medium was discarded, and the cultures were rinsed three times with PBS, then made quiescent by incubating them in RPMI1640 medium for 48 hours. This step was required to ensure that the results obtained were directly associated with protein synthesis and were not the effects related to cell growth. Following the quiescent period, RPMI1640 was decanted, the cultures were washed three times with PBS, then incubated for 24 hours in DMEM devoid of L-proline but supplemented with L-[ C]-proline (250 mCi / mmole, ICN, 1.5 uCi per 14  1.5 ml medium per dish) either in the presence or in the absence of 10% FCS. During this period, cultures were incubated in the presence of ZnCI concentrations of 0 2  (controls), 1.5x10"M, 1.0x10-M, 0.5x10"M, 1.0x10"M, 0.5x10"M, 1.0x10"M, 2  2  2  3  3  4  0.5x10" M, 1.0x10" M, 0.5x10' M and 1.0x10"M (final concentration). Medium 4  5  5  6  was pre-filtered after the addition of ZnCI since it forms a visible precipitate with 2  FCS at high concentrations (>0.5x10 M). _2  At  the end of the labeling period, two protease inhibitors,  phenylmethylsulfonyl fluoride (PMSF) and 4-chloromercuribenzoic acid (PCMB) (50 ug / ml, final concentration), were added to the medium, and the medium was collected. In experiments carried out in the absence of FCS, FCS was added to the medium to 2% concentration to serve as a carrier protein. Then the medium was precipitated by the addition of 10% TCA /1% Tannic acid (final concentration) and then centrifuged (Eppendorf, Model 5142) for 30 minutes at 4°C. Precipitates were washed three times with ice-cold 10% TCA /1% Tannic acid. The [ C]-radioactivity in the final 14  pellets was determined by liquid scintillation counting (LSC) (Philips, PW. 4700).  24  Activities associated with the precipitates represented the total labeled proteins that were synthesized and secreted by fibroblasts during 24 hrs of incubation. Cells were washed five times with ice-cold PBS and scraped into two aliquots of 0.75 ml 10% TCA /1% Tannic acid. Samples were transferred into 1.5 ml Eppendorf centrifuge tubes and centrifuged for 30 minutes at 4°C. Pellets were washed three times with ice-cold 10% TCA / 1% Tannic acid and [ C]-activities in the final 14  precipitates were determined by LSC. They represented total labeled proteins located inside the cells and proteins associated with the cell membranes. The [ C]-activity in 14  both the medium and cellular precipitate fractions collectively represented the total amount of labeled undegraded proteins synthesized during the 24-hr pulsing period. 2.  Measurement of [ Zn] uptake by HGF 65  For determination of zinc uptake by the cells, equal number of fibroblasts (3.5x10 ) were plated in 60 mm Petri dishes and incubated overnight in complete 5  DMEM. After the attachment, fibroblasts were made quiescent in FCS free RPMI 1640 medium for 48 hrs. Following the quiescence, cultures were activated with DMEM supplemented with 10% FCS in the presence of ZnCI concentrations of 0 (control), 2  1.5x10- M, 1.0x10- M, 0.5x10-2 M, 1.0x10 M, 0.5x10 M, LOxlO- M, 0.5x1 O^M, 2  2  -3  3  4  1.0x10- M, 0.5x10- M and 1.0x10- M, and pulsed with [ Zn] (3.3ugZn/mi, 5  5  6  65  0.893 mCi / ml, Amersham, 10ul per dish) for 24 hours. At the end of the reaction, PMSF and PCMB were added to the medium . The medium was collected, then dialyzed exhaustively against 0.9 % NaCl for 24 hours at 4°C (m.w. cut off, 10,000). The [ Zn] activity in non-dialyzable portion was determined by a gamma counter 65  25  (Auto-Gamma 5000 Series Gamma Counter, PACKARD). After the cells were washed five times with ice-cold PBS and trypsinized, their [ Zn] activities were also determined by gamma counting. The actual amount of zinc that was accumulated by the fibroblasts was calculated from the known labeled to cold zinc ratio. 65  3. Effect of zinc on the attachment of the fibroblasts Equal number of fibroblasts (2.5x10) were plated in 60 mm Petri dishes and 5  incubated overnight in DMEM supplemented with 10% FCS in the presence of 0 (control), 1.5x10- M, 0.5x10" M, LOxlO M and 1.0x10"M ZnCI . Following 2  3  -4  5  2  the attachment, cells were washed with PBS, trypsinized and pelleted by centrifugation for 5 minutes at 1,500 rpm. Cell number was determined by electronic counting. 4.  Effect of zinc on the proliferation of the fibroblasts An 11-day growth curve study was conducted to determine whether HGF  proliferation was affected by 1.0x10" to 1.5x10- M ZnCI . 5  2  2  For this experiment, equal number of fibroblasts (2.5x10) were plated in 5  60 mm Petri dishes and incubated overnight in DMEM supplemented with 10% FCS. After the attachment, medium was discarded, cultures were washed with PBS and subsequently incubated in complete DMEM that contained ZnCI of 0 (control), 2  1.5x10- M, 0.5x10"M, 1.0x10- M and 1.0x10"M, which served as growth media. 2  3  4  5  Cell numbers were determined every other day and at the same time the media in the rest of cultures was replaced with identical fresh growth media. The same studies were  26  also performed on quiescent cell lines to determine whether quiescent and non-quiescent cell lines responded differently to ZnCI . Both applied procedures were 2  similar except that for quiescent cell lines, following attachment, the cells were incubated in RPMI 1640 devoid of FCS for 48 hours before they were subjected to growth medium containing various concentrations of ZnCI . 2  5.  Light microscopic (LM) and transmission electron microscopic (TEM) studies of HGF treated with zinc i) Procedures for light microscopic study HGF were attached, made quiescent, and incubated in DMEM in the presence  of 0 (control), 1.5x10" M, 0.5x10" M, LOxlO" M, 0.5x10" M and 1.0x10" M ZnCI . 2  3  4  4  5  2  Following 24-hr incubation, the medium was discarded, the cells were washed once with medium free of FCS, and then fixed for 1 hr by immersion in 2.5% glutaraldehyde (in 0.1 M phosphate buffer). After fixation, the cells were washed three times with distilled water for 15 minutes and stained for 15 seconds with 1% toluidine blue (in 1% Borax. pH, 11). Immediately following staining, the cells were rinsed thoroughly with distilled water before they were examined under the LM. ii) Protocols for transmission electron microscopic study In order to observe any ultrastructural changes in HGF treated with ZnCI , 2  TEM investigations were conducted on equal number of fibroblasts (2.0x10) attached 5  in 30 mm culture dishes and made quiescent for 48 hrs in serum-free medium. Cells  27  were then incubated in DMEM for 24 hrs in the presence of 0 (control), 1.5x10"M, 2  0.5x10- M, 1.0x10"M and 0.5x10* M ZnCI . Subsequently, these cultures were 3  4  4  2  processed for TEM observation as follows: a. Fixation process Cultures were first rinsed twice with serum-free medium. Reagents used in the following procedures were kept at 4°C until use. After rinsing, fibroblasts were fixed in 2.5% glutaraldehyde for 45 minutes at 4°C, then washed twice for 10 minutes with PBS. Subsequent procedures were performed with dishes maintained at 0°C. Following primary fixation, secondary fixation was achieved by treating the cells for 45 minutes with osmium tetraoxide. Then the cells were washed twice for 10 minutes with PBS and once with distilled water. Following washing, the cells underwent dehydration first in 30% ethanol for 5 minutes then in 50% ethanol for an additional 5 minutes followed by treatment with a third fixative, uranyl acetate (1% in 70% ethanol) for 30 minutes. After the third fixation, subsequent procedures were performed at room temperature. Stepwise, fibroblasts were dehydrated by passage through a sequence of solutions of 70% ethanol for 5 minutes, 90% ethanol for 5 minutes and 100% ethanol twice for 10 minutes. Dehydration of fixed specimens was necessary because the embedding media employed was water-insoluble. b. Embedding The employed embedding medium was composed of 30 ml dodecenyl succinic anhydride (DDSA), 10 ml araldite 502 resin and 12.5 Epon 812 resin (EPON) 28  (J.B. EM Service In.), which were mixed thoroughly for 5 minutes before use. Dehydrated specimens were immersed first in a mixture of 100% ethanol and embedding media (v:v=1:1) for 1 hr, then a mixture of 100% ethanol and embedding media (v:v=1:3) for another hour, and finally in 100% embedding media for 1 and 1/2 hours. During the entire 3 and 1/2-hr period, dishes were slowly shaken on a Junior Orbit Shaker (Lab-Line Instruments, Inc.). Specimens were then immersed in the final embedding medium consisting of 100% EPON and catalyst, Tri (dimethylamino methyl) phenol (DMP<30) (J.B. EM Service In, 1.25 ml DMP«30 in 52.2 ml EPON), and first left in a 37°C oven for 24 hrs then in a 60°C oven for two days. c. Sectioning Before sectioning, blocks containing the preserved fibroblasts in embedded specimens were trimmed down to a suitable size and shape. The face of the specimen blocks which contained cells were hand trimmed to a trapezoidal shape. Thick sectioning (1 urn) was performed on a Sorvall ultramicrotome (Model MT-2). The sections were then stained with toluidine blue and examined under the light microscope to ensure that the face of the block from which ultra-thin sections for electron microscope examination would be cut contained high density areas of embedded fibroblasts. Ultra-thin sectioning (60 nm) was performed on a thin sectioning apparatus (Sorvall Instruments, Model MT 6000) by an experienced lab technician. d. Section staining Ultra-thin sections were mounted on a copper grid. Sections were first stained with uranyl acetate (6% in 70% ethanol) for 30 minutes with the sections facing down.  29  Then, the sections were washed sequentially by dipping the grids repeatedly into distilled water in a four specimen tube apparatus (ten dips per tube). After washing, grids were placed with the section side facing up on filter paper to dry. The sections were then stained with lead citrate for 5 minutes. For staining, grids with the section side facing down were placed onto lead citrate droplets in a covered plate that also contained KOH to absorb C0 because lead staining 2  solution reacts with the merest trace of carbon dioxide to form lead carbonate which is insoluble and interferes with TEM observation. The widely-used double staining employed in the procedure with uranyl acetate followed by lead citrate produces more contrast and stains more components than either stain alone. After secondary staining, sections were again washed thoroughly by the previously described washing sequence, then dried on the filter paper with section side facing up. The dried sections were then examined by TEM .  D.  Measurement of the reversal effect of zinc on the metabolism of HGF exposed to CH3SH  1.  CH SH gassing system 3  In all of the below described studies, control and test cultures were incubated simultaneously in separate sealed chambers perfused with 95% air / 5% C0 from a 2  common source. The line leading to the test chamber was equipped with a calibrated  30  permeation tube which emitted a fixed concentration of CH SH to the air mixture. The 3  flow rate of air mixture in both chambers was 60 ml / minute. Additional 600 ml reservoirs were inserted into teflon lines leading to both chambers to preheat gases to 37°C prior to their entry into the culture chambers. Petri dishes containing distilled water were placed at the bottoms of each chamber to humidify the gases and reduce the evaporation of the media (Figure 2). For the test cultures, the air / C0 atmosphere was admixed with 15 ng of 2  CH SH per ml of the air / C0 mixture. This amount corresponds to 30 nmoles CH SH 3  2  3  per 100 ml head space. Although VSC levels in the mouth air of periodontal diseased patients normally reach 2 nmoles per 100 ml, the higher concentration was chosen for experimentation since gas chromatography profiles from patients depict volatiles only present in the mouth air. Such gases are subject to dilution and are unlikely to represent the true concentrations present in the gingival crevice which is the area associated with active periodontal disease. Furthermore, analysis of head-space of putrefied saliva system exhibits VSC levels up to 1000 times greater than those present in the mouth air. Since previous investigations have shown that CH SH is a 3  more potent inhibitor than H S, CH SH was chosen as test agent in all the following 2  3  studies. 2.  Analysis of the reversal effect of zinc on the synthesis of total TCA-precipitable proteins For this study, equal number of fibroblasts (5x10 per dish) were plated in 5  60 mm Petri dishes and incubated overnight in complete DMEM. After the attachment,  31  60 m l / m i n flow  V22///,// $ incubator  121  (37°C) '  —  ^  VX  Control  95% Air 5%  CH SH Permeation Tube  CO2  60 ml / min  3  Mixture  15ng/ml  flow  e Chamber C u l t u r  ^ /  PUS Test  ,  r  Excess Flow Vented  30°C  Figure 2.  600ml Reservoir  Apparatus for incubating control and test cultures  Control and test cultures were enclosed in separate 2000 ml chambers maintained at 37°C in a standard temperature-controlled incubator. Cultures were perfused by a continuous flow of 95% air / 5% C0 at 60 ml / minute. Additional 600 ml reservoirs 2  were inserted into gas lines leading to the chambers to ensure equilibration of air mixtures to 37°C prior to entering the culture chambers. A permeation tube added to the air /C0 line leading to the test chamber delivered a continuous fixed concentration 2  of CH SH. 3  32  cells were made quiescent for 48 hrs in RPMI 1640 devoid of FCS. Following the quiescence, cultures were activated in DMEM deficient in FCS and L-proline but supplemented with L-[ C]-proline (250 mCi / mmole, 1.5 uCi in 1.5 ml medium per 14  dish) and pulsed for 24 hrs. During this period, control cultures were exposed to 95% air / 5% C0 while test cultures received 95% air / 5% C0 admixed with 2  2  15ng/ml ofCH SH in the absence and presence of 0.5x10" M and 1.0x10- M 4  5  3  (final concentration) ZnCI . 2  At the end of the pulsing period, PMSF and PCMB were added to all cultures, medium was collected, to which 2% FCS was added to serve as a carrier protein, then precipitated by the addition of 10% TCA / 1% Tannic acid (final concentration) and centrifuged (Eppendorf 5412, Microfuge) for 30 minutes at 4°C. Precipitates were washed three times with ice-cold 10% TCA /1% Tannic acid before the [ C]-activity in the precipitates was determined by LSC. The cellular fractions were washed five times with ice-cold PBS, then scraped into two aliquots of 0.75 ml 10% TCA / 1% Tannic acid and centrifuged. The pellets were washed and the radioactivity associated with the cellular fractions was also determined by LSC. The combined [ C]-activity found in both medium and cellular fractions represented the total TCA-precipitable proteins synthesized by fibroblasts during the 24-hr incubation period. 14  14  3.  Reversal effect of zinc on proline transport  Amino acid uptake by cells is a membrane-associated active transport process. Measurement of proline transport across the cell membrane yields information on membrane integrity. Alterations in proline uptake would be reflected by changes in the collagen synthesis.  33  For this study, equal number of fibroblasts (7x10 per dish) were attached in 5  60 mm Petri dishes, and made quiescent during 48 hrs of incubation in 2 ml RPMI 1640 devoid of serum. The cultures were then incubated for 30 minutes in DMEM containing 10 % FCS devoid of L-proline but supplemented with L-[ C]-proline (1.5 uCi per 1.5 ml 14  medium per dish). During the pulsing period, control cultures were gassed with 95% air / 5% C0 while the test cultures were exposed to 95% air / 5% C0 admixed 2  2  with 15 ng / ml CH SH in the absence and presence of 0.5x10"M and 1.0x10" M 4  5  3  ZnCI . 2  Following subsequent removal of the media, the cells were washed five times with ice-cold PBS and then scraped into two aliquots of 0.75 ml 10% TCA /1% Tannic acid and transferred into 1.5 ml Eppendorf centrifuge tubes. Following centrifugation for 30 minutes at 4°C, the radioactivity retained in the supernatants was determined by LSC. Since uptake of [ C]-proline would be retained inside the cells during the initial 30 minutes of incubation, unincorporated label in the medium was removed by washing. The activity present in the supernatants from ruptured cells represented L-proline transported into the cells during the labeling period. 14  4.  HPLC analysis of the effect of zinc on collagen and total protein synthesis As hydroxyproline is an identifying amino acid component of the collagen  molecule, quantitation of hydroxyproline is used to determine the amount of collagen in a  sample.  In  recent  years, detection of derivatized amino  34  acids with  phenylisothiocyanate (PITC) using high-performance liquid chromatography (HPLC) has been proven to be sufficiently sensitive to detect hydroxyproline in extremely low concentrations (1 picomole) present in biological systems, such as fibroblast cultures. Furthermore, the method gives good separation of hydroxyproline and proline (105). For this phase of the study, equal number of fibroblasts (5x10 per dish) were 5  plated in Petri dishes, made quiescent for 48 hrs in RPM 11640 free of serum. Then, cultures were incubated in DMEM devoid of L-proline and FCS but supplemented with L-[ C]-proline (1.5 uCi in 1.5 ml medium per dish) and pulsed for 24 hrs. During the 14  pulsing period, control cultures were incubated under 95% air / 5% C0 atmosphere 2  while test cultures were subjected to 95% air / 5% C0 admixed with 15 ng / ml CH SH 2  3  in the absence and presence of 1.0x10" M ZnCI-containing media. 5  2  After 24 hrs of incubation, two protease inhibitors , PMSF and PCMB were added to the media. The media was then collected and cultures were washed twice with 0.5 ml aliquots of ice-cold PBS. The resulting rinsing solutions were added to the original media of corresponding samples. Aliquot samples of media (1 ml) were taken and transferred to 13x100 mm test tubes, then hydrolyzed in 6N HCL (final concentration) at 126°C for 16 hrs. Following hydrolysis, samples were lyophilized (Model 75040 freeze dryer, Labconco, Kansas City, KS, U.S.A.), then redried in 2 ml of redrying solution consisting of 2:2:1 mixture (by volume) of 95% ethanol: water: triethylamine (TEA). This procedure is necessary to make samples basic as the effect of the subsequent derivatization reaction with PITC is maximal under basic conditions. Redried samples were then derivatized in 200ul of reagent for 20 minutes at room temperature. The derivatization reagent consisting of 7:1:1:1 solution (by volume) 35  of 95% ethanol:TEA:water:PITC was mixed thoroughly before use. Then the derivatized samples were finally evaporated to complete dryness. This is an important step in the procedure as incomplete drying will adversely affect the recovery of the derivatized products. Another aliquot sample of media (1ml) was taken and exhaustively dialyzed against 0.5N acetic acid (m.w. cut off, 3,500) for 48 hrs at 4°C. Resulting dialyzed samples were collected, lyophilized, hydrolyzed and again lyophilized. The latter samples were then redried, derivatized and dried thoroughly. The cells were washed three times with ice-cold PBS, then suspended in 1.5 ml of 0.5 N acetic acid. The samples were then transferred to 1.5 ml Eppendorf centrifuge tubes maintained at 4°C, homogenized by sonication for 1 minute (50% out put power, Fisher Model 300) then centrifuged for 15 minutes at 13,000 rpm (Eppendorf). After the supernatants were collected, the pellets were again homogenized in 1.0 ml 0.5N acetic acid and centrifuged. The supernatants were collected and combined with corresponding samples. An aliquot sample of combined first and second supernatants (1 ml) was hydrolyzed and lyophilized. Lyophilized samples were redried, derivatized and again completely dried. Other aliquot samples of combined supernatants (1ml) were exhaustively dialyzed against 0.5N acetic acid for 48 hrs at 4°C. Stepwise, the dialyzed samples were lyophilized, hydrolyzed and again lyophilized. Then, they were redried, derivatized and again dried. All reagents used in the preparation of samples for HPLC analysis were freshly made and all HPLC analysis were performed on a fully-automated  36  high-performance liquid chromatography unit (Water Assoc.) which consisted of a Model 721 programmable system controller, two Model 510 HPLC pumps, a Model 710 WISP (an automatic sample processor and injector), a model 730 data module, and a model 490 programmable multiwavelength detector operating at 254 nm. The unit was connected to a Gilson Model 201 fraction collector and a Pico.Tag reverse phase column (Waters Assoc.) operated at 38°C. The dried samples were reconstituted in 200ul sample diluent solution (Water Assoc.) and filtered through a STHV004NS filter (Millipore, Japan) before sample solutions were analyzed. For HPLC analysis, the mobile phase was programmed at a flow rate of 1.0 ml / min starting with 100% buffer A, followed by a linear gradient to 50% of buffer B for 10 minutes, then increased to 100% buffer B for 30 seconds and maintained at 100% buffer B for an additional 1.5 minutes with the flow rate increased to 1.5 ml / min at the last 30 second time interval of the 12 minute run. The column was then equilibrated with 100% buffer A for the next 8 minutes. During the 12 minute run, post-column fractions of 0.5 minute time interval were collected on Gilson fraction collector and [ C]-activity associated with each fractions was quantitated by LSC . 14  Buffer A of the mobile phase consisted of 60 ml acetonitrile and 940 ml of 138 mM sodium acetate trihydrate buffer (pH, 6.4) containing 0.05% (v/v) triethylamine (TEA). Buffer B consisted of 600 ml acetonitrile and 400 ml Norganic water which was prepared by passage of deionized water through a Norganic cartridge (Waters Assoc.)  37  5.  Effect of zinc on DNA synthesis by CHSH-exposed HGF 3  Equal number of fibroblasts (2.5x10 per well) were plated in Linbro wells and 4  incubated overnight in complete DMEM. Following the attachment, the cells were made quiescent by incubation for 48 hrs in RPMI1640 devoid of FCS. Then, the cultures were first incubated for 22 hours in RPMI 1640 supplemented with 10% FCS and Vit C, followed by an additional 4 hrs of incubation in the same media to which 50ul of labeling solution was added to each well. The labeling solution was composed of 0.5 uCi l-dUrd (1963 Ci / mmole, Amersham), an analog of thymidine, and 10 M 125  -6  5-fluorodeoxyuridine (FURD) in serum-free RPMI 1640 media. As previous studies showed that DNA synthesis by cultured fibroblasts reached its peak between 24-26th hours of incubation, control cultures were incubated for 26 hrs under 95% air / 5% C0  2  in the absence and presence of ZnCI at concentrations of 1.0x10 M, 0.5x10"M, -4  4  2  1.0x10" M, 0.5x10 M and 1.0x10- M in the media while test cultures were incubated 5  -5  6  under 95% air / 5% C0 mixed with 15 ng / ml CH SH in the absence and presence of 2  3  varying concentrations of ZnCI following 48 hr quiescence. 2  Following removal of the media, cells were washed thoroughly with ice-cold PBS and harvested in two aliquots of 0.5 ml of 0.1 N ice-cold NaOH. The samples were then transferred to gamma-counting tubes and immediately precipitated with 1 ml of 20% TCA and capped. Samples were then centrifuged at 2,000 g for 30 minutes at 4°C (Model PR-6, International Refrigerated Centrifuge). After the supernatants were discarded, the precipitates were washed with ice-cold 10%TCA to remove unbounded [ l]-dUrd. The radioactivity associated with the precipitates was determined by gamma counting. 125  38  E.  Statistics Since all experiments involve comparison among multiple means, one-way  analysis of variance (ANOVA) was employed to test for significance of the treatments and a postiori tests (Tukey test or multiple comparison test) were conducted to determine the significance of differences between specific groups.  39  Section III Results  A.  Effect of zinc on the total TCA-precipitable protein synthesis bv HGF The initial experiment was undertaken to establish the maximal concentration  at which ZnCI is non-inhibitory to HGF cultures. Experiments were conducted for 2  24-hr pulsing periods both in the presence and absence of FCS and ZnCI  2  concentrations ranging from 1.0x10" M to 1.5x10' M (final concentration) added to the 6  2  culture media. Comparison between the amount of protein synthesized by fibroblasts in the presence and absence of FCS showed that in the presence of FCS, fibroblasts produced ten-fold more proteins than those synthesized by cells in the absence of FCS (Figures 3 & 4). In both experiments, ZnCI concentrations higher than 1.0x10"M 4  2  significantly reduced the total amount of synthesized TCA-precipitable proteins, and the inhibitory effect was more pronounced on the cellular than the medium fraction (Tables 1 & 2). In the presence of FCS and non-inhibitory levels of ZnCI (<1.0x10- M), the 4  2  distribution of [ C]-labeled protein associated with cells and in the medium was 14  approximately equal except that of cultures exposed to LOxlO^M and 0.5x10' M 4  ZnCI (Table 1). In the absence of FCS and non-inhibitory concentrations of ZnCI , 2  2  approximately two thirds of the total radioactivity was associated with the cells  40  20-y  Zinc Concentration (M, x10 " ) 4  Figure 3:  Total TCA-precipitable protein synthesized by HGF during a 24-hr labeling period in the presence of FCS and various concentrations of ZnCI . 2  The presence of ZnCI at concentrations higher than "I.OxlO M in the culture media -4  2  resulted in an over 92% reduction in total TCA-precipitable proteins synthesized during a 24-hr incubation period (ANOVA, Tukey test, p<0.05). No significant differences were found  between control cultures and those supplemented with 1.0x10" to 6  I.OxlO" M ZnCI (ANOVA, Tukey test, p>0.05). The activity is expressed as dpm per 4  2  Petri dish. The results are based on three separate runs using duplicate samples for each run.  41  Distribution of [ C]-activity between medium and cellular fractions of 24-hr cultures in the presence of FCS and various concentrations of ZnCI. 14  Table 1:  2  Fraction  ZnCUconcen-tration in  the media (M) 0 1.0x10-  6  0.5x10-5 1.0x10-5  0.5x10" 1.0x10" 0.5x101.0x10-  4 4  3  3  0.5x10-2  1.0x10-  2  1.5x10-2  Media (dpm+sd)  Reduction (%)  Cell (dpm+sd)  70454±7372 68070±8815 59580+6409 63553+9173 74956±6918 45940±9324 691911418 6919±1784 3201±397 4027±529 4047±169  3 15 1 +6 35 90 90 95 94 94  89096±6115 77101±12836 81139±14284 83422±12283 105859±17048 101237±14317 3491±913 1778±345 337±62 323±69 272±93  Reduction (%)  13 9 6 +19 +13 96 98 99 99 99  For control and test cultures supplemented with non-inhibitory concentrations of ZnCI  2  (<1.0x10 M), distribution of [ C]-activity was approximately even except for cultures to -4  14  which 0.5x10" M and 1.0x10-M ZnCI were added. The presence of ZnCI at 4  4  2  2  concentrations higher than 1.0x10"M in the media reduced synthesized protein 4  content of both medium and cell fractions, with greater effect on cell associated fractions.  42  0  0.01  0.05  0.1  0.5  1  5  10  50  100  150  Zinc Concentration (M, x10 " ) 4  Figure 4:  Total TCA-precipitable protein synthesized by HGF during a 24-hr labeling period in the absence of FCS but in the presence of various concentrations of ZnCI . 2  Presence of ZnCI in the culture media at concentrations higher than 1.0x10- M reduced the total synthesized TCA-precipitable proteins by over 48% (ANOVA, Tukey test, p<0.05). The higher the ZnCI concentration, the greater the inhibition. No significant differences were found between control cultures and those incubated with 1.0x10- to 1.0x10" M ZnCI added to the media (ANOVA, Tukey test, P>0.05). The activity is expressed as dpm per Petri dish. The results are based on three separate experiments performed in duplicate in each experiment. 2  4  2  6  4  2  43  Table 2:  Distribution of [ C]-activity between medium and cellular fractions of 24-hr cultures in the absence of added FCS but in the presence of various concentrations of ZnCI . 14  2  Fraction  ZnCloConcen-tration in the media (M)  Media (dpm±sd)  0 1.0x100.5x10-  4966±442 3633±913 5050±482 4556±594 5041±644 3319±1208 1024±119 720±208 568±204 481±133 489±124  6  5  1.0x10-5  0.5x10-  4  1.0x10-4  0.5x10-  3  1.0x10-3  0.5x10-  2  1.0x10-2 1.5x10-2  Reduction (%)  27 +2 8 +1 33 79 85 88 90 90  £ejj_ (dpm±sd) 11249±2261 10392±2125 10902±2560 14005±2843 13375±4396 9797±987 7446±388 3601±1994 138±45 77±9 82+2  Reduction  (%)  8 3 +24 +19 13 34 68 99 99 99  Comparison between [ C]-activity in the cellular and media fractions showed that 14  approximately two thirds of the total radioactivity was associated with the cells of control and test cultures incubated in the presence of non-inhibitory concentrations of ZnCI (<1.0x10- M). At higher zinc concentrations, the inhibition was greater on cell 4  2  fractions. As zinc concentration was reduced to a non-inhibitory range, the inhibition was greater on media fraction.  44  (Table 2). When ZnCI was added to the FCS-containing media at concentrations 2  higher than 1.0x1 (H M, most of the [ C]-radioactivity was located in the medium while 14  in the absence of FCS, the major portion of [ C]-activity was also present in the 14  medium except in cultures incubated in the presence of 1.0x10-M and 0.5x10 M 3  _3  ZnCI (Tables 1 & 2). 2  Results of direct flame atomic absorption spectrophotometric analysis of DMEM media showed that the zinc content was between 1.0x10- M and 0.5x10 M in 6  -5  DMEM supplemented with 10% FCS and below 1.0x10"M in the medium devoid of 6  FCS.  B.  Quantitation of zinc transport bv HGF  The kinetics of zinc transport by cultured fibroblasts was biphasic with an initial rapid zinc uptake during the first few minutes of incubation followed by a slower linear phase (80). Table 3 shows that at higher ZnCI concentrations, more zinc was transported 2  into the fibroblasts. Zinc was also capable of binding to the proteins in the media. The higher the concentration of ZnCI in the media, the greater the amount of zinc that was 2  incorporated into the proteins. At the end of the 24-hr incubation, only a small percentage of the total zinc pool (<5%) was transported into the cells and less than 24% of total zinc was incorporated into the proteins in the media regardless  45  Table 3:  The amount of zinc transported into the fibroblasts during a 24-hr labeling period with [ Zn]. 65  Added ZnCI concentration in the media (M)  2  Amount of zinc trans-ported into the cell (ug)  Percentage of total zinc added to the media (%)  Amount of zinc in-corporated into non-dialyzable portion of medium (ug)  Percentage of total zinc added to the media (%)  0.001  4.210.8  0.001  3.510.4  1.0x10-  0.005  3.7±0.2  0.004  3.010.4  0.5x10-  5  0.02  3.0+0.4  0.03  4.111.0  1.0x10-5  0.03  2.1±0.2  0.04  3.510.1  0.5x10-  0.08  1.2+0.04  0.25  3.910.7  1.0x10-  0.31  2.4±0.2  0.43  3.410.4  0.5x10-  3  1.26  1.9±0.2  1.94  3.110.5  1.0x10-3  1.99  1.5±0.3  3.34  2.710.2  0.5x10-  4.18  0.6±0.1  99.96  15.413.6  1.0x10-  2  12.79  1.0+0.04  220.62  17.014.1  1.5x10-2  22.44  1.1±0.2  452.76  23.212.1  0 6  4  4  2  At higher zinc concentrations, greater amount of zinc was transported into the fibroblasts and more zinc was associated with the proteins located in the media. 46  of the different concentrations of ZnCI in the cultures (Table 3). Since in the present 2  study fibroblasts were treated with zinc for 24 hours, zinc transport across the cell membrane should have reached equilibrium.  C.  Effect of zinc on HGF attachment Equal number of fibroblasts (2.5x10) of the same passage were trypsinized, 5  pooled, and cultured in Petri dishes. After overnight incubation in complete DMEM in the absence and in the presence of 1.5x10" M, 0.5x10" M, 1.0x10'M and 1.0x10" M 2  3  4  5  ZnCI , the media was discarded and the adhered cells were trypsinized and their 2  number was determined by electronic cell counting. Comparison between control cultures and those exposed to ZnCI showed 2  that ZnCI at concentrations of 1.5x10' M and 0.5x10" M inhibited fibroblast 2  3  2  attachment by 47% and 21%, respectively (Figure 5). Significant differences were not found between control and the other two test groups with 1.0x10 M and 1.0x10-M _4  5  ZnCI . When fibroblasts were examined before trypsinization under the light 2  microscope, a substantial number of cells incubated in the 1.5x10" M ZnCI-containing 2  2  media failed to attach and were found floating in the media. Fibroblasts incubated in the presence of 0.5x10"M ZnCI appeared rounded. The morphology of fibroblasts 3  2  incubated in the presence of I.OxlO"M and 1.0x10 M ZnCI appeared similar in all 4  -5  2  respects to controls.  47  30n  0  150  5  1  0.1  Zinc Concentration (M, x10 ' ) 4  Figure 5:  The effect of different concentrations of ZnCI on HGF attachment. 2  The presence of 1.5x10-M and 0.5x10" M ZnCI in the culture media reduced the 2  3  2  fibroblast attachment by 47% and 21%, respectively (ANOVA, Tukey test, p<0.05). Significant differences were not found between controls and the two test groups exposed to 1.0x10" M and 1.0x10" M ZnCI (ANOVA, Tukey test, p>0.05). The results 4  5  2  were based on at least three separate runs prepared in duplicate samples for each run.  48  D.  Effect of zinc on HGF proliferation A typical cell growth curve includes three phases: lag phase, exponential  growth phase and plateau phase. Figure 6 shows the growth pattern of the tested quiescent fibroblast cell line. Compared with controls, fibroblasts incubated in the presence of 1.5x10" M ZnCI showed a general reduction in the cell number during the 2  2  thirteen day incubation period. In addition, it took 15 to 20 minutes of incubation in trypsin to detach those fibroblasts from the culture dish while normally it only takes 2 or 3 minutes of trypsinization. Once in trypsin solution, they did not round up as observed with normal fibroblasts. Prolonged treatment of trypsin probably disintegrated some cells, which might contribute to the high cell number counts on the 5th and the 7th day of incubation (Fig. 6). In comparison to controls, fibroblasts incubated in the presence of 0.5x10" M ZnCI also showed inhibition of proliferation and appeared rounded under 3  2  the light microscope. Growth curves of fibroblasts incubated in the presence of 1.0x10" M and 1.0x10' M ZnCI paralleled the growth curve of control cultures. 4  5  2  Compared with controls, they appeared to proliferate at a slower rate, which was probably due to the binding of zinc with some of the growth factors in the FCS which was added to the media (Figure 6). A comparison between growth curves of non-quiescent and quiescent fibroblast systems showed that they both displayed similar patterns in regards of their response to the different concentrations of ZnCI (Figure 7). 2  None of the cell cultures in this experiment reached plateau phase. This  49  O 1 H—i—i—i—i—i—i—i—i—i—i—r—j—i—i 0 2 4 6 8 10 12 14  1 T—i—I—«—I—i—I— —I—i—I— —I—i—I 0 2 4 6 8 10 12 14 1  1  Days  Days  51  o X  S3 E  o H—i—i—•—i—•—i—•—i—i—i—i—i—i—i  "H— —i—•—i—>—i— —i—•—i—i—i—i—i 0 2 4 6 8 10 12 14 1  1  0  2  4  Days  Figure 6:  6  8  10  12  14  Days  HGF proliferation over a 11-day incubation period in the presence of different concentrations of ZnCI following a 48-hr quiescence. 2  Following 48 hrs of quiescence, the number of fibroblasts gradually declined over 11 days of incubation for cultures to which 1.5x10" M ZnCI was added in the media. The 2  2  high cell number on the 5th and 7th day of incubation might be attributable to the prolonged trypsinization. At 0.5x10' M ZnCI concentration, the cell number remained 3  2  approximately the same. The growth curve of fibroblasts treated with 1.0x10" M and 4  1.0x10' M ZnCI paralleled that of the control cultures. However, it appeared that those 5  2  fibroblasts proliferated at a slower rate. 50  T—•—i—•—i—•—i—i—i— —i— —I 1  1-1—1—I—I—I—1—I—1—I—I—I—I—I  0  2  4  6  8 10  0  12  2  4  1-|—i—i—i—|—i—|—i—i—i—i—i—i  2  4  6  8 10  8 10  12  Days  Days  0  6  1  1-|—i—|—i—|—i—|—i—|—i—|—i—|  12  0  Days  2  4  6  8 10  12  Days  Figure 7: HGF proliferation over a 11-day incubation period in the presence of different concentrations of ZnCI . 2  Fibroblasts incubated in the media containing 1.5x10" M and 0.5x10 M added ZnCI 2  -3  2  showed an overall reduction in cell proliferation over a 11-day growth period. The growth curves of fibroblasts incubated in the presence of 1.0x10-M and 1.0x10- M 4  ZnCI in the media paralleled those of the control fibroblasts. 2  51  5  could be ascribed to the low initial seeding cell density, frequent replacement with fresh media and short incubation period.  E.  LM and TEM observations of morphological changes in fibroblasts treated with zinc 1. LM As expected, under LM the fibroblasts of control cultures appeared normal  spindle-shaped and in parallel alignment. Compared with controls, fibroblasts treated with 0.5x10- M ZnCI appeared rounded and aligned irregularly. No discernible 3  2  morphological differences were found among controls and test cultures containing 1.0x10" M, 0.5x10" M and 1.0x10"M ZnCI . Surprisingly, the morphology of 4  4  5  2  fibroblasts incubated in the medium containing 1.5x10"M ZnCI appeared no different 2  2  from that of the controls (Figure 8). The observations obtained from experiments conducted in the presence and in the absence of FCS were essentially alike (Figures 8 & 9). The depicted photographs were based on duplicate samples of three separate experiments and illustrations were taken at two randomly chosen spots . 2. TEM At lower magnification, intact cytoplasmic and nuclear membranes were observed in all fibroblasts. The cytoplasm of fibroblasts of control cultures were  52  53  Figure 8 (Continued)  a) Control d) 1.0X10- MZnCI  b) 1.5x10" M ZnCI e) 0.5x10" M ZnCI 2  2  4  4  2  2  c) 0.5x10 M ZnCI f) 1.0x10-M ZnCI -3  2  5  2  Magnification: 121.68 Confluent human gingival fibroblasts were trypsinized and equal number of fibroblasts per unit volume were attached, made quiescent, then incubated for 24 hrs in the presence of 0 (control), 1.5x10"M, 0.5x10 M, 1.0x10"M, 0.5x10' M and 1.0x10"M 2  3  4  4  5  ZnCI with or without FCS. Fibroblasts were fixed and then stained with Toluidine blue. 2  Compared with the morphology of control fibroblasts, cells incubated with 0.5x10" M 3  ZnCI in the media appeared round. No distinct difference in cell shape was found 2  between the controls and the rest of test groups that were subjected to 1.5x10"M, 2  1.0x10- M, 0.5x10" M and 1.0x10" M ZnCI in the media. 4  4  5  2  54  Figure 9:  Light microscopic examinations of HGF incubated for 24 hrs in the absence of FCS but in the presence of different concentrations of ZnCI. 2  55  Figure 9 (Continued)  a) Control d) 1.0x10- M ZnCI 4  2  b) 1.5x10"M ZnCI e) 0.5x10"M ZnCI 2  2  4  2  c) 0.5x10-M ZnCI f) 1.0x10-M ZnCI 3  2  5  2  Magnification: 121.68 Compared to controls, fibroblasts treated with 0.5x10 M ZnCI appeared round. No -3  2  discernible difference in cell morphology was observed between controls and the other zinc-containing test groups. Visible precipitates were formed following 24-hr incubation in cultures treated with 1.5x10 M ZnCI . -2  2  56  enriched with organelles such as mitochondria and rough endoplasmic reticulum (RERs). The nucleolus that designates active protein synthesis was also prominent (Figure 10). Compared with controls, the cytoplasm of fibroblasts incubated with 1.5x10- M and 0.5x10" M ZnCI appeared disintegrated. In addition, clumping of 2  3  2  nuclear chromatin, which designates later stage of the cell death, was observed. The shape of fibroblasts treated with 0.5x10" M ZnCI appeared round. No discernible 3  2  difference was observed between the cytoplasm of controls and the fibroblasts exposed to I.OxlO- M and 0.5x10" M ZnCI . At higher magnification, the cytoplasm of 4  4  2  1.5x10- M and 0.5x10" M ZnCI-treated fibroblasts was deficient in organelles such as 2  3  2  mitochondria, Golgi bodies and RERs which are essential for protein synthesis and secretion. Instead, disintegration of overall cellular structure and a high frequency of myelin figures were observed. Comparison of the cytoplasm of control fibroblasts with those treated with 1.0x10" M and 0.5x10" M ZnCI showed no distinct difference in 4  4  2  regard to the presence of cytoplasmic organelles (Figure 11).  F.  Reversal effect of zinc on suppression of protein synthesis by CHgSH Previous studies have demonstrated that exposure of fibroblasts to CH SH 3  during a 48-hr quiescent period and additional three to twenty-four hour radiolabeling periods resulted in an approximately 32% reduction in total TCA-precipitable proteins (40). In the present study, the procedure was modified by incubating test cultures in  57  Figure 10:  Lower magnification T E M appearance of H G F incubated for 24 hrs in presence of different concentrations of ZnCI . 2  58  the  Figure 10 (Continued)  a) Control d) 1.0x10" M ZnCI 4  2  b) 1.5x10-M ZnCI c) 0.5x10"M ZnCI e) 0.5x10-M ZnCI 2  3  2  2  4  2  N: nucleolus R: rough endoplasmic reticulum Arrows: show areas of clumped nuclear chromatin Fibroblasts were attached, made quiescent, then incubated for 24 hrs in media containing 0 (control), 1.5x10- M, 0.5x10" M, 1.0x10-M and 0.5x10- M ZnCI , 2  3  4  4  2  respectively, before being processed for TEM investigation. Control fibroblasts and those incubated in the presence of I.OxlO M and 0.5x10 M ZnCI showed prominent -4  _4  2  nucleolus. Organelles such as mitochondria, rough endoplasmic reticulum (RERs.) and lysosomes can be seen in the cytoplasm of these cells. Fibroblasts incubated in the presence of 1.5x10"M and 0.5x10"M ZnCI showed pathologic changes such as 2  3  2  clumping of nuclear chromatin. Fibroblasts exposed to 0.5x10 M ZnCI were round. _3  2  The photographs are based on three separate experiments on duplicate samples.  59  Figure 11:  Higher magnification TEM appearance of HGF incubated for 24 hrs in the presence of different concentrations of ZnCI. 2  60  Figure 11 (Continued)  a) Control d) 1.0x10" M ZnCI 4  2  M: mitochondria My: Myelin figure  b) 1.5x10-2 M ZnCI e) 0.5x10-M ZnCI  2  c) 0.5x10-M ZnCI 3  2  4  2  R: rough endoplasmic reticulum C: cytoskeleton  G: Golgi body  In the cytoplasm of control fibroblasts and cells treated with 1.0x10" M and 0.5x10 M 4  -4  ZnCI , mitochondria, RERs and Golgi bodies are evident while in fibroblasts exposed to 2  1.5x10-2 M and 0.5x10"M ZnCI , the cytoplasm is deficient in organelles and a high 3  2  frequency of myelin figures is observed. The photographs are based on duplicate samples of three separate experiments.  61  an atmosphere of 95% air / 5% C0 during a 48-hr quiescent period followed by 24 hrs 2  of pulsing with [ C]-proline in an atmosphere of 95% air/5%C0 admixed with 14  2  15 ng / ml CH SH. Table 4 shows that exposure of fibroblasts to CH SH for 24 hrs 3  3  under modified conditions resulted in a statistically significant 24% reduction of total TCA-precipitable labeled proteins. Zinc chloride concentrations of 0.5x10- M and 4  1.0x10" M in culture media totally reversed the adverse effect of CH SH. Zinc exerted 5  3  its protective effect largely on cell containing fractions. In other experiments, the presence of 1.0x10"M ZnCI inhibited total protein 4  2  synthesis of both control and test cultures. The combined inhibitory effect of CH SH 3  and ZnCI was greater than the inhibitory effect of either factor alone (Table 5). Hence, 2  0.5x10"M and 1.0x10"M ZnCI were chosen for subsequent protein synthesis and 4  5  2  proline transport experiments.  G.  Reversal effect by zinc on CH^SH-induced inhibition of proline transport Quiescent fibroblast cultures were pulsed for 30 minutes with [ C]-proline in 14  medium devoid of L-proline but supplemented with 10% FCS. During a 30-minute pulsing period, cultures were incubated either as controls exposed to air / C0 or as 2  test systems exposed to a mixture of air / C0 and 15 ng / ml CH SH alone and / or in 2  3  the presence of 0.5x1 O^M and 1.0x10-M ZnCI After the reaction, the medium was 5  2  discarded, cells were washed with PBS, scraped with 10% TCA /1% Tannic acid and  62  Table 4:  Reversal by zinc of CHSH-induced suppression of total protein synthesis 3  Total (media+celh  Fraction media cell (dpm) (dpm)  Samples  Reduction  (dpmisd)  (%)  air/C0  51585  82547  134132+2157  CH SH  37078  64913  101991123267  24*  CH SH+ 1.0x10- MZn2+  41681  84086  1257681380  6(N.S.)  CH SH+ 0.5x10- M Zn+  44225  84503  128728111783  4  2  3  3  5  3  4  2  (N.S.)  Quiescent fibroblasts were cultured for 24 hrs, during which test cultures were subjected to 15 ng / ml CH SH alone and / or in the presence of ZnCI . Exposure of 3  2  fibroblasts to CH SH resulted in a 24% reduction in total TCA-precipitable protein. In 3  the presence of I.OxlO" M and 0.5x10"M ZnCI , protein synthesis was restored to 5  4  2  near control state. In addition, zinc appeared to be more effective in reversing the adverse effect of CH SH on the cellular than on the medium fraction. Activity values are 3  expressed as dpm per 60 mm Petri dish. The results are based on three separate experiments using triplicate samples for each run. *  ANOVA, Tukey test, significantly different from control (p<0.05 ).  N.S. ANOVA, Tukey test, no significant difference from control ( p>0.05).  63  Table 5:  Percent decrease of total TCA-precipitable protein synthesis by HGF in the presence of CH SH and ZnCI . 3  Sample  Air/C02 1.0x10-4 M ZnCI  Cellular fraction (dpm)  Medium fraction (dpm)  TotaKmedia+cein  20304  11997  32301+124  4335  797  5132±252  84(a)  19472  13204  32676±750  +1(b)  18321  7673  25994±1143  19(°)  2204  1349  3553±1047  89()  19233  10171  29404+351  g(e)  (dprrdtsd)  Reduction (%)  2  1.0x10- M 5  ZnCI  2  2  CH SH 3  CH SH+ 3  1.0x10-4 M ZnCl2  d  CH3SH+  1.0x10-5 M ZnCL.2  The addition of 1.0x10"M ZnCI to the media had no adverse effect on the total 5  2  TCA-precipitable protein synthesis and partially reversed the effect of CH SH-exposed 3  cultures. The combined suppressive effect of ZnCI at an inhibitory concentration 2  (1.0x10"M) and CH SH was greater than CH SH alone. Group a, c and d were 4  3  3  significantly different from each other and from other groups (ANOVA, Tukey test, p<0.05). No significant difference was found between control and b (ANOVA, Tukey test, p>0.05). Group e was significantly different from control and b (ANOVA, Tukey test, p<0.05). 64  centrifuged. The radioactivity in the supernatants was quantitated by LSC. The results in Table 6 indicate that exposure of fibroblasts to CH SH resulted 3  in a 21% reduction of [ C]-proline activity in the supernatant. However, with the 14  addition of 0.5x10' M and 1.0x10 M ZnCI to the culture media, the inhibitory effect of 4  5  2  CH SH was nullified. 3  H.  HPLC analysis of the effect of zinc on the total protein and collagen synthesis bv CHSH-exposed HGF a  A study by Tonzetich and Yaegaki (105) has established that during a 12-minute mobile phase, the first radioactive peak that emerged during HPLC (within the first three minutes of elution) represented underivatized proline and hydroxyproline, followed by hydroxyproline which eluted as the second and proline as the third radioactive peak (105). In the present study, the underivatized peak constituted less than 2% of the total radioactivity associated with hydroxyproline and proline. The hydroxyproline peak eluted at approximately the 4th minute followed by proline which eluted at approximately the 7th minute (Figure 12). In control cultures, the hydroxyproline content accounted for approximately 4% of the proline content in both dialyzed medium and cellular fractions (Tables 7 & 8). Compared with controls, cultures exposed to CH SH for 24 hours resulted in a 42% and a 40% reduction in total 3  collagen synthesized  in cellular and medium fractions, respectively. Total protein  associated with cells was also reduced by 30%. Because of the presence of unbound [ C]-proline in the media, the radioactivity of the proline peak was not indicative 14  65  Table 6:  Reversal effect of zinc on CH SH-induced suppression of proline transport by HGF. 3  Control  4  Air/C0  12565±208  2  CH SH  21*  9871±748  3  Test  Reduction (%)  [:L.Cl-activitv (dpm± sd)  Systems  CH SH+ 0.5x1 (HMZn *  12502±1666  1(N.S.)  CH SH+ 1.0x10-5 MZn2+  14023+ 723  +12(N-S.)  3  2  3  Following a 48-hr quiescence, cultures were pulsed for 30 minutes with [ C]-proline 14  under control and test conditions. Medium was then discarded, cells were washed with PBS, scraped into 10% TCA /1% Tannic acid and centrifuged. The radioactivity in the supernatant was quantitated by LSC. In this run, proline transport was reduced by 21% in CHSH-exposed cultures. The addition of 0.5x10"M and 1.0x10-M ZnCI to the 4  5  3  2  culture media totally nullified the effect of CH SH. Activity is expressed as dpm per 3  Petri dish. The results were based on three separate runs using triplicate samples for each run. * ANOVA, Tukey test, significantly different from control ( p<0.05 ). N.S. ANOVA, Tukey test, no significant differences from control (p>0.05).  66  8000-1  time ( minute )  Figure 12:  Retention times for [ C]-labeled hydroxyproline and proline. 14  The first radioactive peak that eluted out in the first two minutes represented underivatized proline and hydroxyproline, which was less than 2% of the radioactivity associated with hydroxyproline and proline peaks. The second peak that eluted at approximately the 4th minute represented hydroxyproline while the third peak was proline.  67  of the total protein synthesized that was secreted into the media during the 24-hr incubation. The reduction of collagen present at the end of the 24-hr incubation was 19% for the medium fraction and 31% for the cellular fraction. The amount of protein present at the end of the 24-hr incubation was decreased by 19% for the medium fraction and 42% for the cellular fraction. It appeared that CH SH had greater inhibitory 3  effect on cell associated fractions. The addition of ZnCI to the media at the 2  concentration of 1.0x10-M in CHSH-exposed culture nullified the adverse effect of 5  3  CH SH on total protein synthesis. However, it did not reverse the effect on collagen 3  synthesis except for the total cellular collagen fractions (Tables 7 & 8). Comparison between dialyzed and non-dialyzed cellular fractions of the test cultures showed that in cultures exposed to CH SH alone, the hydroxyproline of the dialyzed portion was 54% 3  of the non-dialyzed compartment while in cultures exposed to CH SH and 1.0x10"M 5  3  ZnCI , the percentage was 31%. This suggests that there were more smaller peptides 2  (<3,500 m.w.) present in the zinc / CHSH-treated cultures than in cultures treated only 3  with CH SH. 3  The results of the HPLC analysis of the ability of zinc to counteract the effect of CH SH on total protein synthesis confirmed the previous result obtained from the 3  study on the effect of zinc on total TCA-precipitable protein synthesis by HGF exposed to CH SH. 3  I.  Effect of zinc on DNA synthesis bv CHoSH-exposed HGF A previous study with HGF exposed to 10 ng / ml H S has demonstrated that 2  DNA synthesis by HGF was reduced by 47% (93). In the present study, test cultures were exposed to air/C0 admixed with 15 ng/ml CH SH. A comparison between 2  3  68  Table 7:  HPLC analysis of the medium fraction for total protein and collagen synthesis by fibroblasts cultured in the presence of CH SH and ZnCI3  Sample  Non-dialvzed fraction OH-pro (dpm±sd)  Dialed  Reduction  OH-pro  (%)  (dpm±sd)  Redu-ction (%)  fraction  Proline (dpm±sd)  Redu-ction (%)  Control  13216+2608  CH SH  7933±954  40  423±18  19  10535±1741  19  7500±1033  43  367±90  29  12893±1466  0  3  CH SH 1.0x10-M ZnCI  520±4  2  12933±113  3  5  2  Exposure of fibroblasts to CH SH resulted in a 40% reduction of total collagen 3  synthesized found in the medium fraction. The reductions of both collagen and proteins present in the medium at the end of 24-hr incubation was 19%. The addition of 1.0x10"M ZnCI to the media in CHSH-exposed cultures failed to reverse the 5  2  3  effect on collagen synthesis in the medium fractions. It did, however, nullify the adverse effect of CH SH on total protein synthesis. Because of the presence of unbound 3  [ C]-proline in the non-dialyzed medium sample, the radioactivity associated with the 14  proline peak was not indicative of the total protein present in the medium. The activity is expressed as dpm per Petri dish. Results are based on three separate experiments performed in quadriplicate in each experiment.  69  Table 8:  HPLC analysis of the cellular fractions for total protein and collagen synthesis by fibroblasts cultured in the presence of CH SH and ZnCL . 3  Sample  Non-dialvzed fraction OH-pro Redu- Proline Reduction -ction (dpmisd) (%) (dpm±sd) (%)  Control  1640+62  CH3SH  951±60  42  42905±7155  1522±140  7  5841713864  CH SH+ 1.0x10M ZnCI  6089314858  2  Dialvzed fraction OH-pro Redu- Proline Redu-ction -ction (dpm±sd) (%) (dpm±sd) (%) 756±141  22084±915  30  518±64  31 12868±1605 42  4  465+7  38 179151176  3  5  19  2  Exposure of fibroblasts to CH SH resulted in a 42% reduction in total collagen content 3  in the cellular fraction. Total synthesized protein associated with the cells was also reduced by 30%. At the end of 24 hrs of incubation, the collagen content associated with the cellular fraction was reduced by 31%. Proteins associated with cells are decreased by 42%. The addition of 1.0x10-M ZnCI to the media in CHSH-exposed cultures did 5  2  3  not reverse the effect on collagen synthesis except for the non-dialyzed cellular compartment. However, it did nullify the adverse effect of CH SH on total protein 3  synthesis. The activity is expressed as dpm per Petri dish. The results are based on three separate experiments performed in quadriplicate samples in each experiment.  70  control cultures exposed to 95% air / 5% C0 and test cultures exposed to air / C0 2  2  admixed with 15 ng / ml CH SH showed that DNA synthesis was reduced by 38%. 3  DNA synthesis was also reduced in cultures incubated in the presence of non-inhibitory levels of ZnCI even when exposed to air / C0 alone. As the ZnCI 2  2  2  concentration in the media decreased to 0.5x10-M and 1.0x10-M, the amount of 5  6  DNA synthesized was restored to control levels. The reduction of DNA synthesis observed in cultures incubated with 1.0x10-M and 1.0x10-M ZnCI correlated with 4  5  2  the results obtained in the growth curve studies that demonstrated that HGF proliferated at a slower rate when 1.0x10-M and 1.0x10"M ZnCI was supplemented into the 4  5  2  growth media. DNA synthesis by fibroblasts incubated in CHSH-exposed systems in 3  the presence of various concentrations of ZnCI was inhibited to the same degree as 2  fibroblasts exposed to CH SH alone (Table 9). 3  Thus it appears that the addition of non-inhibitory concentrations of ZnCI to 2  the medium was ineffective in counteracting the adverse effect of CH SH on DNA 3  synthesis of cultured HGF.  71  Table 9:  DNA synthesis by HGF cultured in the presence of CH SH and ZnCI . 3  pail-dUrd activitv of cell extract  System Zinc concentration (M)  (cpnrtfcsd)  0 1.0x10-  4  Control  (air/COo)  0.5x10-4  1.0x100.5x10I.OxlO"  5 5 6  0 Test (CH SH) 2  1.0x10-4 0.5x10-4 1.0x10-5 0.5x10-5 1.0x10-6  2  Reduction (%)  9345±1435 7591+450 6681±818 8489±1059 9455±448 9051±1850  19 29 9 0 3  5774±409 4907±1412 5827±962 5562+1275 5671+851 5720+203  38 47 38 40 39 39  Under control conditions, DNA synthesis by fibroblasts treated with 1.0x10-M and 4  0.5x10-4 M ZnCI was reduced by 19% and 29%, respectively. As the ZnCI 2  2  concentration in the media was decreased to 1.0x10-6 M, DNA synthesis was restored to control levels. Exposure of fibroblasts to CH SH resulted in a 38% reduction in DNA 3  synthesis. The addition of ZnCI to the media had no discernible reversal effect and the 2  inhibition was approximately the same as in cultures exposed to CH SH alone. Activity 3  is expressed as cpm per Linbro well. The results are based on three individual runs performed on quadriplicate samples in each run.  72  Section IV Discussion  A.  Molecular mechanisms of the detrimental effect of high levels of zinc on HGF cultures  Zinc is considered to be a trace element. Its concentration in the serum is approximately 1.5x10" M of which ±10" M exists as free Zn . Zinc is relatively harmless in vivo compared to other divalent cations. This may be ascribed to a combination of homeostatic mechanisms which regulate its gastrointestinal absorption and excretion, the action of a variety of hormonal stimuli which control cellular metabolism, its rapid redistribution in the body, and cellular adaptation mechanisms. For cell cultures exposed to relatively high concentrations of zinc, the maintenance of homeostasis is more difficult and the adaptive mechanisms are the only available means of regulating zinc levels, thereby makes them more vulnerable to the exposure of high levels of zinc in the medium. 5  5  2+  The in vitro inhibitory effects of zinc have been demonstrated with various types of cells such as yeast (96), HeLa, human prostate, lymphoid, human fetal lung fibroblasts, B16 melanoma and mammalian epithelial cells (11). The majority of these studies suggest that in vitro zinc becomes inhibitory at above 10 mol /1, which is in accord with the findings of the present study on HGF. Zinc concentrations in the media above LOxlO^M decreased the total protein synthesis by HGF and at concentrations of 1.5x10- M and 0.5x10" M reduced the attachment and proliferation of HGF. -4  2  3  73  The normal cell is believed to be in a homeostatic steady state, able to accommodate changes in environmental conditions. Excessive external agents or stimuli may result in a number of cellular adaptations and a new altered steady state in which cells remain viable. If the limits of the adaptive capability are exceeded, a sequence of events follows, loosely termed "cell injury", which includes two stages of reversible and irreversible injury according to the recovery of the cell upon the removal of the causative agents. It is generally believed that the morphologic changes of cell injury become apparent only after some critical biochemical pathways within the cell have been damaged. Ultrastructural alterations occur earlier than changes visualized by light microscopy (71). In the present study, cultured HGF exposed for 24 hrs to excessive zinc concentrations (>1.0x10" M) in the media, total protein synthesis was significantly 4  reduced with a greater effect on the cell associated fraction. Ultrastructurally, HGF challenged with 1.5x10"M and 0.5x10-M ZnCI showed irregular clumping of nuclear 2  3  2  chromatin and disintegration of cytoplasmic organelles. This indicates cell damage even though under the light microscope HGF exposed to 1.5x10-M ZnCI showed no 2  2  obvious morphologic changes while HGF treated with 0.5x10" M ZnCI appeared to be 3  2  rounded. From this it can be concluded that zinc at concentrations above 1.0x10' M in the culture media for 24 hrs is deleterious to HGF resulting in HGF injury reflected by the inhibitory effect on total protein synthesis by HGF and pathological changes in morphology. The cell damage appeared to be irreversible. This is supported by other investigations which show that cells that had been exposed to high levels of zinc (>1.0x10-M) fail to proliferate when plated in normal growth medium (11). 4  4  74  Four biochemical intracellular systems are particularly vulnerable to detrimental  agents. They are the (a) aerobic respiration involving oxidative  phosphorylation and production of ATP, (b) maintenance of the integrity of the cell membrane on which the ionic and osmotic homeostasis of the cells and its organelles are dependent, (c) synthesis of enzymatic and structural proteins, and (d) preservation of the integrity of the genetic apparatus of the cell. Although the precise molecular mechanisms of zinc suppressive effect is unclear, experimental findings have demonstrated that the inhibition of enzymes that are associated with the membrane by zinc through its binding to functional essential amino acid residues may play an important role. It may resemble the action of mercury which causes cellular damage principally by combining with sulfhydryl groups of the membrane and enzymes (71). Zinc has long been considered a membrane stabilizer. However, higher concentrations (>10"M) induce Na / K leakage (62, 63), probably due to the inhibition of membrane-bound Na / K+ ATPase, which has been shown in pulmonary alveolar macrophages (56). It has been reported that zinc at concentrations between 0.5x10- to 2.0x10-3 M inhibited ATPase system by over 30%. It is possible that the inhibitory effect of zinc is associated with the inhibition of membrane-bound enzymes responsible for maintaining ionic and fluid balance of the cell. Zinc may also exert its adverse effect by inhibiting the mitochondrial respiratory chain through competition with iron for a ligand in the oxidized nonheme iron protein which participates in electron transport in the respiratory chain (17). 4  +  +  +  3  There appears to be a delicate balance between the beneficial and detrimental effects of zinc. My studies have demonstrated that HGF can tolerate up to at least three times the physiological extracellular zinc concentration (1.5x10" M). Is zinc 5  75  uptake by cell necessary for the expression of adversity? Inhibition of cellular membrane-bound enzymes is not, but the inhibition of the mitochondrial respiratory chain reactions clearly depends on the entry of zinc into the cells. The fact that zinc can enter the cells is evident as over 200 enzymes, most of them intracellular, require zinc and contain it in a bound form. Intracellular^, free Zn as low as 10" to 10" M was reported to be harmless (61). What determines the equilibration between intracellular and extracellular zinc is still unclear since an outward-pumping Zn -ATPase has not been found. It is also unclear by what mechanisms zinc is absorbed by cells in vitro. Studies on human diploid fibroblasts from fetal tissue and human forearm skin fibroblasts revealed that the kinetics of zinc transport occurs in a biphasic pattern; an initial rapid phase followed by a slower linear phase (2, 80). The process of uptake appears to be carrier-mediated and saturable (2). The metabolic energy requirements during the process have not been clearly defined. Pronase-sensitive and pronase-resistant enzyme systems have been used in zinc uptake studies to distinguish between surface bound and internalized ligands. They showed that the initial rapid uptake was into the pronase-sensitive fraction, which reached a plateau within 10 minutes, and a linear increase into the pronase-resistant fraction, suggesting the initial binding of zinc is to the cell surface, followed by internalization. The ligand involved in the process has not been identified. The use of N-ethyl maleimide, a sulfhydryl blocking agent, reduced zinc uptake, which implies that sulfhydryl groups may be important for the uptake (2). These results suggest that zinc is not incorporated by a receptor-mediated endocytic pathway as has been described for transferrin and iron. However, the obtained results are based on in vitro experiments conducted with zinc below 4.0x10" M. It is possible that a different mechanism of zinc transport is involved when cells are challenged with higher concentrations of zinc. At least, two in vitro routes of zinc infusion into cells when they were treated with above 1.0x10' M 2+  5  4  2+  5  4  76  zinc have been proposed: (a) one pathway, which utilizes transferrin, can be completely blocked by iron, and (b) a pathway similar to the calcium pathway which can be partially blocked by calcium (11). This implies that if the inhibitory effect of zinc is a result of its entry into cells, its channels may be shared with iron and calcium. Present in vitro studies quantitating zinc uptake by HGF in the presence for 24 hrs of inhibitory and non-inhibitory levels of zinc showed that HGF accumulated zinc in a concentration-dependent manner. It is likely that at the end of the 24-hr incubation, zinc uptake by HGF was saturated or the equilibrium was established. However, these experiments are insufficiently definitive to indicate the relationship between its uptake and adverse effect in HGF cultures.  B.  Preventive and therapeutic aspects of zinc in periodontal diseases: its counteractive effect against CHgSH It is generally accepted that bacteria and their by-products of metabolism in  dental plaque play a primary role in the etiology of periodontal diseases. They are capable of penetrating the epithelial barrier and initiate a series of destructive inflammatory reactions affecting the underlying connective tissue. The pathogenesis of periodontal disease is believed to be multifactorial in nature. Previous investigations have established the role of CH SH, which is produced primarily by gram negative 3  anaerobic oral microflora through putrefaction of sulphur-containing proteinaceous material, as a potential contributor to the disease process. CH SH has been shown to 3  increase the permeability of non-keratinized porcine sublingual mucosa to [ H]-PGE , 3  2  77  [ C]-E. coli endotoxin and [ S]-S0 (59). Both PGE and endotoxin have been 14  35  4  2  implicated as potential agents or mediators in the inflammatory periodontal disease process. Thiols also have the ability to penetrate through all three layers of mucosa consisting of non-keratinized epithelium, basement membrane and connective tissue (59). Furthermore, CH SH has been shown to react with collagen, and thereby alter its 3  structure and increase its solubility (39). In addition, CH SH has also been found to 3  suppress total protein, collagen and DNA synthesis as well as proline transport of cultured HGF (40, 91, 92, 93). Treatment of porcine sublingual mucosa for 15 minutes with ZnCI (0.22%) 2  either prior to or after the exposure of mucosa to CH SH has been shown to totally 3  nullify the effect of CH SH and restore tissue permeability to control state (59). The fact 3  that a 15-minute treatment of mucosa with ZnCI can counteract the CH SH effect 2  3  corroborates the kinetics of zinc uptake by cells and tissue, which is of biphasic pattern with an initial rapid phase, which usually takes first 10 minute of incubation, followed by a slower second phase (2, 58, 80). It has been proposed that when mucosal tissue is exposed to zinc, it can be rapidly incorporated into the cellular and tissue components to form a zinc reservoir. In the presence of CH SH, zinc stored in the 3  tissue can readily react with the mercaptan to form inactive zinc mercaptides before CH SH reacts with the mucosa. This provides a feasible explanation why removal of 3  the ZnCI following 15-minute treatment still nullifies the effect of CH SH on the 2  3  permeability of the mucosa. An in vivo study on rabbits showed that sulcular tissue of animals fed a zinc-deficient diet exhibited statistically significant increase in [ C]-bovine serum albumin and [ C]-phenyltein uptake (42). This implies that zinc 14  14  78  deficiency may increase molecular uptake by periodontal tissues and enhance their susceptibility to periodontal diseases. This present investigation showed that zinc at non-inhibitory levels can also nullify the adverse effect of CH SH on total non-collagenous protein synthesis and 3  proline transport by cultured HGF. The reversal effect of zinc on non-collagenous protein synthesis may be attributable to its protective effect on the cell membranes. The finding that zinc can also counteract the inhibitory effect of CH SH on proline transport 3  supports this hypothesis since proline uptake by fibroblasts is a membrane-associated active transport process. Previous studies using differential staining of HGF exposed to CH SH with fluorescein diacetate and ethidium bromide showed that CH SH 3  3  damaged the membrane integrity of some fibroblasts (59). Sulfhydryl groups have been regarded as functional and structural components of the membrane that contribute to its integrity and stability (17). The observed membrane impairment of fibroblasts may well be the result of interaction between sulfhydryl groups of the membrane and CH SH, which is a reducing agent. Changes in membrane 3  permeability and cell vitality have also been observed in erythrocytes when the sulfhydryl groups are reacted with reducing agents (57). Zinc has long been known as a membrane stabilizer. One of the mechanisms proposed whereby zinc stabilizes the membrane is that the ion interacts with thiol groups of the intrinsic macromolecules of the membrane through formation of zinc mercaptides, thus increasing the integrity and stability of the membrane (43). It is plausible that zinc and CH SH compete for sites on the cell membrane -- the 3  detrimental interaction of CH SH vs the beneficial interaction of zinc. The binding of 3  7 9  zinc with the thiol groups of the membrane seems stronger since treatment of mucosa with zinc after the exposure to CH SH can still restore the tissue permeability to control 3  state (59). Non-collagenous proteins of the extracellular matrix, namely proteoglycans and glycoproteins, are of primary importance in maintaining and regulating cell function and the structural integrity of the connective tissue. Their degradation usually precedes collagen breakdown in periodontal disease. Proteoglycans in their aggregated state serve as a molecular sieve for the passage of both interstitial water and solutes. The aggregation of proteoglycans has been shown to depend on disulfide linkages. Similarly, glycoproteins are also believed to interact with other proteins through disulfide bonds (35). It has been proposed that CH SH enhances the permeation of 3  pathogenic substances via disulphide cleavage mechanisms that results in deaggregation of proteoglycans. Zinc not only stabilizes biomembranes, it also stabilizes a variety of other biological macromolecules. It is involved in the polymerization process of insulin and renders the insulin molecule less soluble (17). Removal of zinc from carbonic anhydrase B decreases its conformational stability against denaturation (17). Apoprotein has been found to be more susceptible to proteolysis than in its zinc-containing form (7). In summary, zinc may counteract CH SH through the above discussed 3  mechanisms: 1) protection of fibroblast membranes, 2) interaction with the structural components of extracellular matrix proteins thereby making them more resistant to proteolysis, and 3) reaction with CH SH to form inactive zinc mercaptides thus 3  80  quantitatively reducing the amount of CH SH available to react against fibroblasts. The 3  observed reversal effect of zinc on non-collagenous protein synthesis by HGF exposed to CH SH could be due to the combination of these mechanisms. 3  ZnCI at a concentration of 1.0x10" M in the HGF cultures exposed to CH SH 5  2  3  did not reverse the CHSH-induced effect on collagen synthesis. The loss of collagen 3  could be resulted from both decreased synthesis which may due to the reduction of proline transport, and the increased extracellular and intracellular degradation as observed in HGF cultures exposed to CH SH. Zinc, while capable of reversing the 3  CHSH-induced reduction of proline transport, may not exert any counteractive effect 3  against increased collagen degradation both inside and outside the cells. This might explain why zinc reversed CHSH-induced reduction of collagen content associated 3  with the cellular non-dialyzed fraction but failed to do so for both dialyzed and non-dialyzed fractions of the media, and the dialyzed fraction of the cells. Zinc also failed to reverse the adverse effect of CH SH on DNA synthesis by 3  cultured HGF. This may due to the presence of FCS in the media or that zinc does not have the ability to protect the cells against DNA damage induced by cytotoxic agent like CH SH, which suggests that the ability of zinc to counteract CH SH might be limited. 3  3  The fact that the previously established in vitro non-inhibitory zinc concentration of 1.0x10 M was later found to be inhibitory to HGF in this study may be -4  ascribed to a number of possibilities. These include HGF cell lines of different origins,  81  since individuals may vary in their tolerance to zinc, and cell lines of different passages since aging fibroblasts have been shown to accumulate more zinc due to the increased cellular permeability to the metal (55, 81). The role of zinc can be preventive as well as therapeutic in periodontal diseases. Mouth rinses that contains ionic zinc have been demonstrated to be strong inhibitors of oral malodor which is primarily attributable to the presence of VSC. The presence of zinc in either mouthwashes or dentifrices has been shown to exhibit anti-plaque activity (76). Oral supplementation with zinc facilitates oral soft tissue wound healing (98). Iontophoresis with zinc ions increases cellular adherence to periodontally diseased roots (45). Our in vitro findings that zinc is capable of counteracting CH SH on total non-collagenous protein synthesis by HGF suggest that 3  zinc may have a beneficial role in the tissue repair. However, its ineffectiveness in reversing CHSH-induced reduction of DNA and collagen synthesis indicates that zinc, 3  if applicable to the prevention and treatment of the periodontal disease, must be used in conjunction with other periodontal hygienic or surgical procedures. 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