Volatile sulphur compounds (VSC) are a family of gases that are formed as a result of tissue breakdown in periodontal pockets. Previous work has indicated that at least some of these gases are directly toxic to cells and can alter collagen structure. The three most abundant VSC in periodontal pockets are methyl mercaptan (CH3SH), hydrogen sulphide (H2S) and dimethyl disulphide (CH3SSCH3). Toxicity appears to be associated with reactive thiols which reduce and, in the case of mercaptan, methylate disulphide bonds in protein. This study proposes to examine the effects of these gases on extracellular matrix (ECM), tissue sections and cultured cells. Since preliminary results have demonstrated that methyl mercaptan is the most reactive, experiments will emphasize the role of this thiol in the pathogenesis of periodontal disease. The study will examine the effects of VSC on the structure and function of molecules in ECM as well as changes in cellular function. The initial experiments will focus on alterations in collagen synthesis and degradation. Collagen synthesis in the presence of thiols will be examined both by Northern blot and ELISA to quantitate changes in the total amount and types of collagen. Synthesis of abnormal collagens will also be investigated. Since collagen degradation is altered as well as synthesis, changes in activity of extracellular proteases will be detected on substrate gels and quantitated in soluble assays. Similar assays will be used to evaluate the role of matrix inhibitors. Analyses of mRNA will determine changes in gene expression of specific proteases and will complement results derived through measurements of protein activity. Phagocytic activity of fibroblasts in the presence of thiol will also be determined since intracellular degradation may be an important factor contributing to the collagen depletion observed in cultures exposed to mercaptan. Alterations in fibronectin structure and function will be analyzed as well as its interrelationship with other ECM proteins. These experiments will examine the effect of VSC on both fibronectin-collagen interactions and matrix assembly using immunofluorescent analysis of ECM from cultured cells as well as basic biochemistry. Alterations in other ECM proteins such as integrins will be investigated by similar methods. Mucosal sections will also be exposed to thiols and will be examined by immunofluorescence to determine if VSC affect tissue architecture or expression of collagens, fibronectin, proteases or integrins. Cell biology experiments will evaluate the effect of CH3SH on the behavior of fibroblasts and epithelial cells in wounding experiments and in migration assays. These assays will also analyze the effect of mercaptan on cell responses to specific substrates. The tests will be conducted using pretreated cells or pretreated matrix as well as in the presence of thiol. These experiments will provide a comprehensive analysis of the influence of VSC on structure-function interactions of important matrix molecules and the effect of CH3SH on the behavior or keratinocytes and fibroblasts. The results will define the role VSC play in the pathogenesis of periodontal disease and may suggest forms of therapy.
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