Effect of zinc on the metabolism of thiol-treated human gingival fibroblasts
Creators: name: Ouyang, Ying
Publisher: name: University of British Columbia
Year: 2010

description: The etiology of periodontal diseases is multifactorial. Cumulative in vivo and in vitro observations implicate volatile sulphur compounds (VSC), namely, hydrogen sulphide (H₂S) and methyl mercaptan (CH₃SH), produced through putrefaction of 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 CH₃SH exerting a more profound inhibitory effect than H₂S. In addition, they have been found to increase the permeability of porcine sublingual mucosa to [3H]-PGE₂, [35S]-SO₄ and E. coli 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% ZnCl₂ totally nullifies 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 CH₃SH on total protein, DNA and collagen synthesis, and proline transport by cultured HGF. While ZnCl₂ at concentration of 0.22% (1.6x10⁻² M) is protective against VSC 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 exposed to various concentrations of ZnCl₂ ranging from 1.0x10⁻⁶M to 1.5x10⁻²M. It was found that zinc at concentrations higher than 1.0x10⁻⁴M significantly decreased the total protein synthesis by HGF over a 24-hr labeling period in DMEM devoid of L-proline but supplemented with L-[′⁴C]-proline. Fibroblast attachment was reduced by 47% and 21% when they were treated with 1.5x10⁻²M and 0.5x10⁻3 M ZnCl₂, respectively. Fibroblasts incubated in the presence of 1.5x10⁻² M and 0.5x10⁻3 M ZnCl₂ 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 ZnCl₂ revealed that cells exposed to higher than 1.0x10⁻⁴M ZnCl₂ underwent pathologic changes characterized by 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 [⁶⁵Zn] resulted in an accumulation of zinc by fibroblasts in a concentration-dependent manner. To assess the ability of zinc to counteract CH₃SH, control HGF cultures were incubated in an atmosphere of 95% air / 5% CO₂ while test cultures were subject to 95% air / 5% CO₂ admixed with 15ng / ml CH₃SH with or without the presence of ZnCl₂ in the culture media. It was found that during a 24-hr L-[′⁴C]-proline pulsing period, exposure of HGF to CH₃SH resulted in a 24% reduction in total protein synthesis which could be totally reversed by the presence of 0.5x10⁻⁴M or 1.0x10⁻⁵ M ZnCl₂ in the media. Furthermore, at these two concentrations, zinc was also found effective in reversing the CH₃SH-induced inhibition of proline transport. HPLC analysis of total protein and collagen confirmed that ZnCl₂ present at a concentration of 1.0x10⁻⁵ M in the media totally nullified the inhibitory effect of CH₃SH and restored total protein synthesis by HGF to control levels. However, at this concentration, ZnCl₂ did not reverse the effect of CH₃SH on collagen synthesis, and at non-inhibitory concentrations (below 0.5x10⁻⁴ M), it was ineffective in reversing the adverse effect of CH₃SH on DNA synthesis by cultured HGF. In summary, zinc at non-inhibitory concentrations in HGF culture has been shown to be capable of counteracting the CH₃SH-induced suppression of total protein 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.