[Effect of heavy metal ions on the cells derived from human periodontal ligament. Effects of Pb and Cd].

Abstract

It has been thought that the incorporation of Pb and Cd into human body, especially calcified tissues including the tooth, has altered the physiological processes of development. But, the biochemical events that initiate this process remain quite unknown. The present study attempt to explore the effect of Pb and Cd on human periodontal ligament fibroblast-like cells of the permanent tooth (HPLF) and the deciduous tooth (HPLF-Y) with respect to the cell growth, ALPase activity and incorporation of 14C-amino acids. HPLF and HPLF-Y migrated from a explant and subcultured according to the previously described method of Saito were inoculated 1.25 x 10(4) cells/cm2 in D-MEM supplemented with 2 mg/ml FCSP, 50 micrograms/ml ascorbic acid and antibiotics. After 24 hrs, HPLF and HPLF-Y were treated every two days for 10 days with 0-200 microM Pb or 0-10 microM Cd. Protein contents, DNA contents and ALPase activity were determined by Bio-Rad protein assay, deaminobenzoic acid assay and p-nitrophenylphosphate (pH 10.15) assay respectively. At 6 days HPLF were incubated with 3H-thymidine (TdR 2.0 microCi/well) and then the incorporation of 3H-TdR into cold TCA precipitates was assayed by a liquid scintillation counter. And also, at 6 days HPLF and HPLF-Y were incubated with 14C-amino acids (2.0 microCi/60 mm dish) for 24 hrs. The cell layers labeled with 14C were extracted with 15 mM Tris-HCl buffer containing 7 M urea (pH 7.4) and applied to the gel permeation chromatography of HPLC system to separate the fractions according to molecular weight. The HPLF and HPLF-Y incubated with Pb and Cd were morphologically identical. Pb stimulated the protein contents of extracellular matrix of HPLF, but not HPLF-Y. Cd inhibited the protein contents of cell layers of HPLF and HPLF-Y. With increasing concentrations of Pb and Cd, the incorporation of 3H-TdR into HPLF was inhibited. On the other hand, Cd stimulated the ALPase activity per DNA content since the ALPase activity of HPLF and HPLF-Y was decreased by the addition of Pb. The distribution of 14C-labeled protein according to molecular weight did not alter the chromatographic pattern of HPLF incubated with Pb and Cd. But, that of HPLF-Y incubated with Pb was relatively shifted to low molecular size. Therefore, these responser concluded that HPLF were not completely identical with HPLF-Y. Pb and Cd not only had a toxic effect on cell growth, but also they may regulate the metabolic alteration.