Ion transport and cadmium-induced inhibition of ciliary activity and induction of swelling of epithelial cells in mouse trachea organ culture.

Swelling of epithelial cells and reduction of ciliary activity in mouse trachea organ culture occurred after incubation for 4 h with a rather low concentration of cadmium acetate (10 microM). Specific inhibitors of ion transport (Na+, K+, Cl-) such as furosemide, amiloride and ouabain did not mimic, abolish or increase the toxic effects induced by cadmium acetate. Exposure to cadmium acetate had no significant effect on electrolyte uptake (22Na+ and 86Rb+). These results suggest that the swelling of epithelial cells induced by cadmium acetate is not due to an osmotic swelling from an accumulation of electrolytes. Ba2+, known to have several biological properties in common with Ca2+, and to influence basolateral K+ flux, counteracted the toxic effects of cadmium acetate, whereas a more rapid and extensive swelling occurred with cadmium acetate in a medium without Ca2+. No effect on the uptake of 109Cd2+ was found with barium chloride, whereas in a medium without Ca2+ the Cd2+ uptake increased by 47%. Trifluoperazine (100 microM), a drug which in vitro binds tightly to calmodulin, imitated the toxic effects of 10 microM cadmium acetate. The combination of 10 microM cadmium acetate and 100 microM trifluoperazine resulted in an additive toxic effect. A possible mechanism for the cadmium acetate-induced swelling and inhibition of ciliary activity could, thus, be a disturbance of the regulatory activity of calmodulin.

Effects of cadmium acetate and sodium selenite on mucociliary functions and adenosine triphosphate content in mouse trachea organ cultures.

The effects of cadmium acetate and sodium selenite in mouse trachea organ culture have been studied separately and in combination. Ciliary activity, morphology, rate of total protein and glycoconjugate (i.e. glycoprotein and proteoglycan) synthesis/secretion and ATP content were investigated. Exposure to 10 microM cadmium acetate or 2000 microM sodium selenite resulted in a complete cessation of ciliary activity within 5 h. With cadmium acetate also a swelling of epithelial cells was observed. Sodium selenite (250-2000 microM) delayed by 2-3 h the inhibitory effect of cadmium acetate (5-20 microM) on ciliary activity. The rate of protein synthesis, as determined by incorporation of [3H]proline, was reduced by 13% and 44% at exposure for 4 h at 37 degrees C to 250 microM and 500 microM sodium selenite respectively, the effect being partly abolished by cadmium acetate. With 5 microM and 10 microM cadmium acetate the rate of glycoconjugate synthesis, as measured by incorporation of [3H]glucosamine, increased by 50% and 69%, respectively, after incubation for 4 h. This increase was partly reduced by sodium selenite. Neither cadmium acetate nor sodium selenite had any effect on the rate of total protein or glycoconjugate secretion. The ATP content in trachea rings was reduced by 48% and 54% after incubation for 4 h with 250 microM and 500 microM sodium selenite, respectively. No significant effect of cadmium acetate was found on ATP content. An antagonistic effect of sodium selenite and cadmium acetate in mouse trachea organ culture is suggested from the present experiments.

Entrance of Actinobacillus actinomycetemcomitans into HEp-2 cells in vitro.

A strain of actinobacillus actinomycetemcomitans, freshly isolated from a juvenile periodontitis patient, and the FDC Y4 laboratory strain of Aa were tested for their capacity to adhere to and enter the epithelial cell line HEp-2 cells in vitro. Immunofluorescence microscopy as well as scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that both strains adhered to the outer surface of the HEp-2 cells. In the TEM studies, the specimens were also treated with Aa specific antibodies and gold labeled protein A. These examinations showed that only the freshly isolated strain of Aa was found within the HEp-2 cells. The intracellular Aa were found to be viable, and in one case one of them was seen to undergo division. It is concluded that freshly isolated Aa has the ability to enter epithelial HEp-2 cells in vitro, and it is tentatively suggested that this may play a role in the pathogenesis of periodontal disease.

Inhibitory effect of NH4Cl on secretion of collagen in human gingival fibroblasts.

The general protein synthesis in human gingival fibroblasts as measured by 14C-proline incorporation was only moderately inhibited by 10 mM NH4C1 during incubation for 36 h. The proportion secreted as noncollagen protein and recovered from the cellular fraction as collagen was not significantly affected, whereas a pronounced inhibitory effect on the secretion of collagen was evident after 24 h. This effect was dose dependent, with a significant inhibition of collagen secretion even at 2 mM ammonia. The applied concentrations of NH4C1 had no significant effect on the hydroxylation of prolyl residues in collagen. Ammonia had no inhibitory effect on the secretion of fibronectin, another major secretory protein from fibroblasts. When comparing different lysosomotropic agents; NH4C1, chloroquine and methylamine, the most prominent effect was consistently found to be an inhibition of the secretion of collagen.

NH4Cl and protein metabolism in human gingival fibroblasts.

The biologic effect of ammonia was studied in cultures of fibroblasts isolated from human gingiva, NH4Cl in the range 2-20 mM was found to exhibit a concentration dependent growth inhibitory effect, with a delayed action which was most pronounced at low concentrations. Concomitant with the growth inhibitory effect a significant cellular accumulation of protein was evident. No effects on protein synthesis in general, as measured by 14C-proline incorporation, was found, whereas some inhibitory effect on collagen biosynthesis was indicated. Secretion of 14C-collagen and other labeled proteins was not affected. The only pronounced effect of ammonia on metabolism of the 14C-labeled proteins was an inhibition of the intracellular degradation of newly synthesized collagen, the lysosomes being suggested as the site for this degradation.

pH and the effect of NH4Cl on human gingival fibroblasts.

pH variations in the range from 6.9 to 7.9 modified the growth rate of human gingival fibroblasts in culture, a pH optimum being found at 7.6-7.8. A pH dependent growth inhibition by ammonia was evident, being particularly prominent in the presence of 2 mM NH4Cl. At this concentration an effect ranging from a complete cessation of growth at pH 8.0 to an indication of a slight stimulatory effect at pH 7.2 was found. Concomitant with this amplification of the growth inhibitory effect, a state of unbalanced growth in the sense of an increased cellular protein content was induced by ammonia upon increasing the pH of the medium. The incorporation of 14C-proline into the total protein fraction decreased rapidly, particularly in the control cultures, with pH; an inhibition by ammonia being found at pH 7.0 and 7.5 with no effect at pH 7.1. Except for part of the inhibition by ammonia, changes in specific activity of 14C-proline in the cellular amino acid pool could ac count for these pH effects. Ammonia reduced the proportion of radiolabeled protein recovered as collagen both from the cells and medium at the lower pHs, whereas the only significant effect on noncollagen protein was an increased fraction being secreted at pH 7.1 in the presence of 10 mM NH4Cl.

Effect of fluoride on protein and collagen biosynthesis in rabbit dental pulp in vitro.

The time course for incorporation of KC-proline into various fractions of rabbit dental pulp in vitro has been measured. In the TCA-soluble precursor pool a steady state level of activity was indicated upon incubation after 3 h, whereas incorporation into protein and 14C-hydroxyproline, i.e. collagen formation, increased linearly for 9 h, leveling off upon further incubation. A lag period of about 3 h was indicated for the appearance of high molecular weight 14C-activity, including 14C-hydroxyproline, in the medium, increasing linearly from 3 h to the end of the incubation period (22 h). In this system, fluoride exhibited a dose-dependent inhibitory effect. At 5.3 mM fluoride the uptake of 14C-proline into the TCA-soluble pool was inhibited by about 50%, and the incorporation into protein and the subsequent conversion to hydroxyproline by about 90 and 60%, respectively. Release of collagen, i.e. 14C-hydroxyproline-containing material, seemed to be the process most sensitive to fluoride; it was inhibited by about 50% at the lowest concentration (1.3 mM) tested.

Effect of some heavy metals on protein and collagen biosynthesis in rabbit dental pulp in vitro.

Cadmium, zinc, copper, and mercury, being components of dental alloys, have been tests at concentrations from 20 to 120 micronM for their effect on the incorporation of 14C-proline into various fractions of rabbit dental pulp incubated in vitro. With cadmium and zinc and increased amount of 14C-activity was recovered from the TCA-soluble pool, whereas the further incorporation into total protein and collagen, i.e. 14C-hydroxyproline, was markedly inhivited. Copper and mercury, however, reduced the amount of label in the TCA-soluble fraction. Both metals exhibited a dose-dependent inhibitory effect on the hydroxylation step in collagen synthesis, whereas for mercury only, an inhibition of the general protein synthesis was indicated. With all four metals the proportion of 14C-labeled protein and hydroxyproline recovered from the medium was reduced, the effect being most prominent with cadmium and zinc.

PH and the effect of fluoride and zinc on protein and collagen biosynthesis in rabbit dental pulp in vitro.

The effect of pH in the range 6.6-8.2 on the incorporation of 14C-proline into the rabbit dental pulp incubated in vitro has been studied. The amount of label in the cold TCA-soluble pool, total protein, and hydroxyproline (i.e. collagen) of the pulp tissue increased linearly with pH in all three fractions. A similar increase was found in the amount of labeled total protein and collagen recovered from the incubation medium. The results indicated that the uptake of 14C-proline into the TCA-soluble pool was the pH-sensitive step, whereas the incorporation into protein, formation of hydroxyproline, and the release of labeled macromolecules into the medium were not affected to any measurable degree by the ambient pH. In this system, zinc (60 micrometer) had less effect on the incorporation of 14C-proline into the different fractions of the pulp tissue at pH 6.6 as compared with pH 7.4, whereas with fluoride (1.3 mM) an increased inhibition of the uptake of 14C-proline into the TCA-soluble pool and the incorporation into total protein was found upon lowering the pH to 6.8. The inhibitory effect of zinc and fluoride on the release of labeled total protein and collagen into the incubation medium was not affected when the pH was lowered.

pH and the cytotoxicity of fluoride in an animal cell culture system.

To investigate the mechanism for the toxicity of silicate cement as observed in a cell culture system, the effects of pH and fluoride were tested on human epithelial cells (NCTC 2544). At pH 7.3, fluoride concentrations from 15 to 25 mug/ml (0.79 to 1.3 mM) had a growth inhibitory effect. When pH of the incubation medium was lowered in the range 7.0 to 6.4, an enhanced cytoxic effect of fluoride was found, and even at 5 to 10 mug/ml growth inhibition occurred. Concomitant with the enhanced cytotoxicity of fluoride at low pH, there was an increased utilization of glucose and formation of lactate. Upon lowering the pH of the incubation medium from 7.4 to 6.7 a twofold increase in the intracellular concentration of fluoride was found.

Fc-binding components: a virulence factor in Actinobacillus actinomycetemcomitans?

Actinobacillus actinomycetemcomitans (ATCC 33384) can produce and release components that bind to the Fc part of IgG. Fc-binding components were observed in whole bacteria, capsular material and medium from broth cultures. The components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with biotinylated Fc-fragments and myeloma proteins. In a phagocytosis assay with human granulocytes and sheep erythrocytes, preincubation of opsonized erythrocytes with protein A reduced phagocytosis by 90%. In contrast, preincubation of the opsonizing antibody with medium components from a culture of A. actinomycetemcomitans enhanced the opsonizing effect of the antibody. The enhanced binding of erythrocytes may be caused by formation of aggregates between opsonizing antibody and bacterial Fc-binding components. Aggregated IgG can bind to low-affinity Fc gamma II and gamma III receptors that cannot bind monomeric IgG. Release of Fc-binding components from bacteria may contribute to the periodontal lesion through interference with the phagocytic activity of granulocytes and with the complement system. Fc-binding components may also interfere with downregulation of the B-cell response.

Factors affecting retention of Cu in the human oral cavity after mouth rinses.

Retention of Cu in the mouth after rinses with aqueous solutions of CuSO4 was measured by atomic absorption spectrophotometry. The retained amount of Cu exhibited a linear relationship against the concentration of the rinsing solution in the range tested. Approximately one third of the Cu retained after a 60 s rinse was retained within the first 15 s, and about 75% within 30 s. Retention of Cu was only little affected by the pH of the rinsing solution in the range from 2.0 to 5.7, being reduced by about 50% at pH 9.0. However, variations in cupric ion activity rather than pH per se could explain these effects. A mutual reduction in retention of either metals was seen when Cu and Zn were applied simultaneously.

Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans.

A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired.

Toxicity of some dental cements in a cell culture system.

A cell culture method has been used to study the effect of zinc phosphate cement (De Trey's Zinc Zement Improved), zinc silicophosphate cement (Fluoro-Thin) and polycarboxylate cement (Durelon) on animal cells. Disks (20 x 1 mm) of the materials were placed in the center of plastic Petri dishes and subsequently incubated with human epithelial cells. Cell multiplication, medium pH and the release of cement constituents were measured. All three cements exhibited a cytotoxic effect, which was most pronounced in the cultures with zinc silicophosphate cement and polycarboxylate cement. The results also indicated that cell growth on the surface of the disks is a more sensitive indicator of cytotoxicity than cell growth around the disks. pH of the medium was only slightly affected in cultures with polycarboxylate cement, whereas a decrease was found in cultures with zinc phosphate cement and especially with zinc silicophosphate cement. A rapid release of phosphate was found in cultures with zinc silicophosphate cement. Zinc was released into the medium from disks of zinc phosphate cement, zinc silicophosphate cement and polycarboxylate cement--exceeding the toxicity level for the present cell line after 24 h. In cultures with zinc silicophosphate cement and polycarboxylate cement the release of fluoride reached toxic levels within the same time interval.

Antibodies against lipopolysaccharide from Bacteroides gingivalis before and after periodontal treatment.

Specific serum antibody activities of the IgG, IgA and IgM isotypes against lipopolysaccharide (LPS) prepared from Bacteroides gingivalis were measured by an enzyme-linked immunosorbent assay (ELISA) in a group of 12 periodontally healthy subjects and a group of 26 patients with periodontitis. The latter group received periodontal therapy, completed within about 1 yr. A serum sample was obtained from each participant at the first periodontal examination; a second sample was taken about 2 yr later. The mean antibody levels calculated for the healthy group did not change significantly between the first and second examination. The correlation coefficients computed between the two sets of measurements were 0.93, 0.90 and 0.96 for IgG, IgM and IgA respectively (P less than 0.05). Periodontal treatment significantly improved the clinical status of the patients and was followed by a statistically significant mean reduction in specific antibody levels to the LPS preparation (IgG: 15%, IgA: 30% and IgM: 15%).

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774.

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.

Fluoride retention in sound and demineralized enamel in vivo after treatment with a fluoride varnish (Duraphat).

The retention of alkali soluble (CaF2) and alkali insoluble (fluorapatite) fluoride in sound enamel and demineralized enamel 2 wk after application of Duraphat was investigated in a group of orthodontic patients from whom pairs of homolog premolars were to be extracted. Demineralization of the enamel was induced during a 4-wk period prior to application of fluoride by applying orthodontic bands to the premolars. The bands also remained attached to the teeth during and after application of fluoride (2 wk) to maintain a cariogenic environment. Three consecutive enamel layers (approximately 5 microns) were subsequently etched off. A significant uptake of fluoride in the first and second layer of sound enamel and in all the three enamel layers of demineralized enamel was found. More fluoride was found in demineralized enamel and a higher proportion of this fluoride was found to be in an alkali insoluble form compared with the fluoride in sound enamel. The SEM study showed a rough enamel surface after three consecutive acid etchings. The etching pattern differed within the etched area. It was suggested that the variation in etching pattern might be due to differences in orientation of the crystallites and the original surface morphology.