Influence of a standard laboratory diet containing nutritionally adequate levels of selenium on renal pathology from mercury released by experimental amalgam tattoos.

Twenty-four guinea pigs were subcutaneously implanted with 2 X 25 mg of powdered dental amalgam for between 1 and 24 months. Four animals served as controls. All animals were fed a standard diet containing 0.2 microgram/g selenium (Se). Mercury (Hg) was released from the implants at an average rate of 21.7 micrograms/d and 4.35-16.94 micrograms of Se were consumed daily in the diet. The renal Hg rose to a maximum of 263 micrograms/g at 11 months in implanted animals and 6.65 mg Hg was excreted over 2 yr. The renal proximal tubular cells of implanted animals contained visible Hg deposits in secondary lysosomes and within nuclei containing both Hg and Se. No other signs of obvious renal pathology were seen in implanted animals as compared with controls. It would seem likely that the low Se levels present within this standard laboratory diet were sufficient to protect against Hg toxicity.

Tissue reactions to the separate implantation of individual constituent phases of dental amalgam, including assessment by energy dispersive X-ray microanalysis.

Soft tissue degradation of the 3 principal amalgam phases have been investigated in relation to their role in the formation of the amalgam tattoo. Each phase, finely powdered, was implanted subcutaneously into the submandibular region of guinea-pigs for periods ranging from 1 week to 1 year. The rates of breakdown were assessed radiographically and the final lesions were examined by light and electron microscopy and X-ray microanalysis. gamma 2 (Sn7Hg) phase degraded rapidly, mainly extracellularly, and did not produce a tattoo. Both mercury and tin disappeared from the lesion. gamma 1 (Ag2Hg3) phase degraded less rapidly, both extra and intracellularly, and produced a small tattoo. Mercury was lost from the lesion. gamma (Ag3Sn) phase degraded slowly, intracellularly, and produced a large tattoo. Tattoos always resulted from persistence of minute particles of silver and sulphur associated with basal lamina and connective tissue.

Renal cortical mercury levels associated with experimental amalgam tattoos: effects of particle size and amount of implanted material.

Powdered dental amalgam that had passed through either a 106 microns or a 45 microns sieve was implanted subcutaneously in guinea pigs for periods of up to 2 yr. The renal cortical mercury levels associated with the 106 microns material were on average 16% of those produced by the 45 microns material. A reduction in the amount of 45 microns powder implanted, by a factor of 75%, resulted in a fall of only 27% in renal mercury concentrations. The marked effect of particle size on mercury release may be explained by the large increase in the proportion of implanted material that was degraded within phagocytic cells in the local lesions.

Further investigations of the soft tissue reaction to the gamma 1 phase (Ag2Hg3) of dental amalgam, including measurements of mercury release and redistribution.

The subcutaneous implantation in guinea pigs of powdered gamma 1 phase induced a severe initial tissue response and the majority of the material was extruded from the healing wounds. This process was accompanied by the release of significant amounts of mercury which appeared in the body organs and excreta. The small numbers of particles which remained in the tissues were handled quite differently, undergoing slow degradation in macrophages and giant cells in chronic granulomata. Minute secondary particles containing silver and sulphur were deposited in the tissues and gave rise to macroscopic tattooing of the skin above the implants.

Further investigations of the soft tissue reaction to the gamma 2 phase (Sn7-8Hg) of dental amalgam, including measurements of mercury release and redistribution.

Following the subcutaneous implantation in guinea pigs of powdered gamma 2 phase with a mercury content only slightly greater than that indicated by the stoichiometric formula Sn7Hg, there was a limited initial release of mercury from extracellular material. Thereafter, chronic granulomata developed around the implants and particles degraded slowly in macrophages and giant cells. Vast numbers of fine secondary particles containing tin were produced and these were associated with macroscopic staining. Mercury seemed to be lost as the result of intracellular particle breakdown but it did not accumulate in the body organs or appear to any great extent in the excreta.

The fate of amalgam implanted in soft tissues -- an experimental study.

The soft tissue reaction to amalgam depended on whether it was finely ground or a solid mass. Finely ground amalgam was actively digested within macrophages and giant-cells with loss of mercury and the formation of the fine particles of silver and sulphur. Larger masses became surrounded by a fibrous capsule.

Localization of active and inactive elastase, alpha-1-proteinase inhibitor, and alpha-2-macroglobulin in human gingiva.

Biochemically, there is usually much less elastase activity in gingival tissue than in crevicular fluid. The tissue distributions of active and inactive elastase and the endogenous inhibitors alpha-1-proteinase inhibitor (alpha 1PI) and alpha-2-macroglobulin (alpha 2M) were therefore compared. Inflamed tissue was obtained from chronic periodontitis patients, and cryostat sections were incubated with the histochemical elastase substrate MeOSuc-Ala-Ala-Pro-Val-MNA. Adjacent sections were examined immunocytochemically with antibodies to neutrophil elastase, alpha 1PI, alpha 2M, and leukocyte differentiation antigens. Antigenic elastase was widely distributed in CD15-positive granulocytes in both the epithelium and lamina propria as well as in granulomatous tissue from infrabony defects. However, there was very limited histochemical staining of these cells, and biochemical activity against the equivalent substrate MeOSuc-Ala-Ala-Pro-Val-AFC could be extracted only from sections with such staining. The pH optimum and effector response of the activity in the extracts were, nevertheless, consistent with those of leukocyte elastase. The large difference between the total elastase content of the tissue, as determined immunocytochemically, and the limited amount of active enzyme, as demonstrated histochemically, indicated that the majority was in an inactive form. The involvement of tissue inhibitors was suggested by the fact that extracts from sections with no histochemical staining reduced biochemical elastase activity in crevicular fluid. alpha 2M was found in many fibroblasts and also some CD68-positive macrophages, which additionally contained alpha 1PI.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between gingivain/gingipain and bacterial dipeptidyl peptidase activity in gingival crevicular fluid and periodontal attachment loss in chronic periodontitis patients. A 2-year longitudinal study.

The aim of this study is to determine whether either gingival crevicular fluid (GCF) bacterial gingivain/gingipain or dipeptidyl peptidase (DPP) levels, total activity (TA) and concentration (EC), predict progressive attachment loss (AL) in 75 patients with moderate periodontitis. GCF was collected from 16 molar and premolar mesiobuccal sites and then clinical attachment level (CAL) and probing depth (PD) were measured with an electronic constant pressure probe. Lastly, gingival, gingival bleeding, and plaque indices were scored. Prior to the baseline visit, patients were given basic periodontal treatment after which the above procedures were repeated. In addition, carefully localized radiographs were taken of the test teeth and repeated annually. Patients were then seen every 3 months for 2 years and the clinical measurements repeated at each visit. In 48 patients, 124 AL sites, 91 rapid AL (RAL), and 33 gradual AL (GAL) were detected. Gingivain/gingipain and bacterial DPP levels (TA and EC) at RAL sites were significantly higher (P < or = 0.0001) than at paired control sites at the attachment loss time (ALT) and prediction time (PT). Mean levels over the study period of both proteases (TA and EC) at GAL sites were significantly higher (P < or = 0.0001) than those at paired control sites. The GCF levels of gingivain/gingipain were always higher than those of DPP. Critical values (CV) of 5 microU/30 seconds (TA) and 30 microU/microL (EC) for both proteases showed high sensitivity and specificity values for TA and EC, which were the same at both ALT and PT. The positive predictive values were higher for gingivain/ gingipain. Mean site levels, over the course of the study, of both proteases (TA and EC) were significantly higher (P < or = 0.0001) at AL, RAL, and GAL sites than non-attachment loss (NAL) sites in AL patients and mean patient levels were significantly higher (P < or = 0.0001) in AL, RAL, and GAL patients than NAL patients. These results indicate that both of these bacterial proteases in GCF may be predictors of periodontal attachment loss.

Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid: a comparison of levels before and after periodontal surgery in chronic periodontitis patients.

Gingival crevicular fluid (GCF) was collected from the deepest probing site of each tooth of 10 chronic periodontitis patients prior to treatment, after scaling and hygiene treatment, and after periodontal surgery. Surgery was carried out at sites which had persistent probing depths in excess of 5 mm. The patients were given a full periodontal examination, including measurements of probing depth, gingival index, bleeding index, and plaque index before each GCF collection. Cathepsin B/L-, elastase-, tryptase-, trypsin-, and dipeptidyl peptidase IV-like activities in the GCF samples were determined by fluorimetric assay with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin. There were reductions in all clinical parameters and all protease activities after scaling and hygiene treatment and further reductions after periodontal surgery. Decreases were recorded for both total enzyme activities and concentrations. The reductions were statistically significant in inter-patient comparisons using mean patient values and also in most intra-patient comparisons using site data from individual patients. GCF protease levels appear to reflect the clinical status of periodontal lesions and may prove to be of value in monitoring disease activity.

Histochemical and immunocytochemical localization of dipeptidyl peptidases II and IV in human gingiva.

Dipeptidyl peptidase (DPP) II and IV activities were demonstrated in unfixed cryostat sections of gingival tissue from chronic periodontitis patients using histochemistry with 2-methoxy-4-naphthylamine (MNA) substrates. In the case of DPP IV, enzyme localization was confirmed by immunocytochemistry with mouse monoclonal antihuman DPP IV (CD26) antibody. Inflammatory cells containing enzyme were identified in adjacent sections with mouse monoclonal antibodies directed against leukocyte differentiation antigens. Lys-Ala-MNA and Ala-Pro-MNA staining in acid buffer for DPP II was only found in a few fibroblasts in superficial tissue. Staining with Gly-Pro-MNA and Ala-Pro-MNA in alkaline buffer for DPP IV was localized in some CD4 and CD8 positive T lymphocytes, CD68 positive macrophages, and fibroblasts and these cells also reacted with the enzyme antibody. DPP IV-containing macrophages and T lymphocytes were seen in the epithelium. In deeper granulomatous tissue Gram positive and negative bacteria stained with the histochemical substrates, but not the DPP IV antibody. Fibroblast DPP II and IV might participate in cellular interactions with collagen, while T lymphocyte DPP IV may be involved in cell signalling.

Comparative histochemical and biochemical studies of mast cell tryptase in human gingiva.

Tryptase-like activity has previously been identified biochemically in gingival homogenates and gingival crevicular fluid (GCF) using substrates linked to the 7-amino-4-trifluoromethyl coumarin (AFC) leaving group. In the present study, activity was demonstrated histochemically in tissue sections with analogous 4-methoxy-2-naphthylamide (MNA) substrates. Z-Ala-Ala-Lys-MNA and D-Val-Leu-Arg-MNA were the most sensitive substrates. Comparison of staining patterns with the MNA substrates and toluidine blue indicated that enzyme activity was localized to mast cell secretory granules. Most stained cells were in the lamina propria, but a few were in the epithelium. The number of stained cells was somewhat greater in inflamed tissue from chronic periodontitis patients than in healthy tissue from controls. However, hardly any staining was seen in inflamed granulomatous tissue. Using high-salt buffer containing heparin, it was possible to extract enzyme activity from tissue sections for biochemical analysis with corresponding AFC substrates. Inhibitors gave similar results in the biochemistry and histochemistry. The inhibitor response and pH profile of the enzyme were the same as that found earlier with gingival homogenates and GCF and were again consistent with mast cell tryptase. The enzyme may have a role in the pathology of chronic periodontitis.

Preliminary studies on cysteine and serine proteinase activities in inflamed human gingiva using different 7-amino-4-trifluoromethyl coumarin substrates and protease inhibitors.

The cysteine proteinases cathepsins B and L have collagenolytic potential and so have been implicated in connective-tissue breakdown in chronic periodontitis. Synthetic peptide substrates are often used to detect proteolytic enzymes. The action of homogenates of inflamed gingiva tissue against three such substrates of cathepsin B have been characterized here by protease inhibitors. Using the selective reagents ZPheAlaCHN2, BzValLysLysArgAFC, ZAlaArgArgAFC and ZPheArgAFC were susceptible to both cysteine and non-cysteine proteinase activity; the two types of enzymes had acidic and alkaline pH optima, respectively. The action of other inhibitors at acidic pH indicated the involvement of cathepsin B and, to a lesser extent, cathepsin L. The enzyme active at alkaline pH was a serine proteinase; it resembled glandular kallikrein in its inhibitor response and its ability to hydrolyse a fourth substrate, DValLeuArgAFC, but its greater reactivity with BzValLysLysArgAFC and ZAlaArgArgAFC was not consistent with kallikrein. ZPheArgAFC, though less sensitive than BzValLysLysArgAFC to cysteine proteinase action, was far less susceptible to hydrolysis by the serine proteinase and thus appears the best choice for selective assays of cathepsins B and L.

Mercury release, distribution and excretion from subcutaneously implanted conventional and high-copper amalgam powders in the guinea pig.

With both types of amalgam there was initial loss of material during healing of the implantation wounds. The rate of Hg release from the healed lesion was estimated by linear regression analysis of the content of implants when removed sequentially over 2 years. There were no significant differences between the amalgams, when expressed in absolute terms or as a percentage of the Hg content of the lesions immediately after healing, probably because gamma 1 (Ag2Hg3) phase breakdown was largely or solely responsible for Hg release with both materials. Hg levels in the blood, liver, renal cortex and faeces were all significantly lower with high-Cu amalgam which might be explained by the interference by Cu with Hg retention. There was no significant difference in urinary Hg excretion by the two groups of animals over the greater part of the study.

Microscopy and X-ray microanalysis of subcutaneously implanted conventional and high-copper dental amalgam powders in the guinea pig.

With each material, there was only a mild early inflammatory response; particles were taken up by macrophages and giant cells. By 1.5 to 3 months, chronic granulomas had developed. With conventional amalgam, there were early changes in the intracellular material, associated with the rapid degradation of Sn-Hg particles, corresponding to the gamma 2 phase. Thereafter, intracellular particles from both types of amalgam underwent progressive degradation, producing halos of secondary material containing Ag and S. Apart from an initial loss of Cu and Sn from some high-Cu amalgam particles, there were no qualitative differences in the later changes between the two materials, although conventional amalgam particles appeared to degrade faster. With both, vast numbers of fine secondary particles containing Ag and S became widely distributed throughout the lesions and gave rise to macroscopic tattooing of the skin. Secondary material and small degrading primary particles from both types of amalgam were detected in the submandibular lymph nodes where they caused localized staining.

A biochemical study of serine proteinase activities at local gingival tissue sites in human chronic periodontitis.

Serine proteinases have the potential to influence the degradation of connective tissue in chronic periodontitis, which may progress episodically at individual tooth sites. Elastase-, chymotrypsin- and tryptase-like proteinase activity in homogenized gingival tissue were measured using, respectively, the selective peptide substrates MeOSuc-Ala-Ala-Pro-Val-AFC. MeOSuc-Phe-Pro-Phe-AFC and Z-Ala-Arg-Arg-AFC. Each tooth site was assayed separately and divided, where appropriate, into gingival tissue and granulomata. Elastase-like activity was detected in only about half of the sites and with large variations. Chymotrypsin-like activity decreased with increasing pocket depth, clinical attachment level, gingival index and gingival bleeding index. Tryptase-like activity did not vary consistently with clinical measures. Chymotrypsin- and tryptase-like proteinase activity were much higher in gingival tissue than in granulomata. These effects are best explained by the likely influence (or lack of influence) of the endogenous serum and tissue inhibitors of serine proteinases, the different cellular origins of the enzymes, and their relative affinities for their substrates.

Identification of a tryptase-like enzyme in extracts of inflamed human gingiva by effector and gel-filtration studies.

Inflamed gingiva contain a serine proteinase which could not previously be identified on the basis of its substrate specificity and inhibitor response. Using the substrate ZAlaArgArgAFC at alkaline pH, the enzyme was shown to be extracted more efficiently in high salt buffer. Inclusion of NaCl in assays, however, caused progressive reduction of activity. There was also inhibition by CaCl2, MgCl2 and 2 mM TosLysCH2Cl but not by 2 mM TosPheCH2Cl. Heparin produced significant activation. In gel filtrations with 1.0 M NaCl, activity appeared in fractions corresponding to a molecular weight of about 135,000. These properties are all consistent with tryptase from human mast cells. The enzyme may participate in both the connective tissue destruction and the inflammatory and immunological processes of gingivitis and periodontitis.

Dipeptidyl peptidase II- and IV-like activities in gingival tissue and crevicular fluid from human periodontitis lesions.

Gingival tissue and gingival crevicular fluid were collected from patients with chronic periodontitis. Gel filtration chromatography of crude tissue extracts yielded separate fractions active against Lys-Ala-7-amino-4-trifluoromethyl-coumarylamide (AFC) at acid pH and Gly-Pro-AFC at alkaline pH. The molecular weights, pH optima and inhibitor responses of these activities were consistent with those of dipeptidyl peptidases (DPP) II and IV, respectively. When tested with the same substrates, crevicular fluid was also found to contain DPP II- and IV-like activities with very similar pH profiles and inhibitor responses to those in tissue. The close resemblance suggested that the crevicular fluid enzymes were derived mainly from inflamed gingival tissues. Slight differences in the DPP II-like activities might be explained by the additional presence in crevicular fluid of enzymes from subgingival bacteria. With use of appropriate buffers, a third substrate, Ala-Pro-AFC, gave selective detection of both DPP II- and IV-like activities in tissue and crevicular fluid. Assays with Ala-Pro-AFC had the advantage of greater sensitivity, especially with DPP II-like activity. Raised levels of this enzyme have previously been found in the gingiva of periodontitis patients and thus DPP II-like activity in crevicular fluid might prove of value in monitoring disease activity.

Comparison of host tissue and bacterial dipeptidyl peptidases in human gingival crevicular fluid by analytical isoelectric focusing.

Earlier work has shown that gingival crevicular fluid (GCF) contains dipeptidyl peptidase (DPP) activities that resemble those in host tissue. Here, further comparisons were made with enzymes from suspected periodontal pathogens. Gingival tissue and GCF were collected from patients with chronic periodontitis. DPP II and DPP IV fractions with acid and alkaline pH optima, respectively, were separated from crude tissue extracts by gel-filtration chromatography. Bacterial cell sonicates were prepared from broth cultures of reference strains. There was moderate to strong DPP activity with Capnocytophaga spp., Porphyromonas gingivalis and Prevotella spp., very weak activity with Treponema denticola and no detectable activity with Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum. Banding patterns in GCF, tissue and bacterial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. In gels washed with acid buffer, GCF had bands corresponding to tissue DPP II. Use of an alkaline washing buffer showed GCF activity which closely matched tissue DPP IV that had been pretreated with neuraminidase, an enzyme found by others in the gingival crevice. P. Gingivalis gave multiple bands and several of these had counterparts in GCF. The apparent presence in GCF of the DPP from P. gingivalis is consistent with the association of this organism with destructive periodontitis.

A comparison of cysteine and serine proteinases in human gingival crevicular fluid with tissue, saliva and bacterial enzymes by analytical isoelectric focusing.

Gingival crevicular fluid (GCF) contains several different proteinase activities and the study sought to clarify their sources. Gingival tissue and GCF were collected from chronic periodontitis patients. Gel-filtration chromatography of crude tissue extracts yielded cathepsin B and tryptase fractions sensitive to cysteine and serine proteinase inhibitors, respectively. Cell sonicates of suspected periodontal pathogens were prepared from broth cultures of reference strains. Of these, Porphyromonas gingivalis showed much the strongest activity and this had an effector response consistent with the metal-dependent cysteine proteinase described by others. Banding patterns in GCF, tissue and bacterial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. On Z-Val-Lys-Lys-Arg-AFC overlays, GCF had bands corresponding to tissue cathepsin B and the enzyme from P. gingivalis, though a contribution from Treponema denticola could not be ruled out. Use of D-Val-Leu-Arg-AFC overlays showed GCF activity similar to tissue tryptase. In GCF there were additional bands that did not correspond to any tissue or bacterial samples and on Z-Ala-Ala-Lys-AFC overlays these closely resembled activity in parotid saliva. The results confirmed that GCF contains tissue cathepsin B and tryptase, while the apparent presence of enzymes from P. gingivalis and possibly T. denticola is consistent with previous reports linking activity to these organisms. The saliva bands demonstrated that contamination of GCF may occur despite rigorous collection procedures.

Correlation of gingival crevicular fluid proteases with clinical and radiological measurements of periodontal attachment loss.

Probing attachment loss and radiographical measurements of bone loss were made on 20 untreated chronic periodontitis patients. At a second visit, gingival crevicular fluid was collected on filter paper strips from the deepest accessible interdental probing site of each tooth. Gingival crevicular fluid volumes were determined and the samples eluted into buffer. Protease activities in the resulting eluates were assayed with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC). Cathepsin B/L-like activity was determined with Bz-Val-Lys-Lys-Arg-AFC, elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC, tryptase-like activity with Z-Ala-Ala-Lys-AFC, trypsin-like activity with Z-Gly-Gly-Arg-AFC and dipeptidyl peptidase IV-like activity with Ala-Pro-AFC. Total enzyme activities and enzyme concentrations correlated positively with probing attachment loss and bone loss in linear regression analysis. This was true at both a patient level, using mean patient values, and a site level, using either individual patient or pooled patient data. All of these correlations were highly statistically significant for site comparisons. In inter- and intra-patient comparisons the proportion of significant correlations was greater for total enzyme activity than concentration. Clinical and radiological measurements of attachment loss showed generally similar levels of correlation. Total enzyme activities had good specificity and sensitivity as indicators of attachment loss in this cross-sectional study. The results support further investigation of the diagnostic potential of gingival crevicular fluid proteases in evaluation of the periodontal condition.