OBJECTIVES:This investigation monitored fluoride ingestion in Mexican children who were susceptible to developing dental fluorosis.METHODS:Total fluoride intake, from dietary and toothpaste samples, was determined in 15-36-month-old children from Mexico City and Veracruz, Mexico. A duplicate plate technique was used. Plasma fluoride levels were also determined in this group of children.RESULTS:The children ingested a mean (+/-SD) of 0.20 +/- 0.08 and 0.18 +/- 0.07 mg fluoride/kg/day, in Mexico City and Veracruz, respectively. There was no statistically significant difference between the fluoride ingested by children in the two cities (P > 0.9). Plasma from children in Mexico City and Veracruz contained a mean of 1.30 +/- 0.81 and 0.87 +/- 0.72 micro mol fluoride/l, respectively, and these values were not significantly different.CONCLUSIONS:Our results lead to the conclusion that mean fluoride ingested from the combination of foods and beverages was within the proposed safe threshold for fluoride intake of 0.05-0.07 mg F/kg/day. Most of the fluoride intake by these children was derived from the ingestion of fluoridated toothpaste. When all sources of ingested fluoride were added and total fluoride intake was calculated, the children, both in Mexico City and Veracruz, were ingesting amounts of fluoride well above the upper limits of the proposed safe threshold for fluoride intake.

In the spring of 1996, foods and beverages most commonly consumed by adolescents were analyzed for fluoride as part of a larger investigation. These foods were selected by interviewing 711 adolescents, 12-14 years of age, who were long-time residents of either an optimally or negligibly fluoridated community. The brand names of the identified foods and beverages most commonly purchased were determined by interviews with the parents. A total of 441 brand-name food and beverage items were purchased from both communities and were individually analyzed for fluoride. These analyses were done in order to estimate the fluoride content of various kinds of foods and beverages and to determine whether or not there was a significant difference between the two communities in the amount of fluoride ingested from these dietary sources. The food and beverage items were classified into dietary groups based on US Department of Agriculture (USDA) guidelines. Overall, the fluoride content of the sampled foods and beverages was low. In addition, there was no significant difference in the fluoride content of the same pre-packaged or ready-to-eat food or beverage items purchased in the two communities. However, a significant difference was found between the two communities in the fluoride content of fountain beverages and in cooked or reconstituted foods prepared using local water from the respective communities. Based on these results, we have estimated the mean daily, dietary fluoride intake for 3-5-year-old children who are more susceptible to developing dental fluorosis.

Previous studies have shown that pretreatment of dental enamel by specific carbon dioxide laser conditions inhibited subsequent progression of caries-like lesions in vitro. The aim of the present study was to use an intra-oral model to determine whether similar inhibition is observed in the human mouth. A cross over study with 23 subjects and three regimens was used. Pre-formed caries-like lesions were made in extracted human enamel and exposed intra-orally in partial dentures in each subject to A) placebo dentifrice [no fluoride added] and no laser treatment {P}, B) placebo dentifrice following laser pretreatment [9.6 mum, 100 mus pulse duration, 4 J/cm(2)] {L}, or C) sodium fluoride dentifrice [1100 ppm F] and no laser treatment {F} during each of three study periods. Samples were assessed by microradiography to compare the mineral loss before and after each treatment and derive a net change in mineral value. Overall (n=23) P was not significantly different from L but both P and L were different from F (mixed effects model of ranks with Bonferroni pairwise tests). For those subjects who demineralized in P (n=10), L and F were significantly better than P, with L showing an 84% inhibition of further demineralization, but no enhancement of remineralization.

Ingested fluoride is incorporated into bone apatite and can affect the structural integrity of bone. Fluoride absorption in the gut and incorporation into bone is affected by the presence of other ions, including calcium. We hypothesized that a low calcium phosphate diet combined with hi-h fluoride intake would have independent detrimental effects on vertebral bone strength. We measured the vertebral biomechanical properties and bone histology for rats given controlled diets with varied fluoride and calcium phosphate intakes for either 16 or 48 weeks. Diets were designed to produce mild calcium deficiency while keeping Ca:P ratio constant. Hence calcium deficiency was accompanied by a proportional phosphate deficiency. Calcium phosphate deficiency (25% of normal) significantly diminished vertebral strength only in younger rats. As the rats Crew older, the effects of calcium phosphate deficiency were mitigated. Calcium phosphate deficiency reduced vertebral strength in young rats primarily by reducing the trabecular bone volume. The highest fluoride intake (50 mg/L) significantly diminished vertebral strength, but there was no effect of lower fluoride intakes on bone strength. In contrast to calcium phosphate deficiency, high fluoride intake had no effect on trabecular bone volume but instead increased the amount of unmineralized osteoid, particularly in older rats. This impairment of mineralization by fluoride appeared to be the primary cause of the diminished vertebral strength. The detrimental effects of fluoride on vertebral bone strength were not amplified by calcium phosphate deficiency.

Currently our intra–oral model uses enamel specimens that have been disinfected by soaking in buffered formalin (pH 6.8). However, because of increasing emphasis on infection control, it is important to identify a way to sterilize these specimens. The aim of this study was to determine if autoclaved, or gas sterilized, lesioned enamel responds to fluoride (F) in the same way alcohol–disinfected enamel lesions do. Seventy–two formalin–disinfected, human enamel specimens (3 mm) were lesioned in demineralizing solution for 96 h and were then divided into three groups. One group was autoclaved; one group was gas sterilized (ethylene oxide), and the remaining 24 specimens were further disinfected in 70% ethanol for 10 min. Specimens in each group were then treated 4 times/day for 4 weeks with 0, 250 or 1,100 ppm F dentifrice slurries in an in vitro cycling, remin/demin model. Following treatment, fluoride uptake was analyzed by microdrill biopsy, and lesion depth and mineral content changes (ΔM) were determined by transverse microradiography. Data were analyzed by one–way ANOVA analysis. In all three groups of specimens there were significant (p<0.05) differences in fluoride uptake in response to different fluoride treatments. Autoclaved lesions failed to provide dose– response data with regard to changes in lesion mineral content. Because formalin and 70% alcohol are only disinfectants, and autoclaving altered the responsiveness of enamel lesions, results from this study suggest that, of the methods tested, gas sterilization is the preferred method for sterilizing enamel specimens that will be used in intra–oral studies.

ABSTRACTThe first step for a pathogenic bacterium to initiate infection is via attachment (i.e., through surface determinants) to a suitable receptor. An in vitro microbial artificial-mouth model was used to test the efficacy of polyclonal antibodies toStreptococcus mutanscell surface proteins (CsAb) and a cell surface 59-kDa protein (59Ab) in preventingS. mutanscolonization and carious lesion formation. In study 1, groups of 12 human teeth specimens were inoculated withS. mutans, which were incubated with different concentrations of CsAb (A1 [positive control], sterile saline, no antibody; A2, 0.007 mg of antibody protein/ml; and A3, 0.7 mg of antibody protein/ml) for 1 h at 37°C. The negative control group (B1) was not infected and was incubated with Trypticase soy broth (TSB) without dextrose supplemented with 5% sucrose (TSBS). In study 2, the same study design was used except that 59Ab was used instead of CsAb, normal rabbit serum was used in the positive control group (A1), and TSB supplemented with 1% glucose was used as the nutrient to control for sucrose-dependent colonization. All groups were exposed for 4 days to circulating cycles of TSBS and TSB (study 1 and study 2, respectively; 30 min each, three times per day) and a mineral washing solution (21 h per day). Prior to each nutrient cycle, 1 ml of the appropriate CsAb or 59Ab solution was administered to each group and allowed to mix for 30 min before cycling was resumed. Data obtained by confocal laser scanning microscopy demonstrated the presence of a significantly smaller (P< 0.05) lesion area and a smaller total lesion fluorescence in group A3 than in group A1 for both studies. In study 1, group A2 had significantly smaller values than A1 for lesion depth and area. There were no significant differences between groups A2 and A3 for lesion area or between groups A1 and A2 for total lesion fluorescence. In study 2, there were no significant differences among groups A1 and A2 for lesion depth or between groups A2 and A3 for all of the parameters studied. In both studies, there were no significant differences betweenS. mutansplaque CFU numbers among any of the groups. These studies demonstrated the efficacy of CsAb and 59Ab in reducing primary caries development in this model, although the underlying mechanism remains unclear.

ABSTRACTStreptococcus mutanshas been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination ofS. mutanssurface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected withS. mutansat 18 to 20 days of age, were intranasally immunized with a mixture ofS. mutanssurface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P< 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recoveredS. mutanscells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture ofS. mutanssurface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.

Fluoride therapy has been used clinically for many years, but its use remains controversial and many basic questions remain unanswered. Accordingly, this study returns to an animal model to study the effects of high doses of fluoride on bone mineral in rabbits. Twelve rabbits, aged 3½ months at the start of the study, received drinking water fluoridated at 100 ppm while their 12 control counterparts drank distilled water. All rabbits were sacrificed after 6 months. Fluoride was readily incorporated into femoral cortical bone (7473 ± 966 ppm F versus 1228 ± 57 ppm in controls; P < 0.00005). Fluoride therapy led to increased mineralization, as measured by density fractionation (P < 0.0005 for the distributions). The bone mineral itself was altered, with a significant increase in the width of crystals (66.2 ± 2.0 Å versus 61.2 ± 0.9 Å; P < 0.01). The microhardness of both cortical and cancellous bone in the femoral head of fluoride-treated rabbits was greater than that in the controls (P < 0.05). The phosphate, calcium, and carbonate contents in the bone was the same in both groups. Finally, fluoride administration did not affect the architecture or connectivity of cancellous bone in the femoral head. Previously published data [1] indicated that the mechanical properties of bone were adversely affected; this suggests that the effect of high doses of fluoride on the strength and stiffness of bone may be mediated by its effect on bone mineral.

UNLABELLED:While the level of fluoride intake that affords optimal cariostatic efficacy without causing dental fluorosis is not precisely known, it has been suggested that the threshold of fluoride exposure above which fluorosis may occur is between 0.05 and 0.07 mg/kg/day.OBJECTIVE:To monitor and compare fluoride intake from diet and dentifrice use (theoretical F: 0.10-0.11%) by three groups of 16- to 40-month-old children: two groups living in the negligibly water-fluoridated communities of San Juan, Puerto Rico, and Connersville, Indiana, and the third group residing in the optimally water-fluoridated region of Indianapolis, Indiana.METHODS:Fluoride intake from diet was monitored by the "duplicate plate" method, and fluoride ingested from dentifrice was determined by subtracting the amount of fluoride recovered after brushing from the amount originally placed on the child's toothbrush.RESULTS:The mean combined amount of fluoride ingested daily by children living in the negligibly fluoridated communities was not significantly different from that ingested by children in the fluoridated community. The major component of fluoride ingested by children in the negligibly fluoridated communities came from fluoridated dentifrice, and in the fluoridated area children ingested as much fluoride from toothpaste as they did from beverages. In San Juan mean daily fluoride intake was within the estimated range for safe fluoride exposure; however, in the "halo" community of Connersville and in Indianapolis, daily fluoride ingested by many of the children may have exceeded this level.CONCLUSION:Attention needs to be given, in negligibly water-fluoridated as well as in optimally water-fluoridated communities, to reducing the daily intake of fluoride by young children in order to avoid putting them at risk of developing dental fluorosis.

OBJECTIVES:To identify risk factors for dental fluorosis that cannot be explained by drinking water fluoride concentration alone.METHODS:Two hundred eighty-four Tanzanian children ages 9 to 19 (mean 14.0+/-SD 1.69), who were lifetime residents at differing altitudes (Chanika, 100 m; Rundugai, 840 m; and Kibosho, 1,463 m; Sites 1, 2, and 3 respectively) were examined for dental fluorosis and caries. They were interviewed about their food habits, environmental characteristics and use of a fluoride-containing food tenderizer known locally as magadi. Meal, urine, water and magadi samples supplied by the participants were analyzed for fluoride content. Urine samples were also analyzed for creatinine concentration. Four magadi samples from Sites 1 and 3 were analyzed for complete element composition.RESULTS:Of the 13 water samples from Site 2, 10 contained > or =4 mg/L F, ranging from 1.26 to 12.36 mg/L with a mean+/-SD of 5.72+/-4.71 mg/L. Sites 1 and 3 had negligible water fluoride of 0.05+/-0.05 and 0.18+/-0.32 mg/L respectively. Mean TFI fluorosis scores (range 0-9) for Site 2 were high: 4.44+/-1.68. In Sites 1 and 3, which both had negligible water fluoride, fluorosis scores varied dramatically: Site 1 mean maximum TFI was 0.01+/-0.07 and Site 3 TFI was 4.39+/-1.52. Mean DMFS was 1.39+/-2.45, 0.15+/-0.73 and 0.19+/-0.61 at Sites 1, 2, and 3, respectively. There were no restorations present. Urinary fluoride values were 0.52+/-0.70, 4.34+/-7.62, and 1.43+/-1.80 mg/L F at Sites 1, 2, and 3, respectively. Mean urinary fluoride values at Site 3 were within the normal urinary fluoride reference value range in spite of pervasive severe pitting fluorosis. Meal and magadi analyses revealed widely varied fluoride concentrations. Concentrations ranged from 0.01 to 22.04 mg/L F for meals and from 189 to 83211 mg/L F for magadi. Complete element analysis revealed the presence of aluminum, iron, magnesium, manganese, strontium and titanium in four magadi samples. There were much higher concentrations of these elements in samples from Site 3, which was at the highest altitude and had severe enamel disturbances in spite of negligible water fluoride concentration. An analysis of covariance model supported the research hypothesis that the three communities differed significantly in mean fluorosis scores (P<0.0001). Controlling for urinary fluoride concentration and urinary fluoride:urinary creatinine ratio, location appeared to significantly affect fluorosis severity. Urinary fluoride:urinary creatinine ratio had a stronger correlation than urinary fluoride concentration with mean TFI fluorosis scores (r=0.43 vs r= 0.25).CONCLUSIONS:The severity of enamel disturbances at Site 3 (1463 m) was not consistent with the low fluoride concentration in drinking water, and was more severe than would be expected from the subjects' normal urinary fluoride values. Location, fluoride in magadi, other elements found in magadi, and malnutrition are variables which may be contributing to the severity of dental enamel disturbances occurring in Site 3. Altitude was a variable which differentiated the locations.

On the basis of observations that endemic fluorosis occurs more often in malnourished populations, a series of studies tested the hypothesis that deficient dietary intake of calcium, protein or energy affects fluoride metabolism so that the margin of safe fluoride exposure may be reduced. The objective of the investigation was to determine whether changes in fluoride metabolism in nutritionally deficient rats resulted in manifestation of any extraskeletal toxic fluoride effects not observed in healthy animals. This investigation included two studies, one that monitored the effect of calcium deficiency on the effects of chronic fluoride exposure, and a second study that observed fluoride effects in rats that were deficient either in protein or in energy and total nutrient intake. Control and experimental rats received drinking water containing 0, 0.26 (5), 0.79 (15) or 2.63 (50) mmol fluoride/L (mg/L) for 16 or 48 wk, Control rats were fed optimal diets and experimental rats were fed diets deficient in calcium (Study 1) or protein (Study 2). An additional group of experimental rats (Study 2) was provided with a restricted amount of diet; thus these rats were deficient in energy and total nutrient intake, The intake, excretion and retention of fluoride were monitored; after the rats were killed, tissue fluoride levels and biochemical markers of tissue function were analyzed. Bone marrow cells were harvested from some of the rats, after 48 wk of treatment, for determining the frequency of sister chromatid exchange, a marker of genetic damage. Although there were significant differences among fluoride treatment groups in fluoride excretion and retention that resulted in significantly greater fluoride levels in tissues of the experimental rats, we were unable to detect any harmful, extraskeletal biochemical, physiologic or genetic effects of fluoride in the nutritionally deficient rats.

Tooth minerals are lost and regained constantly in a normal, human oral environment. Different methods have been developed to measure this gain and loss in enamel minerals; however, these methods deal with different problems, such as being time consuming or involving the use of X–rays. The aim of this study was to determine if remineralization measured in a thin enamel section (TS) by transversal microradiography (MR) can be reliably monitored by measuring lesion parameters (area, total and average dye fluorescence) on the same TS or on half a tooth (HT) with confocal laser scanning microscopy (CLSM). Thirty–six human enamel specimens were demineralized for 96h, and then half of each specimen was covered with an acid–resistant nail varnish. Specimens were divided into three groups (12/group) and subjected for 20 days to a cyclic remineralization regimen with consisted daily of a 4–hour acid challenge, four 1–min treatment periods with 0, 250 or 1,100 ppm F dentifrice slurries (1:2; dentifrice:water) and 20 h in pooled, human saliva, at room temperature. Specimens were cut and analyzed by MR, then stained with a fluorescent dye (0.1mM rhodamine B) for 1 h and analyzed using CLSM. Both MR and CLSM detected significantly greater remineralization (p<0.05) in the specimens treated with the fluoride–containing dentifrices than in the specimens treated with 0 ppm F. Significant differences were detected between specimens treated witht the fluoride–containing dentifrices by MR and CLSM (HT area and total fluorescence). Statistically significant (p<0.05) Pearson correlation coefficients were calculated between the MR and CLSM data: difference in MR mineral content (ΔM) versus HT lesion area = 0.71; ΔM versus HT total fluorescence = 0.70; ΔM versus HT average fluorescence = 0.61; ΔM versus TS lesion area = 0.88; ΔM versus TS total fluorescence = 0.63, and ΔM versus TS average fluorescence = 0.40. It is concluded that confocal microscopy in either TS or HT may provide valid surrogates (area and total fluorescence) for MR measurements in enamel remineralization studies.