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RESEARCH REPORT |
1 Oral-Facial Genetics Division,
2 Department of Oral Facial Development,
3 Graduate Program in Pediatric Dentistry,
4 Department of Preventive and Community Dentistry, and
5 Oral Health Research Institute, Indiana University School of Dentistry, 1121 W. Michigan St., Room 270, Indianapolis, IN 46202;
* corresponding author, eeverett{at}iupui.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dental fluorosis • inbred mouse strains • quantitative light-induced fluorescence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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While it is well-accepted that fluoride interacts with mineralized tissues and, at elevated concentrations, disturbs the mineralization process (Aoba and Fejerskov, 2002), the molecular mechanisms that underlie the pathogenesis of dental fluorosis are not known. We hypothesize that genotype can influence susceptibility or resistance to develop dental fluorosis.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Fluoride Treatment
Three levels of fluoride treatment were delivered ad libitum in the drinking water (0, 25, and 50 ppm F) as NaF. The de-ionized and prepared water samples were periodically analyzed by ion-specific electrode (ISE) for fluoride concentration. The de-ionized water tested from multiple sites within the IUSD Bioresearch Facility showed [F] = 0.02 + 0.01 ppm. Each batch of prepared 25 ppm and 50 ppm water samples used during the study was analyzed, and [F] = 24.49 + 0.27 ppm and 49.68 + 0.51 ppm, respectively.
Three treatment groups consisted of 72 mice each (6 each from the 12 strains studied). Group 1 received distilled water, group 2 received distilled water supplemented with 25 ppm fluoride, and group 3 received distilled water supplemented with 50 ppm fluoride.
Oral Examinations
All mice were uniquely identified, and the two examiners (ETE and MAKM) were blinded to the mouse strains and to the treatment groups. All mice were monitored daily for any changes in health status. Once a week, each mouse was given a complete oral examination where incisor tooth color was assessed along the entire labial surface of the enamel by means of a shade guide (Vita Shade Guide, Vident, Brea, CA, USA). The shade guide was analyzed according to the CMYK (cyan, magenta, yellow, and black) color model in the Photoshop 5.5 software, permitting the selective grouping of shades based upon yellow content. This reduced the observations to an ordinal scale (1-3) where 1 = predominantly shades of white, 2 = yellow/tan, and 3 = shades of deep yellow to yellow-orange. Opacity (adjudged when a periodontal probe was passed behind each incisor), length (in mm with the use of a periodontal probe), enamel wear pattern, and overall development of the upper and lower incisors were also independently judged. The determination of dental fluorosis was made clinically over the entire upper and lower incisor tooth surfaces according to a modified TF index (Thylstrup and Fejerskov, 1978). At day 60 into the treatment, mice were killed, weighed, and examined, and selected mineralized tissues were removed.
Quantitative Light-induced Fluorescence (QLF)
One pair of mandibular central incisors from each animal was dissected, rinsed briefly in 10% formalin, and placed in a labeled microfuge tube. A QLF camera handpiece was attached to a vertical camera stand at a fixed distance of 5 cm above a black cardboard plate. The lower incisors were laid labial side up, flat on the cardboard, and stabilized with wax if needed. An image was taken of the teeth with the QLF handpiece connected to a control-box (Inspektor Research Systems BV, Amsterdam, Netherlands) and analyzed by QLF software, version 2.00d. The software captured an image of the teeth using 10 frames to create an original image in a bitmap format. The areas of the teeth that demonstrated fluorosis by clinical examination showed up as bright areas on the QLF images, indicating an increase in fluorescence. The images were further processed by Photoshop 5.5 software, with the original images inverted such that white lesions appeared dark. The inverted images were then analyzed by the algorithm in the QLF software, version 2.00d, for quantification of the average increase in fluorescence and the size of the affected area of the teeth.
Fluoride Analyses
De-ionized and fluoridated water samples were analyzed directly for fluoride concentration by means of a combination fluoride-specific electrode (Orion #96-909-00) and an Accumet 950 pH/ion meter (Fisher Scientific, Itasca, IL, USA). Analyses of mouse mineralized tissues and mouse chow were performed in duplicate by a modification (Rojas-Sanchez et al., 1999) of the hexamethyldisiloxane (HMDS; Sigma Chemical Co., St. Louis, MO, USA) microdiffusion method of Taves (1968).
Statistical Analyses
Data were analyzed by descriptive statistics, analysis of variance (one-way ANOVA), Student’s t test, or Pearson’s Correlation where appropriate. Values of p < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Dental fluorosis in the A/J strain developed very early, within a couple of weeks, and was manifested at both 25- and 50-ppm-[F]-treated groups. In contrast, the 129P3/J strain (Fig. 1) was the most resistant strain tested, showing minimal dental fluorosis only at the 50-ppm-[F] level. The maxillary and mandibular incisors shown in the 0-ppm-[F] panel illustrate normal tooth development at 13 wks of age for both A/J and 129P3/J mice.
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). When the color scale was reduced to an ordinal scale of 1-3, the examiners’ observations were highly mutually correlative. Inter-examiner variation for upper incisor color determination was measured by Pearson’s Correlation, two-tailed, for each strain, r = 0.649 to 0.832 (mean = 0.740 + 0.06) and significant at p < 0.01. Monitoring these tooth color changes added dimension to the clinical characterization of dental fluorosis in these strains of mice.
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Quantitative Light-induced Fluorescence (QLF) Analysis as a Quantitative Measure of Fluorosis
Mouse mandibular incisors were subjected to QLF analysis. The areas of the teeth that were affected by fluorosis as judged by clinical examination showed up as bright areas on the QLF images, indicating an increase in fluorescence. The amount and area of the fluorescent signal were sufficient to allow us to discriminate among groups of animals with different severities of dental fluorosis (Table 1). There was strong correlation between QLF of the lower incisors and that in the upper incisor exams (Pearson’s Correlation, two-tailed, r = 0.766, p = 0.004), and there was also modest correlation between QLF and the modified TFI scale used in the clinical determination of dental fluorosis (Pearson’s Correlation, two-tailed, r = 0.558, p < 0.001). We show that QLF analysis provides a quantitative measurement of dental fluorosis. For the resistant 129P3/J strain, there was no significant difference between Delta Q at 0 ppm [F] compared with 50 ppm [F] (p = 0.413), whereas, in the susceptible A/J strain, the Delta Q at 0 ppm [F] compared with 50 ppm [F] was significant (p = 0.006). Furthermore, the difference between the most sensitive strain (A/J) and the most resistant strain (129P3/J) is large. At 50 ppm, A/J vs. 129P3/J p = 0.005 (Student’s t test, two-tailed, paired).
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). At 50 ppm [F], there were no significant differences for femur [F] between the 129P3/J and A/J strains (p = 0.559). The total [F] in the entire incisor (dentin and enamel) from the gum line to the tip was measured in the dental-fluorosis-susceptible (A/J) and -resistant (129P3/J) strains (Table 2). The accumulated fluoride in the incisor correlated well with fluoride exposure (Pearson’s Correlation, two-tailed, of drinking water [F] vs. incisor [F], r = 0.799, p < 0.00001), and no significant differences were seen between the fluorosis-susceptible and -resistant strains and at either [F] concentration, p = 0.457.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The clinical presentation of dental fluorosis in mice parallels that which can be seen in mild forms of dental fluorosis in humans. Variations in the onset and severity of dental fluorosis among different inbred strains of mice when age, gender, food, housing, and fluoride exposure are rigorously controlled support the influence of genetic background on dental fluorosis susceptibility/resistance. Mice were grouped into resistant (129P3/J, FVB/NJ, CBA/J, and DBA/1J), intermediate (SWR/J, BALB/cJ, C57BL/10J, and DBA/2J), and sensitive (A/J, SJL/J/ C3H/HeJ, and C57BL/6J) strains. Not all closely related strains behaved similarly. For example, the C57BL/6J and C57BL/10J strains diverged from a common ancestor 
70 yrs ago and show very similar profiles, whereas the DBA/1J and DBA/2J strains were separated more recently and show very different sensitivities. One might also view the differences between related strains as an indication of the actions of a small number of genes in determining susceptibility. The identification of dental-fluorosis-susceptible and -resistant inbred mouse strains will permit the genetic dissection of the genes and pathways involved in enamel formation that underlie the pathogenesis of dental fluorosis. Presently, few studies have been conducted which explore an underlying genetic basis for fluoride resistance. High concentrations of fluoride have been used to isolate fluoride-resistant mutants of Caenorhabditis elegans which have led to the identification of a novel fluoride-resistant gene, an ion channel belonging to the degenerin/epithelial sodium channel superfamily, which regulates defecation rhythm (Katsura et al., 1994; Take-Uchi et al., 1998).
Quantitative light-induced fluorescence (QLF) has emerged in our studies as an innovative tool for the detection, staging, and quantification of dental fluorosis. Fluorescence of teeth and its implications in caries diagnosis have been studied (Angmar-Månsson et al., 1996). Dental fluorosis has been observed on images during caries examinations with the QLF technique, presenting as diffuse dark areas (Ferreira-Zandona et al., 2000). The increased fluorescence seen in the fluorosis lesion is most likely due to the increase in porosity that occurs in fluorosed enamel, similar to the increase in porosity that is found in caries (Angmar-Månsson et al., 1994).
Our investigation of chronic fluoride burden in mineralized tissues (femur and mandibular incisor) in the study animals suggests that active removal of fluoride from the body is not a sufficient explanation for dental fluorosis resistance. Based upon the lack of significant differences between [F] in the erupted incisor and that in the inbred mouse strain (p = 0.457), it is possible that resistant mice are more tolerant to fluoride in their enamel organ microenvironment, whereas susceptible mice are more sensitive to the amount of fluoride within their enamel organ microenvironment. In the erupted mandibular incisors, enamel covers only the labial surface and averages less than 0.1 mm thick (Moinichen et al., 1996). Therefore, it was not possible to separate the enamel from the dentin in our fluoride determinations and provide mapping of fluoride within the incisors’ microanatomy.
Quantitative trait loci (QTL) mapping with the A/J (susceptible) and 129P3/J (resistant) inbred mouse strains can be used in this complex trait dissection to identify the actual genes that directly and/or indirectly contribute to an individual’s susceptibility or resistance to develop dental fluorosis and to an understanding of the cellular roles and functions of these genes during the pathogenesis of fluorosis.
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ACKNOWLEDGMENTS |
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Received May 13, 2002; Last revision August 2, 2002; Accepted August 6, 2002
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REFERENCES |
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