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Dental Fluorosis: Variability among Different Inbred Mouse Strains
1 Oral-Facial Genetics Division, Correspondence: * corresponding author, eeverett{at}iupui.edu
Concurrent with the decline in dental caries has been an increase in the prevalence of dental fluorosis, a side-effect of exposure to greater than optimal levels of fluoride during amelogenesis. The mechanisms that underlie the pathogenesis of dental fluorosis are not known. We hypothesize that genetic determinants influence an individual’s susceptibility or resistance to develop dental fluorosis. We tested this hypothesis using a mouse model system (continuous eruption of the incisors) where genotype, age, gender, food, housing, and drinking water fluoride level can be rigorously controlled. Examination of 12 inbred strains of mice showed differences in dental fluorosis susceptibility/resistance. The A/J mouse strain is highly susceptible, with a rapid onset and severe development of dental fluorosis compared with that in the other strains tested, whereas the 129P3/J mouse strain is least affected, with minimal dental fluorosis. These observations support the contribution of a genetic component in the pathogenesis of dental fluorosis.
Key Words: dental fluorosis • inbred mouse strains • quantitative light-induced fluorescence
According to the Centers for Disease Control (CDC), fluoridation of community drinking water for the prevention of dental caries is considered to be one of the ten most important public health achievements of the 20th century (1999). Concurrent with the decline in dental caries has been an increase in the prevalence of dental fluorosis, a side-effect of systemic exposure to greater than optimal levels of fluoride during amelogenesis. The prevalence of dental fluorosis in recent years ranges from 7.7% to 80.9% of the population in communities with fluoridated water and from 2.9% to 42% in communities with nonfluoridated water (Clark, 1994; Mascarenhas, 2000; Pendrys, 2000). Although the increase in prevalence has occurred primarily in very mild and mild forms of dental fluorosis, there is evidence that the prevalence of moderate and severe fluorosis is increasing as well (Clark, 1994). In addition to those major risk factors previously identified (use of fluoridated drinking water, fluoride supplements, fluoride toothpaste, and infant formulas) (Mascarenhas, 2000), other factors, including critical periods of fluoride exposure during tooth development and individual variation, are important as well. 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.
Mice From The Jackson Laboratory (Bar Harbor, ME), we obtained weanling (three-week-old) male mice representing the following inbred strains: 129P3/J, A/J, BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, CBA/J, DBA1/J, DBA2/J, FVB/NJ, SJL/J, and SWR/J. The selection of these mouse strains was based upon their phenotypic and genetic differences (the degree of unrelatedness from each other) as well as their general availability as normal laboratory strains. All mice were housed within the Indiana University School of Dentistry Bioresearch Facility, a fully AAALAC-accredited unit. Mice were contained in boxed caging and allowed food and water ad libitum. This study was fully approved by the Indiana University School of Dentistry IACUC. Mice were fed a laboratory rodent diet #5001 (PMI® Nutrition International, Richmond, IN, USA). Fluoride determinations of different batches of rodent diet fed to the mice were performed by a modified diffusion method (Rojas-Sanchez et al., 1999). The fluoride concentration in the diet was found to range from 5.0 µg/gm to 8.0 µg/gm (mean 7.2 + 0.8 µg/gm).
Fluoride Treatment 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
Quantitative Light-induced Fluorescence (QLF)
Fluoride Analyses
Statistical Analyses
Weekly oral examinations of study mice showed the onset of fluoride-related color changes in the incisors (dental fluorosis) to be gradual in some strains (i.e., BALB/cJ and FVB/NJ). Other strains (i.e., A/J, SJL/J, C57BL/6J, and C3H/HeJ) developed dental fluorosis more quickly. A few mouse strains, like 129P3/J and CBA/J, developed minimal dental fluorosis. While all strains developed various severities of dental fluorosis at 50 ppm [F], some strains were also responsive at the 25-ppm-[F] level. When dental fluorosis developed, the upper incisors and lower incisors appeared white, chalky opaque, and thin. Striations and erosions of the enamel were particularly prominent on the lower incisors. Quite robust fluorosis was seen in the A/J strain (Fig. 1  ). 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.
Upper Incisor Color as an Index of Fluorosis As the incisors erupted, natural color development was modified during the 60-day course of fluoride treatment. Diminished tooth color, as an indicator of dental fluorosis development, was dose-responsive to fluoride exposure (Fig. 2 ). 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.
Longitudinal study of upper incisor color permitted mice to be 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.
Quantitative Light-induced Fluorescence (QLF) Analysis as a Quantitative Measure of Fluorosis
Fluoride Accumulation in Femurs and Incisors as Indicators of Exposure We analyzed body fluoride burden by determining the quantity of fluoride in mineralized tissues (femurs and mandibular incisors). Femur fluoride (µg F/mg of ashed bone) was determined for all mice across all 12 strains studied. There was significant correlation between fluoride concentration in water and fluoride concentration in the femur, r = 0.869, p < 0.00001 (Pearson’s Correlation, two-tailed). When we compared the femur [F] for each strain at each fluoride treatment dose, there were no significant differences at 0 ppm [F] (p = 0.719) and at 50 ppm [F] (p = 0.06). At 25 ppm [F] (p < 0.01), differences were noted. Overall, all mice accumulated similar amounts of fluoride in their long bone, regardless of whether they exhibited mild to severe dental fluorosis. This was true for the most susceptible (A/J) and the most resistant (129P3/J) strains (Table 2 ). 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.
Many studies have identified the rodent as a useful model for determining the effects of fluoride on dental development (reviewed by Richards, 1990). The mouse remains an important model for aiding our understanding of the pathogenesis of dental fluorosis for several reasons. First, the continual growth of mandibular and maxillary incisors in mice permits the effects of fluoride on new enamel formation to be monitored (Ness, 1965). Second, each inbred mouse strain represents a genetically homogenous group that often differs quite remarkably from mice in another inbred strain (Atchley and Fitch, 1991). Finally, inbred mouse strains have been used for genetic studies because of the isogenicity within a strain and the genetic heterogeneity between inbred strains (Beck et al., 2000).
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 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.
An Indiana 21st Century Research & Technology Grant to GKS supported this investigation. Received for publication May 13, 2002. Revision received August 2, 2002. Accepted for publication August 6, 2002.
Journal of Dental Research, Vol. 81, No. 11,
794-798 (2002) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
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 (
