Fluoride can have deleterious effects on cells, depending upon concentration (micromolar to millimolar), duration of exposure, and cell type (primary cultured cells or established cell lines). Exposures up to 1 mM of NaF failed to induce stress-response RNAs or initiate apoptosis in the mouse odontoblast cell line M06-G3 (Wurtz et al., 2008), whereas 1 mM NaF induced oxidative stress and apoptosis in rat primary hippocampal neurons (M Zhang et al., 2007). Exposures in the 5- to 10-mM range are required to induce apoptosis in rat thymocytes and human gingival fibroblasts, rat primary lung cells, and in the odontoblast cell line MDPC-23 (Thrane et al., 2001; Matsui et al., 2007; Lee et al., 2008; Karube et al., 2009), and micromolar NaF leads to apoptosis in neonatal rat osteoblasts and fetal human ameloblast lineage cells (Q Yan et al., 2007; X Yan et al., 2009). Rats exposed to 0, 10, 50, or 100 ppm F ions (approximately 0 to 5.0 mM F ions) in the drinking water over a period of 10 wks demonstrated increases in reactive oxygen species in the blood at 50 ppm and 100 ppm, without evidence of significant oxidative stress within the brain or liver (Chouhan and Flora, 2008). In the kidney, there was increased oxidative stress only in the 100-ppm-F group. Analysis of these data together suggests tissue-specific sensitivity to oxidative stress following fluoride exposure.
Evidence that fluoride can affect the bone marrow microenvironment comes from ex vivo studies of bone marrow cells collected from several strains of mice treated with NaF in the drinking water. Strain-dependent effects on hematopoietic colony-forming cell unit (CFU) assays were observed (D Yan et al., 2007; Chou et al., 2009). Bone marrow cells leading to the individual colonies (CFU) may have pluripotent characteristics capable of giving rise to mixed colonies containing multiple hematopoietic lineages (e.g., granulocytic, monocytic, and erythroid) or are committed, giving rise to cells from one hematopoietic lineage.
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Resistance and susceptibility (risk factors), defined by host and environment interactions, as well as many quantitative phenotypes are considered complex traits. Complex traits (phenotypes) can be assessed quantitatively and are under the control of multiple genes as well as non-genetic (environmental) factors. Multiple genes that contribute to the variation in a phenotypic trait are called quantitative trait loci (QTL). QTLs can be mapped in mice by traditional genetic approaches. Typically, two strains are selected that have widely different traits or responses. The parental mice are then used in a two-generation cross. First F1 hybrid progeny are generated, then used in sister-brother mating to produce F2 mice (Fig. 3). While all F1 mice are genetically identical, each F2 mouse is unique. This is the result of re-arrangement of the parental alleles during gametogenesis (meiotic recombination) in the F1 animals. Mapping of QTLs associated with DF susceptibility was performed with a dental-fluorosis-resistant strain (129P3/J) and the dental-fluorosis-sensitive (A/J) strain in a two-generation cross to create a panel of F2 mice as described above. All F2 mice were treated with fluoride 50 ppm F in the drinking water and, after 60 days, were phenotyped for DF according to the modified TF scale. Treatment of F2 mice with 50 ppm F in the water yields a mean serum [F] of 12.366 1.713 µM. The serum [F] concentrations between F2 mice with different DF severities were not significantly different and were not significantly different from serum [F] concentrations determined in comparably treated parental mice (11.296 3.984 µM). To maximize the power to detect QTLs contributing to the variation in response to dental fluorosis, only the phenotypic extreme F2 animals (those with TF scores of 1 or 4) were genotyped for 354 SNP-based markers distributed throughout the mouse genome. This panel of mice was composed of equal numbers of males and females. Chi-square analysis was performed to compare the genotypic distributions in the two groups of phenotypically extreme F2 mice. Significant evidence of association was observed on chromosomes 2 and 11 for a series of consecutive markers (p
Narrowing the QTL intervals to fewer genes and, ultimately, the selection of candidate genes remain the challenging aspects of complex trait dissection. This can be accomplished in mice by increasing marker densities within QTLs and using a complementary approach based upon haplotype mapping. Haplotype Association Mapping (HAM) is a phenotype-driven approach to identify genetic loci in mice. This method is similar to Genome-Wide Association Studies (GWAS) in humans. HAM looks for associations between the phenotype and the haplotypes of mouse inbred strains, treating inbred strains as individuals. Since mice within the strain are isogenic, several individuals can be phenotyped to minimize intra-strain variation. The application of haplotype association mapping in mice was first described in 2001 and has developed into a useful tool for QTL mapping (Grupe et al., 2001; Tsaih and Korstanje, 2009). Integrating haplotype-based approaches with traditional mapping tools as described above has great potential for narrowing QTL mapping intervals and prioritizing candidate genes (Pletcher and Wiltshire, 2004; Cervino et al., 2005, 2007; Arbilly et al., 2006). It is conceivable that interval-specific haplotype analysis based upon an a priori knowledge of a QTL interval can reach a resolution of less than 5 Mb (DiPetrillo et al., 2005). Recently, haplotype association mapping in mice identified a haplotype block containing the Cer1 (cerberus 1 homolog) gene that partitions inbred mice strains into high and low bone mineral density groups. The Cer1 gene is important during embryonic development and appears to play a role in bone development. Based upon the discovery in mice, the human CER1 gene was investigated, and a non-synonymous SNP (rs3747532) was identified to be associated with increased risk of low bone mineral density in pre-menopausal women (Tang et al., 2009). 2ff7e9595c
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