5
Musculoskeletal Effects

This chapter evaluates the effects of fluoride exposure on the musculoskeletal system. Topics considered include the effects of fluoride on bone cells (both bone-forming and bone-resorbing cells), on the developing growth plate, and on articular cartilage as it may relate to arthritic changes. New data on the effects of fluoride on skeletal architecture, bone quality, and bone fracture are also considered. Information on bone cancer is provided in Chapter 10. Effects on tooth development and other issues of oral biology are discussed in Chapter 4.

CHEMISTRY OF FLUORIDE AS IT RELATES TO MINERALIZING TISSUES

Fluoride is the ionic form of the element fluorine. Greater than 99% of the fluoride in the body of mammals resides within bone, where it exists in two general forms. The first is a rapidly exchangeable form that associates with the surfaces of the hydroxyapatite crystals of the mineralized component of bone. Fluoride in this form may be readily available to move from a bone compartment to extracellular fluid. Bone resorption is not necessary for the release of fluoride in this form. However, the predominant form of fluoride in bone resides within the hydroxyapatite crystalline matrix.

Hydroxyapatite is the mature form of a calcium phosphate insoluble salt that is deposited in and around the collagen fibrils of skeletal tissues. The formula for pure hydroxyapatite is CA10(PO4)6OH2. It results from the maturation of initial precipitations of calcium and phosphate during the mineralization process. As the precipitate matures, it organizes into



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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards 5 Musculoskeletal Effects This chapter evaluates the effects of fluoride exposure on the musculoskeletal system. Topics considered include the effects of fluoride on bone cells (both bone-forming and bone-resorbing cells), on the developing growth plate, and on articular cartilage as it may relate to arthritic changes. New data on the effects of fluoride on skeletal architecture, bone quality, and bone fracture are also considered. Information on bone cancer is provided in Chapter 10. Effects on tooth development and other issues of oral biology are discussed in Chapter 4. CHEMISTRY OF FLUORIDE AS IT RELATES TO MINERALIZING TISSUES Fluoride is the ionic form of the element fluorine. Greater than 99% of the fluoride in the body of mammals resides within bone, where it exists in two general forms. The first is a rapidly exchangeable form that associates with the surfaces of the hydroxyapatite crystals of the mineralized component of bone. Fluoride in this form may be readily available to move from a bone compartment to extracellular fluid. Bone resorption is not necessary for the release of fluoride in this form. However, the predominant form of fluoride in bone resides within the hydroxyapatite crystalline matrix. Hydroxyapatite is the mature form of a calcium phosphate insoluble salt that is deposited in and around the collagen fibrils of skeletal tissues. The formula for pure hydroxyapatite is CA10(PO4)6OH2. It results from the maturation of initial precipitations of calcium and phosphate during the mineralization process. As the precipitate matures, it organizes into

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards hexagonal, terraced hydroxyapatite crystals. Recent analysis of bone mineral indicates that a significant proportion of the hydroxyapatite crystal is a form of carbonated apatite, where carbonyl groups (CO3−) replace some of the OH− groups. Carbonated apatite is more soluble than hydroxyapatite at acid pH. Fluoride incorporation into the crystalline structure of bone mineral occurs with the creation of a form of apatite known as fluoroapatite (or fluorapatite). The formula for this form of the crystal is Ca10(PO4)6F2 or Ca10(PO4)6OHF. These crystals also take on a hexagonal shape and are found in terraced layers but, depending on the extent of fluoride in the crystal, may be somewhat more elongated than pure hydroxyapatite. Because fluoroapatite is less soluble in acidic solutions than hydroxyapatite, it was expected that fluoride incorporation into bone might actually make the tissue stronger. However, this has proven not to be the case in human studies (see below). Release of fluoride from bone when it is in the form of fluoroapatite requires osteoclastic bone resorption. Acidification of the mineral matrix by the osteoclast is sufficient to solubilize the fluoroapatite and allow free exchange with extracellular fluids. Once released, the effect of fluoride on bone cells may be evident; however, the form in which fluoride has its effect remains under debate. Some investigators contend that fluoride directly affects bone cells, but others claim that the effect must be mediated by fluoride while in a complex with aluminum. Do fluroaluminate complexes exist in biological fluids? The answer to this question depends in large part on pH, protein concentration, and cell composition. However, in general, in the acid environment of the stomach much of the aluminum and fluoride exist in a complex of AlF3 or AlF4−. These forms (mostly AlF3) have been purported to cross the intestine and enter cells (Powell and Thompson 1993). Once inside a bone cell the AlFx form appears to activate a specific protein tyrosine kinase through a G protein and evoke downstream signals. A more complete discussion of this process is presented in a later section of this chapter. The prolonged maintenance of fluoride in the bone requires that uptake of the element occurs at the same or greater rate than its clearance. This appears to be the case. (See Chapter 3 for more detailed discussion of the pharmacokinetic data on fluoride.) Turner et al. (1993) put forward a mathematical model that appears to fit the known pharmacokinetic data. This model assumes that fluoride influx into bone is a nonlinear function. This assumption is supported by pharmacokinetic data (Ekstrand et al. 1978; Kekki et al. 1982; Ekstrand and Spak 1990) and is required for the model to accurately predict fluoride movements. Another reasonable assumption is that the bulk of fluoride that moves between the skeleton and the extracellular fluid is due to bone remodeling. That is, most of the fluoride is either influxing or effluxing as a result of cellular activity. The outcome of the

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Turner model predicts that (1) fluoride uptake is positively associated with the bone remodeling rate and (2) fluoride clearance from the skeleton takes at least four times longer than fluoride uptake. A key correlate to the first prediction is that the concentration of fluoride in bone does not decrease with reduced remodeling rates. Thus, it appears that fluoride enters the bone compartment easily, correlating with bone cell activity, but that it leaves the bone compartment slowly. The model assumes that efflux occurs by bone remodeling and that resorption is reduced at high concentrations of fluoride because of hydroxyapatite solubility. Hence, it is reasonable that 99% of the fluoride in humans resides in bone and the whole body half-life, once in bone, is approximately 20 years (see Chapter 3 for more discussion of pharmacokinetic models). The effects of fluoride on bone quality are evident but are less well characterized than its effects on bone cells. Bone quality is an encompassing term that may mean different things to different investigators. However, in general it is a description of the material properties of the skeleton that are unrelated to skeletal density. In other words, bone quality is a measure of the strength of the tissue regardless of the mass of the specimen being tested. It includes parameters such as extent of mineralization, microarchitecture, protein composition, collagen cross linking, crystal size, crystal composition, sound transmission properties, ash content, and remodeling rate. It has been known for many years that fluoride exposure can change bone quality. Franke et al. (1975) published a study indicating that industrial fluoride exposure altered hydroxyapatite crystal size and shape. Although the measurements in their report were made with relatively crude x-ray diffraction analyses, they showed a shorter and more slender crystal in subjects who were aluminum workers and known to be exposed to high concentrations of fluoride. Other reports documenting the effects of fluoride on ultrasound velocities in bone, vertebral body strength, ash content, and stiffness have shown variable results (Lees and Hanson 1992; Antich et al. 1993; Richards et al. 1994; Zerwekh et al. 1997a; Søgaard et al. 1994, 1995, 1997); however, the general conclusion is that, although there may be an increase in skeletal density, there is no consistent increase in bone strength. A carefully performed comparison study between the effects of fluoride (2 mg/kg/day) and alendronate in minipigs likely points to the true effect: “in bone with higher volume, there was less strength per unit volume, that is, … there was a deterioration in bone quality” (Lafage et al. 1995). EFFECT OF FLUORIDE ON CELL FUNCTION Two key cell types are responsible for bone formation and bone resorption, the osteoblast and osteoclast, respectively. Osteoprogenitor cells give rise to osteoblasts. Osteoprogenitor cells are a self-renewing population of

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards cells that are committed to the osteoblast lineage. They originate from mesenchymal stem cells. Osteoblasts contain a single nucleus, line bone surfaces, possess active secretory machinery for matrix proteins, and produce very large amounts of type I collagen. Because they also produce and respond to factors that control bone formation as well as bone resorption, they play a critical role in the regulating skeletal mass. Osteoclasts are giant, multinucleated phagocytic cells that have the capability to erode mineralized bone matrix. They are derived from cells in the monocyte/macrophage lineage. Their characteristic ultrastructural features allow them to resorb bone efficiently by creating an extracellular lysosome where proteolytic enzymes, reactive oxygen species, and large numbers of protons are secreted. Osteoclastogenesis is controlled by local as well as systemic regulators. Effect of Fluoride on Osteoblasts Perhaps the single clearest effect of fluoride on the skeleton is its stimulation of osteoblast proliferation. The effect on osteoblasts was surmised from clinical trials in the early 1980s documenting an increase in vertebral bone mineral density that could not be ascribed to any effect of fluoride on bone resorption. Biopsy specimens confirmed the effect of fluoride on increasing osteoblast number in humans (Briancon and Meunier 1981; Harrison et al. 1981). Because fluoride stimulates osteoblast proliferation, there is a theoretical risk that it might induce a malignant change in the expanding cell population. This has raised concerns that fluoride exposure might be an independent risk factor for new osteosarcomas (see Chapter 10 for the committee’s assessment). The demonstration of an effect of fluoride on osteoblast growth in vitro was first reported in 1983 in avian osteoblasts (Farley et al. 1983). This study showed that fluoride stimulated osteoblast proliferation in a biphasic fashion with the optimal mitogenic concentration being 10 µM. The finding that fluoride displayed a biphasic pattern of stimulation (achieving a maximal effect at a specific concentration and declining from there) suggests that multiple pathways might be activated. It is possible that low, subtoxic doses do stimulate proliferation, but at higher doses other pathways responsible for decreasing proliferation or increasing apoptosis might become activated. This thinking suggested that fluoride might have multiple effects on osteoblasts and that might be the reason for some paradoxical findings in the clinical literature (see below). Nevertheless, the characteristics of the fluoride effect point clearly to a direct skeletal effect. Some of these characteristics are as follows: (1) the effects of fluoride on osteoblasts occur at low concentrations in vivo and in vitro (Lau and Baylink 1998); (2) fluoride effects are, for the most part, skeletal specific (Farley et al. 1983; Wergedal et al. 1988); (3) fluoride effects may require the presence of a bone-active

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards growth factor (such as insulin-like-growth factor I or transforming growth factor β) for its action (Farley et al. 1988; Reed et al. 1993); and (4) fluoride affects predominantly osteoprogenitor cells as opposed to mature functioning osteoblasts (Bellows et al. 1990; Kassem et al. 1994). Understanding the subcellular signaling mechanisms by which fluoride affects osteoblasts is of paramount importance. Information in this area has the potential to determine whether the fluoride effects are specific, whether toxicity is an issue, and what concentration may influence bone cell function. Moreover, as the pathways become more clearly defined, other targets might emerge. Two hypotheses in the literature describe the effect of fluoride. Both state that the concentration of tyrosine phosphorylated signal pathway intermediates is elevated after fluoride exposure. However, the means by which this occurs differs in the hypotheses. One view is that fluoride blocks or inhibits the activity of a phosphotyrosine phosphatase, thereby increasing the pool of tyrosine-phosphorylated proteins. The other view supports an action of fluoride (along with aluminum) on the stimulation of tyrosine phosphorylation that would also increase the pool of tyrosine-phosphorylated proteins. In the first hypothesis, growth factor activation of the Ras-Raf-MAP kinase pathway would involve stimulation of phosphotyrosine kinase activity. This is mediated by a family of cytosolic G proteins with guanosine triphosphate acting as the energy source. In the presence of fluoride, a sustained high concentration of tyrosine-phosphorylated proteins would be maintained because of the inability of the cell to dephosphorylate the proteins. This theory implicates the existence of a fluoride-sensitive tyrosine phosphatase in osteoblasts. Such an enzyme has been identified and purified. It appears to be a unique osteoblastic acid phosphatase-like enzyme that is inhibited by clinically relevant concentrations of fluoride (Lau et al. 1985, 1987, 1989; Wergedal and Lau 1992). The second hypothesis supports the belief that an AlFx complex activates tyrosine phosphorylation directly. Data from this viewpoint indicate that fluoride alone does not stimulate tyrosine phosphorylation but rather that it requires the presence of aluminum (Caverzasio et al. 1996). The purported mechanism is that the MAP kinase pathway is activated by AlFx, which triggers the proliferation response. A novel tyrosine kinase, Pyk2, has been identified that is known to be activated by AlFx through a G-protein-coupled response and might be responsible for this effect (Jeschke et al. 1998). Two key pieces of evidence that support a G-protein-regulated tyrosine kinase activation step in the fluoride effect are that the mitogenic effect of fluoride can be blocked by genistein (a protein tyrosine kinase inhibitor) and pertussis toxin (a specific inhibitor of heterotrimeric G proteins) (Caverzasio et al. 1997; Susa et al. 1997). At least two other potential mechanisms deserve mention. Kawase and Suzuki (1989) suggested that fluoride activates protein kinase C (PKC),

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards and Farley et al. (1993) and Zerwekh et al. (1990) presented evidence that calcium influx into the cells might be a signal for the fluoride-mediated stimulation of proliferation. In summary, the in vitro effects of fluoride on osteoblast proliferation appear to involve, at the least, a regulation of tyrosine-phosphorylated proteins. Whether this occurs through activation of MAP kinases, G proteins, phosphatases, PKC, or calcium (or a combination) remains to be determined. Whatever the mechanism, however, it is evident that fluoride has an anabolic activity on osteoblasts and their progenitors. The effects of fluoride on osteoblast number and activity in in vivo studies and clinical trials essentially parallel the in vitro findings. Most reports document increased osteoblast number; however, some investigators have documented a complex and paradoxical effect of fluoride in patients with skeletal fluorosis. Boivin et al. (1989, 1990) reported that, in biopsy bone cores taken from 29 patients with skeletal fluorosis of various etiologies (0.79% ± 0.36% or 7,900 ± 3,600 milligrams per kilogram [mg/kg] of bone ash), there is an apparent increase in the production of osteoblasts with a concomitant increase in a toxic effect of fluoride at the cell level. They provided data to indicate that chronic exposure to fluoride in both endemic and industrially exposed subjects led to an increase in bone volume, an increase in cortical width, and an increase in porosity. However, there was no reduction in cortical bone mass. Osteoid parameters (unmineralized type I collagen) were also significantly increased in fluorotic patients. Interestingly, the fluorotic group had more osteoblasts than the control group, with a very high proportion of quiescent, flattened osteoblasts, but the mineral apposition rate was significantly decreased. It appeared as though the increased numbers of quiescent cells were in a prolonged inactive period. Thus, the conclusion drawn by these investigators was that fluoride exposure increased the birth rate of new osteoblasts, but at high concentrations there was an independent toxic effect on the cells that blocked the full manifestation for the increase in skeletal mass. Boivin et al. used a fluoride-specific electrode for measurements in acidified specimens of human bone. As a point of reference to the above findings, they found that normal control subjects (likely not to have lived in areas with water fluoridation) have mean fluoride content in bone ash (from iliac crest samples) ranging from 0.06% to 0.10% (600 to 1,000 mg/kg); untreated osteoporotic patients range from 0.05% to 0.08% (500 to 800 mg/kg); NaF-treated osteoporotic patients range from 0.24% to 0.67% (2,400 to 6,700 mg/kg) depending on duration of therapy; and skeletal fluorosis patients range from 0.56% to 1.33% (5,600 to 13,300 mg/kg) depending on the source and level of exposure (Boivin et al. 1988). All these ranges are of mean concentrations of fluoride and not individual measurements.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Effect of Fluoride on Osteoclasts The effects of fluoride on osteoclast activity, and by extension the rate of bone resorption, are less well defined than its effects on osteoblasts. In general, there appears to be good evidence that fluoride decreases osteoclastogenesis and osteoclast activity in in vitro systems; however, its effect in in vivo systems is equivocal. This may be due, in part, to the systemic effects of fluoride in whole animals or humans. A further discussion on this point appears below. Most reports in the literature studying the effect of fluoride on osteoclast function indicate an inhibition. In fact, the effect might be mediated through G-protein-coupled pathways as in the osteoblast. Moonga et al. (1993) showed that fluoride, in the form of AlF4− resulted in a marked concentration-dependent inhibition of bone resorption. In association with this inhibition, they found a marked increase in the secretion of tartrate-resistant acid phosphatase (TRAP). TRAP presumably originated from the osteoclast; however, its function as a secreted enzyme is not known. The fluoride effect was reproduced with cholera toxin, another Gs stimulator. This effect does not appear to be mediated solely by an AlFx complex because studies using NaF have reported similar findings (Taylor et al. 1989, 1990; Okuda et al. 1990). Further evidence that fluoride might blunt osteoclastic bone resorption was reported in a study that investigated acid production as a critical feature of osteoclastic function. The pH within osteoclasts can be measured with the proton-sensitive dye acridine orange. Studies in which osteoclasts were observed found that parathyroid hormone induced osteoclast acidity but that calcitonin, cortisol, and NaF all blocked the effect. As acidification of the matrix is required for normal osteoclast function, fluoride, in this case, would act as an inhibitor to bone resorption (Anderson et al. 1986). The effects of fluoride on bone resorption and osteoclast function in vivo present a complex picture. Some well-controlled animal studies document a decrease in osteoclast (as well as odontoclast) activity. In these studies, rodents and rabbits were exposed to doses of fluoride ranging from clinically relevant to high. Time courses ranged from days to weeks, and the findings indicated a statistically significant decrease in the number and activity of resorbing cells (Faccini 1967; Lindskog et al. 1989; Kameyama et al. 1994). Other studies documented little or no statistically significant effect of fluoride on osteoclast activity (Marie and Hott 1986; Huang 1987). Yet other work that utilized skeletal turnover and remodeling showed an increase in resorption after fluoride therapy (Kragstrup et al. 1984; Snow and Anderson 1986). These studies based their conclusions on the initiation of basic multicellular units (BMUs) and extent of remodeling surface. In the field of skeletal research, it has been accepted that adult bone remodels

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards itself through the generation of BMUs. This unit is a temporal description of remodeling starting with osteoclastic bone resorption and progressing through a coupled stimulation of bone formation. All BMU activity, thus, is initiated with the action of an osteoclast. An increase in remodeling surface also implies an increase in BMUs. Snow and Anderson (1986) and Kragstrup et al. (1984) demonstrated an increase in resorption under the influence of fluoride by measuring BMU numbers and remodeling surface, respectively. Because these data were derived from intact in vivo animal models, the investigators could not conclude that the effects of fluoride on osteoclastic bone resorption were direct. It is interesting that only a single report has appeared that links fluoride exposure to the receptor activator of NF kappaB (RANK) ligand, RANK receptor, or osteoprotegerin (OPG) concentrations. These molecules have recently been characterized as end-stage regulators of osteoclast formation and activity (Lee and Kim 2003). RANK ligand is produced by a variety of cells, with osteoblasts being the most prominent. In its usual form, it is a membrane-associated factor that binds to the RANK receptor on preosteoclasts and induces their further differentiation. OPG is a decoy RANK receptor that is an endogenous inhibitor of bone resorption by virtue of its ability to bind RANK ligand. A clinical trial by von Tirpitz et al. (2003) showed that both fluoride and bisphosphonate therapy decreased OPG concentrations. If this were a direct effect of fluoride, one would expect to see an increase in bone resorption. Conversely, if fluoride blocked bone resorption, the decrease in OPG concentrations could be due to a compensatory feedback pathway. Unfortunately, there were not enough histologic or biochemical marker data in this report to determine whether the fluoride effect was direct or indirect. EFFECTS OF FLUORIDE ON HUMAN SKELETAL METABOLISM Bone Strength and Fracture Cellular and Molecular Aspects Inducing a permanent alteration of skeletal mass in an adult human (or experimental animal) is quite difficult, because bone, as an organ system, possesses an innate mechanism for self-correction. That is, rates of bone formation are controlled, for the most part, by rates of bone resorption. As osteoclastic bone resorption increases or decreases, there is a compensatory increase or decrease in the rate of osteoblastic bone formation. This coupling between the two cell activities was first described by Hattner et al. (1965), and is responsible for the maintenance of a steady-state skeletal mass in adults. These early results indicate that effective management of skeletal

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards mass would require controlling both cell processes. However, until recently, the only therapies approved by the U.S. Food and Drug Admnistration for treating osteoporosis in the United States targeted only osteoclastic bone resorption. They included molecules such as the bisphosphonates, estrogen and its analogs, and calcitonin derivatives. Currently, teraparitide is available as the only approved treatment that acts to stimulate osteoblastic bone formation. Fluoride falls into this category and that is the reason why there was such great interest in this ion as a potential therapy for osteoporosis. Unfortunately, fluoride did not prove to be an effective treatment for two major reasons. First, although it showed robust stimulation of bone mineral density (see below), its effects as an agent to reduce fractures have never been unequivocally documented. Second, because this naturally occurring element cannot be protected with a patent, the pharmaceutical industry has not been interested in investigating all its potential. The first clinical trials of fluoride in humans were performed by Rich and Ensinck (1961). Since then many hundreds of reports have appeared in the medical literature. The overwhelming weight of evidence in these reports documents the effect of fluoride, at therapeutic doses, to be that of an increase in bone mineral density. The lowest dose of NaF to show a clear increase in bone mineral density was 30 mg/day, although there may be effects at lower doses (Hansson and Roos 1987; Kleerekoper and Balena 1991). Response was linear with time for at least 4 to 6 years (Riggs et al. 1990). This linear relationship was confirmed in another study lasting more than 10 years (Kleerekoper and Balena 1991). The observation that bone mineral density continues to increase with time is not surprising in and of itself; however, it differs from the action of the antiresorptive bisphosphonates. Whereas agents that depress bone resorption are most effective when the rate of bone remodeling is high, there appears to be no relationship between the rate of remodeling and the response to fluoride. Also, in contrast to the recent data demonstrating a persistence of bone density with the discontinuance of bisphosphonate therapy, discontinuance of fluoride therapy leads to immediate resumption of bone density loss (Talbot et al. 1996). The dose and duration of fluoride exposure are critical components in determining the effects of the ingested ion on bone. In addition, approximately 30% of patients do not respond to fluoride at any dose (Kleerekoper and Mendlovic 1993). Moreover, there are wide variations in bioavailability among patients and fluoride preparations, and individual responses to the ion also vary widely (Boivin et al. 1993; Erlacher et al. 1995). Whereas the daily dose of fluoride in randomized therapeutic trials (20 to 34 mg/day) exceeds that for people drinking water with fluoride at 4 mg/L (4 to 8 mg/ day for 1 to 2 L/day), the latter may be exposed much longer, leading to comparable or higher cumulative doses and bone fluoride concentrations (see discussion later in this chapter.)

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Allolio and Lehmann (1999)noted that the peak blood concentrations of fluoride after swallowing 8 oz of water (at 1.0 µg/L)all at once will reach 8.75 µg/L. If peak blood concentrations are proportional to water concentration,then consumption of 8 oz of water containing fluoride at 4 mg/L would produce peak concentrations below the threshold for effects on osteoblasts examined in vitro (95 ng/mL)(Ekstrand and Spak 1990). Assuming that the blood fluoride concentrations decline between each episode of water consumption of 8 oz or less, such exposures may not achieve a concentration of fluoride in the extracellular fluids sufficient to affect bone cells. A caveat to this analysis is that bone cells may be exposed to potentially higher (bbut unknown) concentrations because of their proximity to the mineralized bone compartment. There have been no direct measurements of the local fluoride concentration around a site of bone resorption. However, a calculation based on estimated rates of resorption,diffusion kinetics, and starting concentration indicates that bone cells and other cells in the immediate vicinity may experience high concentrations of fluoride. The conditions for an estimate of the fluoride concentration as a function of distance from the osteoclast are as follows: The bone being resorbed has a fluoride content of 3,000 mg per kg of bone ash. Bone ash is assumed to include 65% of the volume of viable bone and the density of viable bone is 1.2 g/cm3. Thus,the concentration of fluoride in the bone compartment is approximately 5,500 µg/cm3. An osteoclast resorbs bone at an average rate of about 30,000 µm3 in 2.5 weeks. The osteoclast is delivering fluoride to the extracellular fluid space from a point source with a radius of 20 µm. Diffusion occurs into a three-dimensional spherical space around the osteoclast. The diffusion coefficient of fluoride in extracellular fluid is approximately 1.5 × 10−5 cm2/s. Under these conditions, the following equation describes the concentration of fluoride as a function of time and distance from the site of bone resorption (Saltzman 2004): where C is the concentration of fluoride as a function of distance and time, S is the delivery rate of fluoride from the resorption site, A is the radius of the point source from which the fluoride is delivered, D is the diffusion

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards coefficient of the fluoride, r is the distance from the resorption site, and t is the time after commencement of the resorption. A graphical representation of this function is presented in Figure 5-1. An examination of the curves in Figure 5-1 indicates that the fluoride concentration around a site of bone resorption can be quite high immediately adjacent to the osteoclast. The theoretical maximum concentration at 20 µm from the site (at the surface of the osteoclast) would be about 5,500 µg/cm3. The concentration rapidly decays to zero in very short times at distances greater than 100 µm from the site. However, it appears that a sustained fluoride concentration is achieved in the range of hours and persists for the entire resorption process. Thus, by 2.5 weeks, the concentration of fluoride will be about 500 µg/cm3 at a distance of 250 µm from the resorption site. FIGURE 5-1 Concentration of fluoride plotted as a function of time and distance from the site of bone resorption. Release of fluoride from a site of bone resorption can achieve a near steady state concentration in a matter of hours. Twenty microns was defined as the radius of the point source from which fluoride was delivered to the extracellular fluid. Acknowledgement: Hani Awad, University of Rochester, Rochester, New York, assisted in this analysis.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards ratio.) The committee found that because no data were presented on the distribution of fluoride exposure within the different groups, because data on gender and age were not reported separately, and because no parameters for assessing cumulative exposure were provided, reliable conclusions could not be drawn from this study. Fabiani et al. (1999) conducted a study in two sociodemographically similar regions in central Italy. One region had fluoride concentrations in drinking water of 0.05 mg/L and the second region had fluoride at 1.45 mg/L. A significantly greater rate of fracture incidence, particularly femur fractures, were found in the low-exposure community. The relative risk was 4.28 (95% CI, 4.16 to 4.40) for males and 2.64 (95% CI, 2.54 to 2.75) for females. These risks were based on age-adjusted rates per 1,000 person-years. However, the number of cases was not provided and the mean age of cases in the two towns varied greatly in some instances. The investigators relied on similarity of regions to control for confounding, but it should be noted that the high-fluoride area included seven towns near Rome, whereas the lower-fluoride area included 35 towns further from Rome. Because of the serious design and analysis limitations of the study, the committee placed little weight on this study. Overall, the committee finds that the available epidemiologic data for assessing bone fracture risk in relation to fluoride exposure around 2 mg/L is suggestive but inadequate for drawing firm conclusions about the risk or safety of exposures at that concentration. There is only one strong report to inform the evaluation, and, although that study (Kurttio et al. 1999) indicated an increased risk of fractures, it is not sufficient alone to base judgment of fracture risk for people exposed at 2 mg/L. It should be considered, however, that the Li et al. (2001) and Alarcón-Herrera et al. (2001) studies reported fracture increases (although imprecise with wide confidence intervals) between 1 and 4 mg/L, giving support to a continuous exposure-effect gradient in this range. Skeletal Fluorosis Excessive intake of fluoride will manifest itself in a musculoskeletal disease with a high morbidity. This pathology has generally been termed skeletal fluorosis. Four stages of this affliction have been defined, including a preclinical stage and three clinical stages that characterize the severity. The preclinical stage and clinical stage I are composed of two grades of increased skeletal density as judged by radiography, neither of which presents with significant clinical symptoms. Clinical stage II is associated with chronic joint pain, arthritic symptoms, calcification of ligaments, and osteosclerosis of cancellous bones. Stage III has been termed “crippling” skeletal fluorosis because mobility is significantly affected as a result of excessive calcifications

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards in joints, ligaments, and vertebral bodies. This stage may also be associated with muscle wasting and neurological deficits due to spinal cord compression. The current MCLG is based on induction of crippling skeletal fluorosis (50 Fed. Reg. 20164 [1985]). Because the symptoms associated with stage II skeletal fluorosis could affect mobility and are precursors to more serious mobility problems, the committee judges that stage II is more appropriately characterized as the first stage at which the condition is adverse to health. Thus, this stage of the affliction should also be considered in evaluating any proposed changes in drinking-water standards for fluoride. Descriptions of skeletal fluorosis date back to the 1930s, when the pathology was first recognized in India in areas of endemic fluoride exposure (Shortt et al. 1937) and in occupationally exposed individuals in Denmark (Roholm 1937). From an epidemiological standpoint, few cases of clinical skeletal fluorosis have been documented in the United States. Stevenson and Watson (1957) performed a large retrospective study involving 170,000 radiologic examinations1 in people from Texas and Oklahoma, where many communities have fluoride water concentrations above 4 mg/L. They radiographically diagnosed only 23 cases of fluoride osteosclerosis in people consuming fluoride at 4 to 8 mg/L and no cases in people exposed to less (the number of people exposed in these categories was not provided). The cases (age 44 to 85) did not have unusual amounts of arthritis or back stiffness given their age (details not provided). Eleven had bone density of an extreme degree, and nine had more than minimal calcification of pelvic ligaments. The authors found no relationship between radiographic findings and clinical diagnosis or symptoms (details not provided). Cases were not classified as to the stage of the fluorosis (using the scheme discussed earlier). Based on the information in the paper, the committee could not determine whether stage II fluorosis was present. In a study of 253 subjects, Leone et al. (1955a) reported increased bone density and coarsened trabeculation in residents of a town with fluoride at 8 mg/L relative to another town with fluoride at 0.4 mg/L. Radiographic evidence of bone changes occurred in 10% to 15% of the exposed residents and was described as being slight and not associated with other physical findings except enamel mottling. The high-fluoride town was partially defluoridated in March 19522 (Maier 1953; Leone et al. 1954a,b; 1955b), a detail not mentioned in the radiographic study (Leone 1 The number of patients represented by the 170,000 radiological examinations is not given. 2 Maier (1953) indicates that “regular operation” of the defluoridation plant began March 11, 1952. At least one small pilot plant was operated for an unspecified period prior to that date (Maier 1953). Leone et al. (1954a,b) indicated initial defluoridation to 1.2 mg/L. Likins et al. (1956) reported a mean daily fluoride content of treated water in Bartlett of 1.32 mg/L over the first 113 weeks (27 months), with average monthly fluoride concentrations of 0.98-2.13 mg/L over the 18-month period referred to by Leone et al. (1954a,b; 1955b).

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards et al. 1955a) but which could have affected its results and interpretation. Leone et al. (1954a,b; 1955b) state that “any significant physiological manifestations of prolonged exposure would not be expected to have regressed materially in the 18 months of partial defluoridation.” However, Likins et al. (1956) reported that urinary fluoride concentrations in males fell from means of 6.5 (children) and 7.7 (adults) mg/L before defluoridation to 4.9 and 5.1 mg/L, respectively, after 1 week, 3.5 and 3.4 mg/L, respectively, after 39 weeks, and 2.2 and 2.5 mg/L, respectively, after 113 weeks. These results indicate that, following defluoridation of the water supply, substantial changes in fluoride balance were occurring in the residents, including the apparent remobilization of fluoride from bone. In patients with reduced renal function, the potential for fluoride accumulation in the skeleton is increased (see Chapter 3). It has been known for many years that people with renal insufficiency have elevated plasma fluoride concentrations compared with normal healthy persons (Hanhijärvi et al. 1972) and are at a higher risk of developing skeletal fluorosis (Juncos and Donadio 1972; Johnson et al. 1979). In cases in which renal disease and skeletal fluorosis were simultaneously present, it still took high concentrations of fluoride, such as from daily ingestion of 4 to 8 L of water containing fluoride at 2 to 3 mg/L (Sauerbrunn et al. 1965; Juncos and Donadio 1972), at least 3 L/day at 2 to 3 mg/L (Johnson et al. 1979), or 2 to 4 L/day at 8.5 mg/L (Lantz et al. 1987) to become symptomatic. Most recently, the Institute of Medicine evaluated fluoride intake and skeletal fluorosis and was able to find only five reported cases of individuals with stage III skeletal fluorosis in the United States from approximately 1960 to 1997 (IOM 1997). Interestingly, however, a recent report has documented an advanced stage of skeletal fluorosis in a 52-year-old woman consuming 1 to 2 gal of double-strength instant tea per day throughout her adult life (Whyte et al. 2005). Her total fluoride intake was estimated at 37 to 74 mg/day from exposure to fluoride from well water (up to 2.8 mg/L) and instant tea. The report also documented the fluoride content of commercial instant teas and found substantial amounts in most brands. This illustrates the possibility that a combination of exposures can lead to higher than expected fluoride intake with associated musculoskeletal problems. Another case, documented by Felsenfeld and Roberts (1991), indicates the development of skeletal fluorosis from consumption of well water containing fluoride at 7 to 8 mg/L for 7 years. Renal insufficiency was not a factor in this case, but water consumption was considered likely to have been “increased” because of hot weather. Both cases mention joint stiffness or pain, suggesting at least stage II skeletal fluorosis. From reports from the 1950s through the 1980s, it appears that preclinical bone changes and symptoms of clinical stages I and II may occur with bone concentrations between 3,500 and 12,900 mg/kg (Franke et al.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards 1975; Dominok et al. 1984; Krishnamachari 1986). The Public Health Service (PHS 1991) has reported that patients with preclinical skeletal fluorosis have fluoride concentrations between 3,500 and 5,500 mg/kg by ash weight. Clinical stage I patients have concentrations in the range of 6,000 to 7,000 mg/kg, stage II patients range from 7,500 to 9,000 mg/kg, and stage III patients have fluoride concentrations of 8,400 mg/kg and greater.3 However, a broader review of the literature on bone fluoride concentrations in patients with skeletal fluorosis revealed wider and overlapping ranges associated with different stages of the condition. Tables 5-6 and 5-7 show the reported concentrations of fluoride in bone ash and in bone (dry fat-free material) in cases of skeletal fluorosis. Most authors reported ash concentrations; others reported the dry weight concentrations or both types of results. Because ash contents (fraction of bone remaining in the ash) range widely,4 the committee did not convert dry weight concentrations to ash concentrations. As reported ranges for various bones in individuals can differ, the tables list the type of bone sampled, distinguishing between measurements of iliac crest or pelvis and other bones. On the basis of data on fluoride in the iliac crest or pelvis, fluoride concentrations of 4,300 to 9,200 mg/kg in bone ash have been found in cases of stage II skeletal fluorosis, and concentrations of 4,200 to 12,700 mg/kg in bone ash have been reported in cases of stage III fluorosis. The overall ranges for other bones are similar. These ranges are much broader than those indicated by PHS (1991). Baud et al. (1978) showed an overlap in the fluoride content in iliac crest samples between their controls (mean 1,036 mg/kg, range <500 to >2,500) and their cases (mean 5,617 mg/kg, range <2,500 to >10,000). The above ranges overlap the measurements reported by Zipkin et al. (1958), for which no evidence of fluorosis was reported (4,496 ±­ 2015 and 6,870 ± 1629 mg/kg ash in iliac crest at 2.6 and 4 mg/L, respectively). The expected degree of skeletal fluorosis was not found in two small groups of patients dialyzed with fluoride-containing water, who accumulated average bone-ash fluoride concentrations of 5,000 mg/kg and 7,200 mg/kg (Erben et al. 1984). Some of the cases with the lowest values (e.g., Teotia and Teotia 1973; Pettifor et al. 1989) were known to have hypocalcemia or secondary hyperparathyroidism; many of the industrial case reports described no hypocalcemia. Thus, it appears that fluoride content in bone may be a marker of the risk of skeletal fluorosis. In other words, the likelihood and severity of clinical skeletal fluorosis increase with the 3 According to the sources cited by PHS (1991), these concentrations are based on measurements in iliac crest samples. 4 From 38% to 60%, calculated from 100% minus the reported fraction lost during ashing (Franke and Auerman 1972); (41.8% standard error 1.94%) for the affected group and 49.9% (standard error 5.34%) for the control group (Krishnamachari 1982); and 32.7% to 68.4% (Zipkin et al. 1958).

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards TABLE 5-6 Reported Concentrations of Fluoride in Bone Ash in Cases of Skeletal Fluorosis   Fluoride Concentration in Bone Ash, mg/kg in Bone Ash     Stage of Skeletal Fluorosis Iliac Crest or Pelvis Other Bones Number of Individuals Reference Preclinical stage Vague symptoms 4,100   2 Franke and Auermann 1972   4300     Vague symptoms 3,500 to 4,500   Authors’ summary Franke et al. 1975 Stage 0 to 1 Stage 0 to I 5,000   1 Franke and Auermann 1972 Stage 0 to I 6,900 (mean)   2 Schlegel 1974 Stage 0 to I 5,000 to 5,500   Authors’ summary Franke et al. 1975 Stage 1 Stage I 6,000   2 Franke and Auermann 1972   6400     Stage I 5,200 (mean)   8 Schlegel 1974 Stage I 6,000 to 7,000   Authors’ summary Franke et al. 1975 Stage 2 Second phase 9,200 3,100 to 9,900 1 Roholm 1937 Stage I to II 8,700   1 Franke and Auermann 1972 Stage II 7,700   2 Franke and Auermann 1972   7800     Stage II 7,500 (mean)   9 Schlegel 1974 Stage II 7,500 to 9,000   Authors’ summary Franke et al. 1975 Stage II 4,300 2,500 to 5,000 1 Dominok et al. 1984   4,700a     Stage II 8,800 4,900 to 11,100 1 Dominok et al. 1984   8900a     Stage II   2,900 to 4,400 1 Dominok et al. 1984 Stage 3 Third phase   7,600 to 13,100 1 Roholm 1937 Stage 3   6,300 1 Singh and Jolly 1961 Stage III   11,500 1 Franke and Auermann 1972 Crippling fluorosis 4,200   1 Teotia and Teotia 1973 Stage III 8,400   1 Schlegel 1974 Stage III >10,000   Authors’ summary Franke et al. 1975

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards   Fluoride Concentration in Bone Ash, mg/kg in Bone Ash     Stage of Skeletal Fluorosis Iliac Crest or Pelvis Other Bones Number of Individuals Reference Stage III 10,000 9,000 to 11,700 1 Dominok et al. 1984 Stage III 9,100 4,200 to 11,000 1 Dominok et al. 1984 Stage III 12,700 7,600 to 12,900 1 Dominok et al. 1984 Stage III 8,600 8,500 to 12,400 1 Dominok et al. 1984   8,700a       Stage not given, or range of stages Skeletal fluorosis   700 to 6,800b (mean, 3,430) 10 Singh and Jolly 1961; see also Singh et al. 1961 Old fluorosis, 7 years without fluoride exposure 3,000   1 Franke and Auermann 1972 Skeletal fluorosis 2,650   4 Teotia and Teotia 1973   3,780       4,750       5,850     Industrial fluorosis 5,617 (2,143)c   43 (54 samples) Baud et al. 1978; Boillat et al. 1980 Endemic genu valgum   7,283 (416)d 20 (37 samples) Krishnamachari 1982 Skeletal fluorosis 4,200 to 10,100   9 Boivin et al. 1986 Skeletal fluorosis 13,300 (2,700)c   6 Boivin et al. 1988 (summary of studiese)   8,900 (3,400)c   5   6,900 (1,900)c   13   5,600 (2,100)c   54   6,600 (2,700)c   4   7,600 (4,800)c   14 Skeletal fluorosis 7,900 (3,600)c (range: 4,200 to 22,000)   29 Boivin et al. 1989; 1990 f Admitted to hospital for skeletal pain or skeletal deformities 5,580 (980)c (range: 4,430 to 6,790)   7 Pettifor et al. 1989 aSamples from right and left sides in same individual. bTibia or iliac crest; includes 1 case of stage III fluorosis listed separately above. cIndicates mean and standard deviation. dIndicates mean and standard error. eIncludes some studies (or individuals from studies) listed separately above. fProbably includes individuals from other studies listed above.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards TABLE 5-7 Reported Concentrations of Fluoride in Bone (Dry Fat-Free Material) in Cases of Skeletal Fluorosis   Fluoride Concentration in Bone, mg/kg in Dry Fat-Free Material     Stage of Skeletal Fluorosis Iliac Crest or Pelvis Other Bones Number of Individuals Reference Preclinical stage Vague symptoms 1,700 and 2,100   2 Franke and Auermann 1972 Stage 0 to 1 Stage 0 to I 1,900   1 Franke and Auermann 1972 Stage 0 to I 3,000 (mean)   5 Schlegel 1974 Stage 1 Early   5,000 to 7,000 1 Wolff and Kerr 1938 (cited in Jackson and Weidmann 1958) Early   6,260 and 7,200 2 Sankaran and Gadekar 1964 Stage I 2,300 and 2,900   2 Franke and Auermann 1972 Stage I 3,200 (mean)   15 Schlegel 1974 Stage 2 Moderate   7,680 1 Sankaran and Gadekar 1964 Stage I to II 4,300   1 Franke and Auermann 1972 Stage II 4,100 and 4,600   2 Franke and Auermann 1972 Stage II 3,000 (mean)   18 Schlegel 1974 Stage 3 Skeletal fluorosis   8,600 1 Sankaran and Gadekar 1964 Advanced   8,800 and 9,680 2 Sankaran and Gadekar 1964 Stage III 3,600 (mean)   4 Schlegel 1974 Stage not given Old fluorosis, 7 years without fluoride exposure 1,700   1 Franke and Auermann 1972

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards bone fluoride content, but a given concentration of bone fluoride does not necessarily correspond to a certain stage of skeletal fluorosis in all cases. Other factors (e.g., calcium intake) appear to influence fluorosis severity at different concentrations of bone fluoride. Overall, the committee finds that the predicted bone fluoride concentrations that can be achieved from lifetime exposure to fluoride at 4 mg/L (10,000 to 12,000 mg/kg bone ash) fall within or exceed the ranges of concentrations that have been associated with stage II and stage III skeletal fluorosis. Based on the existing epidemiologic literature, stage III skeletal fluorosis appears to be a rare condition in the United States. As discussed above, the committee judges that stage II skeletal fluorosis is also an adverse health effect. However, the data are insufficient to provide a quantitative estimate of the risk of this stage of the affliction. The committee could not determine from the existing epidemiologic literature whether stage II skeletal fluorosis is occurring in U.S. residents who drink water with fluoride at 4 mg/L. The condition does not appear to have been systematically investigated in recent years in U.S. populations that have had long-term exposures to high concentrations of fluoride in drinking water. Thus, research is needed on clinical stage II and stage III skeletal fluorosis to clarify the relationship of fluoride ingestion, fluoride concentration in bone, and clinical symptoms. EFFECT OF FLUORIDE ON CHONDROCYTE METABOLISM AND ARTHRITIS The two key chondrocyte cell types that are susceptible to pathological changes are articular chondrocytes in the joint and growth plate chondrocytes in the developing physis. The medical literature on fluoride effects in these cells is sparse and in some cases conflicting. From physical chemical considerations, it might be expected that mineral precipitates containing fluoride would occur in a joint if concentrations of fluoride and other cations (such as Ca2+) achieved a high enough concentration. A single case report by Bang et al. (1985) noted that a 74-year-old female who was on fluoride therapy for osteoporosis for 30 months had a layer of calcified cartilage containing 0.39% fluoride (or 3,900 mg/kg) by ash weight in her femoral head. The calcification was also visible on x-ray. Unfortunately, the limitation of this observation in a single patient is the lack of information on the preexistence of any calcified osteophytes. Nevertheless, it does indicate that at high therapeutic doses fluoride can be found in mineralizing nodules in articular cartilage. Studies evaluating patient groups with a greater number of subjects found that the use of fluoride at therapeutic doses in rheumatoid patients showed a conflicting result. In one report (Duell and Chesnut 1991), fluoride exacerbated symptoms of rheumatoid arthritis, but, in another case

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards (Adachi et al. 1997), it was “well tolerated” with no evidence of worsening of the arthritis. No indications from either study implied that fluoride had a causal relationship with the rheumatoid arthritis. Perhaps the only study in the literature that attempts to link fluoride exposure to the induction of arthritis (osteoarthritis) is from Savas et al. (2001), who indicated that Turkish patients with demonstrated endemic fluorosis had a greater severity of osteoarthritic symptoms and osteophyte formation than age- and sex-matched controls. The veterinary literature also contains a report indicating that, in 21 dairy herds consuming fluoride-containing feed and water, of the 100 cows examined and determined to have arthritic changes, the bone fluoride concentrations ranged from 2,000 to 8,000 mg/kg (Griffith-Jones 1977). There are no data from which a dose-response relationship can be drawn regarding fluoride intake and arthritis in humans. However, in a rat study, Harbrow et al. (1992) showed articular changes with fluoride at 100 mg/L in drinking water but no effect at 10 mg/L. The changes with fluoride at 100 mg/L were a thickening of the articular surface (rather than a thinning as would be expected in arthritis) and there were no effects on patterns of collagen and proteoglycan staining. There are no comprehensive reports on the mechanism of fluoride effects in articular chondrocytes in vitro. The effect of fluoride on growth plate chondrocytes is even less well studied than the effect on articular chondrocytes. It has been demonstrated that chronic renal insufficiency in a rat model can increase the fluoride content in the growth plate and other regions of bone (Mathias et al. 2000); however, this has not been known to occur in humans. Fluoride has also been shown to negatively influence the formation of mineral in matrix vesicles at high concentrations. Matrix vesicles are the ultrastructural particles responsible for initiating mineralization in the developing physis (Sauer et al. 1997). This effect could possibly account, in part, for the observation that fluoride may reduce the thickness of the developing growth plate (Mohr 1990). In summary, the small number of studies and the conflicting results regarding the effects of fluoride on cartilage cells of the articular surface and growth plate indicate that there is likely to be only a small effect of fluoride at therapeutic doses and no effect at environmental doses. FINDINGS Fluoride is a biologically active ion with demonstrable effects on bone cells, both osteoblasts and osteoclasts. Its most profound effect is on osteoblast precursor cells where it stimulates proliferation both in vitro and in vivo. In some cases, this is manifested by increases in bone mass in vivo.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards The signaling pathways by which this agent works are slowly becoming elucidated. Life-long exposure to fluoride at the MCLG of 4 mg/L may have the potential to induce stage II or stage III skeletal fluorosis and may increase the risk of fracture. These adverse effects are discussed separately below. The current MCLG was designed to protect against stage III skeletal fluorosis. As discussed above, the committee judges that stage II is also an adverse health effect, as it is associated with chronic joint pain, arthritic symptoms, slight calcification of ligaments, and osteosclerosis of cancellous bones. The committee found that bone fluoride concentrations estimated to be achieved from lifetime exposure to fluoride at 2 mg/L (4,000 to 5,000 mg/kg ash) or 4 mg/L (10,000 to 12,000 mg/kg ash) fall within or exceed the ranges historically associated with stage II and stage III skeletal fluorosis (4,300 to 9,200 mg/kg ash and 4,200 to 12,700 mg/kg ash, respectively). This suggests that fluoride at 2 or 4 mg/L might not protect all individuals from the adverse stages of the condition. However, this comparison alone is not sufficient evidence to conclude that individuals exposed to fluoride at those concentrations are at risk of stage II skeletal fluorosis. There is little information in the epidemiologic literature on the occurrence of stage II skeletal fluorosis in U.S. residents, and stage III skeletal fluorosis appears to be a rare condition in the United States. Therefore, more research is needed to clarify the relationship between fluoride ingestion, fluoride concentrations in bone, and stage of skeletal fluorosis before any firm conclusions can be drawn. Although a small set of epidemiologic studies were useful for evaluating bone fracture risks from exposure to fluoride at 4 mg/L in drinking water, there was consistency among studies using ecologic exposure measures to suggest the potential for an increased risk. The one study using serum fluoride concentrations found no appreciable relationship to fractures. Because serum fluoride concentrations may not be a good measure of bone fluoride concentrations or long-term exposure, the ability to shown an association might have been diminished. Biochemical and physiological data indicate a biologically plausible mechanism by which fluoride could weaken bone. In this case, the physiological effect of fluoride on bone quality and risk of fracture observed in animal studies is consistent with the observational evidence. Furthermore, the results of the randomized clinical trials were consistent with the observational studies. In addition, a dose-response relationship is indicated. On the basis of this information, all members of the committee agreed that there is scientific evidence that under certain conditions fluoride can weaken bone and increase the risk of fractures. The majority of the committee concluded that lifetime exposure to fluoride at drinking-water concentrations of 4 mg/L or higher is likely to increase fracture rates in the population, compared with exposure at 1 mg/L, particularly in some

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards susceptible demographic groups that are more prone to accumulate fluoride in their bones. However, three of the 12 members judged that the evidence only supported a conclusion that the MCLG might not be protective against bone fracture. They judge that more evidence that bone fractures occur at an appreciable frequency in human populations exposed to fluoride at 4 mg/L is needed before drawing a conclusion that the MCLG is likely to be not protective. Few studies have assessed fracture risk in populations exposed to fluoride at 2 mg/L in drinking water. The best available study was from Finland, which provided data that suggested an increased rate of hip fracture in populations exposed to fluoride at >1.5 mg/L. However, this study alone is not sufficient to determine the fracture risk for people exposed to fluoride at 2 mg/L in drinking water. Thus, the committee finds that the available epidemiologic data for assessing bone fracture risk in relation to fluoride exposure around 2 mg/L are inadequate for drawing firm conclusions about the risk or safety of exposures at that concentration. RECOMMENDATIONS A more complete analysis of communities consuming water with fluoride at 2 and 4 mg/L is necessary to assess the potential for fracture risk at those concentrations. These studies should use a quantitative measure of fracture such as radiological assessment of vertebral body collapse rather than self-reported fractures or hospital records. Moreover, if possible, bone fluoride concentrations should be measured in long-term residents. The effects of fluoride exposure in bone cells in vivo depend on the local concentrations surrounding the cells. More data are needed on concentration gradients during active remodeling. A series of experiments aimed at quantifying the graded exposure of bone and marrow cells to fluoride released by osteoclastic activity would go a long way in estimating the skeletal effects of this agent. A systematic study of stage II and stage III skeletal fluorosis should be conducted to clarify the relationship of fluoride ingestion, fluoride concentration in bone, and clinical symptoms. Such a study might be particularly valuable in populations in which predicted bone concentrations are high enough to suggest a risk of stage II skeletal fluorosis (e.g., areas with water concentrations of fluoride above 2 mg/L). More research is needed on bone concentrations of fluoride in people with altered renal function, as well as other potentially sensitive populations (e.g., the elderly, postmenopausal women, people with altered acid-balance), to better understand the risks of musculoskeletal effects in these populations.