9
Effects on the Gastrointestinal, Renal, Hepatic, and Immune Systems

This chapter evaluates the effects of fluoride on the gastrointestinal system (GI), the kidney, the liver, and the immune system, focusing primarily on new data that have been generated since the earlier NRC (1993) review. Studies that involved exposures to fluoride in the range of 2-4 milligrams per liter (mg/L) are emphasized, so that the safety of the maximum-contaminant-level goal (MCLG) can be evaluated.

GI SYSTEM

Fluoride occurs in drinking water primarily as free fluoride. When ingested some fluorides combine with hydrogen ions to form hydrogen fluoride (HF), depending on the pH of the contents of the stomach (2.4% HF at pH 5; 96% HF at pH 2). HF easily crosses the gastric epithelium, and is the major form in which fluoride is absorbed from the stomach (see Chapter 3). Upon entering the interstitial fluid in the mucosa where the pH approaches neutrality, HF dissociates to release fluoride and hydrogen ions which can cause tissue damage. Whether damage occurs depends on the concentrations of these ions in the tissue. It appears that an HF concentration somewhere between 1.0 and 5.0 mmol/L (20 and 100 mg/L), applied to the stomach mucosa for at least 15 minutes, is the threshold for effects on the function and structure of the tissue (Whitford et al. 1997). Reported GI symptoms, such as nausea, may not be accompanied by visible damage to the gastric mucosa. Thus, the threshold for adverse effects (discomfort) is likely to be lower than that proposed by Whitford et al. This review is concerned primarily with the chronic ingestion of fluoride in drinking wa-



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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards 9 Effects on the Gastrointestinal, Renal, Hepatic, and Immune Systems This chapter evaluates the effects of fluoride on the gastrointestinal system (GI), the kidney, the liver, and the immune system, focusing primarily on new data that have been generated since the earlier NRC (1993) review. Studies that involved exposures to fluoride in the range of 2-4 milligrams per liter (mg/L) are emphasized, so that the safety of the maximum-contaminant-level goal (MCLG) can be evaluated. GI SYSTEM Fluoride occurs in drinking water primarily as free fluoride. When ingested some fluorides combine with hydrogen ions to form hydrogen fluoride (HF), depending on the pH of the contents of the stomach (2.4% HF at pH 5; 96% HF at pH 2). HF easily crosses the gastric epithelium, and is the major form in which fluoride is absorbed from the stomach (see Chapter 3). Upon entering the interstitial fluid in the mucosa where the pH approaches neutrality, HF dissociates to release fluoride and hydrogen ions which can cause tissue damage. Whether damage occurs depends on the concentrations of these ions in the tissue. It appears that an HF concentration somewhere between 1.0 and 5.0 mmol/L (20 and 100 mg/L), applied to the stomach mucosa for at least 15 minutes, is the threshold for effects on the function and structure of the tissue (Whitford et al. 1997). Reported GI symptoms, such as nausea, may not be accompanied by visible damage to the gastric mucosa. Thus, the threshold for adverse effects (discomfort) is likely to be lower than that proposed by Whitford et al. This review is concerned primarily with the chronic ingestion of fluoride in drinking wa-

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards ter containing fluoride at 2-4 mg/L. Single high doses of ingested fluoride are known to elicit acute GI symptoms, such as nausea and vomiting, but whether chronic exposure to drinking water with fluoride at 4 mg/L can elicit the same symptoms has not been documented well. The primary symptoms of GI injury are nausea, vomiting, and abdominal pain (see Table 9-1). Such symptoms have been reported in case studies (Waldbott 1956; Petraborg 1977) and in a clinical study involving double-blind tests on subjects drinking water artificially fluoridated at 1.0 mg/L (Grimbergen 1974). In the clinical study, subjects were selected whose GI symptoms appeared with the consumption of fluoridated water and disappeared when they switched to nonfluoridated water. A pharmacist prepared solutions of sodium fluoride (NaF) and sodium silicofluoride (Na2SiF6) so that the final fluoride ion concentrations were 1.0 mg/L. Eight bottles of water were prepared with either fluoridated water or distilled water. Patients were instructed to use one bottle at a time for 2 weeks. They were asked to record their symptoms throughout the study period. Neither patients nor the physician administering the water knew which water samples were fluoridated until after the experiments were completed. The fluoridation chemicals added to the water at the time of the experiments were likely the best candidates to produce these symptoms. Despite those well-documented case reports, the authors did not estimate what percentage of the population might have GI problems. The authors could have been examining a group of patients whose GI tracts were particularly hypersensitive. The possibility that a small percentage of the population reacts systemically to fluoride, perhaps through changes in the immune system, cannot be ruled out (see section on the immune system later in this chapter). Perhaps it is safe to say that less than 1% of the population complains of GI symptoms after fluoridation is initiated (Feltman and Kosel 1961). The numerous fluoridation studies in the past failed to rigorously test for changes in GI symptoms and there are no studies on drinking water containing fluoride at 4 mg/L in which GI symptoms were carefully documented. Nevertheless, there are reports of areas in the United States where the drinking water contains fluoride at concentrations greater than 4 mg/L and as much as 8 mg/L (Leone et al. 1955b). Symptoms of GI distress or discomfort were not reported. In the United Kingdom, where tea drinking is more common, people can consume up to 9 mg of fluoride a day (Jenkins 1991). GI symptoms were not reported in the tea drinkers. The absence of symptoms might be related to the hardness of the water, which is high in some areas of the United Kingdom. Jenkins (1991) reported finding unexpectedly high concentrations of fluoride (as high as 14 mg/L) in soft water compared with hard water when boiled. In contrast, in India, where endemic fluorosis is well documented, severe GI symptoms are common (Gupta et al. 1992; Susheela et al. 1993; Dasarathy et al. 1996). One cannot rule out the

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards TABLE 9-1 Studies of Gastrointestinal Effects in Humans Approximate Concentration of Fluoride in the Stomacha Study Design Findings Application/Proposed Mechanisms Comments Reference Water Fluoridation 1.0 mg/L Case reports of patients (n = 52) drinking artificially fluoridated water. Stomach cramps, abdominal pain, and nausea resolved when patients stopped drinking fluoridated water. Possible gastrointestinal hypersensitivity. Low daily dose of fluoride; cluster of subjects selected on the basis of symptoms. Waldbott 1956 1.0 mg/L Double-blinded test of patients (n = 60) drinking artificially fluoridated water in Haarlem, Netherlands. 50% of subjects had stomach and intestinal symptoms; 30% had stomatitis. Possible gastrointestinal hypersensitivity. Low daily dose of fluoride; self-reporting of symptoms. Grimbergen 1974 1.0 mg/L Case reports of symptoms in subjects (n = 20) drinking fluoridated water in Milwaukee. Fatigue, pruritis, polydipsia, headaches, and gastrointestinal symptoms. Possible gastrointestinal hypersensitivity. Low daily dose; cluster of subjects selected on the basis of symptoms. Petraborg 1977 Water Fluoridation Accidents 75-300 mg/Lb (range due to differences found in 2 fluoride feeders) Symptoms reported in 34 children during accidental overfeed in school water supply. Fluoride concentrations in water were 93.5 and 375 mg/L. 68% of the children had gastrointestinal upset. Acute fluoride toxicity of the gastric epithelium. Symptoms resolved after problem was corrected; doses of fluoride in mg/ kg were not reported. Hoffman et al. 1980 250 mg/L, (based on 50-mL ingestion) Symptoms reported in 22 subjects during accidental overfeed in school water supply. Fluoride concentration in water was 1,041 mg/L. 91% of the subjects had nausea and vomiting. Acute fluoride toxicity of the gastric epithelium. Only small amounts of the beverages made with the school’s water were consumed. Vogt et al. 1982

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards 41 mg/L Symptoms reported in 321 subjects during accidental overfeed in water supply. Of the 160 persons who drank water; 52% had gastroenteritis. Only 2% of subjects who did not drink water reported gastroenteritis. Itching and skin rash also reported. Fluoride concentration in water peaked at 51 mg/L. Acute fluoride toxicity of the gastric epithelium.   Petersen et al. 1988 150 mg/L (assuming no dilution with stomach fluid) Symptoms reported in 47 residents of a town during accidental fluoride overfeed of the water supply. 90% had nausea, vomiting, diarrhea, abdominal pains, or numbness or tingling of the face or extremities. One person in the town died. Fluoride concentration in water was 150 mg/L. Acute fluoride toxicity of the gastric epithelium. Death occurred due to nausea, vomiting, diarrhea, and repeated ingestion of water (large acute dose). Gessner et al. 1994 20-30 mg/L (based on 100-mL ingestion) Symptoms reported in 39 patrons of a restaurant who consumed water or ice during an overfeed accident. 34 subjects had acute gastrointestinal illness in a 24-hour period after exposure. Fluoride concentration in water was 40 mg/L. Acute fluoride toxicity of the gastric epithelium. Symptoms resolved after problem was corrected; dose of fluoride was not reported but was estimated to be 3 mg/kg. Penman et al. 1997 46-69 mg/L Symptoms reported in 7 school children during accidental overfeed. Nausea and vomiting. Fluoride concentration in water was 92 mg/L. Acute fluoride toxicity of the gastric epithelium. Dose in mg/kg was not reported. Sidhu and Kimmer 2002

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Approximate Concentration of Fluoride in the Stomacha Study Design Findings Application/Proposed Mechanisms Comments Reference Other Exposures 5 ppm Symptoms reported in pregnant women and their children from birth to 9 years taking NaF (1.2 mg) supplements. 672 cases (461 controls) 1% of cases had dermatologic, gastrointestinal, and neurologic effects. Comparisons with controls treated with binder placebo tablets established the effects to be from fluoride and not the binder. Chronic or acute toxicity. Details of clinical trial (e.g., randomization, stratification) not reported; dose in mg/kg was not reported; gastrointestinal systems were probably worse in small children (due to higher dose per kilogram of body weight). Feltman and Kosel 1961 20 ppm, (assuming 100 of mL stomach fluid) Symptoms observed in 10 adult volunteers who ingested 3 g of gel containing fluoride at 0.42% (4,200 mg/L). Petechiae and erosion found in 7 of 10 subjects. Surface epithelium was most affected portion of the mucosa. Acute fluoride toxicity of the gastric epithelium. Approximately 10% of a probably toxic dose. Spak et al. 1990 136 ppm (calculated from on 30 mg of NaF ingested in 100 mL of stomach fluid) Symptoms observed in 10 patients with otosclerosis treated with NaF at 30 mg/ day for 3-12 months. 7 subjects had abdominal pains, vomiting, and nausea. Endoscopy revealed petechiae, erosion, and erythema. Histological exams showed chronic atrophic gastritis in all patients and in only one of the controls. Acute fluoride toxicity of the gastric epithelium.   Das et al. 1994

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards 200 ppm (using the 0.05% NaF mouthwash example) Evaluation of reports to the American Association of Poison Control Centers of suspected overingestion of fluoride to estimate toxic amounts of home-use fluoride products. Authors estimate a “probably toxic dose” of fluoride to children less than 6 years of age to be 50 mg. That dose was based on examples of a 10-kg child ingesting 10.1 g of 1.1% NaF gel; 32.7 g of 0.63% SnF2 gel; 33.3 g of toothpaste with 1,500 ppm of fluoride; 50 g of toothpaste with 1,000 ppm of fluoride; or 221 mL of 0.05% NaF rinse. Acute fluoride toxicity of the gastric epithelium. Similar total acute doses as the water fluoridation overfeed accidents. Shulman and Wells 1997 aIn most studies, the concentration of fluoride in the stomach was not determined, so estimates were made by the committee. The actual concentrations could vary widely depending on the volume in the stomach and the rate of gastric secretions. The latter could also vary depending on the effect of fluoride (or any other agent) on the secretory process. bEstimated from ingesting 400 mL of fluoridated water (unless dose was reported) diluted 0.8 with 100 mL of stomach fluid with fluoride at 1 mg/: (empty stomach).

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards influence of poor nutrition (the absence of dietary calcium in the stomach) contributing to the GI upset from fluoride ingestion. Chronic ingestion of drinking water rich in fluoride on an empty stomach is more likely to elicit symptoms. GI Symptoms Relating to the Concentration of Fluoride Intake It is important to realize that GI effects depend more on the net concentration of the aqueous solution of fluoride in the stomach than on the total fluoride dose in the fluid or solid ingested. The presence of gastric fluids already in the stomach when the fluoride is ingested can affect the concentration of the fluoride to which the gut epithelium is exposed. The residual volume of stomach fluid ranges between 15 and 30 mL in people fasting overnight (Narchi et al. 1993; Naguib et al. 2001; Chang et al. 2004). Such volumes would decrease the fluoride concentration of a glass of drinking water by only about 10%. In Table 9-1, the concentrations of fluoride in the stomach were estimated from the mean reported fluoride exposures. A dilution factor was used when it was clear that the subjects already had fluid in their stomach. The results from the water fluoridation overfeed reports (concentrations of fluoride in the stomach between 20 and 250 mg/L) indicate that GI symptoms, such as nausea and vomiting, are common side effects from exposure to high concentrations of fluoride. Fluoride supplements are still routinely used today in areas where natural fluoride in the drinking water falls below 0.7 mg/L. In an early clinical trial using fluoride supplements, Feltman and Kosel (1961) administered fluoride tablets containing 1.2 mg of fluoride or placebo tablets to pregnant mothers and children up to 9 years of age. They determined that about 1% of the subjects complained of GI symptoms from the fluoride ingredient in the test tablets. If it is assumed that the stomach fluid volume after taking the fluoride supplement was approximately 250 mL, the concentration to which the stomach mucosal lining was exposed was in the neighborhood of 5 mg/L. GI effects appear to have been rarely evaluated in the fluoride supplement studies that followed the early ones in the 1950s and 1960s. Table 9-1 suggests that, as the fluoride concentration increases in drinking water, the percentage of the population with GI symptoms also increases. The table suggests that fluoride at 4 mg/L in the drinking water results in approximately 1% of the population experiencing GI symptoms (see Feltman and Kosel 1961). Chronic Moderate Dose Ingestion of Fluoride It is clear from the fluoride and osteoporosis clinical trial literature (also see Chapter 5) that gastric side effects were common in these studies (e.g.,

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Mamelle et al. 1988; Hodsman and Drost 1989; Kleerekoper and Mendlovic 1993). Slow-release fluorides and calcium supplementation helped to reduce GI side effects (Kleerekoper and Mendlovic 1993; Das et al. 1994; Haguenauer et al. 2000). In areas of endemic fluorosis, such as parts of India, most subjects suffer from GI damage and adverse GI symptoms (Gupta et al. 1992; Susheela et al. 1993; Dasarathy et al. 1996). In one study (Susheela et al. 1993), every fourth person exposed to fluoride in drinking water (<1 to 8 mg/L) reported adverse GI symptoms. The results from these studies cannot be compared with the water fluoridation studies summarized in Table 9-1, because in the osteoporosis trials fluoride was nearly always administered as enteric coated tablets along with calcium supplements and the nutrition status of populations in endemic fluorosis areas is different from that in the United States. Fluoride Injury Mechanisms in the GI Tract Because 1% of the population is likely to experience GI symptoms, and GI symptoms are common in areas of endemic fluorosis, especially where there is poor nutrition (Gupta et al. 1992; Susheela et al. 1993; Dasarathy et al. 1996), it is important to understand the biological and physiological pathways for the effects of fluoride on the GI system. Those mechanisms have been investigated in many animal studies. In those studies, the concentrations of fluoride used were generally 100- to 1,000-fold higher than what occurs in the serum of subjects drinking fluoridated water. Although some tissues encounter enormous elevations in fluoride concentrations relative to the serum (e.g., kidney, bone), it is unlikely that the gut epithelium would be exposed to millimolar concentrations of fluoride unless there has been ingestion of large doses of fluoride from acute fluoride poisoning. During the ingestion of a large acute dose of fluoride such as fluoride-rich oral care products, contaminated drinking water during fluoridation accidents, and fluoride drugs for the treatment of osteoporosis, the consumption of large amounts of drinking water containing fluoride at 4 mg/L would serve only to aggravate the GI symptoms. Animal studies (see Table 9-2) have provided some important information on the mechanisms involved in GI toxicity from fluoride. Fluoride can stimulate secretion of acid in the stomach (Assem and Wan 1982; Shayiq et al. 1984), reduce blood flow away from the stomach lining, dilate blood vessels, increase redness of the stomach lining (Fujii and Tamura 1989; Whitford et al. 1997), and cause cell death and desquamation of the GI tract epithelium (Easmann et al. 1984; Pashley et al. 1984; Susheela and Das 1988; Kertesz et al. 1989; NTP 1990; Shashi 2002). Because fluoride is a known inhibitor of several metabolic intracellular enzymes, it is not surprising that, at very high exposures, there is cell

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards TABLE 9-2 Animal Studies of Gastrointestinal Effects and Mechanisms of Fluoride Species Study Findings Possible Mechanisms/Comments Reference In Vitro Studies Rat Circular muscle strips from the colons of colitic rats were treated with 10 mM NaF. Colitis was experimentally induced by intracolonic instillation of acetic acid. NaF-induced contractions were significantly reduced in tissues from colitic rats compared with controls on days 2 and 3 postenema but not 14 days after enema. Results suggest that colitis alters smooth muscle contractility by disturbing elements in the signal transduction pathway distal to receptor activation of the G proteins. Purpose of the study was to investigate whether colitis-induced decreases in the contraction of colonic smooth muscle is due to alteration in the excitation-contraction-coupling process at a site distal to receptor occupancy. Decrease in contractility might be due to impaired utilization of intracellular calcium. Myers et al. 1997 Mouse Isolated distended stomachs were treated with NaF at various concentrations (1-10 mM NaF). Dose-related stimulation of H+ ion secretion. Stimulation of H+ ion secretion might be due to histamine release and increased formation of cyclic AMP (cAMP) in the gastric mucosa. Fluoride might contribute to excess acid production in gastrointestinal tract. Assem and Wan 1982 Guinea pig Isolated gastric chief cells treated with NaF (0-30 mM). NaF increased intracellular diacylglycerol and Ca2+; 0.1 mM AlCl3 increased the effect of NaF. Possible activation of a pertussis-toxin insensitive G protein coupled to a signal transducing mechanism. Action appears to be distinct from that activated by cholecystokinin. Nakano et al. 1990 Rabbit Electronic chloride secretion by the jejunum was assessed by measuring short-circuit current variations (ΔIsc) due to alterations in ionic transport. NaF induced a transient increase in Isc at >5 mM; inhibited the antisecretory effect of peptide PYY and its analog P915 at 2 mM and decreased the stimulation of secretion by forskolin and dibutyryl cAMP by 50% at 2 mM. At 5 mM, inhibition of protein kinase C by bisindolylmaleimide caused a sustained increase in Isc. NaF might reduce PYY-induced inhibition via a G-protein-dependent and a G-protein-independent functional pathway. Eto et al. 1996

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Rabbit Fluoride transport in intestinal brush border membrane vesicles examined. Fluoride uptake by brush border membrane vesicles occurred rapidly and with an overshoot only in the presence of an inward-directed proton gradient. Fluoride transport occurs via a carrier-mediated process that might involve cotransport of fluoride with H+ or exchange of fluoride with OH− He et al. 1998 Rabbit Effect of NaF on the transport of bovine serum albumin across the distal and proximal colonic epithelium. Transport of bovine serum albumin was significantly reduced by NaF. Fluoride affected transport mechanisms in the colon. Hardin et al. 1999 In Vivo Studies Rat 25 mg/kg in drinking water for 60 days. Increased gastric acidity and output. Elevation of cAMP concentrations in the gastric mucosa can stimulate H+ output, which might account for gastric symptoms reported in endemic fluorosis areas or from occupational exposure by inhalation. Shayiq et al. 1984 Rat 1 or 10 mM NaF (in 0.1 M HCl) placed in rat stomach for 1 hour. Concentration- and time-dependent histological damage to the surface mucous cells. The higher concentration of NaF increased gastric permeability to small but not large molecules. Pashley et al. 1984 Rat 1, 10, or 50 mM NaF (in 0.1 M HCl) placed in rat stomach. At 10 mM, desquamation of the surface mucous epithelial cells. At 50 mM, substantial damage to cells around the gastric gland openings and interfoveolar cell loss. Possible toxicity of the gut epithelium. Easmann et al. 1984

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Species Study Findings Possible Mechanisms/Comments Reference Rat 100 mM NaF and 50 mM CaF2 intragastrically NaF-treated rats had extensive desquamation and cell injury.CaF2-treated animals showed some desquamation and decrease in secretory activity. Injury to stomach lining might affect secretion. Kertesz et al. 1989 Rat Single oral dose of NaF at 300 mg/kg. Reduced blood flow from the stomach, reduced blood calcium, dilated blood vessels in the stomach, and redness. Redness in the pyloric region of the stomach and intestine is likely due to a relaxation of the small veins, resulting in an accumulation of circulating blood in the mucosa of the intestinal tract. Fujii and Tamura 1989 Rat 300 mg/L in drinking water for 6 months. Gross lesions of the stomach in male rats. Diffuse mucosal hyperplasia with cellular necrosis in female rats. Chronic fluoride toxicity of the gut epithelium. NTP 1990 Rat Stomachs of rats were instilled with 5 and 20 mM NaF for 1 hour. Increased output of fluid, fucose, and galactose; marked reduction of titratable acidity of the lumen was pH dependent; and reduced amount of Alcian blue was bound to adherent mucus in a pH-independent manner. Authors suggest that NaF accumulates with acid and acts as a barrier-breaking agent, rather than as a mucus-secretion stimulating agent. Gharzouli et al. 2000 Mouse NaF at 100 mg/L in drinking water for 30 days. Organosomatic index decreased. Histopathologic changes of the intestine included increased number of goblet cells in the villi and crypts, cytoplasmic degranulation and vacuolation, nuclear pyknosis, abnormal mitosis, and lymphatic infiltration of submucosa and lamina propria.   Sondhi et al. 1995

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards fatty acid components increased and polyunsaturated fatty acids decreased. Liver cholesterol and dolichol were unchanged. The authors concluded that fluoride-induced alteration in liver membrane lipids could be an important factor in the pathogenesis of chronic fluorosis. Whether any of these changes has relevance to the long-term daily ingestion of drinking water containing fluoride at 4 mg/L will require careful analysis of liver function tests in areas with high and low concentrations of fluoride in the drinking water. The clinical trials involving fluoride therapy for treating osteoporosis require that subjects be administered fluoride at concentrations approaching 1.0 mg/kg/day. Although such studies are rarely carried out for more than 5 years, this period of time should be sufficient to measure any changes in hepatic function. Jackson et al. (1994) reported that there was a significant increase in liver function enzymes in test subjects taking 23 mg of fluoride a day for 18 months, but the enzyme concentrations were still within the normal range. It is possible that a lifetime ingestion of 5-10 mg/day from drinking water containing fluoride at 4 mg/L might turn out to have long-term effects on the liver, and this should be investigated in future epidemiologic studies. Finally, because the liver is the primary organ for defluorinating toxic organofluorides, there is a concern that added fluoride body burden that would be experienced in areas where the drinking water had fluoride at 4 mg/L might interfere with the activity of the cytochrome P450 complex (Baker and Ronnenberg 1992; Kharasch and Hankins 1996). IMMUNE SYSTEM Hypersensitivity In the studies by physicians treating patients who reported problems after fluoridation was initiated, there were several reports of skin irritation (Waldbott 1956; Grimbergen 1974; Petraborg 1977). Although blinded experiments suggested that the symptoms were the result of chemicals in the water supply, various anecdotal reports from patients complaining, for example, of oral ulcers, colitis, urticaria, skin rashes, nasal congestion, and epigastric distress, do not represent type I (anaphylactic), II (cytotoxic), III (toxic complex), or IV (delayed type reactivity) hypersensitivity, according to the American Academy of Allergy (Austen et al. 1971). These patients might be sensitive to the effects of silicofluorides and not the fluoride ion itself. In a recent study, Machalinski et al. (2003) reported that the four different human leukemic cell lines were more susceptible to the effects of sodium hexafluorosilicate, the compound most often used in fluoridation, than to NaF. Nevertheless, patients who live in either an artificially fluoridated com-

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards munity or a community where the drinking water naturally contains fluoride at 4 mg/L have all accumulated fluoride in their skeletal systems and potentially have very high fluoride concentrations in their bones (see Chapter 3). The bone marrow is where immune cells develop and that could affect humoral immunity and the production of antibodies to foreign chemicals. For example, Butler et al. (1990) showed that fluoride can be an adjuvant, causing an increase in the production of antibodies to an antigen and an increase in the size and cellularity of the Peyer’s patches and mesenteric lymph nodes. The same group (Loftenius et al. 1999) then demonstrated that human lymphocytes were more responsive to the morbilli antigen. Jain and Susheela (1987), on the other hand, showed that rabbit lymphocytes exposed to NaF had reduced antibody production to transferrin. At the very early stages of stem cell differentiation in bone, fluoride could affect which cell line is stimulated or inhibited. Kawase et al. (1996) suggested that NaF (0.5 mM for 0-4 days) stimulates the granulocytic pathway of the progenitor cells in vitro. This was confirmed by Oguro et al. (2003), who concluded that “NaF [<0.5 mM] induces early differentiation of bone marrow hemopoietic progenitor cells along the granulocytic pathway but not the monocytic pathway.” It has long been claimed that cells do not experience the concentrations of fluoride that are used in vitro to demonstrate the changes seen in cell culture. Usually millimolar concentrations are required to observe an effect in culture. Because serum fluoride normally is found in the micromolar range, it has been claimed that there is no relevance to the in vivo situation. However, studies by Okuda et al. (1990) on resorbing osteoclasts reported that: “NaF in concentrations of 0.5-1.0 mM decreased the number of resorption lacunae made by individual osteoclasts and decreased the resorbed area per osteoclast. We argue that the concentration of fluoride in these experiments may be within the range ‘seen’ by osteoclasts in mammals treated for prolonged periods with approximately 1 mg of NaF/kg body weight (bw) per day.” Sodium fluoride intake at 1 mg/kg/day in humans could result in bone fluoride concentrations that might occur in an elderly person with impaired renal function drinking 2 L of water per day containing fluoride at 4 mg/L (see Chapters 3 and 5 for more information on bone fluoride concentrations). Cellular Immunity Macrophage function is a major first line of defense in immunity. When macrophage function is impaired, the body could fail to control the invasion of foreign cells or molecules and their destructive effects. The studies that have investigated the function of the cells involved in humoral immunity are summarized in Table 9-4.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Fluoride, usually in the millimolar range, has a number of effects on immune cells, including polymorphonuclear leukocytes, lymphocytes, and neutrophils. Fluoride interferes with adherence to substrate in vitro. The variety of biochemical effects on immune cells in culture are described in Table 9-4. Fluoride also augments the inflammatory response to irritants. Several mechanisms have been proposed, and the main route is thought to be by means of activation of the G-protein complex. It appears that aluminum combines with fluoride to form aluminum fluoride, a potent activator of G protein. In a study by O’Shea et al. (1987), for example, AlF4 had a greater influence on lymphocyte lipid metabolism than did fluoride in the absence of aluminum. On the other hand, Goldman et al. (1995) showed that the aluminofluoride effect of activating various enzymes in macrophages is independent of the G-protein complex. There is no question that fluoride can affect the cells involved in providing immune responses. The question is what proportion, if any, of the population consuming drinking water containing fluoride at 4.0 mg/L on a regular basis will have their immune systems compromised? Not a single epidemiologic study has investigated whether fluoride in the drinking water at 4 mg/L is associated with changes in immune function. Nor has any study examined whether a person with an immunodeficiency disease can tolerate fluoride ingestion from drinking water. Because most of the studies conducted to date have been carried out in vitro and with high fluoride concentrations, Challacombe (1996) did not believe they warranted attention. However, as mentioned previously in this chapter, bone concentrates fluoride and the blood-borne progenitors could be exposed to exceptionally high fluoride concentrations. Thus, more research needs to be carried out before one can state that drinking water containing fluoride at 4 mg/L has no effect on the immune system. FINDINGS The committee did not find any human studies on drinking water containing fluoride at 4 mg/L where GI, renal, hepatic, or immune effects were carefully documented. Most reports of GI effects involve exposures to high concentrations of fluoride from accidental overfeeds of fluoride into water supplies or from therapeutic uses. There are a few case reports of GI upset in subjects exposed to drinking water fluoridated at 1 mg/L. Those effects were observed in only a small number of cases, which suggest hypersensitivity. However, the available data are not robust enough to determine whether that is the case. Studies of the effects of fluoride on the kidney, liver, and immune system indicate that exposure to concentrations much higher than 4 mg/L can affect renal tissues and function and cause hepatic and immunologic alterations

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards TABLE 9-4 Effects of Fluoride on Immune System Cells Species Study Findings In vitro Human Metabolism factors measured in cultured PMNs incubated with mM concentrations of fluoride. Significant inhibition of PMN metabolic activity at 0.1 mM fluoride for O2 generation. Activity was also inhibited at 0.5 mM for 14CO2 release from labeled glucose and at 1.0 mM for nitroblue tetrazolium-reduction. Human Leukocyte capillary migration inhibition assay. 8% inhibition with 0.5 ppm fluoride and 20% inhibition with 20 ppm fluoride. Various Evaluated signal transduction in cultured macrophages exposed to NaF with or without aluminum. NaF reduced intracellular ATP concentrations, suppressed agonist-induced protein tyrosine phosphorylation and reactive oxygen species formation. There was in situ activation of nitrogen-activated protein kinase, phospholipase A2, and phosphatidylinositol-phospholipase C. Little or no effect on NaF-mediated enzyme action was observed when cells were treated with AlCl3 or deferoxamine. Human Cell migration assay and micropore filter assay used to assess effect of NaF on locomotion and chemotaxis of human blood leukocytes. Significant reduction in chemotaxis and locomotion observed with 1 mM fluoride. Human Cultured neutrophils treated with fluoride. Fluoride activated diacylglycerol generation and phospholipase D activity. Increased diradylglycerol mass, with kinetics similar to superoxide generation. Human Electropermeabilized neutrophils treated with fluoride. O2 production was increased by electropermeabilization. That effect was antagonized by GDP[β-S], required Mg2+, and was blocked by staurosporine and H-7. Human Adherence assay of PMNs cultured with 0.0625-4.0 µM with or without autologous serum. No effect in the absence of serum. With serum, adherence significantly decreased at 0.5 µM. Decrease was 1.1% at 0.125 µM and 52.7% at 1.5 µM.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Application/Proposed Mechanisms Comments Reference Inhibition was primarily due to suppression of nonoxidative glucose metabolism. Peak effect was at 20 mM, a lethal dose to the cells.   Gabler and Leong 1979   Effect at 0.5 ppm fluoride likely not significant. 20 ppm fluoride is 100 times higher than serum fluoride concentrations expected if 1.5 L of 4 ppm fluoride in water is consumed. Gibson 1992 Authors suggest that some of the pleiotropic effects of NaF in intact cells might be due to depletion of ATP and not by G-protein activation.   Goldman et al. 1995   1 mM fluoride is a high concentration relative to blood fluoride, but such a concentration might be possible within the Haversian canal system of bone, restricting migration of leukocytes through bone. Wilkinson 1983 Data are consistent with the activation of phosphatidic acid and diglyceride generation by both phopholipase D-dependent and independent mechanisms.   Olson et al. 1990 Supports the hypothesis that fluoride activates G protein, most likely Gp, by interacting with the nucleotide-binding site on the G α subunit.   Hartfield and Robinson 1990 Effect is not direct and is probably modulated by a seric factor. Concentrations of fluoride tested are similar to those found in blood. Gomez-Ubric et al. 1992

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Species Study Findings Human Promyelocytic HL-60 cells treated with 0.5 mM NaF for 0-4 days. Cell proliferation was inhibited by NaF and was augmented by the addition of 1,25-dihydroxyvitamin D3. Other observations were changes in cellular morphology, increased cellular adhesion to plastic, reduced nuclear/ cytoplasmic ratio, and increased cellular expression of chloroacetate esterase. No effect on cellular nonspecific esterase activity. Human Blood lymphocytes incubated with NaF at 0.31, 0.62, or 1.2 mM. NaF augmented lymphocyte response to a mitogen (PHA) or a specific antigen (morbilli antigen from infected cells). Simultaneous incubation of NaF at 0.62 mM with PHA significantly increased cytokine INF-γ release from activated T and/or NK cells compared with treatment with PHA alone (P < 0.01). Human CD34+ cells isolated from umbilical cord blood were incubated with 1, 10, and 50 mM NaF for 30 and 120 minutes. At 10 and 50 mM NaF, there was damage to CFU-GM and significantly decreased cloning potential of these cells. Growth of BFU-E was also inhibited. Rat Liver macrophages treated with fluoride. Arachidonic acid and prostaglandins were released (required extracellular calcium), but there was no formation of inositol phosphates or superoxide. Those effects were inhibited by staurosporine and phorbol ester. Protein kinase C was translocated from the cytosol to membranes. Mouse Cultured lymphocytes treated with NaF and AlCl3. With NaF, there was a breakdown of polyphosphoinositides, decreased production of phosphoinositols, increased cytosolic Ca2+, and start of phosphorylation of the T-cell receptor. Effects were potentiated by addition of AlCl3. Mouse Bone marrow progenitor cells cultured with 0.1-0.5 mM NaF. Upregulation in the activities of intracellular enzymes (LDH, β-glucuronidase, acid phosphatase), cellular reduction of nitroblue tetrazolium, and nitric oxide production.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Application/Proposed Mechanisms Comments Reference NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell. 1,25-Dihydroxyvitamin D3 acts as a cofactor with NaF, primarily through interaction with an endogenous NaF-induced cyclooxygenase product(s), possibly PGE2.   Kawase et al. 1996 Authors concluded that NaF’s effect on INF-γ release during an immune response might be one of the primary ways that fluoride ion influences the immune system.   Loftenius et al. 1999     Machalinski et al. 2000 Calcium-dependent protein kinase C appears to be involved in fluoride’s action on liver macrophages.   Schulze-Specking et al. 1991 The active moiety is AlF4-. AlF4−induced effects were insensitive to cyclic adenosine monophosphate.   O’Shea et al. 1987 Authors suggest that NaF induces early differentiation of bone marrow hemopoietic progenitor cells along the granulocytic pathway but not the monocytic pathway linked to osteoclast formation.   Oguro et al. 2003

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Species Study Findings In vivo Rabbit Rabbits immunized with transferrin before or after 9 months treatment with 10 mg/kg/day. Circulating anti-transferrin titers were measured during the 9 months. DNA and protein synthesis were determined by [3H]thymidine and [14C]leucine incorporation. NaF inhibited antibody formation and had a threshold of 0.78 ppm in circulation. DNA and protein synthesis were also inhibited. Rat Sensitization assay performed with rats administered 5 mL of a 100-mmol solution of NaF twice a week for 2-3 weeks and given ovalbumin in drinking water. Significant increase in surface immunoglobulin expression on lymphocytes from the Peyer’s patches and mesenteric lymph nodes. Rat 0.1, 0.2, and 0.4 mg of fluoride administered intratracheally. Significant PMN-leukocyte infiltration in the lungs observed 24 hours after treatment with 0.2 and 0.4 mg. mRNA of chemokines and proinflammatory cytokines was increased. Increased adhesion of PMNs to plastic dish. Mouse Antibacterial defense mechanisms and lung damage were assessed in mice exposed to 2, 5, 10 mg/m3 of a fluoride aerosol in an inhalation chamber for 4 hours per day for 14 days. Suppression of pulmonary bactericidal activity against Staphylococcus aureus at 5 and 10 mg/m3. Significant decrease in the number of alveolar macrophages in bronchoalveolar lavage fluid at 10 mg/m3 in mice not bacterially challenged. Significant increase in PMNs and lymphocytes at 10 mg/m3. ABBREVIATIONS: BFU-E, burst forming unit of erythrocytes; CFU-GM, colony-forming unit of granulocyte-macrophages; GDP[β-S], guanosine 5′-[β-thio]diphosphate; INF-γ, interferon γ; LDH, lactate dehydrogenase; PGE2, prostaglandin E; PHA, phytohemaggultinin ; PMN, polymorphonuclear leukocyte.

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards Application/Proposed Mechanisms Comments Reference Antibody formation appears to be inhibited because of the decrease in lymphocyte proliferation and inhibition of protein synthetic ability of immunocytes. General inhibition of metabolic function. Jain and Susheela 1987 Microulcerations of the gastric mucosa. Authors note that the concentrations tested were within the range that could be inadvertently ingested by infants/ children or adults from fluoride supplements or gels. Butler et al. 1990     S. Hirano et al. 1999 Authors concluded that inhalation of fluoride can cause cellular alterations in the lung that diminish the ability to respond to infectious bacteria.   Yamamoto et al. 2001

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards in test animals and in vitro test systems. For example, a few studies suggest that fluoride might be associated with kidney stone formation, while other studies suggest that it might inhibit stone formation. Some effects on liver enzymes have been observed in studies of osteoporosis patients treated with fluoride, but the available data are not sufficient to draw any conclusions about potential risks from low-level long-term exposures. Little data is available on immunologic parameters in human subjects exposed to fluoride from drinking water or osteoporosis therapy, but in vitro and animal data suggest the need for more research in this area. As noted earlier in Chapters 2 and 3, several subpopulations are likely to be susceptible to the effects of fluoride from exposure and pharmacokinetic standpoints. With regard to the end points covered in this chapter, it is important to consider subpopulations that accumulate large concentrations of fluoride in their bones (e.g., renal patients). When bone turnover occurs, the potential exists for immune system cells and stem cells to be exposed to concentrations of fluoride in the interstitial fluids of bone that are higher than would be found in serum. From an immunologic standpoint, individuals who are immunocompromised (e.g., AIDS, transplant, and bone-marrow-replacement patients) could be at greater risk of the immunologic effects of fluoride. RECOMMENDATIONS Gastric Effects Studies are needed to evaluate gastric responses to fluoride from natural sources at concentrations up to 4 mg/L and from artificial sources. Data on both types of exposures would help to distinguish between the effects of water fluoridation chemicals and natural fluoride. Consideration should be given to identifying groups that might be more susceptible to the gastric effects of fluoride. The influence of fluoride and other minerals, such as calcium and magnesium, present in water sources containing natural concentrations of fluoride up to 4 mg/L on gastric responses should be carefully measured. Renal and Hepatic Effects Rigorous epidemiologic studies should be carried out in North America to determine whether fluoride in drinking water at 4 mg/L is associated with an increased incidence of kidney stones. There is a particular need to study patients with renal impairments. Additional studies should be carried out to determine the incidence, prevalence, and severity of renal osteodystrophy in patients with renal im-

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Fluoride in Drinking Water: A Scientific Review of EPA’S Standards pairments in areas where there is fluoride at up to 4 mg/L in the drinking water. The effect of low doses of fluoride on kidney and liver enzyme functions in humans needs to be carefully documented in communities exposed to different concentrations of fluoride in drinking water. Immune Response Epidemiologic studies should be carried out to determine whether there is a higher prevalence of hypersensitivity reactions in areas where there is elevated fluoride in the drinking water. If evidence is found, hypersensitive subjects could then be selected to test, by means of double-blinded randomized clinical trials, which fluoride chemicals can cause hypersensitivity. In addition, studies could be conducted to determine what percentage of immunocompromised subjects have adverse reactions when exposed to fluoride in the range of 1-4 mg/L in drinking water. More research is needed on the immunotoxic effects of fluoride in animals and humans to determine if fluoride accumulation can influence immune function. It is paramount that careful biochemical studies be conducted to determine what fluoride concentrations occur in the bone and surrounding interstitial fluids from exposure to fluoride in drinking water at up to 4 mg/L, because bone marrow is the source of the progenitors that produce the immune system cells.