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Keywords:

  • Caenorhabditis elegans;
  • Toxicity testing;
  • Fluoride toxicity;
  • Drinking water;
  • Silicofluoride

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

Fluorides are commonly added to drinking water in the United States to decrease the incidence of dental caries. Silicofluorides, such as sodium hexafluorosilicate (Na2SiF6) and fluorosilicic acid (H2SiF6), are mainly used for fluoridation, although fluoride salts such as sodium fluoride (NaF) are also used. Interestingly, only the toxicity of NaF has been examined and not that of the more often used silicofluorides. In the present study, the toxicities of NaF, Na2SiF6, and H2SiF6 were compared. The toxicity of these fluorides on the growth, feeding, and reproduction in the alternative toxicological testing organism Caenorhabditis elegans was examined. Exposure to these compounds produced classic concentration–response toxicity profiles. Although the effects of the fluoride compounds varied among the 3 biological endpoints, no differences were found between the 3 compounds, relative to the fluoride ion concentration, in any of the assays. This suggests that silicofluorides have similar toxicity to NaF. Environ Toxicol Chem 2013;33:82–88. © 2013 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

Fluorides are commonly added to public water supplies in the United States to decrease the incidence of dental caries. This practice has been controversial since its inception nearly 70 years ago. Chronic exposure to fluoride can lead to enamel fluorosis and, at levels higher than those added to drinking water, can lead to skeletal defects [1, 2]. In addition, several animal studies have reported fluoride toxicity to reproductive, nervous, gastrointestinal, endocrine, and renal systems [3].

One complicating factor in animal studies has been the source of fluoride. Nearly all of the published studies used sodium fluoride (NaF), even though the majority of public water fluoridation uses silicofluorides. A census of water-treatment plants found that 63% of the population received water fluoridated with fluorosilicic acid (H2SiF6), 28% with sodium hexafluorosilicate (Na2SiF6), and only 9% with NaF [4]. A question frequently raised is whether NaF and silicofluorides have comparable toxicological profiles [5].

Many have speculated that silicofluoride-treated water may be more toxic than NaF-treated water. Fluoride salts such as NaF completely dissociate in water to produce the important fluoride anion that prevents dental caries. For decades, it was assumed that silicofluorides also completely dissociated and hydrolyzed to form fluoride anions; however, there has been strong disagreement with this assumption [6]. One concern is based on the observations that silicofluorides may not completely hydrolyze, leaving trace amounts of silicofluoride in the drinking water, which may be more toxic than the fluoride anion alone [7]. Others claim that all of the silicofluorides hydrolyze; therefore, the toxicity of silicofluorides should have the same level of toxicity as NaF [8]. The question of whether silicofluorides completely hydrolyze has long been studied and has been difficult to determine experimentally.

Given that 86% of the US population consumes public-supply water, 90% of which is treated with silicofluorides, both the National Academy of Sciences and the National Toxicology Program have recommended that the in vivo toxicity of silicofluorides be determined [3, 9]. Despite these recommendations, there has been little progress toward understanding the toxicity of silicofluorides. This may partially be because of the time and cost involved in conducting traditional in vivo toxicity studies, which use large numbers of animals. In fact, there is a backlog of thousands of chemicals from several US Environmental Protection Agency programs that need toxicity testing [10].

There has been a clear need to develop methods to rapidly evaluate chemicals like silicofluorides to rank their relative toxicity. The nematode Caenorhabditis elegans has emerged as a popular alternative toxicological test organism because of its small size, ease of handling, reproductive prowess, low cost of maintenance, short life cycle, and high level of genomic conservation with higher organisms [11, 12]. Three quantitative, high-throughput toxicological assays have been developed to rapidly assess chemical toxicity in C. elegans including growth (change in nematode length), reproduction (number of offspring), and feeding (ingestion of fluorescent microspheres) [13]. Each of these assays uses the Complex Object Parametric Analyzer and Sorter, which sorts and dispenses individual nematodes while simultaneously measuring nematode size and fluorescence [14]. The growth, feeding, and reproduction assays have been used to evaluate the toxicities of hundreds of chemicals including metals, pesticides, and failed drugs (W.A. Boyd and J.H. Freedman, personal communication). In the present study, the relative toxicities of NaF, H2SiF6, and Na2SiF6 were evaluated using these C. elegans toxicity biological endpoints.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

Nematode culture

Bristol N2 (wild-type) C. elegans (Caenorhabditis Genetics Center) were maintained at 20 °C on K-agar plates (2% bacto-agar, 0.25% bacto-peptone, 51 mM sodium chloride, 32 mM potassium chloride, 13 µM cholesterol) seeded with Escherichia coli OP50 as the food source [15]. Age-synchronized cultures were prepared using an alkaline sodium hypochlorite solution (250 µM sodium hydroxide, 1% bleach) as previously described [16]. After a final rinse with K-medium (51 mM sodium chloride, 32 mM potassium chloride) [17], embryos were transferred to a complete K-medium (K-medium plus 3 mM calcium chloride, 3 mM magnesium sulfate, 13 µM cholesterol) [18] to arrest as L1s for the growth assay. Alternatively, for the reproduction and feeding assays L1 nematodes were placed on K-agar plates with food and allowed to develop to the appropriate life stage.

Growth, feeding, and reproduction assays

Growth, feeding, and reproduction assays were performed as previously described using the Complex Object Parametric Analyzer and Sorter Biosort (Union Biometrica) [13]. For the 3 C. elegans assays, age-synchronized nematodes were added to 96-well plates containing the fluoride toxicant, E. coli, and either K-medium or complete K-medium. After the appropriate incubation period, nematodes were aspirated using the Complex Object Parametric Analyzer and Sorter ReFlx option, where 2 size characteristics of individual nematodes—the length or time of flight and optical density or extinction—and red fluorescence were measured. Three biological replicates were conducted for each assay.

To measure growth, 50 synchronized L1 larvae were placed in each well of a 96-well plate as described above and incubated in the presence of toxicant for 48 h at 20 °C, after which time of flight and extinction of each nematode were measured. To assay reproduction, 5 L4 larvae were placed into each well of a 96-well plate and allowed to grow and lay embryos in the presence of toxicant for 48 h at 20 °C [19]. The total numbers of adults and offspring were then quantified. For the feeding assay, 25 young adult nematodes were loaded into each well of a 96-well plate and incubated for 24 h at 20 °C in the presence of toxicant. To measure feeding, 5 µL fluoresbrite polychromatic red 0.5-µm microspheres (Polysciences) diluted 20-fold in deionized water were added to each well [20]. Nematodes were allowed to feed for 15 min, after which 5 µL of 170 mM sodium azide was added to arrest feeding. Nematodes were collected, and the levels of red fluorescence in the nematode pharynx and intestine were measured [20].

Fluoride compounds

The 3 test compounds, NaF (CAS no. 7681-49-4), H2SiF6 (CAS no. 16961-83-4), and Na2SiF6 (CAS no. 16893-85-9), were purchased from Sigma-Aldrich. To maintain the proper pH, NaF, and Na2SiF6 were prepared in M9 buffer (22 mM monobasic potassium phosphate, 42 mM dibasic sodium phosphate, 86 mM sodium chloride, 1 mM magnesium sulfate) instead of complete K-medium. The pH of the highest concentrations tested was 6.37 for NaF at 50 mM and 5.99 for Na2SiF6 at 5 mM. To prevent precipitation in complete K-medium or M9 buffer, H2SiF6 was made as a 1-M solution in deionized water. The H2SiF6 solution was further diluted using M9 buffer with no precipitation to a final pH of 5.13. Boyd et al. reported no detrimental effects on nematodes that were maintained in solutions at pH >4.5 [19, 20].

To determine the fluoride ion concentrations, NaF (50 mM), H2SiF6 (5 mM), and Na2SiF6 (5 mM) were dissolved in M9 buffer and the fluoride ion concentration was measured using a fluoride ion-specific electrode (Mettler Toledo). Standards and samples were diluted 10-fold with total ionic–strength adjustment buffer (TISAB III; Sigma) according to the manufacturer's instructions. Fluoride ion concentrations in the test solutions were determined by comparing the millivolt standard curve for the standards with that of the samples. The electrode was rinsed and placed into standards bracketing the sample concentration, and the fluoride concentrations of the samples were determined by linear regression.

To determine concentration ranges for each of the compounds in each C. elegans toxicity assay, range-finding tests were performed. Final compound concentration ranges encompassed 2 orders of magnitude to capture the entire concentration response, from no effect to full effect. The only exception was the Na2SiF6 feeding assay, which was tested over a single order of magnitude.

Statistical analysis

Statistical analyses were performed using Matlab software (MathWorks). Three replicates of the growth assay were analyzed using the extinction measurement in log scale. After noise observations were removed, the mean of the measurements at time 0 for each replicate was subtracted so that only growth during the exposure was analyzed [13]. Plate adjustments for the 3 replicate plates of each chemical were made by subtracting overall plate means and adding the mean over the 3 replicate plates. The 3-parameter Hill function with a 0 lower asymptote was fit to the well means using weighted nonlinear regression. The number of observations in each well divided by the mean number of observations per well for each well used in the regression were used as weights. The Hill function was fit to each replicate separately, as well as to the combined replicates for each chemical. The data presented in the figures represent the overall weighted means for each replicate/concentration group. Exposed samples were compared with the control group using a weighted t test on the well means with the Bonferroni correction.

The 3 replicates of the feeding assay were analyzed using red fluorescence intensity in log scale. For each chemical, the 3 replicate plates were adjusted by subtracting overall plate fluorescence intensity means and adding the mean over the 3 replicate plates. A 4-parameter Hill function was fit to well means, weighted by the normed number of observations in each well, as detailed for the growth assay. For each chemical, the replicates were fit separately as well as combined. Analyses were performed twice, once using all 12 concentrations and again without using the highest concentration. The data presented in the figures show the weighted means for each replicate/concentration group. Exposed samples were compared with the control group using a weighted t test on the well means with the Bonferroni correction.

The number of offspring observations from the reproduction assay was fit to a 3-parameter Hill function (the lower asymptote was fixed at 0) as individual replicates and with the combined replicates for each chemical. Exposed samples were compared with the control group using Dunnett's test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

Fluoride ion concentrations

The fluoride ion concentration in the 50 mM NaF in M9 solution was measured as 900 ppm. The fluoride ion has a molecular mass of 19; therefore, 900 ppm fluoride would be 47.4 mM fluoride or NaF. At 5 mM, Na2SiF6 resulted in a fluoride ion concentration of 627 ppm fluoride, or 33 mM fluoride. Because H2SiF6 has 6 fluoride ions, this molarity is consistent with a fully hydrolyzed and dissolved solution of 5 mM Na2SiF6. Similarly, 5 mM Na2SiF6 contained 569 ppm fluoride, which is equivalent to 30 mM fluoride.

Effect of fluorides on C. elegans growth

The growth of C. elegans from the first larval stage (L1) was determined during 48-h exposures to the 3 fluoride compounds. The concentration range tested for NaF included higher concentrations (0.5–50 mM) compared with either H2SiF6 (0.05–5 mM) or Na2SiF6 (0.05–5 mM) (Supplemental Data, Figure S1). Visual observations of nematodes exposed to NaF indicated no effects on growth at concentrations up to 2 mM, complete growth inhibition at 20 mM, and lethality at 32 mM. Similar effects were observed after exposure to Na2SiF6 and H2SiF6 but at lower concentrations. Both showed effects at 0.32 mM, with no growth at 3.2 mM and lethality at 5 mM. All compounds led to concentration-dependent decreases in nematode size, with good agreement between replicates (Figure 1). As shown in Table 1, the median effective concentrations (EC50) for growth were 5 times and 7 times higher for NaF (2.87 mM) than Na2SiF6 (0.56 mM) and H2SiF6 (0.41 mM), respectively. Likewise, the lowest-observed-effect concentrations (LOECs) were 6.25 times higher for NaF (2.0 mM) than for either H2SiF6 or Na2SiF6, both of which had an LOEC of 0.32 mM. The results for NaF (EC50 2.87 mM and LOEC 2.0 mM) concurred with previous observations in C. elegans in which there was a significant reduction in the growth of L1 larvae on agar plates at 3.6 mM but no effect at 0.95 mM [21].

image

Figure 1. Effects of fluoride compounds on Caenorhabditis elegans growth. Groups of 50 L1 nematodes were exposed to varying concentrations for 48 h of NaF, H2SiF6, or Na2SiF6. Each panel presents the weighted mean sizes (log[EXT]) and standard errors for 3 replicate experiments (red, blue, and green dots). The black line is the Hill equation fit of the combined data. Full concentration–response curves—0 mM to 50 mM for NaF and 0 mM to 5 mM for H2SiF6, and Na2SiF6—are presented in Supplemental Data, Figure S1. EXT = extinction; NaF = sodium fluoride; H2SiF6 = fluorosilicic acid; Na2SiF6 = sodium hexafluorosilicate.

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Table 1. Effects of fluoride compounds on Caenorhabditis elegans growth
ChemicalEC50 (95% CI)aLECaEC50 (95% CI)LEC
 (mM)(mM)(ppm fluoride)(ppm fluoride)
  • a

    The median effective concentration (EC50) and lowest effective concentration (LEC) results are shown for the individual replicates and the combined data for the growth assay.

  • CI = confidence interval.

Sodium fluorideCombined2.87 (2.80–2.95)2.055 (53.19–56.04)38
 Replicate 13.28 (3.20–3.36) 62 (60.79–63.83) 
 Replicate 22.65 (2.61–2.69) 50 (49.58–51.10) 
 Replicate 33.04 (2.94–3.13) 58 (55.85–59.46) 
Fluorosilicic acidCombined0.41 (0.40–0.42)0.3247 (45.59–47.87)36
 Replicate 10.36 (0.35–0.36) 41 (39.89–41.03) 
 Replicate 20.47 (0.46–0.49) 54 (52.43–55.85) 
 Replicate 30.43 (0.42–0.44) 49 (47.87–50.15) 
Sodium hexafluorosilicateCombined0.56 (0.55–0.58)0.3264 (62.69–66.11)36
 Replicate 10.76 (0.73–0.80) 87 (83.21–91.19) 
 Replicate 20.51 (0.50–0.52) 58 (56.99–59.27) 
 Replicate 30.50 (0.50–0.51) 57 (56.99–58.13) 

Effect of fluorides on C. elegans feeding

Figure 2 shows the concentration–response plots for the 3 compounds using the feeding assay. This assay measures the effects of toxicant exposure on the ingestion of fluorescent microspheres by adult nematodes after 24-h exposure. Like growth, the concentration range for NaF included higher concentrations (0.5–50 mM) than H2SiF6 (0.05–5 mM) or Na2SiF6 (0.5–5 mM; Supplemental Data, Figure S2). Marked decreases in pharyngeal pumping after NaF exposures were visually observed at 13 mM fluoride, with almost no pumping at concentrations of 20 mM and higher. A decline in feeding after exposure to Na2SiF6 was noted at 0.79 mM, with very slow pumping at 2 mM and above. Similarly for H2SiF6, almost no pharyngeal pumping was observed visually at 2 mM and above (Figure 2). For the 3 compounds, the highest concentration tested led to an increase in fluorescence even though no signs of pharyngeal pumping or intestinal accumulation of fluorescent microspheres were visually observed. This may be a result of beads attaching to dying nematodes, leading to higher red fluorescence levels. Therefore, the highest concentrations were not used to calculate the EC50 and LOEC (Table 2). Another observation of note is that the higher H2SiF6 concentrations in the feeding study were lethal to offspring that hatched during the incubation but not to the adults. This was not true for NaF or Na2SiF6. Interestingly, the LOEC for H2SiF6 (0.2 mM) was lower than values for NaF (5 mM) and Na2SiF6 (0.79 mM; Table 2). However, the EC50 estimates for Na2SiF6 and H2SiF6 were similar. The EC50 for NaF (6.3 mM) was 5 times higher than that for H2SiF6 (1.26 mM) and 6.7 times higher than that for Na2SiF6 (0.94 mM).

image

Figure 2. Effects of fluoride compounds on Caenorhabditis elegans feeding. Groups of 25 young adult nematodes were exposed for 24 h to varying concentrations of NaF, H2SiF6, and Na2SiF6. Feeding was measured as ingestion of red fluorescent microspheres at the end of the exposures. Each panel presents the weighted mean fluorescence values and standard errors for 3 replicate experiments (red, blue, and green dots). The black line is the Hill equation fit of the combined data. Hill equation fits of all the data for replicate experiments. Full concentration response curves—0 mM to 50 mM for NaF and 0 mM to 5 mM for H2SiF6 and Na2SiF6—are presented in Supplemental Data, Figure S2. A.U. = arbitrary units; NaF = sodium fluoride; H2SiF6 = fluorosilicic acid; Na2SiF6 = sodium hexafluorosilicate.

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Table 2. Effects of fluoride compounds on Caenorhabditis elegans feeding
ChemicalEC50 (95% CI)aLECaEC50 (95% CI)LEC
 (mM)(mM)(ppm fluoride)(ppm fluoride)
  • a

    The median effective concentration (EC50) and lowest effective concentration (LEC) results are shown for the individual replicates and the combined data for the feeding assay.

  • CI = confidence interval.

Sodium fluorideCombined6.30 (5.66–6.93)5.0120 (107.53–131.66)95
 Replicate 15.75 (4.88–6.62) 109 (92.71–125.77) 
 Replicate 25.91 (4.75–7.08) 112 (90.24–134.50) 
 Replicate 37.18 (6.16–8.19) 136 (117.03–155.59) 
Fluorosilicic acidCombined1.26 (0.89–1.64)0.20144 (101.45–186.94)23
 Replicate 11.03 (–0.20–2.25) 117 (–22.80–256.47) 
 Replicate 20.93 (0.78–1.08) 106 (88.91–123.11) 
 Replicate 31.35 (1.01–1.70) 154 (115.13–193.78) 
Sodium hexafluorosilicateCombined0.94 (0.89–0.99)0.79107 (101.45–112.85)90
 Replicate 11.00 (0.91–1.10) 114 (103.73–125.39) 
 Replicate 20.84 (0.80–0.88) 96 (91.19–100.31) 
 Replicate 31.02 (0.95–1.08) 116 (108.29–123.11) 

Effect of fluorides on C. elegans reproduction

Figure 3 shows the concentration–response plots for the 3 compounds using the reproduction assay, which measures the number of offspring produced during 48-h chemical exposure. The concentration range for NaF included higher values (0.5–50 mM) than for either H2SiF6 (0.04–4 mM) or Na2SiF6 (0.04–4 mM; Figure 3). For the reproduction assay, it was noted by visual observations that there was a reduction in offspring of approximately 15% at 3.2-mM NaF exposure and no offspring present by 32 mM. In contrast to NaF, a decrease in the number of offspring was observed after exposure to Na2SiF6 and H2SiF6 at concentrations as low as 0.4 mM, with only a few offspring in each well at 4 mM (Figure 3). Estimated EC50 and LOEC values were slightly higher for NaF (6.74 and 5 mM, respectively) than for either H2SiF6 (0.9 and 0.63 mM, respectively) or Na2SiF6 (0.87 mM and 0.63 mM, respectively; Table 3).

image

Figure 3. Effects of fluoride compounds on Caenorhabditis elegans reproduction. Groups of 5 L4 nematodes were exposed for 48 h to varying concentrations of NaF, H2SiF6, and Na2SiF6. Reproduction was measured by counting the total number of nematodes at the end of the exposure, at which time the untreated group consisted of adults and their offspring. Each panel presents the weighted mean numbers of recovered nematodes and Hill equation fits of all the data for 3 replicate experiments (red, blue, and green dots). NaF = sodium fluoride; H2SiF6 = fluorosilicic acid; Na2SiF6 = sodium hexafluorosilicate.

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Table 3. Effects of fluoride compounds on Caenorhabditis elegans reproduction
ChemicalEC50 (95% CI)aLECaEC50 (95% CI)LEC
 (mM fluoride)(mM fluoride)(ppm fluoride)(ppm fluoride)
  • a

    The half-maximal effective concentration (EC50) and lowest effective concentration (LEC) results are shown for the individual replicates and the combined data for the reproduction assay.

  • CI = confidence interval.

Sodium fluorideCombined6.74 (5.04–8.43)5.0128 (95.75–160.15)95
 Replicate 17.06 (6.23–7.89) 134 (118.36–149.89) 
 Replicate 26.42 (5.14–7.69) 122 (97.65–146.09) 
 Replicate 36.75 (4.26–9.24) 128 (80.93–175.54) 
Fluorosilicic AcidCombined0.90 (0.77–1.03)0.63103 (87.77–117.41)72
 Replicate 10.97 (0.78–1.17) 111 (88.91–133.36) 
 Replicate 20.75 (0.63–0.88) 85 (71.81–100.31) 
 Replicate 30.90 (0.80–1.00) 103 (91.19–113.99) 
Sodium hexafluorosilicateCombined0.87 (0.68–1.06)0.6399 (77.51–120.83)72
 Replicate 10.77 (0.43–1.12) 88 (49.01–127.67) 
 Replicate 20.86 (0.67–1.04) 98 (76.37–118.55) 
 Replicate 31.01 (0.81–1.21) 115 (92.33–137.92) 

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

The addition of fluorides to drinking water to prevent dental caries remains controversial, mostly because of unresolved toxicological questions. The majority of previous fluoride toxicity studies utilized NaF as the source of fluoride rather than silicofluorides, which are more often used to fluoridate the public water supplies. Some have questioned whether Na2SiF6 and H2SiF6 may be more toxic than NaF. In the present study, the relative toxicity of NaF, Na2SiF6, and H2SiF6 on 3 biological endpoints—growth, feeding, and reproduction—were compared using the alternative toxicological testing organism C. elegans. Within each assay, the toxicities of these compounds varied considerably when measured in millimolar concentrations. These results appear to indicate that NaF is less toxic in regard to nematode development and growth than Na2SiF6 or H2SiF6. However, Na2SiF6 and H2SiF6 each contain 6 fluoride ions per molecule, and when toxicity is expressed in parts per million of fluoride, as opposed to millimoles of compound, the apparent differences in toxicity disappear (Table 1). For the growth assay, the 2.87 mM EC50 for NaF is equivalent to 54.52 ppm fluoride, whereas the H2SiF6 EC50 of 0.41 mM is equivalent to 46.73 ppm fluoride and the 0.56 mM Na2SiF6 EC50 is equivalent to 63.83 ppm fluoride. Sodium fluoride, when measured by millimoles of compound, is 7-fold less toxic than H2SiF6 and 5-fold less toxic than Na2SiF6. Once again, when the concentrations are expressed relative to fluoride ion, the EC50 values are similar. While the confidence intervals do not overlap, there is a difference of only 17 ppm between the lowest and highest values and the LOECs are all within 2 ppm of each other. In the reproduction study, H2SiF6 and Na2SiF6 have very similar EC50s and identical LOECs, with NaF results being approximately 7.5 times higher when compared by compound, yet when they are compared by parts per million fluoride the NaF results are only slightly higher and the confidence intervals of the EC50s overlap for all of the compounds (Table 3).

The results of the NaF assay also correspond well to another reproduction assay. As previously stated, there was an observed decrease of 15% in the brood size at 3.2 mM NaF and no offspring at 32 mM. This result is similar to a decrease in brood size of 28% at 3.8 mM (72 ppm) NaF, which was reported by Li et al. [22] using a different methodology. The feeding study results indicate that Na2SiF6 has the lowest EC50 (0.94 mM), followed by H2SiF6 (1.26 mM) and NaF (6.30 mM). Once again, the confidence intervals overlap for all of the compounds. Biologically, these numbers are very close, well within an order of magnitude, and the differences are minimal. When measured in parts per million, the results show very little difference between the compounds. This would appear to indicate that silicofluorides have similar toxicity to NaF in C. elegans (Table 2).

Fluoride alone is toxic at high levels, and some studies suggest that it can be even more toxic when complexed with other agents in the drinking water. For example, aluminum, arsenic, and lead have additive effects on fluoride toxicity [23-25]. Silicofluorides may interact differently with these species. Masters et al. [7] claimed that trace amounts of undissolved fluorosilicates could complex with lead and facilitate its transport from the gastrointestinal tract to the bloodstream. In these initial experiments, the effects of NaF, SiF6, and H2SiF6 were tested alone and not in conjunction with other metals. This may be a reason that no differences were found in toxicity between compounds. Future studies in which a combination of fluoride and other toxicants is tested would be informative and practical using the C. elegans model.

Fluorides have long been known to affect calcified tissues in the body, such as teeth and bone. Recently, it has become clear that fluoride also has cellular effects [26]. In this regard, C. elegans can be a valuable toxicity model in fluoride research, with many purported cellular mechanisms of fluoride toxicity, such as oxidative stress and apoptosis [11, 26]. As many signaling molecules and pathways are conserved in C. elegans, it could serve as an excellent complement to cell culture–based systems and vertebrate models to further understand the toxicology of fluoride.

Acknowledgment

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

The authors have no conflicts of interest. This work was supported in part by the National Toxicology Program and by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (Z01ES102045 and Z01ES102046).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information
  • 1
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    National Toxicology Program. 2001. Review of toxicological literature: Sodium hexafluorosilicate [CASRN 16893-85-9] and fluorosilicic acid [CASRN 16961-83-4]. Research Triangle Park, NC, USA.
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    Dix DJ, Houck KA, Martin MT, Richard AM, Setzer RW, Kavlock RJ. 2007. The ToxCast program for prioritizing toxicity testing of environmental chemicals. Toxicol Sci 95:512.
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    Leung MC, Williams PL, Benedetto A, Au C, Helmcke KJ, Aschner M, Meyer JN. 2008. Caenorhabditis elegans: An emerging model in biomedical and environmental toxicology. Toxicol Sci 106:528.
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    Hope IA. 1999. C.elegans: A Practical Approach. Oxford University Press, Oxford, UK; New York, NY, USA.
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgment
  9. REFERENCES
  10. Supporting Information

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