Intracellular Fluoride in Cultured Mammalian Cells

Abstract

Most investigations of the sensitivity of cultured mammalian cells to fluoride have been concerncd with a determination of the levels of fluoride which inhibit growth. While some studies (1, 2 ) have suggested that the growth of mammalian cells can be inhibited by from 0.045 to 2 ppm F, most reports (3)(4)(5)(6)(7)(8) have indicated that a minimum of 5-15 ppm F (0.3-0.8 mM) is required to decrease the rate of cell division or accumulation of cell protein. At higher levels of fluoride, there appears to be a sigmoidal dose-response relationship with slight inhibition a t 10 to 20 ppm and compkte inhibition of growth a t 40 to 50 ppm (6, 7 ) . Although i t has been observed that cellular ATP levels are decreased (9), and glucose uptake per milligram of cellular protein is increased (8,9) in fluoride-treated cells, the mechanism by which fluoride inhibits growth is not understood. To further characterize the toxicity of fluoride in mammalian cell cultures, the distribution of fluoride between cells and medium was determined. A comparison of intracellular fluoride concentrations with the concentrations of fluoride reported (10) to inhibit various enzymes in vitro may allow a prediction as LO enzyme inhibitions of physiological significance.Methods. Suspension cultures of HeLa CCL 2 cells and L cells (mouse fibroblasts) with generation times of 36 and 19 hr, respectively, were utilized for these experiments. The L cells were grown in medium B of Medappa et al. (1 1) and the HeLa cells were grown in the medium described by Carlson and Suttie (9) modified by the substitution of a Ca-Mg-free salt mixture (12) and the addition of 0.1 % Pluronic F-68 detergent. The cells were resuspended in fresh medium at a cellular concentration of 4 x 10" cells/ml for an equilibration period of 1 hr before measuring fluoride distribution. A mixture of carrier-free ISF prepared by neutron activation (13) and NaF in a total volume of 3 ml was added to 200 ml of cell suspension in a 500 ml Erlenmeyer flask, and the cells were incubated for 1 or 2 hr at 37" on a rotatory shaker. The cell suspensions were then centriiuged a t 5OOg for 1-2 min; and the cell pellets were resuspended in 10 ml of supernatant. After the addition of 10 pCi of 14C-inulin, 2.5 ml aliquots of cell suspension were injected into specially designed glass centrifuge tubes (14) having a reservoir of 2.5 ml capacity connected with a capillary 6 CM long and 1 mm in diameter and centrifuged at 2000g for 20 min a t 4 ' . The cell pellets and supernatant medium recovered from centrifugation were analyzed for lSF and 14C-inulin according to standard procedures. Since inulin cannot penetrate mammalian cells, the I4C in the cell pellets was used to calculate the amount of entrapped medium. Intracellular fluoride concentrations were calculated from the amounts of 18F in cell pellets and entrapped medium, the specific activity of lXF in the medium, and the amount of intracellular water in pellets. Intracellular water values were calculated from the wet and dry weights of cell pelle...

pH and the cytotoxicity of fluoride in an animal cell culture system

“…This increased uptake of fluoride might therefore be a possible explanation of the potentiating effect of a lowered pH. A distribution ratio of about 0.4 has also been reported for HeLa and L 929 cells (DRESCHER & SuTTiE 1972), being the same whether the cells were incubated with 10 or 50 \ig fluoride/ml. The pH of their incubation medium was not given, but was most probably about 7.2-7.4, as standard culturing conditions were used.…”

“…To measure the cellular uptake of fluoride, a slight modification of the procedure described by DRESCHER & SUTTIE (1972) was used. Monolayer cultures of human epithelial cells (NGTC 2544) were trypsinized, centrifuged and suspended to a den.sity of 0.4-10'' cells'ml in 50 ml of Eagle's Minimum Essential Medium, supplemented with 10% calf serum and buffered at pH 6.7 and 7.4 with 20 mM HEPES (Af-2-hydroxyethylpiperazine-A'"-2-ethanesulfonic acid (WIL- LIAMSON & Cox 1968).…”

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

“…The cell volumes were determined using the cell displacement method and inulin as previously described (35). Thirty mL of ∼20 OD 600 cells were pelleted and resuspended in 200 μL water with 10,000 counts/μL 14 C purified inulin.…”

Small Molecule Fluoride Toxicity Agonists

“…A number of methods exist for the detection of fluoride, including small-molecule sensors (Cametti and Rissanen, 2009, 2013), direct monitoring of 18 F accumulation (Drescher and Suttie, 1972; Li et al, 2013; Quissell and Suttie, 1972), and YFP variants that fluoresce in the presence of fluoride (Jayaraman et al, 2000). However, none of these methods are sufficiently selective or sensitive to rapidly and easily detect intracellular fluoride concentrations in bacteria.…”