Murarion Research, 279 (1992) 109-l 1.5 0 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00

MUTGEN 01756

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells

D. Slameiiovii, A. Gibelov6 and K. Ruppovi Department of Mutagenesis and Chemical Carcinogenesis, Cancer Research Institute of the SloLsak Academy of Sciences.

BratislaLla (Czechoslovakia)

(Received 17 January 1991) (Revision received 19 August 1991)

(Accepted 26 September 1991)

Keywords: Sodium fluoride; V79 cells; Human EUE cells

Summary

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-re- sistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of lo-150 pg of NaF per ml of culture medium induced lO-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentra- tions (200-600 pg/ml). Growth of both cell types with 100 and 200 pg of NaF per ml caused inhibition of r4C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macro- molecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of lo-400 pg of NaF per ml of culture medium.

Fluorine represents the 13th most plentiful element in the earth. The human population is exposed to inorganic fluorides from fluoridation of water and dental products as well as the natu- ral fluoride occurring in water, soil and food. It is generally assumed that NaF is safe to add to

Correspondence: Dr. D. Slamefiovl, Cancer Research Insti- tute, Slovak Academy of Sciences, Spitalska 21, 81232 Bratislava (Czechoslovakia).

drinking water and to solutions for mouth-rinsing as a preventer of dental caries. Because of its ability to reduce the development of dental caries by about 50%, the addition of small amounts of fluoride (about 1 ppm = 1 pg/g) to drinking wa- ter was initiated in the U.S.A. in 1945. Now this measure is generally accepted and recommended by health authorities and dentists (McClure, 1970; Horowitz, 1973; WHO, 1970, 1984). In many countries of the world tablets of sodium fluoride are given to children until the age of 12-13 years.

The daily dose given in this way is about 1 mg of sodium fluoride (i.e., four tablets of sodium fluo- ride containing 0.25 mg of NaF in each).

Since the effects of sodium fluoride seem to be controversial at present (Li et al., 1988), we de- cided to study this chemical compound by mea- suring colony-forming ability, macromolecular synthesis and mutagenicity of mammalian cells of two lines.

Material and methods

Cell lines We used heteroploid human EUE fibroblasts

and quasidiploid Chinese hamster V79 cells (both obtained from Dr. A. Abbondandolo, Laboratory of Mutagenesis, Pisa, Italy). Cells were cultured in MEM medium with the addition of 6% fetal calf serum, penicillin (200 U/ml), streptomycin (100 pg/ml), kanamycin (100 wg/ml), a mixture of non-essential amino acids, and 0.1 g/l sodium pyruvate in a humidified 5% CO, atmosphere at 37°C in glass petri dishes. The cells were checked under an electron microscope for the presence of mycoplasma, no contamination was noted.

Chemicals Sodium fluoride (NaF) is a white powder pro-

duced by Slovakofarma Hlohovec (Czecho- slovakia). It was kept at room temperature and dissolved in MEM medium. Freshly prepared so- lutions of the drug were used in each experiment.

ZV-Methyl-N’-nitro-N-nitrosoguanidine (MNNG, Koch Light Laboratories) was dissolved in DMSO and diluted immediately before use.

Plating eficiency test On to 6 cm diameter petri dishes we plated

3 x 10’ V79 cells and on to 8 cm diameter petri dishes we plated 5 x lo2 EUE cells. After the cells were attached to the bottom of the petri dishes NaF in culture medium was added at different concentrations. Both kinds of cells were incubated with NaF for 20 h and EUE cells were in addition incubated with NaF for 24 and 48 h. Then the cells were washed and cultured in fresh growth medium. On day 7-8 after treatment,

they were stained with methylene blue (1% solu- tion) and the number of colonies was counted. From the ratio of the number of colonies to cells plated the plating efficiency (%) of cells was calculated.

Intensity of DNA, RNA and protein synthesis It was investigated on the basis of 60-min

incorporation of ‘“C-thymidine, 14C-uridine or ‘“C-L-leucine (1 pCi/ml = 40 kBq/ml) in cells in single phases of continual culturing in the pres- ence of NaF in culture medium. Radioactively labeled cells were washed with SSC buffer (0.15 M sodium chloride + 0.015 M sodium citrate cooled to O’C) and the high-molecular fraction of cells was precipitated by 5% TCA (trichloroacetic acid) in which were the cells kept for 24 h at 0°C. Then the cells were mechanically removed from the glass with a silicone scraper, resuspended, filtered through a Synpor membrane filter with a porosity of 0.44 pm and washed with 5% TCA, distilled water and ethanol. The amount of ra- dioactivity of dried membranes was measured on a Beckman LS 1801 scintillation counter.

Detection of 6-thioguanine-resistant (6TG’) muta- tions

V79 cells (3 x 10’) were plated in petri dishes (0 10 cm) and cultivated for 24 h. The medium was removed and replaced with growth medium containing 0, 10, 50, 100, 150, 200, 250, 300, 350 or 400 pg of NaF/ml in which cells were cul- tured for 24.h. As a positive mutagenic factor we used MNNG (1 pg/ml). The cells were culti- vated in the presence of MNNG for 24 h. For detection of 6-TG’ mutations we used the re- spreading mutation assay proposed by Chasin (1973) and modified by Abbondandolo et al. (1976) and Slamefiovi and Ggbelov& (1980). In brief, treated cells and control cells were plated on 5 petri dishes (0 10 cm) at 0.15-l X 10h in dependence on NaF concentration. Next, 3 X 10’ cells were plated to determine cloning efficiency. After several subcultures mutagenized and con- trol cells were used for measurement of 6-TG’ mutations on the 5th, 7th and 12th day of expres- sion and for the estimation of cloning efficiency necessary to calculate the frequency of 6-TG’ mutations.

Results

When investigating the prolonged (20-h) influ- ence of NaF (lo-150 ~g/mlI on the cloning efficiency of EUE and V79 cells we found that the studied concentrations of NaF acted cytotoxi- tally on hamster V79 cells but they had no cyto- toxic effect on human EUE cells. Higher concen- trations of NaF (200-600 pg/ml) were needed to produce cytotoxic effects in human EUE cells (Fig. 1). Prolongation of treatment of human cells from 24 to 48 h did not change the toxic effect of NaF significantly (data not shown). Equal con- centrations of two different sodium salts (NaCI and NaN,) added to the culture medium did not show any cytotoxic effect on mammalian cells (data not shown).

Fig. I. Colony-forming ability of V79 cells (0) and WE cells (01 after 20-h incubation with various concentrations of NaF

in complete medium.

Incorporation of “C-thymidine mto V79 cells and EUE cells cultured in the presence of NaF (100 and 200 I_cg/ml) is presented in Fig. 2. Control cells were cultured and labeled in the absence of NaF. Fig. 3 presents the percentage of Control incorporation of “C-uridine in both cell

lines and Fig. 4 presents the percentage of con- trol incorporation of ‘“C-L-leucine. During the 60-min labeling of cells with “C-thymidine, “C- uridine and ‘%-L-leucine an equivalent amount of NaF was added to the radioactive medium so as not to disturb continual NaF treatment.

The result of mutagenicity testing is presented in Table 1, where the levels of 6-TG’ mutations per 1 X 10’ cells are shown. Mutations were scored on the Sth, 7th and 12th day of expression. The bottom line of Table 1 presents the muta- genic activity of MNNG, used as a positive con- trol.

Discussion

Mutagenic chemical compounds are consid- ered to be genotoxic agents which can be de- tected on the basis of their ability to react with DNA. It has been hypothesized that mutagens play an important role in the initiation phase of multi-stage carcinogenesis. However, from the point of view of carcinogenesis, not only the DNA-damaging, but also the cytotoxic effects of chemicals can be very dangerous. Many non- mutagenic chemical compounds can induce tu- mors in animals exposed to high concentrations during long-term bioassays. A cytotoxic effect is very often connected with the induction of hyper- plasia and neoplasia. Cytotoxicity has very often a co-carcinogenic effect (Reitz, 1987; Swenberg and Short, 1987).

The cytotoxic effects of NaF have at least two main reasons: (1) fluoride inhibits several metal- loproteins (Wiseman, 1970) including Escherichia

coli DNA polymerase (Lehman, 1963) and en- dolase and hence glycolysis; (2) inhibition of gly- colysis could reduce the cellular pools of nu- cleotide triphosphates needed for normal DNA synthesis and repair.

We studied all macromolecular synthesis, i.e., DNA, RNA and protein synthesis, in cells CUl- tured in the presence of NaF, by means of ra-

V79 EUE

n 7 20 u

TIME OF TREATMENT (HRS) _

Fig. 2. Intensity of DNA synthesis in V79 cells (left) and EUE cells (right) during 24-h cultivation of cells in the presence of 100 (O)or200~gofNaF/ml(~).

dioisotope techniques. Concentrations of NaF EUE cells (Fig. 1). As is clear from Fig. 2, at 200 (100 and 200 pg/ml) acted cytotoxically on V79 pg/ml the level of DNA synthesis dropped in cells and non-cytotoxically or mildly toxically on V79 cells to 10% of control during the first 3 h

b

EUE

20 u TlMF OF TREATMENT (Hf3S.j c

Fig. 3. Intensity of RNA synthesis in V79 cells (left) and EUE cells (right) during 24-h cultivation of cells in the presence of 100 (0) or 200 pg of NaF/ml ( n 1.

113

v 79 EUE

TlME OF TREATMENT IHRS: I -

Fig 4. Intensity of protein synthesis in V79 cells (left) and EUE cells (right) during 24-h cultivation of the cells in the presence of

100(nlor200~gofNaF/ml(mI.

and then gradually decreased to 5% and less and 70% in EUE cells cultured with 100 and 200 (90-100% decrease of DNA synthesis), which pg of NaF per ml was sufficient for nearly full was fatal for most cells. On the other hand, survival (90% and 80% survival of EUE cells; Fig. decreases in DNA synthesis of approximately 50% 1). A gradual decrease in DNA synthesis in V79

TABLE 1

OCCURRENCE OF 6-TG’ MUTATIONS PER lo5 SENSITIVE CELLS IN V79 CELLS TREATED FOR 24 h WITH NaF OR MNNG

Concen- Cloning Expt. No. 1 Cloning Expt. No. 2 M+SD R tration efficiency a expression time efficiency ’ expression time

bg/mIl 5 days 7 days 5 days 7 days 12 days

- 69.6 0.44 + 0.66 1.77 f 1.08 73.0 2.64k2.16 5.94 f 2.64 2.69 f 0.63 2.69 + 2.02 1 .OO NaF 10 54.8 1.29+ 1.02 2.13 f 1.53 67.5. 3.14f2.24 1.62k 1.31 2.08~0.71 2.05 + 0.69 0.70

SO 58.6 1.33k 1.26 0.80 f 0.32 44.4 1.47f0.89 3.03 f 1.95 5.64 f 2.35 2.45 f 1.96 0.91 100 51.6 2.60 f 1.77 2.90 + 0.65 36.8 2.67 f 0.96 6.02 f 2.88 3.78 + 1.65 3.59 f 1.43 1.33 150 - 1.76 f 0.75 2.64kO.57 21.1 3.12k2.01 2.90 + 1.50 2.97 f 1.46 2.67 f 0.54 0.99 200 32.2 2.69 f 0.78 3.05 + 0.69 20.8 I .85 rt 0.64 1.40* 1.00 2.36k 1.21 2.26 k 0.65 0.84 250 17.9 3.15 *0.74 3.02& 1.07 23.4 1.67k0.62 0.91 f 0.91 1.09 f 0.79 1.96* 1.05 0.72 300 23.6 3.49 f 2.48 4.55 + 2.81 28.1 1.78 f 0.69 0.33 f 0.49 1.28 f 0.82 2.28? 1.70 0.84 350 29.5 0.54 f 0.49 1.33f0.30 21.5 4.24 f 2.76 5.25 f 1.39 3.53 * 1.58 2.97* 1.98 1.10 400 26.1 2.24 f 0.85 6.53f3.93 21.5 0.99 f 0.63 2.11k2.16 2.22+0>99 2.81 f 2.13 1.04

MNNG 1 24.1 37.20 f 3.98 22.1 45.11 rt6.92 41.15k5.59 15.29

a Immediately after treatment. M, average number of 6-TGr mutations per to* cells; R, ratio of mutations in treated samples to SPontaneous mutations in

controls.

~~11s cultured with 100 k_e of NaF (Fig. 2) allowed about 50’;; survival of the treated cells (Fig. I). v&es of protein synthesis (Fig. 4) in cells cul- tured with NaF were similar to values of DNA synthesis (Fig. 2). The least sensitive to sodium fluoride treatment in both cell tines was RNA synthesis (Fig. 3) whose intensity dropped signifi- cantly only in V79 cells after long treatment times (20 and 24 h) when viabihty was probably low.

These results confirm that the cytotoxic effects of sodium fluoride on V79 and EUE cells are in direct correlation with its inhibitory effects on DNA and protein synthesis; however, it seems that human EUE cells are able to recover to a certain extent from relatively long (24 h) inhibi- tion of macromolecular synthesis. Increasing the NaF concentration to > 200 pg/ml led to a loss of viability of human EUE cells as well.

Although sodium fluoride has been active in many tests which are considered special tests of genotoxicity (chromosomal aberrations, sister- chromatid exchanges, unscheduled DNA synthe- sis: see Tsutsui et aI., 1984a.b), it is not expected to covalently modify DNA. Emsley et al. (1982) have proposed that the fluoride ion could disrupt DNA structure by interfering with adenine- thymine base pairing. Crespi et al. (1990) sug- gested NAF to induce gene-locus mutations at the thymidine kinase and hypoxanthine-guanine phosphoribosyl transferas, (HGPRT) loci in hu- man lymphoblastoid cz!ls treated with cytotoxic concentrations. The mechanism of the mutagenic effect of fluoride was explained by ‘indirect muta- genesis’ (Caspary et al., 1987), where defects of normal DNA replication induced by fluoride ion interfere with adenine-thymine base pairing. We tested the mutagenic activity of NaF in Chinese hamster V79 cells, (Table 1). The cells were treated without the enzymatic fraction S9 as Mar- tin et al. (1979) and Caspaty et al. (1987) proved that NaF cannot be activated by this microsomal fraction. Concentrations were tested in the range of lo-400 pg/ml of culture medium. Concentra- tions > 400 pg of NaF/ml of culture medium proved extremely cytotoxic for V79. The results obtained were compared to the mutagenic activ- ity of MNNG. As is clear from Table 1, all tested concentrations of NaF were ineffective after sin- gle treatment of cells. In addition to single treat-

ment we therefore tried to induce HGPRT muta- tions in V79 cells by repeated treatment (data not shown), as many chemical compounds manifest an additive mutagenic effect (Slameiiovs et al., 1983; Slameiiov~ and Ggbelovi, 1986). We used a 168-h interval between individual treatments. It is noteworthy that neither single nor double NaF treatment of cells brought about any positive effects. The well-known super-mutagen MNNG increased the number of induced mutations by 15.29. On the basis of our results we suppose that NaF added to culture medium for 24 h is unable to induce gene mutations in the HGPRT locus of Chinese hamster V79 cells, despite its cytotoxic effect.

Cytotoxic effects of sodium fluoride on mam- malian cells in vitro have been described in ex- planted human foreskin fibroblasts (Tsutsui et al., 1984a), in human lymphoblastoid cells (Crespi et al., 1990), in Syrian hamster embryo cells (Tsutsui et al., 19&k), etc. A study of the cytotoxicity of sodium fluoride on Chinese hamster V79 cells and human fibroblastoid EUE cells is presented in this paper. On the basis of our results we can say that after 20-24-h NaF treatment of mam- malian cells of different origin the average LD,, concentration is approximately 150-200 pg/ml. Human intake of fluoride as a combination of several fluoride sources can be as high as 2.9 mg/day (children) and 6.2 mg/day (adult) and in special cases even more (Li et al., 1988). From the aspect of the average human intake of NaF on the one hand and the average toxicity of NaF on mammalian cells on the other hand, we cannot regard the toxic effects of NaF as unimportant. It was, however, stated (Singer and Armstrong, 1964) that plasma fluoride concentrations in rats given up to 50 ppm fluoride in drinking water remained constant at 0.25 pg/ml. Provided that the fluoride concentration in human cells in vivo is likewise low, the perilous effects of rather high NaF doses on the human organism need not be considered as a serious threat.

Acknowledgement

The authors express their appreciation to Mrs. A. Moravkova for her able technical help.

115

References

Abbondandolo, A., S. Bonatti, C. Colella. G. Corti, F. Mat- teucci. A. Mazacarro and G. Reinaldi (1976) A compara- tive study of different experimental protocols for mutagen- esis assay with 8-azaguanine resistant system in cultured Chinese hamster cells, Mutation Res.. 37, 293-306.

Caspary, W.J., B. Myhr. L. Bowers, D. McGregor, C. Riach and A. Brown (1987) Mutagenic activity of fluorides in mouse lymphoma cells, Mutation Res.. 187, 165-180.

Chasin, L.A. (1973) The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells, J. Cell Physiol., 82, 299-308.

Crespi. Ch.L., GM. Seixas. T. Turner and B.W. Penman (1990) Sodium fluoride is a less efficient human cell muta- gen at low concentrations, Environ. Mol. Mutagen., 15, 71-77.

Emsley, J., D.J. Jones and R.E. Overill (1982) The uracil-fluo- ride interaction: ab initio calculations including solvation, J. Chem. Sot. Chem. Commun., 476-478.

Horowitz, S.H. (1973) A review of systemic and topical fluo- rides for the prevention of dental caries, Community Dent. Oral Epidemiol., 1, 104-114.

Lehman, I.R. (1963) DNA synthesis bacterial, in: S.P. Colow- ick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. VI, Academic Press, New York, pp. 34-39.

Li, Y., A.J. Dunipace and G.K. Stookey (1988) Genotoxic effects of fluoride: a controversial issue, Mutation Res.. 195, 127-136.

Martin, G.R., K.S. Brown, D.W. Matheson, H. Lebowitz, L. Singer and R. Ophaug (1979) Lack of cytogenetic effects in mice or mutations in Salmonella receiving sodium fluo- ride, Mutation Res., 66, 159-167.

McClure, F.J. (1970) Water Fluoridation, U.S. Dept. Health, Education and Welfare, NIH, Bethesda, MD, pp. l-302.

Reitz. R.H. (1987) Role of cytotoxicity in the carinogenic process, Banbury Rep., 25, 107-l 17.

Singer. L., and W.D. Armstrong (1964) Regulation in plasma fluoride in rats, Sot. Exp. Biol. Med., 117. 686-689.

Slameiiova. D., and A. Gibelova (1980) The effects of sodium azide on mammalian cells cultivated in vitro. Mutation Res., 71. 253-261.

Slameiiovi. D., and A. GabelovL (1986) The influence of multiple mutagenic treatment on the occurrence of 6- thioguanine-resistant mutants in dividing V79 cells. Muta- tion Res., 159, 91-97.

Slameiiova, D., M. DuSinski, E. Budayova and A. Gibelovi (1983) The genetic effects of the cytostatic drug TS,,, on Chinese hamster fibroblasts in vitro. Mutation Res.. 116. 431-440.

Swenberg. J.A., and B.G. Short (1987) Influence of cytotoxic- ity on the induction of tumors, Banbury Rep.. 25, 151-161.

Tsutsui, T., N. Suzuki, M. Obmori and H. Maizumi (1984a) Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts in- duced by sodium fluoride. Mutation Res., 139. 193-198.

Tsutsui, T., K. Ide and H. Maizumi (1984b) Induction of unscheduled DNA synthesis in cultured human oral kera- tocytes by sodium fluoride, Mutation Res., 140,43-48.

Tsutsui. T.. N. Suzuki and M. Ohmori (1984c) Sodium fluo- ride induced morphological and neoplastic transformation. chromosome aberrations, sister chromatid exchanges and unscheduled DNA synthesis, in cultured Syrian hamster embryo cells, Cancer Res.. 44. 938-941.

World Health Organization (1970) Fluorides and Human Health, Monogr. 59, WHO, Geneva, pp. l-364.

World Health Organization (1984) Fluorine and Fluorides. Environmental Health Criteria 36. WHO. Geneva, pp. l-136.

Wiseman. A. (1970) Pharmacology of fluorides, in: F.A. Smith (Ed.), Handbook of Experimental Pharmacology, Vol. XX/Z, Springer, Berlin, pp. 48-97.