Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride

Mutation Research, 139 (1984) 193-198

193

Elsevier

MRLett 0527

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride Takeki Tsutsui, Nobuko Suzuki, Manabu Ohmori and Heiji Maizumi Department of Pharmacology, Nippon Dental University Tokyo, 1-9-20, Fujimi, Chiyoda-ku, Tokyo 102 (Japan) (Accepted 22 November 1983)

Summary The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 #g/ml) or exposure time (1-24 h). Treatment of the cells with 50/~g/ml NaF for 24 h resulted in a lethality (-70°7o) similar to that obtained with 100 #g/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-I cells treated with 20-50 #g/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence o f 10 mM hydroxyurea. Treatment with 100-400 #g N a F / m l for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

Fluoride is a ubiquitous substance found naturally in food and water, and extensively utilized for industrial purposes. It is generally assumed that NaF is safe to add to drinking water and to solutions for mouth-rinsing as a preventer of denAbbreviations: B(a)P, benzo[a]pyrene; FCS, fetal calf serum; 1% FCS medium, medium containing 1% fetal calf serum; 1% FCS-HU medium, medium containing 1% fetal calf serum and 10 mM hydroxyurea; HU, hydroxyurea; MMC, mitomycin C; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; 4NQO, 4-nitroquinoline 1-oxide; PBS, phosphate-buffered saline [0.14 M NaC1, 3 mM KC1, 8 mM Na2HPO4, l mM KHzPO4 (pH 7.4)]; [3H]TdR, [3H]thymidine; UDS, unscheduled DNA synthesis. 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.

tal caries (Horowitz, 1973). However, conflicting results on the cytogenetic effects of NaF on mammalian cells in vivo and in vitro have been reported (Obe and Slacik-Erben, 1973; Jagiello and Lin, 1974; Jachimczak and Skotarczak, 1978; Kram et al., 1978; Martin et al., 1979). To examine possible damage to the genetic apparatus at chromosome and DNA levels caused by treatment of normal human diploid fibroblasts with NaF, we analyzed the treated cells for chromosome aberrations and unscheduled DNA synthesis (UDS). The present studies provide the first evidence that NaF caused chromosome aber-

194

rations and unscheduled DNA synthesis cultured human diploid fibroblasts.

in

Materials and methods

Cells, growth medium and chemicals Explanted human foreskin fibroblasts (JHU-1 cells) (Lin et al., 1980) were generously provided by Dr. P . O . P . T s ' o (the Johns Hopkins Univ., Baltimore, MD) and were routinely cultured in Dulbecco's modified Eagle's medium (Grand Island Biological Co., Grand Island, NY), supplemented with 10o70 fetal calf serum (FCS: Gibco) in a humidified atmosphere with 5°70 CO2 in air at 37°C. Upon reaching confluence, cells were transferred by gentle trypsinization with 0.25°70 trypsin (1:250, Gibco) for 5 min at room temperature. The number of times the population of JHU-1 cells used in this study doubled was between 15 and 20. Sodium fluoride (more than 99o70 pure) was purchased from Koso Chemical Co. (Tokyo), dissolved in medium containing 1 or 10O7o FCS at 1 mg/ml and filter sterilized immediately before use. Hydroxyurea (HU) and benzo[a]pyrene (B(a)P) were obtained from Sigma Chemical Co. (St. Louis, MO). 4-Nitroquinoline 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were acquired from Wako Pure Chemical Ind. Ltd. (Tokyo). Mitomycin C (MMC) and [3H]thymidine ([3H]TdR: specific activity, 49 Ci/mmole) were purchased from Sankyo Corp. (Tokyo) and Amersham-Searle Corp. (Arlington Heights, IL), respectively.

Cytotoxicity of NaF Cytotoxicity of NaF on JHU-1 cells was determined by their colony-forming ability. 200-500 cells were plated on 60-mm dishes (Falcon Labware, Oxnard, CA). After overnight incubation, the cells were treated with NaF at the desired concentrations for 1, 2, 6, 12 or 24h. The population doubling time of JHU-1 cells was approximately 24 h; therefore, a 24-h treatment with NaF encompassed at least one cell cycle. The cells were then

washed twice with fresh medium and incubated for 7 days to form colonies. The cells were fixed with absolute methanol and stained with 3°7o aqueous Giemsa solution. The number of colonies was then counted and the °7o cell survival expressed as the number of colonies in the treated plates divided by the number in the untreated plates x 100. The plating efficiency of control cells was 30-40°7o of the plated cells.

Chromosome aberrations JHU-1 cells (1.5 × 106) were seeded in 75-cm 2 flasks. After overnight incubation, NaF was added and the cultures incubated for an additional 12 or 24 h. 3 h before the end of treatment time, colcemid (Gibco) was administered at 0.05 #g/ml and metaphase chromosomes were prepared as described previously (Tsutsui et al., 1979, 1983a). After trypsinization, the ceils were treated with 0.8°7o sodium citrate at room temperature for 20 min, fixed in Carnoy's solution (methanol/acetic acid = 3:1) and spread on glass slides by the air-drying method. The specimens were stained with 3°70 Giemsa in 0.07 M phosphate buffer (pH 6.8) for 10 min.

Detection of unscheduled DNA synthesis (UDS) Cells (1 × 105) in the logarithmic growth phase were plated in triplicate on 15-mm-diameter Thermanox coverslips (2 cm z) (Lux, Miles Lab. Inc. Napirville, IL) in 16-mm tissue culture dishes (Coster, Cambridge, MA) with medium containing 10°70 FCS. The cells reached confluence after 24 h incubation, since the saturation density was - 3 . 8 × 104 cells/cm 2. The medium of the confluent cultures was replaced with medium containing 1 °70 FCS (1 °70 FCS medium) and the cultures incubated for an appropriate time (1-72 h) to reduce DNA synthesis for replication. The cells were then treated with the indicated dose of chemicals for 1-24 h in 1 o70 FCS medium containing 10 mM H U (1°70 F C S - H U medium). To measure UDS, the cells were washed after chemical treatment with 1 ml of 1o70 F C S - H U medium and incubated in 1°70 F C S - H U medium containing 10/zCi/ml [3H]TdR for 6 h at 37°C. The uptake o f [3H]TdR was stop-

195

ped by rinsing the coverslips with cold 0.15 M NaC1 and immersing 3 times in 5°7o cold trichloroacetic acid for 15 min. The coverslips were put into scintillation vials to determine the radioactivity in these samples. The background levels of [3H]TdR incorporation per coverslip in cultures treated with solvent alone were <200 cpm, and these were subtracted from each determination. All experiments were repeated at least twice with similar results. The number of cells on a coverslip was counted using the culture cluster dishes treated with the same conditions used in the experiments for detection of UDS with [3H]TdR. No significant difference in the number of cells was found between the control and experimental groups.

ted against the product of NaF dose (50-150 #g/ml) and exposure time (1-24 h), the cell survival also decreased linearly with the product of dose and exposure time (Fig. 2). Treatment of the cells with 50/~g/ml NaF for 24 h resulted in a lethality (about 70o7o) similar to that obtained with 100 /xg/ml NaF for 12 h.

Chromosome aberrations To study the effect of NaF on chromosome aberrations, cells treated with between 20 and 50 #g/ml NaF for 12 or 24 h were analyzed. In control cultures, few metaphases contained chromosome aberrations. A significant increase in chromosome aberrations at the chromatid level was, however, observed in treated cells in dose- and treatment time-dependent fashions (Table 1).

Results

Cytotoxicity o f NaF on JHU-1 cells Treatment with NaF was cytotoxic to JHU-1 cells grown as described in Fig. 1. Cell survival of NaF-treated cultures decreased linearly with increasing dose of NaF or exposure time. When plot100-

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Development o f an unscheduled DNA synthesis assay with human diploid fibroblasts In order to reduce replicative DNA synthesis, confluent cells were cultivated with 1°70 FCS medium for an appropriate time and subsequently treated with HU. To determine the optimal incubation time in 1°70 FCS medium, the incorporation of [3H]TdR into DNA of cells cultivated for varying times with 1% FCS medium was measured. Following cultivation for 1, 24, 48 and 72 h in 1 °7o FCS medium, the cells were labeled for 2 h with 10 /zCi/ml [3H]TdR in 1°70 F C S - H U medium. As

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Fig. 1. Cytotoxicity of sodium fluoride. To assay cytotoxicity, 2-5 X l 0 2 cells were plated overnight in 60-mm dishes and treated with NaF at 50-150 #g/ml for 1-24 h. After washing twice with fresh medium, the cells were incubated for 7 days for colony formation. The O7ocell survival was calculated relative to the survival of untreated cells. ©, 1 h treatment; o, 2 h treatment; ta, 6 h treatment; B, 12 h treatment; ,x, 24 h treatment. Bars indicate S.D.

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Fig. 2. Survival of cells exposed to sodium fluoride. Cell survival shown in Fig. 1 was plotted against the product of NaF dose and exposure time. Bars denote S.D.

196 TABLE 1 C H R O M O S O M E A B E R R A T I O N S OF H U M A N D I P L O I D FIBROBLASTS I N D U C E D BY SODIUM F L U O R I D E Dose of NaF (/zg/ml)

Treatment period (h)

N u m b e r of metaphases scored

Type of aberrations a (%) G

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Aberrant metaphases (%)

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100 100 100

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0 0 0

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0 0 0

0 0 0

aG, gap; B, break; E, exchange; D, dicentric; O, ring; F, fragmentations.

shown in Fig. 3, DNA synthesis decreased with increasing time in 1% FCS medium from 1 to 48 h. The level of inhibition of DNA synthesis of cells cultivated for 48 h was similar to that of cells cultivated for 72 h. On the other hand, there was no significant difference in the number of cells among 1, 24, 48 and 72 h cultures. From these results, the ceils in subsequent experiments were cultivated for 48 h with 1°70 FCS medium to reduce replicative DNA synthesis. Even though the cultures were inhibited for growth and uptake of [3H]TdR by high density and use of low serum medium, there was still considerable incorporation

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of radioactivity into the cells (see the control group in Table 2). To examine further inhibition of DNA synthesis, confluent cells cultivated with 1°70 FCS medium for 48 h were exposed to different doses of H U in medium containing 1°7o FCS and 10 #Ci/ml [3H]TdR. As shown in Table 2, DNA synthesis of cells exposed to H U for 2 h was inhibited in a dosedependent manner. By treatment with 10 mM HU, DNA synthesis was reduced to less than 5°70 of control. Thus, we decided to apply 10 mM HU to cells following cultivation with 1°/0 FCS medium for 2 days. Cells were exposed to B(a)P, 4NQO, MNNG and MMC, which are well known chemical DNAdamaging agents, to examine whether the cells have the ability to repair DNA damage under our conditions. In the experiments, the cells were treated with the chemicals at the designed concen-

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Fig. 3. Effect of incubation period in m e d i u m containing 1% FCS on the number of JHU-1 cells and incorporation of [3H]thymidine into D N A . The number of cells and incorporation of [~H]thymidine into D N A were analyzed as described in Materials and methods, and Text. Bars denote S.D. W h e n not indicated, S.D. are within the symbols.

TABLE 2 E F F E C T OF H Y D R O X Y U R E A C O N C E N T R A T I O N ON D N A SYNTHESIS IN H U M A N D I P L O I D FIBROBLASTS FOLLOWING CULTIVATION WITH MEDIUM CONTAINING 1070 FCS FOR 2 DAYS Concentration of hydroxyurea (mM)

D N A synthesis for 2 h in 1070 FCS medium ([3H]TdR cpm/culture well)

0 1 3 10 30

3014.5 299.3 173.9 147.3 150.7

aMean _+ S.D. of 4 cultures.

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Fig. 4. Unscheduled D N A synthesis induced by several chemicals. Confluent cells were cultivated for 48 h with medium containing 1°7o FCS and treated with chemicals at the indicated doses for 1 h. UDS was detected as described in Materials and methods. O, 4NQO; m, B(a)P; zx, MMC; v , M N N G . Bars denote S.D. When not indicated, S.D. are within the symbols.

trations for 1 h, washed once with 1 ml of 1°70 F C S - H U medium and labelled for 6 h with 10 /xCi/ml [3H]TdR in 1°70 F C S - H U medium. As shown in Fig. 4, induction of UDS in JHU-1 cells was observed in a dose-dependent fashion by all the chemicals except B(a)P which requires oxidative metabolic conversion not found in human diploid fibroblasts (Gelboin, 1969).

Unscheduled DNA synthesis induced by NaF JHU-1 cells were exposed to various concentrations of NaF for varying treatment times in 1°70 F C S - H U medium. Thereafter, the cells removed from NaF were incubated for 6 h with 1070 F C S - H U medium containing 10/zCi/ml [3H]TdR. As shown in Fig. 5, UDS was not induced by treatment with NaF over the dose range 50-5000/zg/ml for 1 h. No significant UDS was detected until the cells were treated for 4 h. When the treatment time was extended beyond 8-16 h, increased UDS was elicited in a dose-related fashion. The inducibility, however, markedly decreased in the cells treated for 24 h, probably due to cytotoxicity.

sodium fluoride. Confluent cells were cultivated for 48 h with medium containing 1070FCS and treated with the indicated doses of NaF for varyingtimes. UDS was detected as described in Materials and methods. O, l h treatment; O, 4 h treatment; t~, 8 h treatment; i, 12 h treatment; zx, 16 h treatment; A, 24 h treatment.

Discussion The results in this report demonstrate that NaF induces chromosome aberrations and unscheduled D N A synthesis in human diploid fibroblasts in vitro. The mechanism by which NaF induces chromosome aberrations and UDS are as yet unknown. It is plausible that NaF causes D N A damage. In fact, Emsley et al. (1982) have investigated the interaction between nucleic acid and fluoride by means of ab initio calculations and proposed that F - could completely disrupt the thymine-adenine link in D N A duplex. In addition, our recent studies showed that D N A single-strand breaks detected by the alkaline elution method were elicited in JHU-1 cells treated with NaF (100-200 #g/ml) for 16 h (unpublished data). NaF treatment resulted in a linear increase in cytotoxicity as a fraction of the product of treatment time and dose (Fig. 2). The minimum significant level of chromosome aberrations or UDS was induced by treatment with 20 #g/ml NaF for 24 h or 100/~g/ml for 8 h, respectively (Table 1 and Fig. 5). According to Fig. 2, the cell survival rates resulting from both treatments were similar (ap-

198

proximately 40-50% o f untreated cultures). On the basis of the cytotoxicity, this indicates little difference in the sensitivity in identification of the genotoxicity of NaF between the assays used for detection of chromosome aberrations and UDS. No or poor inducibility of UDS by NaF (Fig. 5) was observed when JHU-1 cells were treated for 1 or 4 h. Significantly high levels o f UDS were induced in the cells treated for over 8 h. Two possible explanations may be considered with regard to the delay in the response of DNA repair in NaFtreated cells. The first possibility is that DNAdamaging activity of fluoride may be weak or insufficient to induce detectable DNA damage during a short treatment time under our conditions. Secondly, inhibition of protein synthesis by fluoride may retard the progression of DNA repair following DNA damage. It has been known for some time that fluoride inhibits a number of metalloproteins (Wiseman, 1941), including DNA polymerase of E. coli (Lehman, 1963). Further experiments are necessary to elucidate these possibilities. In conclusion, the present studies provide the first evidence that NaF induces chromosome aberrations and UDS in human diploid fibroblasts in vitro. Our recent experiments show that exposure of Syrian hamster embryo cells to concentrations of NaF between 75 and 125 #g/ml for 24 h resulted in approximately 90-40% cell survival and a dosedependent increase in the frequency of morphological transformation of the cells. Mass cultures of cells treated with 75 or 100 #g/ml NaF for 24 h, followed by continuous cultivation were transformed to the tumorigenic state. Furthermore, a significant increase in chromosome aberrations at the chromatid level, sister-chromatid exchanges and UDS was induced by treatment of the same cells with 40-100 #g/ml NaF for 24 h (Tsutsui et al., 1983b). These results suggest that NaF is potentially dangerous to humans.

Acknowledgement We thank Dr. J. Carl Barrett for his critical reading of the manuscript and Dr. M. Miyaki for

her helpful discussions. This work was supported in part by a grant for Cancer Research from the Ministry of Education, Science and Culture of Japan.

References Emsley, J., D.J. Jones and R.E. Overill (1982) The uracilfluoride interaction: Ab initio calculations including solvation, J. Chem. Soc. Chem. Commun., 476-478. Gelboin, H.V. (1969) A microsome-dependent binding of benzo[a]pyrene to DNA, Cancer Res., 29. 1272-1276. Horowitz, S.H. (1973) A review of systemic and topical fluorides for the prevention of dental caries, Community Dent. Oral Epidemiol., 1, 104-114. Jachimczak, D., and B. Skotarczak (1978) The effect of fluoride and lead ions on the chromosomes of human leucocytes in vitro, Genet. Polon. 19, 353-357. Jagiello, G., and J. Lin (1974) Sodium fluoride as potential mutagen in mammalian eggs, Arch. Environ. Health, 29, 230-235. Kram, D., E.L. Schneider, L. Singer and G.R. Martin (1978) The effects of high and low fluoride diets on the frequencies of sister chromatid exchanges, Mutation Res., 57, 51-55. Lehman, I.R. (1963) DNA synthesis (bacteria), in: S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. VI, Academic Press, New York, pp. 34--39. Lin, S.L., P.O.P. Ts'o and M.D. Hollenberg (1980) The effects of interferon on epidermal growth factor action, Biochem. Biophys. Res. Commun., 96, 168-174. 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 fluoride, Mutation Res., 66, 159-167. Obe, G., and R. Slacik-Erben (1973) Suppressive activity by fluoride on the induction of chromosome aberrations in human cells with alkylating agents in vitro, Mutation Res., 19, 369-371. Tsutsui, T., J.C. Barrett and P.O.P Ts'o (1979) Morphological transformation, DNA damage, and chromosomal aberrations induced by a direct DNA perturbation of synchronized Syrian hamster embryo cells, Cancer Res., 39, 2356-2365. Tsutsui, T., H. Maizumi, J.A. McLachlan and J.C. Barrett (1983a) Aneuploidy induction and cell transformation by diethylstilbestrol: A possible chromosomal mechanism in carcinogenesis, Cancer Res., 43, 3814-3821. Tsutsui, T., N. Suzuki and M. Ohmori (1983b) Sodium fluoride-induced morphological and neoplastic transformation, chromosome aberrations, sister-chromatid exchanges and unscheduled DNA synthesis in cultured Syrian hamster embryo cells, Cancer Res., in press. Wiseman, A. (1970) Pharmacology of Fluorides, in: F.A. Smith (Ed.), Handbook of Experimental Pharmacology, Vol. XX/2, Springer, Berlin, pp. 48-97.