In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity

Mutation Research 652 (2008) 139–144

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In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity S. Jantova´ a,∗ , M. Theiszova´ a , S. Letaˇsiova´ a , L. Biroˇsova´ a , T.M. Palou b a Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinsk´eho 9, SK-81237 Bratislava, Slovakia b Institute of Inorganic Chemistry, Technology and Materials, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinsk´eho 9, SK-81237 Bratislava, Slovakia

a r t i c l e

i n f o

Article history: Received 7 September 2007 Received in revised form 19 December 2007 Accepted 26 January 2008 Available online 18 March 2008 Keywords: Fluor-hydroxyapatite Fluorapatite Cell colony formation Cell number/colony Genotoxicity Mutagenicity

a b s t r a c t The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH− groups and F− ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA < FHA < FA. None of the biomaterials induced mutagenic effects compared with the positive control (N-methyl-N -nitro-N-nitrosoguanidine), and DNA breakage was probably the reason for the inhibition of cell division in V79 cell colonies. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Bone is considered essentially as a nanocomposite of minerals and proteins. The minerals are mostly apatites such as hydroxyapatite (HA: Ca10 (PO4 )6 (OH)2 ), fluorapatite (FA: Ca10 (PO4 )6 F2 ) and carbonate-apatite [1]. Bone disease is a serious health condition that directly impacts on the quality of life of patients, particularly among the aged. In most cases, the treatment of bone defects requires a bone graft. Bone harvesting can cause postoperative complications and sometimes does not provide a sufficient quantity of bone. Therefore, synthetic biomaterials have been investigated as an alternative to autogenous bone grafts [2]. Hydroxyapatite has been studied extensively and prepared for clinical applications. HA has also attracted much attention for use as a substitute for teeth, due to its excellent biocompatibility with human tissues. Nevertheless, hydroxyapatite has intrinsically poor mechanical properties (strength, toughness and hardness), which

∗ Corresponding author. Tel.: +421 2 59325455; fax: +421 2 52493198. ´ E-mail address: [email protected] (S. Jantova). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.01.010

have restricted wider applications in load-bearing implants. Therefore, composite HA materials have been developed by different synthetic routes and techniques in order to improve both bioactivity and mechanical properties of various orthopaedic prosthesic and dental implants. Bioactive ceramics, bioactive glass or glassceramics, bio-inactive ceramics, polymers and metals have all been used to fabricate HA composites [3]. Many of them have demonstrated an excellent biocompatibility and ideal bioactivity in in vitro as well as in vivo tests [4–7]. Fluorapatite is considered as a biomedical material due to its structure similar to that of hydroxyapatite. Some studies in the last 10 years were extended to the synthesis of solid solution fluorhydroxyapatite (FHA). The fluoride ion (F− ) is partially or totally replaced by hydroxide in the OH− lattice position in HA, thus forming a large range of solid solutions (composites) of FHA, with a formula Ca10 (PO4 )6 (OH)2−x Fx . The importance of such an approach is related to the presence of partially fluorided HA found in bone and mainly in tooth enamel. It was found that fluoride is uniformly distributed within the bone tissue or within the thin tooth enamel outer layer. It is clear that fluor-hydroxyapatite composites are homogeneous solid solutions and not a simple mixture of HA

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and FA. Many recent studies on the synthesis and characterization of solid solutions between hydroxyapatite and fluorapatite have shown that FA has a higher thermal stability than HA. Therefore, it is expected that the introduction of FA will retard the decomposition of HA. In addition, F− itself retains advantages over other ions in that it protects teeth from dental caries, especially in the environment of the oral cavity; it also enhances mineralization and crystallization. Some studies have revealed that FA has good biocompatibility [8–10] and this gives a good opportunity to extend the research to fluor-hydroxyapatite. The composite can be used in different fields of surgery medicine as surface coating on various orthopaedic prostheses and dental implants. In our previous work we have prepared FHA, FA and HA by a precipitation method [11]. We subsequently examined the cytotoxic activity of FHA, FA and HA discs/eluates by direct/indirect contact [12,13]. Cytotoxic effects of all tested biomaterials on murine NIH-3T3 cells were very weak. On the other hand, 5-day eluates of FHA and FA inhibited the growth of leukemia L1210 cells and induced programmed cell death through a mitochondrial/caspase9/caspase-3-dependent pathway [11]. In the present study, the cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates were investigated on Chinese hamster V79 cells and compared with the effects of an HA eluate. Cytotoxicity of the biomaterial eluates tested was evaluated with the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity of the biomaterials was evaluated with the single-cell gel electrophoresis (comet) assay. Mutagenicity was investigated by use of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene-mutation assay on mammalian cells and by bacterial mutagenicity tests using Salmonella typhimurium TA100. 2. Materials and methods 2.1. Chemicals 2.1.1. Preparation of biomaterials Hydroxyapatite was prepared by a homogeneous precipitation method using Ca (NO3 )2 ·4H2 O and (NH4 )2 HPO4 as starting materials and ammonia solution as a reagent for pH adjustment. The following equation illustrates the chemical reaction sequence leading to the precipitation of HA: 10Ca(NO3 )2 ·4H2 O + 6(NH4 )2 HPO4 + 8NH4 OH → Ca10 (PO4 )6 (OH)2 + 20NH4 NO3 + 43H2 O A suspension of Ca(NO3 )2 ·4H2 O powder was diluted in deionized water and stirred at 25 ◦ C. A solution of (NH4 )2 HPO4 was slowly added drop-wise to the Ca(NO3 )2 ·4H2 O solution. In all experiments, the pH of the Ca(NO3 )2 ·4H2 O solution was kept at 10 with ammonia solution. The final solution was stirred at room temperature for 3 h. Then, the precipitate formed was filtered off, washed with deionized distilled water several times to neutral pH, and finally dried under an infrared lamp for 24 h. After drying, the sample was powdered and treated at 900 ◦ C for 1 h. The obtained product was checked by powder X-ray diffraction (XRD). HA has been identified as Hap JCPDS 9-438. Fluorapatite was obtained using Ca(NO3 )2 ·4H2 O and (NH4 )2 HPO4 according to the following equation: 10Ca(NO3 )2 ·4H2 O + 6(NH4 )2 HPO4 + 8NH4 F → Ca10 (PO4 )6 F2 + 20NH4 NO3 + 6HF + 40H2 O The solid solutions of FHA, Ca10 (PO4 )6 (OH)F, were prepared by a precipitation method according to the following equation: 10Ca(NO3 )2 ·4H2 O + 6(NH4 )2 HPO4 + 4NH4 OH + 4NH4 F → Ca10 (PO4 )6 (OH)F + 20NH4 NO3 + 3HF + 43H2 O 2.1.2. Preparation of biomaterial eluates FHA, HA and FA powders (porous size < 125 ␮m) were used for preparation of 5-day concentrated eluates (non-diluted). Culture medium supplemented with penicillin and streptomycin was used. Biomaterial powders were sterilized for 30 min at 130 ◦ C, the cultivation medium was added (1 mL to 100 mg powder) and samples were shaken on reciprocal shaker for 5 days at 37 ◦ C. After the 5-day elution the concentrated samples were centrifuged (10 min, 1100 × g), the culture medium

was aspirated with a syringe and filtered (∅ 0.22 ␮m). This procedure led to preparation of 100% (concentrated, non-diluted) of biomaterial eluates (FHA, FA, HA) with a pH in the range 6.8–7.1. Eluates were stored at −20 ◦ C. Before the experiments these concentrated eluates were diluted by culture medium to the test concentrations (75%, 50%, 25%, 10%, 5% and 1%). 2.1.3. Other materials Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin and antibiotics were purchased from Biocom Company (Bratislava, Slovakia). Low melting-point agarose (CAS registry number 9012-36-6) was obtained from Invitrogen (Paisley, Scotland, UK) and agarose, d-biotin (58-85-5), l-histidine (71-60-1), Triton-X-100 (9002-93-1), ethidium bromide (EtBr) and 6-thioguanine (6-TG) (15442-7) from Sigma (St. Louis, MO, USA). N-Methyl-N -nitro-N-nitrosoguanidine (MNNG) (70-25-7) was obtained from Serva (NY, USA). Glucose (50-99-7) was obtained from Mikrochem (Bratislava, Slovakia). Ethylene-diamine-tetraacetic acid disodium salt (Na2 EDTA) was obtained from Lachema (Brno, Czech Republic). V79 Chinese hamster lung fibroblasts were obtained from A. Abbodandolo, National Institute for Cancer Research, Genova, Italy. The histidine-dependent strain S. typhimurium TA100 was received from the Collection of Microorganisms, Masaryk University, Brno (Czech Republic). It was stored at −80 ◦ C and regularly checked for its genetic markers. 2.2. Cell culture The cells (starting inoculum 3.5 × 104 cells/mL) were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 ␮g/mL) at 37 ◦ C in an atmosphere containing 5% CO2 . Before a confluent monolayer was formed, the cells were harvested from the culture surface by incubation with a 0.25% solution of trypsin. When a suitable cell concentration was reached, the suspension was used for the experiments. The cells were just in the exponential phase of growth and doubled within 12–16 h. 2.3. Cytotoxicity evaluation on V79 cells V79 cells were set up at densities of 200 cells per 60-mm Petri dish in a total volume of 5 mL. The growth proceeded for 7 days without changing medium at 37 ◦ C in an atmosphere of 5% CO2 in air. Test concentrations (100%, 75%, 50%, 25%, 10%, 5%, 1%) of eluates of the biomaterials FHA, FA and HA were added after 2 h of cell attachment. After a 7-day incubation period, the medium was discarded and the colonies were stained with 1% methylene blue for 10 min, then the number of colonies was counted. The plating efficiency was in the range from 85% to 76%. For the determination of cell number, the cells were harvested in triplicate from Petri dishes by trypsinization and then suspended in 0.9% NaCl. The colony-forming ability (CFA, %) was calculated from the ratio of the number of colonies/cells plated for each tested biomaterial and its concentrations. 2.4. Comet assay Detection of DNA damage by alkaline comet assay was used for measurement of the genotoxic activity of the biomaterials tested. The procedure of Singh et al. [14] ˇ a´ et al. [15] and Gabelov ´ was used with minor changes, as suggested by Slamenov a´ et al. [16]. A base layer of 1% NMP agarose (100 ␮L) in PBS buffer (Ca2+ - and Mg2+ -free) was placed on microscope slides. V79 cells were treated with the biomaterial eluates in concentrations of 100%, 75%, 50%, 25%, 10%, 5% and 1% for 24 h. Treated cells and untreated control cells were suspended in 0.75% low melting-point agarose. Of this suspension, 85 ␮L containing 2 × 104 cells was spread on the base layer. Triplicate slides were prepared per sample. The agarose was allowed to solidify and the slides were placed in lysis mixture (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10.0 and freshly added 1% Triton X-100) at 4 ◦ C. After lysis the slides were transferred to an electrophoresis box containing an alkaline solution (300 mM NaOH, 1 mM Na2 EDTA, pH > 13) and kept in this solution for 40 min at 4 ◦ C for the DNA strands to unwind. A voltage of 19 V (current of 300 mA) was applied for 30 min. The slides were removed, neutralized by 2× 10 min washing in Tris–HCl (0.4 M, pH 7.5), and stained with 20 ␮L EtBr, 10 ␮g/mL. EtBr-stained nucleoids were evaluated with a Carl Zeiss Axion Imager (magnification 200×). Three independent experiments were done and three separate Petri dishes were used for each concentration of biomaterials. For each sample 100 comets were scored by computerized image analysis (Komet 5.5 Europe, Kineting Imaging, Liverpool, UK) for determination of DNA in the tail, linearly related to the frequency of DNA strand breaks [17]. On the basis of observed DNA tails from 100 images, the comets according to the degree of damage [18] were divided into five classes (0–4) and the percentage of DNA damage representing DNA strand breaks was calculated. In statistical analysis, each value was compared with the corresponding control value and the percentage of DNA damage was calculated from the following formula: % DNA damage =

arbitrary unit × 100% 400

where arbitrary unit = a × 0 + b × 1 + c × 2 + d × 3 + e × 4 (a = number of cells in class of DNA damage 0, b = number of cells in class of DNA damage 1, c = number

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of cells in class of DNA damage 2, d = number of cells in class of DNA damage 3, e = number of cells in class of DNA damage 4) and 400 is maximum of DNA damage related to 100 analyzed cells. In statistical analysis, we used Student’s t-test to detect the statistical significance of differences between the control values (cells without any biomaterial treatment) and values obtained from individual concentration values (cells treated with biomaterials). 2.5. Hprt test ˇ a´ and The mutation assay proposed by Chasin [19] and modified by Slamenov ´ Gabelov a´ [20] was used for detection of 6-thioguanine resistant (6-TGr ) mutants. Briefly, after the treatment the cells were trypsinized and diluted as follows: (a) 3 × 102 V79 cells were plated on Petri dishes (diameter 60 mm, in triplicate per sample) to determine the cytotoxic effect of the biomaterials as described above, (b) 3.5 × 105 V79 cells per dish were plated on three Petri dishes (diameter 100 mm) for further cultivation. V79 were kept by regular subculture at a certain cell density per surface unit in order to avoid overcrowding. On the seventh and ninth day of expression, the yield of 6-TGr mutations was measured. At that time V79 cells from each sample were plated (a) on five Petri dishes (diameter 100 mm) at a density of 2 × 105 cells per dish for detection of 6-TGr mutants; 1-h later, after cell attachment, the selective agent 6-TG was added to these dishes at a final concentration 5 ␮g/mL, (b) on three Petri dishes (diameter 60 mm) at density 3 × 102 cells per dish for estimation of cell viability (necessary for calculation of the frequency of TGr mutants at appropriate expression times), (c) at the seventh day only 3.5 × 105 V79 cells per plate from each sample were plated on three Petri dishes (diameter 100 mm) for the analysis of 6-TGr mutants at the next expression time. Colonies of mutants were stained by methylene blue (1% solution) and counted 10 days after addition of 6-TG. The frequency of 6-TGr mutants per 1 × 105 viable cells was calculated at the sixth and ninth day of sampling. Three separate experiments were done and for each concentration of biomaterials five separate Petri dishes were used. The results were statistically evaluated by the Student’s t-test. 2.6. Bacterial mutagenicity test Bacterial strain S. typhimurium TA100 was cultivated on nutrient agar. Sixteen hours before the experiment an overnight culture was started on nutrient broth. The TA100 strain was used because of its sensitivity to a broad spectrum of chemical compounds. The plate-incorporation method according to Maron and Ames [21] was used. To 2 mL of molten top agar containing 50 ␮M of l-histidine-biotin, 0.1 mL of a cell suspension (overnight cultivation at 37 ◦ C, approximate cell density 2–5 × 108 cells/mL) and 0.1 mL of a solution of the test compound were added. The mixture was poured onto a minimal glucose agar plate and incubated for 48 h at 37 ◦ C and the number of histidine-independent revertants was counted. Data points represent at least three separate experiments, each run in triplicate. A positive response was defined as a reproducible, twofold increase of revertants with dose–response relationship and statistical evaluation using Student’s t-test.

Fig. 1. Cytotoxic effect of biomaterials FHA, FA and HA on colony formation (A) and division (B) of V79 cells. Each data point represents means ± S.D. of three individual experiments (n = 3): *<0.05, **<0.01, ***<0.001 when compared with control (dishes containing only V79 cells).

3. Results 3.1. Cytotoxicity evaluation on V79 cells Results from the study of 24 h, 48 h and 72 h treatment with FHA, FA and HA on colony formation of V79 cells are shown in Fig. 1. We determined the number of cell colonies per dish (Fig. 1a) and simultaneously the number of cells per colony (Fig. 1b). A slight inhibition of cell-colony formation was observed in V79 cells treated with FHA, FA and HA. The cytotoxicity increased with increasing concentration of test biomaterials and it was in the range from 1.4% to 10.7%. The biomaterials tested also reduced the cell number per colony. This reduction was in the range from 2.1% to 42.9%. 3.2. Comet assay Fig. 2 shows the effects of FHA, FA and HA on induction of DNA damage in V79 cells measured by the comet assay. DNA damage represented by DNA breaks arose in control and biomaterial-treated cells that formed the tail of a comet when visualized by fluorescence microscopy. Induction of DNA damage was dose-dependent with increasing concentration of biomaterial tested. HA generated a statistically significant amount of DNA damage at concentrations from 10% to 100% in the range of 13.1–14.2%. FHA induced statistically significant DNA damage in the range of 11.0–13.1% at concentrations

Fig. 2. FHA, FA and HA effects on the level of DNA damage in V79 cells after 24 h treatment. DNA damage was represented by DNA strand breaks which were detected as described in Section 2. The percentage of DNA damage at each biomaterial concentration was calculated, based on the formula given in Section 2, for 100 cells counted on each slide. Each value represents the arithmetic means ± S.D. of three separate experiments (n = 3): *<0.05, **<0.01, ***<0.001 when compared with control (dishes containing only V79 cells).

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Table 1 The level of 6-TGr mutations per 1 × 105 viable V79 cells treated for 24 h with biomaterials FHA, FA and HA Level of mutations × 100a

Platting efficiency (%)a

Biomaterial

Concentration (%)

HA

0 1 5 10 25 50 75 100

72.78 65.22 64.84 66.67 62.78 63.50 69.32 66.00

± ± ± ± ± ± ± ±

3.15 1.17 2.60 0.00 1.35 2.59 2.81 2.41

100 89.61 89.09 91.61 86.26 87.25 95.24 90.69

0.92 1.86 1.45 0.66 1.02 1.02 1.11 1.19

± ± ± ± ± ± ± ±

0.00 0.46 0.46b 0.01 0.12b 0.14b 0.14b 0.00

1.00 2.03 1.59 0.72 1.07 1.12 1.21 1.30

1.09 1.53 1.08 1.12 1.55 1.85 1.11 1.92

± ± ± ± ± ± ± ±

0.00 0.00 0.00 0.10 0.43 0.00 0.14 0.19

1.00 1.21 0.85 1.03 1.42 1.46 1.02 1.76

FA

0 1 5 10 25 50 75 100

96.44 76.44 73.78 71.22 65.22 69.11 65.22 61.33

± ± ± ± ± ± ± ±

1.84 3.56 4.02 5.27 4.03 0.51 0.19 3.53

100 79.26 76.49 73.85 67.63 71.67 67.63 63.59

0.67 1.00 1.02 0.95 0.91 1.13 1.24 1.43

± ± ± ± ± ± ± ±

0.00 0.20 0.05 0.07 0.06 0.16 0.15 0.05

1.00 1.50 1.53 1.42 1.36 1.69 1.85 2.14

0.88 0.91 0.99 0.85 0.99 1.33 1.35 2.19

± ± ± ± ± ± ± ±

0.08 0.08 0.06 0.06 0.16 0.38 0.17 0.38

1.00 1.03 1.13 0.97 1.13 1.52 1.54 2.50

FHA

0 1 5 10 25 50 75 100

83.00 83.50 80.17 80.17 78.17 74.00 77.83 72.17

± ± ± ± ± ± ± ±

3.12 4.44 4.04 6.90 3.40 3.12 2.02 2.08

100 100 96.59 96.59 94.18 89.16 93.78 86.95

0.23 0.11 0.12 0.12 0.25 0.64 1.50 1.22

± ± ± ± ± ± ± ±

0.01 0.00 0.00 0.00 0.00 0.01 0.09 0.08

1.00 0.50 0.50 0.50 1.00 2.50 5.50 4.50

0.36 0.12 0.13 0.13 0.27 0.39 0.97 1.11

± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.06

1.00 0.33 0.33 0.33 0.33 0.67 1.00 2.33

50.83 ± 1.26

57.23

48.47 ± 3.75*

69.88

0 min

MNNG

1b

R

7 days

MF

57.66 ± 1.43*

146.5

9 days

MF

R, ratio of platting efficiency in treated to untreated cells and MF, ratio of induced to spontaneous mutations. a Values represent means ± S.D. of three separate experiments (n = 3). b Value is in ␮g/mL. * p < 0.001 when compared with control (dishes containing only V79 cells without MNNG).

from 5% to 100% and FA damaged DNA at all concentrations tested, in the range 12.2–15.5%. The weak genotoxic effects observed for the biomaterials increased in the order HA < FHA < FA. 3.3. Hprt test Mutations at the Hprt locus were measured by selection of 6TG-resistant colonies. An endpoint of biological activity of tested biomaterials FHA, FA and HA was determined at the seventh and ninth day of expression. N-methyl-N -nitro-N-nitrosoguanidine (the stock solution was 1 mg/mL) was used as a positive control. Under these conditions of treatment, none of the tested biomaterials significantly increased the frequency of 6-TGr mutants over the untreated control (Table 1).

recommended and appropriate steps for the biological assessment of potential medical biomaterials consists of an in vitro evaluation of cytotoxicity and genotoxicity. In our previous experiments, we comparatively evaluated the cytotoxicity of FHA, FA and HA discs and 5-day eluates on murine fibroblast NIH-3T3 cells [12,13] and murine leukemia L1210 cells [11]. A direct contact study on NIH-3T3 cells [12] demonstrated that measuring cell number, LDH level, cell protein and DNA content

3.4. Bacterial mutagenicity test The results of monitoring the potential of the biomaterials FHA, FA and HA to induce mutations in the bacterial strain S. typhimurium TA100 are presented in Fig. 3. MNNG (the stock solution was 1 mg/mL) was used as a positive control. The highest test concentration (100%) of all biomaterials did not induce growth of revertants. No mutagenic activity was observed. 4. Discussion Biomaterials may have low, medium or high potential risk to human safety, depending on the type and extent of patient contact. Safety assessments of medical biomaterials are guided by the toxicological guidelines recommended by the International Organization of Standardization (ISO 10993-1/EN 30993-1). One of the

Fig. 3. Mutagenic effect of biomaterials FHA, FA and HA on Salmonella typhimurium TA 100. Mutagenic activity is expressed as number of revertant colonies. MNNG (1 ␮g/mL) was used as a positive control (PC). Each data point represents means ± S.D. of three separate experiments (n = 3). ***p < 0.001 when compared with control (plate containing only bacterial strain).

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showed a very slight cytotoxicity of biomaterial discs. The cytotoxicity was in the range of 3.1–25.9%. The eluate assay on NIH-3T3 cells [13] showed that none of the tested 5-day eluates of biomaterials caused the total inhibition of cell division. Biomaterials induced different antiproliferative effects increasing in the order HA < FHA < FA, which were time- and concentration-dependent. None of the tested biomaterials induced necrotic/apoptotic death of NIH-3T3 cells. On the other hand, after 72 h we found that while FHA and FA induced G0 /G1 arrest of NIH-3T3 cells, HA did not affect any of cell cycle phases. The comet assay showed that while HA demonstrated weaker genotoxicity, DNA breaks induced by FHA and FA caused G0 /G1 arrest of NIH-3T3 cells. The eluate assay on leukemia L1210 cells [11] showed that biomaterial eluates induced a concentration- and time-dependent inhibition of cell growth. Cytotoxicity of eluates increased in the order FA < HA < FHA. Viability staining and flow-cytometric analysis revealed that eluates did not induce significant changes in cell-cycle profile. Instead, apoptotic cell death, detected as sub-G0 cell population, apoptotic DNA fragmentation and apoptotic bodies, was responsible for the decreased number of cells treated with eluates of FHA and FA. FA induced caspase-3 activity comparable with the caspase-3 activity induced by 1.8 mg/mL cisplatin. On the other hand, the 75–100% eluates of FHA caused higher caspase-3 activity compared with cisplatin. Furthermore, the increase in caspase-9 activity was detected in cells treated with 50–100% eluates of FA and FHA, activation of caspase-8 was not detected. Our comparative studies on murine fibroblasts NIH-3T3 showed that fluoridated biomaterials FHA and FA caused higher cytotoxicity than HA. It could probably be caused by the presence of fluoride in the structure of FHA and FA. It is known that fluoride is the substance with significant biological activity. Fluoride stimulates bone formation and is capable of substantially increasing cancerous bone mass. Therefore, it is used for treatment of osteoporosis [22]. Further, fluoride has been widely used in dentistry as a caries-prophylactic agent [23]. NaF was the first and still most recommended fluoride compound for fluoridation of drinking water [24]. Fluoride has shown considerable variation as to its cytotoxicity against different cultured cells [25,26]. The cytotoxicity against normal cells [27] has spurred debate on the use of fluoride in dentistry [28]. Fluoride showed slightly higher cytotoxicity against tumor cell lines than against normal cells (tumor-specificity ratio, 1.8) [29]. Other manifestations of the biological activity of fluoride include toxic effects—skeletal fluorosis and damage to kidney liver, parathyroid glands and brain [30,31]. Based on these effects of fluoride, there has been some discussion that excess fluoride could cause an impact on genome integrity and may represent a hazard to human health. Therefore, the potential DNA damage associated with exposure to fluoride was assessed in cells growing in in vitro and in vivo conditions. Data obtained this way are controversial. Some authors reported that fluoride does not induce DNA damage in vitro [32–34] and in vivo [23,35]. Other authors [36–39] have observed the mutagenic potential (induction of damage to DNA and chromosome) of fluoride. Human studies showed that fluoride had no detectable clastogenic potential (chromosomal aberrations and micronucleus formation) in peripheral lymphocytes [22] and long-term exposure of humans to fluoride in drinking water does not cause chromosomal damage [35]. On the basis of the data presented above, the aim of the present study was to compare cytotoxic, genotoxic and mutagenic effects of FHA, FA and HA. V79 Chinese hamster fibroblasts were used for cytotoxicity, genotoxicity and mutagenicity tests. Cytotoxicity of the biomaterials was measured by a colony-formation assay, genotoxicity was determined by the comet assay and mutagenicity was assessed by the Hprt gene-mutation assay. Mutagenicity

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of the tested biomaterials was also examined in primary screening by gene-mutation analyses on bacteria. Because MNNG was used as a positive control in the mammalian gene-mutation assay, S. typhimurium TA100 (which is sensitive for MNNG) was used in the bacterial cell gene-mutation assay. The colony-formation assay is often used as a sensitive test for cytotoxic examination in which the individual cells are treated. As shown in Fig. 1, the tested biomaterials induced very weak cytotoxicity. The highest reduction of cell colony number (about 10%) was found in V79 cells treated with the highest test concentrations (100% and 75%) of all tested biomaterials, while the lowest reduction was caused by HA. Other concentrations of all tested biomaterials induced cytotoxicity that was less than 8.6%. On the other hand, higher cytotoxicity of FHA, FA and HA was observed on the basis of cell number per colony. The highest reduction in number of cells per colony (42.9–18.8%) was found at five tested concentrations (100%, 75%, 50%, 25% and 10%). The weakest cytotoxicity of the biomaterials (about 14%) was found at the lowest two test concentrations (1% and 5%). While comparing the cytotoxic effects of the test biomaterials we found that HA had the smallest reducing effect on cell number per colony, while the effects of FHA and FA were comparable. From these results we can conclude that all biomaterials tested here influenced cell division in colonies. The single-cell gel electrophoresis (SCGE) assay, better known as the comet assay, is a sensitive technique for detecting singleand double-strand breaks and/or alkali-labile sites at the singlecell level in DNA of any kind of cell. This test is based on the ability of DNA fragments to migrate in a weak electric field in the direction of the anode, giving the nucleolus the appearance of the comet tail when visualized by fluorescence microscopy. In our experiments, we used the comet assay to detect induction of DNA damage in V79 cells after a 24-h treatment with FHA, FA and HA (Fig. 2). We found that all tested biomaterials induced DNA damage, increasing in the order HA < FHA < FA. The genotoxic effect increased with increasing concentration. Next we studied the ability of FHA, FA and HA to induce mutagenic effects. We used the assays that are able to detect gene mutations: the Hprt test and the bacterial mutagenicity assay. The results presented in Table 1 show that the positive control MNNG induced mutations in V79 cells. On the other hand, FHA, FA and HA did not show a mutagenic effect. Although higher test concentrations of all biomaterial eluates induced a genotoxic effect in V79 cells, the DNA damage led to growth inhibition and did not result in mutations. The results presented in Fig. 3 demonstrate that the positive control MNNG induced mutations in S. typhimurium TA100. On the other hand, FHA, FA and HA did not show a mutagenic effect. In summary, the highest test concentrations (100% and 75%) of all tested biomaterials induced a very weak reduction of cell-colony number (about 10%). On the other hand, a reduction of the number of cells per colony was found (42.9–18.8%) at these concentrations. Biomaterials induced DNA breaks that increased with the test concentration in the order HA < FHA < FA. From the previous results obtained with adherent-growing fibroblasts NIH-3T3 [12,13] and from the results realized in the present work on adherent-growing V79 fibroblasts it can be stated that both fluoridated biomaterials induced DNA breaks to a greater extent than did HA. At higher concentrations of FHA and FA eluates, DNA breaks were probably the cause of the G0 /G1 arrest of NIH-3T3 cells, which was manifested by inhibition of cell proliferation [13]. In V79 cells, DNA breaks induced by FHA and FA eluates were probably the cause of the reduced cell division in cell colonies. On the other hand, DNA breaks induced by higher concentrations of FHA and FA caused apoptotic death of L1210 cells [8]. In contrast, the low level of DNA damage induced by HA eluates were probably repaired in NIH-3T3 and V79 cells,

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