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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 1  |  Page : 12-15

Genotoxicity evaluation of locally produced nano-hydroxyapatite-silica: An in vitro study using the bacterial reverse mutation test


Human Genetic and Molecular Biology Unit, School of Dental Sciences, Health Campus, Universiti Sains Malaysia, Kelantan, Malaysia

Date of Web Publication12-Apr-2019

Correspondence Address:
Nik Rozainah Nik Abdul Ghani
School of Dental Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/dmr.dmr_39_18

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  Abstract 


Background: Nanohydroxyapatite-silica (nanoHA-Silica) has been produced by one-pot sol-gel technique. The material when incorporated into commercial Glass Ionomer Cement (GIC) was found to exhibit higher Vickers hardness, compressive strength, and flexural strength compared to conventional GIC. However, before starting to be used and exposed to the human cell, every material product should undergo for genotoxic evaluation. Thus, the objective of this in vitro study was to evaluate the genotoxicity of locally produced nanoHA-Silica under bacterial reverse mutation assay (Ames test). Materials and Methods: Four Salmonella typhimurium strains TA98, TA102, TA1535, and TA1537 were incubated with nanoHA-Silica in the presence and absence of exogenous metabolic activation system (S9) at five different concentrations (0.3125, 0.625, 1.25, 2.5, and 5 mg/plate) along with appropriate positive and negative controls. The assessment of the results was based on the number of revertant colonies in each plate, and the results were regarded as mutagenic when the number of revertant colonies was more than two-fold of the negative control. Results: There was no significant increase in the number of revertant colonies corresponding to the increase in the concentrations of the test substance for all the five bacterial strains treated with or without S9. Conclusion: NanoHA-Silica-GIC was non-genotoxic and had no mutagenic potential under present test conditions.

Keywords: Ames assay, genotoxicity, nano-hydroxyapatite silica, Salmonella typhimurium


How to cite this article:
Abdul Ghani NR, Muhammad Sazri AN, Yee CY, Luddin N, Ponnuraj KT. Genotoxicity evaluation of locally produced nano-hydroxyapatite-silica: An in vitro study using the bacterial reverse mutation test. Dent Med Res 2019;7:12-5

How to cite this URL:
Abdul Ghani NR, Muhammad Sazri AN, Yee CY, Luddin N, Ponnuraj KT. Genotoxicity evaluation of locally produced nano-hydroxyapatite-silica: An in vitro study using the bacterial reverse mutation test. Dent Med Res [serial online] 2019 [cited 2019 Apr 26];7:12-5. Available from: http://www.dmrjournal.org/text.asp?2019/7/1/12/256024




  Introduction Top


Nanotechnology in dentistry has precipitated the vast variety of production and applications of biomaterials. The nanosized biomaterials particle in dental materials, was proven to increase their Vickers hardness, compressive and flexural strength, shear bond strength, and polishability which contribute to their esthetic appearance.[1],[2],[3] Hydroxyapatite (HA) in another hand is a natural mineral form of calcium apatite. The mineral has an excellent biological behavior[4] and its hardness is very much similar to the natural tooth.[5] HA has been used in many fields of dentistry including for caries prevention[6] restoration of periodontal defect[7] repair of mechanical furcation perforations[8] desensitizing agent and remineralization agent in toothpastes.[9] Nano-Hydroxyapatite (nano-HA) is a combination of both nanosized biomaterials and HA. The combination has been used as an additive material to improve the quality of existing dental materials. Recently, nanohydroxyapatite-silica (nanoHA-Silica) was synthesized by one-pot sol-gel technique.[10] This nanoHA-Silica was incorporated into commercially available Glass Ionomer Cement (GIC) Fuji IX GP (GC International, Japan)[10],[11] which was named as nanoHA-Silica-GIC The nanoHA-Silica-GIC was found to have better hardness compared to conventional Glass Ionomer Cement.[11] This material was believed to have a higher content of nano-silica which result in more denser cement to produce stronger GIC.[11],[12] The development of every single biomaterial requires approval from mutagenicity testing to be considered safe on the human dental tissue. The mutagenicity testing conducted for nanoHA-Silica was based on the microsome mutagenicity assay (Ames test) which specifically designed to detect a wide range of chemical substances that can induce DNA damage and leads to gene mutations.[13] In thisin vitro study, mutant strains of the bacteria  Salmonella More Details typhimurium (S. typhimurium) have been chosen for the test.


  Materials and Methods Top


Test material

NanoHA-Silica which comprising of 89% HA and 11% silica was synthesized by one-pot-sol-gel technique.[10]

Mutagenicity test

The highest dose of 5 mg/plate and four lower doses of 2.5, 1.25, 0.625, and 0.3125 mg/plate and all of these doses were obtained by a two-fold dilution.[13] The extracts were prepared according to ISO 10993-5. First, the locally produced nanoHA-silica powder was weighed and then autoclaved for 15 min at 121°C. 1 mL of sterile distilled water was then mixed with 0.1 g of the test materials and incubated at 37°C for 48 h. The extract of the test materials was the filtered through a 0.2 μm filter (Sartorius, USA). The extracts were prepared fresh, before every experiment procedure.

The tester strain

Four strains of S. typhimurium were used – TA98, TA102, TA1535, and TA1537. The tester strains TA1535 and TA1537 were obtained from the American Type Culture Collection (ATCC, USA), whereas, the tester strains TA98 and TA102 were obtained from Riken Com. Japan. Before the experiments, a genetic analysis was done to check their genetic integrity for histidine/biotin dependence, marker (crystal violet), and the presence of the plasmid pKM101 (ampicillin resistance).[13]

Chemicals, reagents, and equipment

The chemicals used in this study were citric acid monohydrate (CAS No. 5949-29-1) obtained from Mallinckrodt (Mexico), magnesium sulfate (CAS No. 1.05886.1000) were purchased from Merck (Darmstadt, Germany), sodium ammonium hydrogen phosphate tetrahydrate (CAS No. 1.06682.1000) and potassium phosphate, dibasic, anhydrous for the Vogel-Bonner (VB salts) medium. D-glucose (dextrose) anhydrous (CAS No. 50-99-7) from RandM Marketing (UK) was used for the glucose solution. Agar powder (CAS No. 9002-18-0) from HIMEDIA (India) used for making the GM agar. D-biotin (CAS No. 58-85-5) obtained from Calbiochem (Darmstadt, Germany) and the L-Histidine (CAS No. 1.04351.0100) were purchased from Mallinckrodt (Mexico) were used for Histidine/biotin solution (0.5 mM). Oxoid nutrient broth No. 2 (CAS No. CM0067) were also purchased from Mallinckrodt (Mexico) used to make the nutrient broth. D-glucose-6-phosphate (CAS No. G-7879) from Sigma (USA), magnesium chloride anhydrous (5958), potassium chloride (CAS No. 7447-40-7), and sodium phosphate monobasic hydrate (CAS No. 10049-21-5) were purchased from Mallinckrodt (Mexico). Sodium phosphate dibasic anhydrous (CAS No. 7558-79-4) was purchased from Sigma (USA) and used for the preparation of Co-factor for S9 mix. Ampicillin sodium salt (CAS No. 69-52-3) from Amresco (USA) and liver microsomal enzymes (S9 homogenate) (CAS No. S-2067) were purchased from Sigma (USA). Sterile distilled water was used as the negative control in the Ames assay. Four types of positive controls were used in the Ames assay. 2-Aminoanthracene (2AA) (CAS No. 017-06851, Wako Pure Chemical Industries, Japan) was used with all the tester strains of Salmonella bacteria at a concentration of 5 μg/plate with the metabolic activation (S9). 4-Nitro-o-phenylenediamine (CAS No. 99-56-9, ACROS ORGANICS, USA) was used as a positive control at a concentration of 2.5 μg/plate without metabolic activation (S9) with the TA98 tester strain, sodium azide (NaN3) (CAS No26628-22-8, ACROS ORGANICS, USA) was used as a positive control at a concentration of 5 μg/plate without metabolic activation (S9) with the TA1535 and the TA1537 tester strains and Mitomycin C was used as positive control at a concentration of 0.5 μg/plate without metabolic activation (S9) with the TA102 tester strain. The top agar, consisting of 0.6% agar and 0.6% NaCl, is one of the most critical medium components in the Ames test as it contains the trace amount of histidine (0.05 mM) for limited growth. It also contains biotin at a concentration of 0.05 mM which is in excess of what is needed for the growth of the Salmonella strains. Too little histidine may result in the background lawn looking sparse, which might be taken as evidence that toxicity is present even on the solvent negative control plates. Too much histidine will cause heavy growth that may obscure the revertant colonies.[13]

The preincubation assay

The preincubation assay is a modification of the standard plate incorporation assay with few exceptions. It is believed that this assay is more sensitive as compared to the plate incorporation assay. This is due to the tester strains in the small volume of preincubation mixture may have a better chance reacting with the short-lived mutagenic metabolites and the effective concentration of S9 mix in the preincubation volume is higher than on the plate.[13]

To the sterile 15 mL centrifuge tube, 0.05 mL of the test material dilution, 0.1 mL positive control overnight culture of Salmonella strain, and 0.5 mL of metabolic activation (S9) mix or sodium phosphate buffer (pH 7.4) was added in order. The contents of the test tubes were then mixed using vortex and incubated 37°C for 20 min. After that, 2 mL of molten top agar maintained at 43–48°C was added into the mixture. The contents of the test tubes were mixed again with vortex and poured slowly onto the surface of glucose minimal agar plates. The top agar was left for a while, and when it was already hardened, the plate was inverted and incubated for 48 h at 37°C incubator. The colonies were then counted using colony counter (aCOLyte, SYMBIOSIS, UK). The result was expressed as the number of revertant colonies per plate where mean value was obtained twice in triplicates experiment.[10],[13],[14],[15] In this study, the two-fold rule was used for interpretation of the results.

Interpretation of the results

In this study, a non-statistical procedure was used to evaluate the results of Salmonella experiments.

Positive

A compound is considered a mutagen if it produces a reproducible, dose-related increase in the number of revertant in one or more strains. A minimum fold increase, usually 2–3 folds, in revertant (over the solvent control) is the cutoff between a mutagenic and nonmutagenic response. A compound is considered a weak mutagen if it produces a reproducible, dose-related increase in the number of revertant in one or more strains, but the number of revertant is not double of the background.

Negative

A compound is considered a nonmutagen if no dose-related increase in the number of revertant is observed in at least two independent experiments.

Inconclusive

If a compound cannot be identified clearly as a mutagen or a nonmutagen, the results are classified as inconclusive.


  Results Top


The test substance is considered to be mutagenic when the number of counted colonies exceeds the number of colonies in the negative controls by at least double and a relationship between dose and response can be observed.

In this study, the results showed that the number of revertant in each plate in the presence and absence of an exogenous metabolic activation system were less than twice of the negative control at all tested. No dose-related increase was observed. The results in the Salmonella tester strains are presented in [Table 1] and [Table 2].
Table 1: Mutagenicity Ames test of nanohydroxyapatite-silica in Salmonella typhimurium strains in the presence of S9 mix

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Table 2: Mutagenicity Ames test of nanohydroxyapatite-silica in Salmonella typhimurium strains in the absence of S9 mix

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  Discussions Top


Locally produced Nano-Hydroxyapatite-Silica which comprised 89% hydroxyapatite and 11% silica had undergone for Mutagenicity Ames test and the result shown they were nongenotoxic and had no mutagenic potential.

According to the Organization for Economic Cooperation and Development (OECD) Guidelines 1986, the best two tests were the S. typhimurium reverse mutation assay (471) and mammalian cytogenetic test 473.[16],[17] Positive controls showed much more than two-fold the number of colonies than the negative controls imply that the substance induces frameshifts or base-pair substitution mutations and therefore, the test is valid. Negative results denote that the test substance is non-mutagenic under the test conditions. Although all known assays can yield false-positive and false-negative results, experience shows that the combination of two different test methods is a reliable parameter for determining carcinogens which are a risk to human health.[15]

In humans and lower animals, the cytochrome-based P450 metabolic oxidation system, which is present mainly in the liver and to a lesser extent in the lung and kidneys, is capable of metabolizing a large number of carcinogens chemicals to DNA-reactive, electrophilic forms.[13] Since S. typhimurium resembles a prokaryote and does not have the metabolic capability, a mixture of exogenous metabolic activation system (S9) was introduced to the test substance to improve the quality of the results. To ensure the comparability of results, the extraction temperature should preferably be 37 ± 0.5°C that mimicked the human body temperature and the time must be at least 24 h.

The number of revertant colonies of S. typhimurium were counted by using Computerized Colony Analyzer (aCOLyte counter) after 48 h of incubation. However, hand-counting is required when strains TA102 and TA104 are used because of the high number of spontaneous revertant colonies, usually above 200 colonies/plate.[18] Hand counting is also required when the precipitate is present on the plate when there is poor contrast between the colony and the agar, or when a test chemical discolor the agar which prevents sufficient light from passing through it. In our study, all plates, including the control plates for the selected tester strain are hand counted where the agar plates are divided into quadrants with appropriate adjustment of the counts. In the future, more strains are preferentially used in the future ofin vitro genotoxicity testing, and eukaryotic cells (mammalian cells) should be alternatively used as the target tester strains. A nanotechnology measurement technique with high specificity and sensitivity of the colony counter which are capable of detecting the high density of colonies should be utilized in the future.


  Conclusion Top


Within the limitation of the study, the nanoHA-silica was proven to have non-genotoxic and have no mutagenic potential. There is a need for more reliable biocompatibility test systems including MTT assay, filter diffusion test or pulp and dentine usage test. The choice and interpretation of the methodologies used are essential for the results analysis and the success or failure of for the evaluation of dental materials product.

Acknowledgments

Special appreciation to Professor Ismail Abdul Rahman and team for preparing the Nano-Hydroxyapatite-Silica, the staff of Craniofacial Science Laboratory, USM and USM Grant FRGS 2/2014-203/PPSG/6171173 for funding this research.

Financial support and sponsorship

Universiti Sains Malaysia, FRGS 2/2014-203/PPSG/6171173.

Conflicts of interest

There are no conflicts of interest.



 
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Mitra SB, Wu D, Holmes BN. An application of nanotechnology in advanced dental materials. J Am Dent Assoc 2003;134:1382-90.  Back to cited text no. 1
    
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Saunders SA. Current practicality of nanotechnology in dentistry. Part 1: Focus on nanocomposite restoratives and biomimetics. Clin Cosmet Investig Dent 2009;1:47-61.  Back to cited text no. 2
    
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Terry DA. Direct applications of a nanocomposite resin system: Part 1 – The evolution of contemporary composite materials. Pract Proced Aesthet Dent 2004;16:417-22.  Back to cited text no. 3
    
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Arcís RW, López-Macipe A, Toledano M, Osorio E, Rodríguez-Clemente R, Murtra J, et al. Mechanical properties of visible light-cured resins reinforced with hydroxyapatite for dental restoration. Dent Mater 2002;18:49-57.  Back to cited text no. 4
    
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Arita K, Lucas ME, Nishino M. The effect of adding hydroxyapatite on the flexural strength of glass ionomer cement. Dent Mater J 2003;22:126-36.  Back to cited text no. 5
    
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Kaushick BT, Jayakumar ND, Padmalatha O, Varghese S. Treatment of human periodontal infrabony defects with hydroxyapatite + β tricalcium phosphate bone graft alone and in combination with platelet rich plasma: A randomized clinical trial. Indian J Dent Res 2011;22:505-10.  Back to cited text no. 7
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Kakani AK, Veeramachaneni C, Majeti C, Tummala M, Khiyani L. A review on perforation repair materials. J Clin Diagn Res 2015;9:ZE09-13.  Back to cited text no. 8
    
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Kantharia N, Naik S, Apte S, Kheur M, Kheur S, Kale B. Nano-hydroxyapatite and its contemporary applications. Bone 2014;34:1-71.  Back to cited text no. 9
    
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Abdul RI, Sam'an MS, Norhayati L, Ahmad SR. One-pot synthesis of hydroxyapatite silica nanopowder composite for hardness enhancement of glass ionomer cement (GIC). Bull Mater Sci 2014;37:213-9.  Back to cited text no. 10
    
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Sheikh R, Ab Rahman I, Luddin N. Modification of glass ionomer cement by incorporating hydroxyapatite-silica nano-powder composite: Sol-gel synthesis and characterization. Ceramics Int 2014;40:3165-70.  Back to cited text no. 11
    
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Moheet I, Luddin N, Ab Rahman I, Kannan TP, Ghani NR. Evaluation of mechanical properties and bond strength of nano-hydroxyapatite-silica added glass ionomer cement. Ceramic Int 2018;44:9899-906.  Back to cited text no. 12
    
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Mortalmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res 2000;455:26-60.  Back to cited text no. 13
    
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Hassan A, Omar ZA, Ariffin Z. An in vitro genotoxicity study of silver amalgam on Ames test. Indones J Dent Res 2010;1:55-60.  Back to cited text no. 14
    
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Katzer A, Marquardt H, Westendorf J, Wening JV, von Foerster G. Polyetheretherketone – Cytotoxicity and mutagenicity in vitro. Biomaterials 2002;23:1749-59.  Back to cited text no. 15
    
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Organization for Economic Cooperation and Development Guidelines. Introduction to the OECD Guidelines on Genetic Toxicology Testing and Guidance on the Selection and Application Assay. Guidelines on Genetic Toxicology; 1986. p. 1-17.  Back to cited text no. 16
    
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Organization for Economic Cooperation and Development. TG 471. Mutagenicity: Reverse Mutation Test Using Bacteria. Bacterial Reverse Mutation Test. Organization for Economic Cooperation and Development; 1997. p. 1-11  Back to cited text no. 17
    
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Noushad M, Kannan TP, Husein A, Abdullah H, Ismail AR. Genotoxicity evaluation of locally produced dental porcelain – Anin vitro study using the Ames and comet assays. Toxicol In Vitro 2009;23:1145-50.  Back to cited text no. 18
    



 
 
    Tables

  [Table 1], [Table 2]



 

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