Evaluation of genotoxic effect of amalgam restorations in oral cavity

Chennoju Sai Kiran; Reddy Sudhakara Reddy; Rajesh Nallakunta

doi:10.4103/0975-8844.169756

ORIGINAL ARTICLE

Year : 2015 | Volume : 7 | Issue : 2 | Page : 100-103

Evaluation of genotoxic effect of amalgam restorations in oral cavity

Chennoju Sai Kiran¹, Reddy Sudhakara Reddy², Rajesh Nallakunta²
¹ Department of Oral Medicine and Radiology, St. Joseph Dental College, Eluru, Andhra Pradesh, India
² Department of Oral Medicine and Radiology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India

Date of Web Publication

17-Nov-2015

Correspondence Address:
Dr. Chennoju Sai Kiran
Department of Oral Medicine and Radiology, St. Joseph Dental College, Eluru, Andhra Pradesh
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/0975-8844.169756

Abstract

Background: Mercury a popular heavy metal used in dentistry in the form of amalgam is a known clastogen. The assessment of micronuclei in cells is a promising tool for studying the genotoxic effect of mercury on them. Hence, a study was conducted to evaluate the frequency of micronuclei in exfoliated buccal cells of subjects with amalgam restorations. Materials and Methods: A total of 60 subjects (age and gender-matched sample of 30 study group and 30 control group) were included in this study. Smears were obtained with moistened wooden spatula from buccal mucosa in close contact with amalgam restoration and fixed with 100% ethyl alcohol. After staining with Papanicolaou stain, all the slides were examined under ×40 and 1,000 cells were counted for the presence of micronuclei. The data were entered into a spread sheet and subjected to statistical analysis. Results: A statistically significant increase in the number of micronuclei containing cells was observed in the study group when compared to control group (P < 0.05). A positive correlation was observed between the duration of restoration and frequency of micronuclei (P < 0.05). Conclusion: The results showed a definite genotoxic effect of amalgam restorations on the oral cavity which can be attributed to the clastogenic action of mercury in amalgam restorations.

Keywords: Amalgam, biocompatibility, genetics, genotoxicity, mercury, micronuclei, Papanicolaou stain

How to cite this article:
Kiran CS, Reddy RS, Nallakunta R. Evaluation of genotoxic effect of amalgam restorations in oral cavity. J Orofac Sci 2015;7:100-3

How to cite this URL:
Kiran CS, Reddy RS, Nallakunta R. Evaluation of genotoxic effect of amalgam restorations in oral cavity. J Orofac Sci [serial online] 2015 [cited 2023 Jun 9];7:100-3. Available from: https://www.jofs.in/text.asp?2015/7/2/100/169756

Introduction

Cytogenetic biomonitoring using biomarkers has been used in health sciences for many years in diagnosing and staging diseases, as well as in risk assessment and providing information regarding the susceptibility status. Biomarkers serve as internal indicators of genotoxicity. Mucronuclei is the potential biomarkers and traditionally been used for biomonitoring genotoxic effects of various materials. ^[1] Micronuclei are expressed in cells when they contain chromosomal breaks, lacking centromeres or when a whole chromosome is unstable to travel to the spindle poles during mitosis. They are included by "clastogens," materials which cause breakage of chromosomes. ^[2] Mercury a popular heavy metal used in dentistry in the form of amalgam is a known clastogen. Inorganic mercury mainly acts on the cytoskeletal proteins like tubulin or kinesin, which play a major role in chromosomal segregation thus resulting in the formation of micronuclei. ^[3] However, the genotoxic effects of amalgam restorations was not fully evaluated.

Hence, a study was conducted to evaluate the genotoxic effects of amalgam restorations on exfoliated oral buccal cells in the oral cavity.

Materials and Methods

After obtaining approval from the institutional ethical committee (IRB NO: VDC/RP/2012-17), a total of 30 subjects who were willing to participate in the study were selected from the outpatient department of a dental institution. An age and gender-matched sample of 30 control subjects were recruited from the same institution who were absolutely free from any restorations.

Inclusion criteria

Healthy patients with amalgam restorations for a period of >6 months were included in the study.
The restorations must be involving >one-third of the tooth surface, with or without amalgam tattooing on the adjacent mucosal surface.

Exclusion criteria

Patient based - (a) Medically compromised patients, (b) patients with oral lesions including impacted teeth or any inflammatory conditions.

Cytological - (a) Cells in the form of clumps, (b) other chromosomal aberrations like nuclear blebs, necrotic cells, apoptotic cells, karyolysis, karyorrhexis and nuclear fragmentation were excluded in the study.

After obtaining informed consent, the subjects were asked to rinse their mouths with water, and a moistened wooden spatula was used to collect cells from the buccal mucosa which was in close contact with the restoration. The spatula was applied to a precleaned microscope slide. Smears were air dried and fixed in 100% ethyl alcohol. Slides were stained by the Papanicolaou technique using a commercially available staining kit RAPIDPAP. In each slide, 1,000 cells were counted at ×40 using random Z technique on a light microscope (OLUMPUS 3X-21) and the number of cells containing the micronuclei were counted [Figure 1]. Scoring was done using Tolbert's criteria for micronuclei. ^[4]

Figure 1: Cells with micronuclei at ×40

Click here to view

Rounded smooth perimeter suggestive of a membrane
Less than one-third, the diameter of associated nucleus, but large enough to discern shape and color
Staining intensity similar to nucleus
Texture similar to nucleus
Same focal plane as nucleus
The absence of overlap or bridge to the nucleus.

The collected data was entered into a spreadsheet (Excel 2007, Microsoft office 2007, Version: 13.0.6023.1000 (32 BIT), ©2007 Microsoft Corporation. all rights reserved) and was analyzed using, statistical analysis software (SPSS version 16.01, SPSS inc., Chicago, USA, 1989-2007). Pearson correlation and t-test were used to determine any significance between the groups. Significance was set at 0.05 level (P < 0.05).

Results

A total of 60 buccal smears (30 study group and 30 control group) were obtained from subjects who were willing to participate in the study. The number of cells containing micronuclei out of 1,000 cells was counted and recorded. The demographic details of the study are tabulated in [Table 1].

Table 1: Demographic details of the study

Click here to view

The mean age ± standard error of the whole sample was 31.46 ± 2.08 years. For the study and control groups, the means were 32.20 ± 2.14 and 30.73 ± 2.02 years, respectively. The mean number of micronuclei containing cells in study and control group was 29.23 ± 8.30 (26.26-32.2 at 95% confidence interval) and 9.40 ± 3.46 (8.16-10.64 at 95% confidence interval), respectively. An independent sample t-test was conducted to compare the frequency of micronuclei in study and control group. Results showed a significant increase in the frequency in study group when compared to control group (t = 12.076, P = 0.001 at 95% confidence interval). The groups did not differ in gender (P = 0.114). The maximum and minimum frequencies of micronuclei obtained in the study were 47 and 16 in the study group and 14 and two in the control group respectively. A Pearson product moment correlation coefficient was computed to assess the relation between duration of restoration and frequency of micronuclei. A statistically significant weak positive correlation was obtained (r = 0.572, P = 0).

Discussion

The occurrence of chromosomal damage and their association with carcinogenesis has been well established by many studies. Since 1937, micronuclei have been considered a marker for genome damage in the initiation stage. ^[5] In oral cavity, micronuclei are considered to be important biomarkers for analyzing the genotoxic effects of various substances with clastogenic and aneugenic modes of action. In this study, the micronucleus assay was applied to verify the effect of a well-known dental restorative material, amalgam.

Mercury is a commonly used heavy metal, in the form of amalgam restorations. The interaction of inorganic mercury with microtubule network of cells was studied in depth by Thomas et al. who described its genotoxic effects. ^[3] Interestingly they found that the induction of micronuclei was dose dependent starting at minimum concentrations of 0.05 ΅M of mercury. The other actions mainly include binding of free mercury to sulfhydryl groups in tubulin which inhibits polymerization of microtubules during cell division. ^[6],[7] This clastogenic action of mercury on the cytoskeletal proteins during cell division is the main etiology for the production of micronuclei in the cells. ^[8] This was supported by the results of the present study where a statistically significant increase in the frequency of micronuclei was observed in the study group when compared to control group.

Di pietro et al. reported that mercury-containing amalgam restorations cause impairment of cellular pro and anti-oxidant redox balance resulting in genotoxic effects. The association between dental fillings and DNA damage was enhanced by the number of fillings and the exposure time. ^[9] Similar results were found in our study. A statistically significant correlation was obtained between duration of restoration and frequency of micronuclei.

Akiyama et al. have done an in vitro study to evaluate the genotoxic effects of mercury in amalgam restorations at various concentration levels. He observed that the normal concentrations of mercury in conventional and high copper amalgams were 17.64 µM and 20.63 µM respectively. High copper amalgam restorations were found to induce high-level chromosomal aberrations at two-third dilution. ^[10] These results support the possible genotoxic effect of mercury in amalgam restorations on the oral mucosa which can be hypothesized in [Figure 2].

Figure 2: Hypothetical explanation of the possible effect of mercury in the amalgam restorations on the oral epithelium. Note the genotoxic effect of mercury by its possible clastogenic action on the tubulin assembly of spindle fibres during cell division resulting in the formation of micronuclei

Click here to view

Ekstrand et al. reported that the genotoxic effect of mercury may vary from person to person. This is because the mercury's tissue accumulation is very much dependent on glutathione production in the body, which in turn is regulated by genes. ^[11] The results of the present study were in favor of the above statement. As the duration of restoration increases, there is more chance for the mercury in the amalgam restoration to get accumulated in the tissues. However, we found only a weak positive correlation between the duration of restoration and the frequency of micronuclei.

Interestingly, Uçar and Brantley et al. reported that the release of mercury from amalgam restorations decrease significantly as the time progresses, and the amount of mercury release at the beginning was par below the limits, that can cause health hazard. ^[12] But, our results showed that the frequency of micronuclei increased with duration of the restoration. This can be attributed to the fact that the initial period of amalgam restoration, and the amount of mercury released from the restoration at the time of restoring the tooth is very crucial. The higher the amount of mercury released, the higher, it will be accumulated in the tissues and the severe will be its genotoxic effects. Hence, our study confirms that proper trituration of amalgam with the removal of excess mercury and proper condensation can produce good restorations with minimal genotoxic effects.

Conclusion

Our findings with the micronuclei essay to indicate the chromosomal mutations, add valuable information to elucidate the cytotoxic activity of amalgam restorations. However, more research on a large scale is required regarding the concentrations of mercury in saliva with amalgam restorations, to establish its potential cytotoxic effects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Guzmán P, Sotelo-Regil RC, Mohar A, Gonsebatt ME. Positive correlation between the frequency of micronucleated cells and dysplasia in Papanicolaou smears. Environ Mol Mutagen 2003;41:339-43.
2.	Ghosh P, Basu A, Singh KK, Giri AK. Evaluation of cell types for assessment of cytogenetic damage in arsenic exposed population. Mol Cancer 2008;7:45.
3.	Stoiber T, Bonacker D, Böhm KJ, Bolt HM, Thier R, Degen GH, et al. Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II). Mutat Res 2004;563:97-106.
4.	Jois HS, Kale AD, Mohan Kumar KP. Micronucleus as potential biomarker of oral carcinogenesis. Indian J Dent Adv 2010;2:1-5.
5.	Bloching M, Reich W, Schubert J, Grummt T, Sandner A. Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors. Oral Oncol 2008;44:220-6.
6.	Kuriyama R, Sakai H. Role of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization. J Biochem 1974;76:651-4.
7.	Wallin M, Larsson H, Edström A. Tubulin sulfhydryl groups and polymerization in vitro. Effects of di- and trivalent cations. Exp Cell Res 1977;107:219-25.
8.	Thier R, Bonacker D, Stoiber T, Böhm KJ, Wang M, Unger E, et al. Interaction of metal salts with cytoskeletal motor protein systems. Toxicol Lett 2003;140-141:75-81.
9.	Di Pietro A, Visalli G, La Maestra S, Micale R, Baluce B, Matarese G, et al. Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings. Mutat Res 2008;650:115-22.
10.	Akiyama M, Oshima H, Nakamura M. Genotoxicity of mercury used in chromosome aberration tests. Toxicol In Vitro 2001; 15:463-7.
11.	Ekstrand J, Nielsen JB, Havarinasab S, Zalups RK, Söderkvist P, Hultman P. Mercury toxicokinetics - Dependency on strain and gender. Toxicol Appl Pharmacol 2010;243:283-91.
12.	Uçar Y, Brantley WA. Biocompatibility of dental amalgams. Int J Dent 2011;2011:981595.