|Year : 2014 | Volume
| Issue : 6 | Page : 772-776
|A study of sister chromatid exchange in patients with dental amalgam restorations
E Lakshmi Priya1, K Ranganathan2, Uma Devi K Rao2, Elizabeth Joshua2, Deepu George Mathew3, Kavitha Wilson2
1 Department of Oral and Maxillofacial Pathology, Al Azhar Dental College, Thodupuzha, India
2 Department of Oral and Maxillofacial Pathology, Ragas Dental College, Chennai, Tamil Nadu, India
3 Department of Oral and Maxillofacial Pathology, Mar Baselios Dental College, Kothamangalam, Kerala, India
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|Date of Submission||02-Apr-2014|
|Date of Decision||28-Apr-2014|
|Date of Acceptance||11-Aug-2014|
|Date of Web Publication||02-Mar-2015|
| Abstract|| |
Study Background: Dental amalgam is still widely used as a restorative material in developing countries due to its low cost and ease of manipulation. The health risks associated with the components of this restorative material has always been a matter of concern. Our study was designed to address this question regarding dental amalgam.
Objective: To study sister chromatid exchange (SCE) as an indicator of systemic genotoxicity, due to the exposure from the components of amalgam restorations during its placement and chronic use.
Materials and Methods: Systemic genotoxicity in subjects exposed to amalgam during its placement (Group II; n = 5) and subjects with chronic exposure to amalgam (Group III; n = 5) were compared with controls (Group I; n = 5) by SCE assay in cultured peripheral blood lymphocytes.
Result: Subjects exposed to amalgam during its placement and subjects having chronic exposure to amalgam showed an increase in the frequency of SCE, but the change was not statistically significant (P = 0.84, P = 0.123 respectively).
Conclusion: Systemic genotoxicity was not observed due to the components of amalgam restorations released during its placement and chronic use. The findings of this study can be considered as preliminary information on the systemic toxicity due to the components of amalgam restorations.
Keywords: Dental amalgam, genotoxicity, mercury exposure, sister chromatid exchange
|How to cite this article:|
Priya E L, Ranganathan K, Rao UK, Joshua E, Mathew DG, Wilson K. A study of sister chromatid exchange in patients with dental amalgam restorations. Indian J Dent Res 2014;25:772-6
Dental amalgam is still the most frequently used material for restorative dental treatment especially for posterior teeth. , Amalgam has been in use for >150 years and is considered to be cost effective, easy to use, and durable.  It consists of 50% mercury combined with other metals like silver and copper. There have always been periodic concerns about the assumed health risk due to the mercury released from these restorations.  During the functional life of amalgam restorations, a pacifying layer of corrosive products is formed on the surface. Factors that disrupt this pacifying layer such as tooth brushing and chewing can potentially increase the release of mercury and ions of copper and zinc. , Though in vitro studies have been done to assess the genotoxicity of organic and inorganic mercury, in vivo studies on genotoxicity due to exposure from dental amalgams are very few. , There are no studies in English literature on systemic genotoxicity due to exposure to amalgam components during placement evaluated by sister chromatid exchange (SCE), a highly sensitive technique to detect DNA damage. , Even though 53% of dental practitioners are carrying out amalgam restorations in their clinics in India, (metha) no studies have been done in Indian population regarding the genotoxic effects of amalgam restorations. The following study was undertaken to provide preliminary data on DNA (Chromosomal) instability from exposure to components of amalgam restorations during its placement and chronic use by studying the frequency of SCEs in peripheral blood lymphocytes (PBL).
|How to cite this URL:|
Priya E L, Ranganathan K, Rao UK, Joshua E, Mathew DG, Wilson K. A study of sister chromatid exchange in patients with dental amalgam restorations. Indian J Dent Res [serial online] 2014 [cited 2019 Sep 17];25:772-6. Available from: http://www.ijdr.in/text.asp?2014/25/6/772/152203
| Materials and methods|| |
The study comprised of three groups: Group I (n = 5) was the control group that comprised of subjects with two or more tooth surfaces affected with dental caries but without any restorations (Group I; n = 5). Subjects from Group I after receiving amalgam restoration served as subjects with exposure during amalgam placement or placement exposure group (Group II, n = 5). Subjects who had two or more amalgam restorations for a minimum period of 1 year formed the subjects with chronic exposure to amalgam or chronic exposure group (Group III; n = 5). All subjects were within the age group of 15-30 years and were from the same socio-economic background with no known exposure to genotoxic substances. Subjects with the habit of tobacco chewing or smoking and alcohol consumption were excluded from the study.
Peripheral venous blood was collected from all the subjects. The peripheral blood from subjects of group II was collected, 2 weeks after the placement of amalgam restorations. The study was approved by an Institutional Review Board. Informed consent was obtained from all subjects who participated in the study.
Heparinized whole blood was collected by venous puncture and used for the PBL cultures. 0.5 ml of peripheral blood was added to 5 ml of RPMI™ 1640 (Roswell Park Memorial Institute medium) cell culture media supplemented with 20% Fetal bovine serum, 0.2 ml of phytohemagglutinin and incubated for 72 h at 37°C. 
Sister chromatid exchange test
Chromatid differentiation in PBL cultures was initiated by adding 1 ml of 5-bromo-2-deoxyuridine (BrdU, Sigma, USA) (which was prepared by adding 200 μg of BrdU in 0.8 ml of sterile water) on the 2 nd day of culture. Metaphase inhibitor colchicine was added at 691/2 h and incubated for 1/2 h. The cells were collected by centrifugation, hypotonic solution was added and left in an incubator for 15 min. Slides were stained with Bis Benzamide H 33258 (Sigma) and exposed to sunlight and finally stained with giemsa for 30 min. 25 s division metaphase plates were analyzed per sample and mean SCE's/cell/metaphase plate was calculated. 
All data were entered and analyzed using Statistical Package for Social Science version 11.5. The mean SCE's between Group I and II was compared with paired t-test. Independent Student's t-test was used to compare, the mean SCE's between Group I and III and the mean SCE's of the male and female subject's in the control group (Group I). Pearson's correlation was performed to correlate the mean SCE's of the study Group III with number, duration of amalgam restorations and mean SCE's of the control group with subject's age. A P < 0.05 was considered to be statistically significant.
| Results|| |
The average period of exposure to amalgam in group III patients was 5.2 years (range 5-6 years), and the average number of restorations in this group was 4.4 (range 4-6 numbers). The average age of the subjects in this study was 21 years (range 15-28 years) [Table 1].
Comparison of sister chromatid exchange's between study and control groups
When subjects in placement exposure group (Group II) and chronic exposure group (Group III) were compared with the control group (Group I), there was an increase in SCE's in the study group, but the change was not statistically significant (P = 0.137, P = 0.98 respectively) [Table 2], Graph 1 and [Figure 1] [Figure 2] [Figure 3].
|Figure 1: Metaphase plate of a subject from group I. Arrow points chromosomes showing exchanges|
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|Figure 2: Metaphase plate of a subject from group II. Arrow points chromosomes showing exchanges|
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|Figure 3: Metaphase plate of a subject from group III. Arrow points chromosomes showing exchanges|
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A positive correlation was observed between the number, duration of restorations and frequency of SCE's in chronic exposure group (Group III). This correlation was not found to be statistically significant (P = 0.84, P = 0.123 respectively) [Table 3].
|Table 3: Correlation of Group III subject's mean SCE with number and duration of restorations|
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Sister chromatid exchange frequency regarding confounding factors
Confounding factors such as age and gender were analyzed in the control group (Group I). The average age of subjects in Group I was 18 years (range 15-20 years). Gender distribution in Group I showed, males comprising 60% and females 40%. Age of the control group (Group I) was related to the frequency of SCE's, and no significant correlation was observed (P = 0.731). When the mean SCE's between males and females in Group I were compared, even though there was an increase in the frequency of SCE's in males the difference was not statistically significant (P = 0.323) [Table 4] and [Table 5].
| Discussion|| |
Dental amalgam is defined by American Dental Association as an alloy composed of mercury, silver, tin and copper along with other metallic elements added to improve physical and mechanical properties.  It constitutes approximately 75% of all the restorative materials used by dentists.  Mercury and other metallic ions released from the restoration, even though in trace amounts has always been a health concern. , Among the different metals released from amalgam restoration, mercury is a major constituent and has often been associated with various health risks. 
Various studies on mercury cytotoxicity have found that gene polymorphisms regulating the production of glutathione, and its conjugation with mercury can influence mercury's tissue accumulation. Hence, human population can be heterogenous in mercury toxicity due to genetic variations. 
Studies on in vivo assessment of genotoxicity due to amalgam restorations are few in number. The techniques often used to assess amalgam-induced genotoxicity are mironuclei and comet assays which reflect chromosomal aberrations. , In our study, we have used SCE frequency to evaluate the amalgam restoration induced systemic genotoxicity. Analyzing SCE frequency is found to be a more sensitive genotoxicity assay than the conventionally used ones, as the frequency of SCE's are found to be increased by mutagens at a concentration much lower than what is required to produce chromosomal aberrations and breaks. 
Engle et al. in 1992 had found that patients receiving amalgam restorations have a high level of elemental mercury vapor exposure during its placement amounting to 6-8 μg but these levels are found to be well below the daily threshold limits established by regulatory agencies. , In our study when we compared the frequency of SCE's in subjects before and after receiving amalgam restorations (2 posterior occlusal restorations), there was an increase in the frequency of SCE's 2 weeks after receiving the restoration but the difference was not statistically significant [Table 2].
Patients bearing amalgam restorations are reported to be exposed to a constant and steady release of mercury vapors.  Metallic ions other than mercury in amalgam like copper and zinc ions were also found to be released in trace amounts to saliva from the restorations.  In our study, we did not see a significant increase in the SCE frequency in patients having amalgam fillings for more than a year; neither could we find a significant correlation between the frequency of SCE's with duration or number of restorations. The number of SCE's in chronic exposure group (Group III) was also found to be lesser when compared to placement exposure group (Group II) [Table 1] [Table 2] [Table 3]. Uηar and Brantley has reported that there is a steady decrease in the release of mercury as the restoration ages and the amounts released were well below the limits that can cause health hazard.  This reduction was found to be up to 90% within the first 30 days of restoration placement.  It has been proved to be due to the microstructural changes associated with solid state transformations resulting in the formation of biounavailable forms of mercury like β-HgS.  The release of other metallic ions like Copper and Zinc present in the amalgam were also found to be steadily decreasing with aging of the restoration. 
Confounding factors like age and gender did not show any significant correlations [Table 4] and [Table 5]. Many studies have also found that SCE frequencies are independent of the subject's age and gender. ,
| Conclusion|| |
In the present study, the genotoxicity due to amalgam restorations during placement and chronic use were analyzed, and there was no statistically significant increase in the SCE frequency. Since gene polymorphisms can influence the toxicokinetics of mercury; a major component of amalgam restorations, it is important to have a population specific information on genotoxicity to assess the risk of exposure to amalgam restoration.  No such data on genotoxicity due to amalgam restoration exists for Indian population, even though it's the most prevalent restoration carried out in this part of the world.  The findings of this study can be considered as preliminary data. Information on subject variability and effect size can be derived from this study so that further work on a larger sample size can be planned in order to add more information on the systemic effects due to the components of amalgam restorations.
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E Lakshmi Priya
Department of Oral and Maxillofacial Pathology, Al Azhar Dental College, Thodupuzha
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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