Skip to main content
NCBI home page
Acta Stomatologica Croatica logoLink to Acta Stomatologica Croatica
. 2023 Sep;57(3):216–228. doi: 10.15644/asc57/3/2

Assessment of Cytotoxic and Genotoxic Effect of Modern Dental Materials in vivo

Milena Trutina Gavran 1,✉, Davor Željezić 2, Lara Vranić 3, Dubravka Negovetić Vranić 4, Lana Grabarević 5, Danijela Jurić-Kaćunarić 6, Antonija Tadin 7, Sanja Šegović 8, Nada Galić 8
PMCID: PMC10557110  PMID: 37808410

Abstract

Objectives

The aim of the study was to assess the biocompatibility of modern composite and amalgam dental fillings.

Material and Methods

The research was conducted on 150 healthy patients between the ages of 10 and 20 who had amalgam and composite fillings between 6 and 12 months. Under in vivo conditions, a swab of buccal cells near the fillings was taken, and the cytotoxic and genotoxic impact of composite and amalgam fillings on these cells was analyzed using the extended micronucleus test (cytomeassay).

Results

The results showed statistically significant differences between the groups of subjects with amalgam and composite fillings and subjects without fillings for the following parameters: number of micronuclei (p=0.006), number of buds (p<0.001), number of binuclear cells (p<0.001), number of nucleoplasmic bridges (p<0.001).The number of micronuclei was statistically significantly higher in the group of subjects with amalgam and composite fillings compared to the group without fillings. The results for nuclear buds, for the number of binuclear cells and the number of nucleoplasmic bridges showed that the group with amalgam fillings had a statistically significantly higher number of these changes compared to other groups.The results of the analysis of the relationship between the parameters of the micronucleus test and the number of amalgam and composite surfaces did not show statistically significant values. Parameters indicating cell cytotoxicity were not statistically significantly elevated in subjects with fillings. The results of the analysis of the influence of the patients' lifestyle on the results of the micronucleus test showed statistically significant results for certain predictors (diagnostic X-ray radiation, coffee consumption, consumption of cooked, dried meat and baked food).

Conclusion

Based on the results, it can be concluded that the buccal cells of subjects with amalgam fillings showed the highest degree of genotoxic changes, followed by those with composite fillings and the least buccal cells of patients without fillings.

Keywords: MeSH Terms: Materials Testing, Composite Resins, Dental Amalgam, Micronucleus Tests, Cytotoxins

Author Keywords: cytotoxicity, genotoxicity, composite materials, buccal epithelial cells

Introduction

Composite materials and dental amalgams, as materials for dental fillings, come into direct contact with oral tissues and, due to this close and permanent contact, must have the highest degree of biocompatibility. Biocompatibility is defined as the ability of a material to stimulate a favorable host response after application within the host organism (1-3). Before receiving a license for use, biomaterials, including dental materials, must pass a series of tests and regulations (1). The biocompatibility of materials is evaluated through numerous parameters: (a) cytotoxicity (systemic and local), (b) genotoxicity, (c) mutagenicity, (d) carcinogenicity and (d) immunogenicity (1, 4). Cytotoxicity is a term that describes how toxic an agent is to cells; hence it can cause cell damage or death, mostly through necrosis or apoptosis (5). The term genotoxicity refers to harmful effects of a certain substance (genotoxin) on the genetic material of cells (DNA, RNA), that is, chromosomes, causing mutations. To assess the level of toxicity of materials that lead to DNA damage, numerous sophisticated techniques, i.e. in vitro and in vivo/ex vivo tests, have been developed. The most important in vivo tests include three cytogenetic procedures: the comet test, the chromosomal aberration test, and various types of micronucleus (MN) tests (1, 6). When the toxicity of dental amalgams is discussed, the toxicity of mercury (Hg) is mainly thought of. The dominant forms of Hg include: elemental Hg (Hg0); the ionic form of Hg also called inorganic Hg (II) or Hg2+ and the organic form of Hg with methylmercury (MeHg) (7).

Mercury in the body can come from food, air, industry, dental medicine, some medicines and other products. Dental amalgam contains elemental mercury, which is lipophilic, but as soon as it enters the body, as a result of the action of the enzyme hydrogen peroxide catalase, it changes to an inorganic form, which is not lipophilic and is more difficult to resorb into cells (7, 8). If mercury enters the body, it has affinity towards sulfhydryl groups and damages DNA (9), especially in people with certain genetic variants (10). The genotoxicity of Hg and its derivatives is mainly due to their ability to generate ROS (reactive oxygen species), which are formed when Hg enters the cell through the plasma membrane or via protein transporters (11, 12). Some scientific papers stated that the tripeptide glutathione is decreased in the population exposed to Hg (12, 13). Possible toxicity of dental amalgam was the reason for constant doubts about its danger to the health of amalgam filling holders, which stimulated numerous researches in in vitro and in vivo conditions (14, 15) and numerous articles in scientific and popular magazines (16, 17). Although the World Health Organization (WHO) and FDI, as an international dental organization, have a plan to reduce and gradually withdraw dental amalgam from use (18), the use of amalgam in dental medicine is still not completely prohibited (19). However, as all dental materials in the oral cavity are subject to mechanical, chemical, thermal, microbiological, enzymatic and other influences, their biocompatibility may change over time due to the release of ingredients from the material (20-22). After polymerization, composite fillings are never completely polymerized (23). The cytotoxicity and genotoxicity of the composite mainly depends on the chemical composition of the organic component and is, most often, the result of the release of free, residual monomers of HEMA, TEGDMA, UDMA and Bis-GMA from the filling due to the action of the aforementioned factors (20). Free monomers can generate ROS compounds and reduce glutathione levels, which can promote oxidative stress. They can stimulate the formation of inflammatory factors (25), increase the number of micronuclei with a clastogenic effect (26), cause cell necrosis (22) and can have numerous other toxic effects (16, 17). In the case of modern nanofilled or nanohybrid composites, in addition to the toxicity of free monomers, the potential toxicity of nanofillers from such fillings is also analyzed (27, 28). For this reason, constant monitoring and ex vivo and in vivo research of all dental materials is needed, even though they have passed all tests and received permission for official use (29-32). Since numerous studies have reported a correlation between the age of fillings and the degree of cell damage (13, 33, 34), in this study a swab of buccal cells was taken near composite and amalgam fillings aged six to twelve months. The goal was to evaluate the potential cytotoxic and genotoxic effects of fillings on the cells of the oral mucosa using the extended micronucleus (MN) test (cytomeassay). The cells of the oral mucosa are excellent indicators of the cytotoxic and genotoxic effects of dental materials and other factors within the oral cavity because they are directly and permanently exposed to them. An analysis of micronuclei on human buccal cells using the MN test is an effective and minimally invasive procedure for assessing the cytotoxic and genotoxic impact of dental materials and other factors on these cells. The aforementioned impact is measured by evaluating the findings of MN and other parameters for monitoring genetic damage (number of cells with micronuclei, with binuclear cells, with nucleoplasmic bridges and with nuclear buds („broken egg“)) and cell death (number of cells with pyknosis, with condensed chromatin, with karyolysis and with karyorrehxis) (35-37). Numerous papers have described the assessment of cytotoxic and genotoxic effects on oral epithelial cells using the MN test (38-41).

Materials and Methods

Composite materials 3MTM FiltekTM Z550 (3M) and dental amalgam Amalgam ANA 2000 (Nordiska Dental AB, Ängelholm, Sweden) were used in this research.

In the production of composite fillings, in addition to the composite material Filtek Z 550, an appropriate adhesive system from the same manufacturer was used (ScotchbondTM Universal Adhesive, 3M Espe), and for etching hard dental tissues a 37% orthophosphoric acid (Total Etch, Ivoclar Vivadent, Schaan, Liechtenstein) was used

Subjects

The research was carried out on 150 voluntary respondents aged between 10 and 20, who are patients of the Dental Practice of the Health Center Vrgorac, School of Dental Medicine University of Zagreb

Only completely healthy subjects were included in the research. Individuals who consumed two or more units of alcohol three or more times per week were excluded from the study, as well as the patients who smoke, patients with oral lesions, patients with a history of malignancy, and those exposed to materials used in orthodontics/or mobile and fixed prosthetics.

After the patient's or parent's/guardian's consent, a swab of the buccal mucosa was taken from each patient in order to assess the biocompatibility of modern restorative materials. The subjects were divided according to the number of filling areas and the age of the filling.

Group 1 consisted of 50 subjects aged between 10-20 years, who had amalgam fillings aged 6 to 12 months, and the number of amalgam surfaces was counted for each patient.Group 2 consisted of 50 subjects aged between 10-20 years, who only had composite fillings between 6 and 12 months old, and the number of composite surfaces was counted for each patient. Group 3 consisted of 50 subjects aged between 10-20 years who had healthy teeth and did not have a single dental filling.

Sample collection

Each patient was asked not to drink alcohol, smoke or eat for 1 hour before sampling. Before taking the swab, the subjects rinsed their mouths three times with water, then the superficial, dead layer of cells was removed with sterile gauze, and a swab of buccal cells was gently taken with a sterile cytological brush (Cytobrush Plus, GmbH, Dietramszell-Linden, Germany). The cell suspension was carefully applied to the slide, then fixed with methanol (80% v/v) at 40C for 20 minutes and air-dried. After that, the cells were stained with Giemsa solution (Sigma) for 10 minutes, washed with distilled water and air-dried and analyzed with a light microscope. In order to evaluate the cytotoxic and genotoxic effect of the material, an expanded micronucleus test (cytomeassay) was used.

Micronucleus assay in buccal epithelial cells

As a measure of cytotoxicity and genotoxicity, using the MN test, the number of micronuclei and other morphological changes in the nucleus was determined in the cells. The analysis was carried out with an Olympus CX 40 light microscope (Olympus, Tokyo, Japan) under a magnification of 400x, whereby each micronucleus and other chromatin anomalies were additionally checked under a magnification of 1000x. For each subject, one thousand of epithelial cells were analyzed.The frequency of occurrence of certain parameters of the micronucleus test (number of micronuclei, buds, morphological changes of the broken egg type, binuclear cells, nucleoplasmic bridges, pyknosis, karyolysis, karyorexia and morphological changes of the condensed chromatin type) was evaluated and systematized according to Tolbert et al. (37).

Statistical analysis

The obtained results were processed with the Shapiro-Wilk and Kolmogorov-Smirnov test to assess the normality of the data distribution, while the statistical analysis of the obtained data was carried out by Kruskal-Wallis non-parametric analysis using Kruskal-Wallis one-way analysis of variance (ANOVA) with Bonferroni adjustment for multiple comparisons (Table 1). A multivariate regression analysis was performed for the dependence of the parameters of the micronucleus test. Statistical analysis was performed in the SPSS 25.0 software package (IBM, Armonk, NY, USA) with a significance level of 0.05.

Table 1. Results of the regression analysis of the dependence of the parameters of the micronucleus test as an independent variable and the number of amalgam and composite surfaces as predictor variables.

Independent variable R2 Predictor variables β t p-value
MN 0,066 Number of amalgam surfaces 0,113 1,396 0,165
Number of composite surfaces 0,233 2,864 0,005
Broken egg 0,007 Number of amalgam surfaces 0,068 0,803 0,423
Number of composite surfaces 0,038 0,455 0,650
Binucleated cells 0,183 Number of amalgam surfaces 0,394 5,181 <0,001
Number of composite surfaces 0,127 1,675 0,096
Nucleoplasmic bridges 0,199 Number of amalgam surfaces 0,451 6,000 <0,001
Number of composite surfaces 0,026 0,352 0,726
Pyknosis 0,004 Number of amalgam surfaces -0,124 -1,483 0,140
Number of composite surfaces 0,078 0,932 0,353
Karyolysis 0,013 Number of amalgam surfaces -0,060 -0,714 0,476
Number of composite surfaces -0,086 -1,028 0,306
Karyorrexis 0,033 Number of amalgam surfaces -0,035 -0,427 0,670
Number of composite surfaces 0,219 2,647 0,009
Condensed Chromatin 0,034 Number of amalgam surfaces -0,016 -0,198 0,843
Number of composite surfaces 0,220 2,659 0,009
Open in a new tab

An analysis for multiple comparisons was not required in cases where the omnibus Kruskal-Wallis one-way ANOVA result was not statistically significant in subjects with infills.

Results

In accordance with the deviations from the normal distribution, the results are presented using boxplots, which better emphasize the features of non-normal distributions compared to the display of mean values and standard deviations.

The results in Figure 1 show that the number of micronuclei was statistically significantly higher in the group of subjects with amalgam fillings compared to the group without fillings (p=0.006). A marginally significant increase in the number of micronuclei was also observed in the group with composite fillings compared to the group without fillings (p=0.050). The groups with amalgam fillings and composite fillings did not statistically significantly differ from each other.

Figure 1.

Figure 1

Open in a new tab

Boxplots for the number of micronuclei. Boxes represent 25% and 75% quartiles, black lines in boxes represent medians, and upper and lower horizontal lines represent 1.5 x interquartile range. Statistically significant differences and corresponding p-values are shown by horizontal lines above the box.

The results for core buds in Figure 2 show that the group with amalgam fillings had a statistically significantly higher number of these morphological changes compared to the group without fillings (p<0.001) and the group with composite fillings (p=0.003). The group with composite fillings did not differ statistically significantly from the group without fillings.

Figure 2.

Figure 2

Open in a new tab

Boxplots for the number of buds.Boxes represent 25% and 75% quartiles, black lines in boxes represent medians, and upper and lower horizontal lines represent 1.5 x interquartile range. Statistically significant differences and corresponding p-values are shown by horizontal lines above the box.

The results of the number of binuclear cells, shown in Figure 3, showed statistically significant differences between all three groups of subjects. The obtained p-values were highly significant for the comparison of the group with amalgam fillings and the group without fillings (p<0.001), that is, for the comparison of the group with amalgam fillings and the group with composite fillings (p<0.001). A significant difference was also observed between the group without fillings and the group with composite fillings (p=0.029).

Figure 3.

Figure 3

Open in a new tab

Boxplots for the number of binuclear cells. Boxes represent 25% and 75% quartiles, black lines in boxes represent medians, and upper and lower horizontal lines represent 1.5 x interquartile range. Statistically significant differences and corresponding p-values are shown by horizontal lines above the box.

Figure 4 shows a statistically significantly higher number of nucleoplasmic bridges in the group of subjects with amalgam fillings compared to the group without fillings (p<0.001) and the group with composite fillings (p<0.001).

Figure 4.

Figure 4

Open in a new tab

Boxplots for the number of nucleoplasmic bridges.Boxes represent 25% and 75% quartiles, black lines in boxes represent medians, and upper and lower horizontal lines represent 1.5 x interquartile range. Statistically significant differences and corresponding p-values are shown by horizontal lines above the box.

The results of the regression analysis of the relationship between the parameters of the micronucleus test as an independent variable and the number of amalgam and composite surfaces as predictor variables shown in Table 1 generally showed low R2 values, which points to the fact that a relatively small proportion of the total variance can be explained by the predictor variables, i.e. the number of amalgam/composite surfaces surface.

In order to determine the connection between potential genotoxic factors related to the patients' lifestyle and the parameters of the micronucleus test, a multivariate regression analysis was performed. For each of nine parameters of the micronucleus test, the following variables were examined as predictors: diagnostic radiation (x-ray), drugs, consumption of dried meat, consumption of cooked food, consumption of baked food, frequency of meat consumption, frequency of consumption of baked meat, frequency of consumption of dried meat, consumption of vegetables, frequency consumption of vegetables, frequency of fruit consumption, frequency of coffee consumption, frequency of tea consumption and frequency of soda consumption.By reorganizing the order of individual predictors according to the decreasing values of the test statistic t, i.e. the corresponding increase in the p-value, Pareto diagrams were obtained that facilitate the visualization of the relative influence of predictor variables on the parameters of the micronucleus test Figures 5-10. Only predictors with t-values higher than the limit values defined at the significance level of 0.05 can be considered statistically significant. They are shown as dashed red vertical lines on the Pareto diagrams.

Figure 5.

Figure 5

Open in a new tab

Pareto diagram of the dependence of the number of micronuclei on the predictor variables. The dashed red line marks the significance limit. Predictor variables with t-values greater than this limit have a statistically significant effect in the regression model.

Figure 6.

Figure 6

Open in a new tab

Pareto diagram of the dependence of the number of buds on the predictor variables. The dashed red line marks the significance limit. Predictor variables with t-values greater than this limit have a statistically significant effect in the regression model.

Figure 7.

Figure 7

Open in a new tab

Pareto diagram of the dependence of the number of micronuclei broken eggon the predictor variables. The dashed red line marks the significance limit. Predictor variables with t-values greater than this limit have a statistically significant effect in the regression model.

Figure 8.

Figure 8

Open in a new tab

Pareto diagram of the dependence of the number of binuclear cells on the predictor variables. The dashed red line marks the significance limit. Predictor variables with t-values greater than this limit have a statistically significant effect in the regression model.

Figure 9.

Figure 9

Open in a new tab

Pareto diagram of the dependence of the number of nucleoplasmic bridgeson the predictor variables. The dashed red line marks the significance limit. Predictor variables with t-values greater than this limit have a statistically significant effect in the regression model.

Figure 10.

Figure 10

Open in a new tab

Pareto diagram of the dependence of the number of pyknosis on the predictor variables. The dashed red line marks the significance limit. Predictor variables with t-values greater than this limit have a statistically significant effect in the regression model.

Discussion

Numerous papers (13, 31-34) stated the influence of time on the cytotoxicity and genotoxicity of dental materials. The purpose of the current in vivo study was to use the MN test to analyze buccal epithelial cells from the vicinity of amalgam and composite fillings that were exposed to the conditions of the oral cavity for longer than 6 months but not longer than 12 months. The MN test is very important for the monitoring, diagnosis and timely treatment of diseases caused by genetic damage because it can detect the activity of clastogenic (chromosomal breakage) and aneugenic (loss of chromosomes) genotoxic factors (2). There are numerous articles in scientific and popular magazines dealing with the harmful effects of amalgam fillings on the entire body, mostly referring to the toxicity of mercury from amalgam (14-17). However, the findings of some studies of cytogenetic effects in humans exposed to Hg and its compounds from various sources have been negative, controversial or uncertain as to the actual role of Hg in some positive results; therefore standardization of cytotoxicity and genotoxicity tests is recommended (12). Composite fillings, due to the action of various factors inside the oral cavity, can release monomers and various compounds which can have a toxic effect on the surrounding tissues and the entire body of the composite filling wearer (20-26).

The results of this research showed statistically significant differences between the groups of subjects with amalgam fillings, with composite fillings and without fillings, for the following parameters of the micronucleus test: number of micronuclei (p=0.006), number of buds (p<0.001), number of binuclear cells (p< 0.001), the number of nucleoplasmic bridges (p<0.001). For the other parameters of the micronucleus test (morphological changes of the broken egg type, pyknosis, karyorexia, karyolysis, condensed chromatin), no statistically significant differences were observed between the groups with the mentioned fillings.

The results show that the number of micronuclei was statistically significantly higher in the group of subjects with amalgam fillings compared to the group without fillings (p=0.006). A marginally significant increase in the number of micronuclei was also observed in the group with composite fillings compared to the group without fillings (p=0.050). The groups with amalgam fillings and composite fillings did not statistically significantly differ from each other. Despite the high variability within the groups, the results showed statistically significant effects of amalgam and composite fillings on the morphological changes of cells of the oral mucosa indicative of genome damage.

The results for nuclear buds showed that the group with amalgam fillings had a statistically significantly higher number of this morphological change compared to the group without fillings (p<0.001) and the group with composite fillings (p=0.003).The results of the number of binuclear cellsshowed statistically significant differences between all three groups of subjects.The number of nucleoplasmic bridges was significantly higher in the group of subjects with amalgam fillings compared to the group without fillings (p<0.001) and the group with composite fillings (p<0.001). The appearance of this morphological characteristic indicates a significant damage to the genome. Parameters indicating cell cytotoxicity (number of pyknosis, number of karyolysis, number of karyorrehxis and number of morphological changes of the condensed chromatin type) show that the presence of amalgam and composite fillings did not lead to a measurable increase in these morphological anomalies. Reichl et al. (42) based on their in vitro study on the cytotoxicity of dental composite monomers and amalgam component Hg2 in human gingival fibroblasts concluded that Hg from amalgam is more toxic than composite components. Visalli et al. (13) monitored the genotoxic effect of amalgam and composite fillings on the cells of the buccal mucosa. They observed that the frequency of MN in the cells of the oral mucosa was significantly higher in subjects with restorative fillings compared to that in subjects without fillings. Ahmed et al. (33) stated that in subjects with composite fillings, cytotoxic changes on human buccal and labial cells become more pronounced the longer the filling is in the oral cavity, while in amalgam fillings the greatest toxic damage was observed in the first few hours after the filling was placed. Mary et al. (34) reported that in their study that the average number of MNs in the cells of amalgam filling carriers was statistically significantly higher than that of composite fillings. Likewise, the average number of MNs in the cells of subjects with amalgam and composite fillings was statistically significantly higher compared to the cells of participants without fillings. In addition to the aforementioned studies, there are numerous other studies on the toxicity, that is, the biocompatibility of restorative dental materials, especially dental amalgams and composite materials, in relation to local tissues and the entire organism of the dental filling holder (43, 44).

The results of the regression analysis of the relationship between the parameters of the MN test as an independent variable and the number of amalgam and composite surfaces as predictor variables generally showed low R2 values, which points to the fact that a relatively small proportion of the total variance can be explained by the predictor variables, i.e. the number of amalgam/composite surfaces.Despite the low values of R2, the regression results were statistically significant for certain parameters of the micronucleus test. Among all examined parameters, the number of buds stands out, for which the regression showed statistical significance with the number of surfaces for both types of fillings, i.e. amalgam (p=0.003) and composite (p=0.006) with beta-coefficients of 0.237 and 0.221. The above mentioned results are consistent with the findings of previous research (13, 33, 34), which stated that a higher level of DNA damage in the cells was correlated with a higher number of fillings. A multivariate regression analysis was performed to determine the associations between potential genotoxic factors related to patients' lifestyle and micronucleus test parameters. The goal of such an analysis was to examine which of the subjects' dietary habits and other factors could influence the results of the MN test, in addition to the influence of the previously discussed main factors related to the presence of amalgam and composite fillings.A number of biological, environmental and demographic factors can interfere with in vivo research. Lifestyle factors most often associated with genetic damage include smoking, alcohol consumption, diet, lack of vitamins and supplements (35). In this paper, the effect of some habits such as smoking and alcohol consumption could not be estimated by regression model, since the vast majority of subjects did not smoke (98%), nor did they consume alcohol (96%), which is expected, since the research was conducted on subjects aged between 10 and 20 years.The results of some studies (13, 31, 32, 35) did not find any effects of smoking and alcohol on the appearance of micronuclei in the cells of the oral cavity, while some other studies (35, 45) describe the influence of the synergistic interaction of alcohol consumption and smoking on buccal cell damage.

In this research, the Pareto diagram for the number of micronuclei showed a statistically significant effect of diagnostic radiation, while other predictors did not show a significant effect.The regression model for the number of buds showed a statistically significant effect of a number of predictors: diagnostic radiation, consumption of cooked food, consumption of dried meat and consumption of baked food.The regression model for morphological changes of the broken egg type showed the frequency of consumption of roasted meat and the frequency of fruit consumption as statistically significant predictors for the appearance of this morphological anomaly. The opposite signs of the beta coefficients (0.275 for the frequency of consumption of baked meat, i.e. -0.191 for the frequency of consumption of fruit) indicate that these two factors worked in opposite directions.A positive sign of the beta coefficient for the frequency of consumption of roasted meat is consistent with the known genotoxic effect of this type of food, while a negative sign of the beta coefficient for the frequency of fruit consumption indicates a possible protective effect, probably mediated by antioxidants from fruits that protect against genome damage. Some studies (35, 46, 47) indicate that a number of micronutrients, including beta-carotene and some other vitamins and N-acetylcysteine, significantly reduce MN levels in healthy smokers, as well as in people with precancerous lesions. The regression model for the dependence of the number of binuclear cells on the subjects' habits did not show statistical significance for any of the predictor variables. The high discriminatory value of the number of binuclear cells on exposure to potential harmful substances from restorative materials and at the same time relative insensitivity to variability in the habits and lifestyle of the test subjects could enable high sensitivity and specificity of this parameter in future research on the genotoxicity of restorative materials.The number of nucleoplasmic bridges according to the results of the regression model showed a statistically significant effect of the predictor diagnostic radiation and the frequency of coffee consumption. The frequency of coffee consumption was also a statistically significant predictor in the regression model for the number of pyknosis. Exposure to diagnostic radiation (X-radiation) was a significant predictor of three parameters of the micronucleus test: the number of micronuclei, the number of buds and the number of nucleoplasmic bridges. Ionizing radiation plays an important role in diagnosis and treatment, but it can also cause DNA damage. Some authors (41) did not find a statistically significant increase in MN in patients exposed to X-rays and CBCT (Cone-beam computed tomography) with an effective dose of 12mSv, however, they reported an increase in MN parameters that indicate cytotoxicity (karyorrehxis, pyknosis, karyolysis). In this study, for the number of karyolysis and for morphological changes of the condensed chromatin type, none of the predictor variables showed a statistically significant effect, while in the case of karyorrehxis, the variable of frequency of fruit consumption showed a protective effect.

Conclusions

Amalgam fillings showed a genotoxic effect on buccal mucosa cells, composite fillings showed a limited genotoxic effect, while the number of surfaces of amalgam and composite fillings in the oral cavity did not significantly affect their genotoxic effect on buccal cells. Cytotoxic effects have not been proven for either amalgam or composite fillings.

Due to the limited number of respondents who voluntarily participated in this research, the obtained effects of the material are indicative values and should be confirmed on a larger study group over a longer period of time.

Funding Information

No funding was received for this article.

Conflict of interest

The authors declare no conflict of interest related to this study.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  • 1.Schmalz G, Arenholt-Bindslev D. Biocompatibility of Dental Materials. Berlin Heidelberg; Springer-Verlang; 2009. [Google Scholar]
  • 2.Anusavice KJ. Phillips science of dental materials. Philadelphia: Saunders; 2003. [Google Scholar]
  • 3.Ratner BD. The Biocompatibility Manifesto: Biocompatibility for the twenty-first century. J Cardiovasc Transl Res. 2011;4:523–7. 10.1007/s12265-011-9287-x [DOI] [PubMed] [Google Scholar]
  • 4.Elshahaw W. Biocompatibility. In: Sikalidis C, editor. Advances in Ceramics-Electric and Magnetic Ceramics, Bioceramics, Ceramics and Environment. Rijeka; InTech; 2011. [Google Scholar]
  • 5.Elmore S. Apoptosis: A Reviw of Programmed Cell Death. Toxicol Pathol. 2007;35(4):495–516. 10.1080/01926230701320337 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Saks M, Upreti S, Rajendra SV, Dang R. Genotoxicity: Mechanisms, Testing Guidelines and Methods. GlobJ Pharmaceut Sci. 2017;555575:1–6. [Google Scholar]
  • 7.World Health Organization (WHO) Guidance for Identifying Populations at Risk from Mercury Exposure. IOMC Inter-Organization Programme for the Sound Management of Chemicals (UNEP. UNEP DTIE Chemicals Branch and WHO Department of Food Safety, Zoonoses and Foodborne Diseases); Geneva, Switzerland: 2008. [(accessed on 22 May 2023)]. Mercury Publications. Available online:https://www.who.int/foodsafety/publications/chem/mercuryexposure.pdf [Google Scholar]
  • 8.Galić N, Prskalo K, Prpić-Mehičić G, Šutalo J, Anić I, Prester Lj. Toksičnost dentalnog amalgama I. Acta Stomatol Croat. 1997;31:243–51. [Google Scholar]
  • 9.Ajsuvakova OP, Tinkov AA, Aschner M, Rocha JBT, Michalke B, Skalnaya MG, et al. Sulfhydryl groups as targets of mercury toxicity. Coord Chem Rev. 2020;417:213343. 10.1016/j.ccr.2020.213343 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Homme KG, Kern JK, Haley BE, Geier DA, King PG, Sykes LK, et al. New science challenges old notion that mercury dental amalgam is safe. Biometals. 2014;27:19–24. 10.1007/s10534-013-9700-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.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. 10.1016/j.mrgentox.2007.10.023 [DOI] [PubMed] [Google Scholar]
  • 12.Sánchez-Alarcón J, Milić M, Bustamante-Montes LP, Isaac-Olivé K, Valencia-Quintana R, Ramírez-Durán N. Genotoxicity of mercury and its derivatives demonstrated in vitro and in vivo in human populations studies. Systematic review. Toxics. 2021;9(12):326. 10.3390/toxics9120326 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Visalli G, Baluce B, La Maestra S, Micale RT, Cingano L, De Flora S, et al. Genotoxic damage in the oral mucosa cells of subjects carrying restorative dental fillings. Arch Toxicol. 2013;87:179–87. 10.1007/s00204-012-0915-2 [DOI] [PubMed] [Google Scholar]
  • 14.Siblerud R, Mutter J. An Overview of Evidence that Mercury from Dental Fillings may be an Etiological Factor in Many Health Disorders. J Biomed Res Environ Sci. 2021;2:472–85. [Google Scholar]
  • 15.Bartel-Steinbach M, Lermen D, Gwinner F, Schäfer M, Göen T, Conrad A, et al. Long-term monitoring of mercury in young German adults: Time trend analyses from the German Environmental Specimen Bank, 1995–2018. Environ Res. 2022;207:112592. 10.1016/j.envres.2021.112592 [DOI] [PubMed] [Google Scholar]
  • 16.Shahi S, et al. A review on potential toxicity of dental material and screening their biocompatibility. Toxicol Mech Methods. 2019;29(5):368–77. 10.1080/15376516.2019.1566424 [DOI] [PubMed] [Google Scholar]
  • 17.Schmaltz G, et al. Biocompatibility of amalgam vs composite-a review. Oral Health Prev Dent. 2022;20(1):149–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Chemicals of Public Health Concern. World Health Organisation. 2020. Available from: https://www.who.int/news-room/photo-story/photo-story-detail/10-chemicals-of-public-health-concern
  • 19.Amalgam (Part 2): Safe Use and Phase Down of Dental Amalgam. Adopted by the FDI General Assembly: 27-29 September 2021, Sydney, Australia. Available from: www.fdiworlddental.org/amalgam-part-2-safe-use-and-phase-down-dental-amalgam [DOI] [PMC free article] [PubMed]
  • 20.Knežević A, Ristić M, Tarle Z, Pichler G, Musić S. Degree of Conversion and Temperature Increase During composite polymerisation with LED units of different intensity. Acta Stomatol Croat. 2008;42(1):19–29. [Google Scholar]
  • 21.Urcan E, Scherthan H, Styllou M, Haertel U, Hickel R, Reichl FX. Induction of DNA double-strand breaks in primary gingival fibroblasts by exposure to dental resin composites. Biomaterials. 2010;31(8):2010–4. 10.1016/j.biomaterials.2009.11.065 [DOI] [PubMed] [Google Scholar]
  • 22.Samuelsen JT, Dahl JE, Karlsson S, Morisbak E, Becher R. Apoptosis induced by the monomers HEMA and TEGDMA involves formation of ROS and differential activation oft he MAP-kinases p38, JNK and ERK. Dental Materials: official publication oft he Academy of Dental Materials. 2007; 23(1):34-9. [DOI] [PubMed]
  • 23.Kuan Y-H, Huang F-M, Lee S-S, Li Y-C, Chang Y-C. BisGMA stimulates prostaglandin E2 production in macrophages via cyclooxygenase-2, cytosolic phospholipase A2, and mitogen-activated protein kinases family. PLoS One. 2013;8:e82942. 10.1371/journal.pone.0082942 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Schweikl H, Schmalz G, Spruss T. The induction of micronuclei in vitro by unpolymerized resin monomers. J Dent Res. 2001;80(7):1615–20. 10.1177/00220345010800070401 [DOI] [PubMed] [Google Scholar]
  • 25.Bakopoulou A, Papadopoulos T, Garefis P. Molecular toxicology of substances released from resin-based dental restorative materials. Int J Mol Sci. 2009;10(9):3861–99. 10.3390/ijms10093861 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Schmalz G, Hickel R, van Landuyt KL, Reichl F-X. Scientific update on nanoparticles in dentistry. Int Dent J. 2018;68:299–305. 10.1111/idj.12394 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Schubert A, Ziegler C, Bernhard A, Burgers R, Miosge N. Cytotoxic effects to mouse and humangingival fibroblasts of a nanohybrid ormocer versus dimethacrylate-based composites. Clin Oral Investig. 2019;23:133–9. 10.1007/s00784-018-2419-9 [DOI] [PubMed] [Google Scholar]
  • 28.Gosavi SS, Gosavi SY, Alla RK. Local and systemic effects of unpolymerised monomers. Dent Res J (Isfahan). 2010;7(2):82–7. [PMC free article] [PubMed] [Google Scholar]
  • 29.Prica D, Galić N, Želježić D, Prica A, Šegović S. Genotoksični potencijal dentinskih adheziva. Acta Stomatol Croat. 2007;41(2):104–14. [Google Scholar]
  • 30.Galić N, Prpić-Mehičić G, Prester LJ, Krnić Ž, Blanuša M, Erceg D. Elimination of mercury from amalgam in rats. J Trace Elem Med Biol. 2001;15:1–4. 10.1016/S0946-672X(01)80018-3 [DOI] [PubMed] [Google Scholar]
  • 31.Tadin A, Galić N, Mladinić M, Marović D, Kovačić I, Želježić D. Genotoxicity in gingival cells of patients undergoing tooth restoration with two different dental composite materials. Clin Oral Investig. 2014;18(1):87–96. 10.1007/s00784-013-0933-3 [DOI] [PubMed] [Google Scholar]
  • 32.Tadin A, Marović D, Galić N, Kovačić I, Želježić D. Composite-induced toxicity in human gingival and pulp fibroblast cells. Acta Odontol Scand. 2014;72(4):304–11. 10.3109/00016357.2013.824607 [DOI] [PubMed] [Google Scholar]
  • 33.Ahmed RH, Aref MI, Hassan RM, Mohammed NR. Cytotoxic effect of composite resin and amalgam filling materials on human labial and buccal epithelium. Nat Sci. 2010;8(10):48–53. [Google Scholar]
  • 34.Mary SJ, Girish KL, Joseph TI, Sathyan P. Genotoxic Effects of Silver Amalgam and Composite Restorations: Micronuclei-Based Cohort and Case–Control Study in Oral Exfoliated Cells. Contemp Clin Dent. 2018;9(2):249–54. 10.4103/ccd.ccd_849_17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, et al. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: The HUMN project perspective on current status and knowledge gaps. Mutat Res. 2008;659(1-2):93–108. 10.1016/j.mrrev.2008.03.007 [DOI] [PubMed] [Google Scholar]
  • 36.Sommer S, Buraczewska I, Kruszewski M. Micronucleus Assay: The State of Art, and Future Directions. Int J Mol Sci. 2020;21(4):1534. 10.3390/ijms21041534 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: methods development. Mutat Res. 1992;271(1):69–77. 10.1016/0165-1161(92)90033-I [DOI] [PubMed] [Google Scholar]
  • 38.Gavić L, Goršeta K, Buterin A, Glavina D, Želježić D, Tadin A. Assessment of Cytotoxic and Genotoxic Effect of Fissure Sealants in Buccal Epithelial Cells. Acta Stomatol Croat. 2021;55(1):10–7. 10.15644/asc55/1/2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vladislavić Zorica N, Franić I, Gavić L, Tadin A. Assessement of cytotoxic and genotoxic effect of whitening toothpastes in buccal mucosal cells. Acta Stomatol Croat. 2022;56:441–6. [Google Scholar]
  • 40.Radović M, Gavić L, Jerković D, Želježić D, Puizina J, Srzentić I, et al. Clinical Prospective Assessment of Dental Implants in Gingival Epithelial Cells. Acta Stomatol Croat. 2022;56(3):222–34. 10.15644/asc56/3/1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ribeiro DA. Cytogenetic biomonitoring in oral mucosa cells following dental X-ray. Dentomaxillofac Radiol. 2012;41:181–4. 10.1259/dmfr/14555883 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Reichl FX, Simon S, Esters M, Seiss M, Kehe K, Kleinsasser N, et al. Cytotoxicity of dental composite (co) monomers and the amalgam component Hg(2+) in human gingival fibroblasts. Arch Toxicol. 2006;80:465–72. 10.1007/s00204-006-0073-5 [DOI] [PubMed] [Google Scholar]
  • 43.Rathore M, Archana Singh A, Pant VA. The Dental Amalgam Toxicity Fear: A Myth or Actuality. Toxicol Int. 2012;19(2):81–8. 10.4103/0971-6580.97191 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Gupta SK, Saxena P, Pant VA, Pant AB. Release and toxicity of dental resin composite. Toxicol Int. 2012;19(3):225–34. 10.4103/0971-6580.103652 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ramirez A, Saldanha PH. Micronucleus investigation of alcoholic patients with oral carcinomas. Genet Mol Res. 2002;1:246–60. [PubMed] [Google Scholar]
  • 46.Stich HF, Stich W, Rosin MP, Vallejera MO. Use of the micronucleus test to monitor the effect of vitamin A, beta-carotene and canthaxanthin on the buccal mucosa of betel nut/tobacco chewers. Int J Cancer. 1984;34:745–50. 10.1002/ijc.2910340602 [DOI] [PubMed] [Google Scholar]
  • 47.Van Schooten FJ, Besaratinia A, De Flora S, D’Agostini F, Izzotti A, Camoirano A, et al. Zandwijk. Effects of oral administration of N-acetyl-L-cysteine: a multibiomarker study in smokers. Cancer Epidemiol Biomarkers Prev. 2002;11:167–75. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Acta Stomatologica Croatica are provided here courtesy of University of Zagreb: School of Dental Medicine

RESOURCES