Oxidative genomic damage in pediatric patients exposed to mercury released by dental amalgam

Abstract

Background

Mercury (Hg) is the major component of dental amalgam which has been utilized for decades because it is durable, inexpensive and easy to manipulate and position, as well as having a relatively low cost. Hg from dental amalgam fillings has been shown to be released into the buccal cavity due to processes such as abrasion and corrosion, and which could represent a continuous source of oxidative damage to mouth tissues. Therefore, this study assessed the impact of Hg-containing dental amalgams exposure on the oxidative genomic damage using a means of the buccal micronucleus cytome assay by counting the nuclear abnormalities (NAs) in buccal mucosa cells and by analyzing in whole saliva the molecules 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in whole saliva.

Methods

This study was conducted on 120 healthy children divided into two groups-group 1 (or the control group) (n = 60), including participants without dental caries, no history of dental amalgam (Hg-containing) restorations, and no other dental treatments, and group 2 (the intervention group) (n = 60), including participants with dental caries involving enamel or dentin, who received dental amalgam restorations in two posterior teeth. Oral epithelial cell and whole saliva samples were taken at baseline and 30 days later for group 1, while for group 2, samples were obtained at different pre-treatment (baseline) and post-amalgam placement time points at 15 and 30 days.

Results

An increased frequency of NAs (p < 0.05) and higher levels of 8-OHdG (p < 0.05) and MDA (p < 0.05) were observed after Hg-dental amalgams exposure. In addition, a positive correlation existed between NAs and oxidative stress induced by Hg from dental amalgam.

Conclusions

The presents study shows that individuals exposed to Hg-containing dental amalgam exhibit increased NAs in oral epithelial cells, as well as increased levels of 8-OHdG and MDA in saliva, which are directly related to genotoxicity, oxidative DNA damage and lipid peroxidation, respectively. Exposure to Hg- containing dental amalgams may contribute to oxidative stress and genetic damage, potentially leading to long-term implications for both oral and systemic health.

Introduction

The World Health Organization (WHO) includes mercury (Hg) among the ten pollutants of particular concern for public health [1]. Amalgam that contains this element is a principal source of human exposure to Hg and they contribute to the load of this metal in human body tissues [2]– [3]. Hg constitutes the primary component of this type of amalgam, making up 50% of its weight, of dental amalgam [4–6]. Despite the presence of alternative materials, dental amalgam has been utilized for decades due to its durability, cost-effectiveness, ease of manipulation and placement, and relatively low overall cost [7]– [8]. Researchers have documented the harmful health effects of Hg exposure, demonstrating an increased risk of neurological and neurodegenerative, metabolic, and renal conditions, multiple sclerosis; autism; and cardiovascular diseases [9–11].

Some studies have evaluated the safety of dental amalgam restorations in children, and they reported no differences in neuropsychological, renal, neurobehavioral, or nerve conduction velocity values between children with caries restored using dental amalgam and those with caries restored without dental amalgam [12]– [13].

Given that Hg can be released from dental amalgam filling in to the oral cavity and subsequently enter the bloodstream, in vivo studies have demonstrated the genotoxic effect of Hg dental amalgam in oral mucosa cells [14]– [15] and in vitro studies have demonstrated its genotoxic effects in salivary glands and peripheral blood lymphocyte [16]– [17]as well as its potential to induce chromosome aberration [18]. The Hg contained in dental amalgam is lipophilic, but once it enters the body, due to the action of the enzyme hydrogen peroxide catalase, it changes to an inorganic form, which is not lipophilic and cannot as easily resorb into cells [19]– [20]. The Hg has an affinity for sulfhydryl groups, and it is proposed that the attachment of Hg to sulfhydryl groups has a significant effect on DNA damages [21]. In addition, Hg has been to cause enzyme inactivation, inflammation, and oxidative stress [22]– [23]. The genotoxicity of Hg is mainly due to its ability to produce reactive oxygen species (ROS) [24]– [25] (Figure 1). ROS can induce oxidative injury in tissues and affect cells integrity and genetic material [26].

Fig. 1.

Oxidative genomic damage caused by Hg-containing dental amalgams. The mercury contained in amalgam is elemental Hg, which is transformed, due to the action of the enzyme catalase in hydrogen peroxide, into an inorganic form. Once it enters the cell through the plasma membrane, it causes enzyme inactivation, inflammation, the production of ROS, and it bonds to sulfhydryl groups. These affect cellular integrity and genetic material. Because of DNA damage, the production of several products increases, 8-OHdG, along with MN production. Also, MDA levels rise as a product of lipid peroxidation

The use of biomarkers represents a valuable approach to addressing public health concerns, serving as an essential tool for examining the health effects associated with chronic exposure to xenobiotics. A buccal micronucleus cytome (BMcyt) assay is a minimally invasive method and is one of validated cytogenetic methods used to monitor genetic damage induced by an agent [27]– [28].

A BMcyt assay allows us to measure micronuclei (MN) and nuclear buds (NBs) as biomarkers of genome damage; binucleated (BN) cells as biomarkers of cytokinetic defects; and karyolitic (KL), karyorrhexis (KR), condensed chromatin (CC) and pyknotic (PYK) cells as biomarkers of cell death in exfoliated buccal mucosa cells [29]. An increase in these biomarkers is associated with genotoxic and cytotoxic processes, which are associated with a high risk of cancer. Moreover, considering that a high percentage (90%) of cancers have an epithelial origin, BMcyt is an important tool for monitoring the early genotoxic effects of carcinogens, especially when exposed directly to the tissue [29–31].

On the other hand, an important biomarker of oxidative stress is 8-hydroxy-2′-deoxyguanosine (8-OHdG), which is formed through the oxidation of guanine from damaged DNA, and increased body fluid levels of this biomarker have been reported in diseases such as diabetes mellitus, cancer, rheumatoid arthritis, and periodontitis [32–35].

It has been shown that Hg induces membrane associated oxidative stress by enhancing lipid peroxidation [36]. Malondialdehyde (MDA), a widely used marker for lipid peroxidation, is a secondary oxidation product derived from polyunsaturated fatty acids in body fluids or tissues that can attack DNA [37]– [38].

According to the Global Burden of Disease study, dental caries is the most widespread dental condition, ranking first among diseases affecting permanent teeth, with 2.3 billion people impacted, including 560 million children [39]. Despite ongoing efforts to phase out dental amalgam, it remains one of the most widely used restorative materials and a key component of dental services in many countries, including Mexico. In Mexico, the use of Hg-containing dental amalgam is permitted in children and regulated by official standards (NOM-BB-61-1981). Additionally, the high prevalence of dental caries (88.5%) among primary school children [40] contributes to the continued reliance on dental amalgam as a restorative material.

The aim of this study was to analyze the effects of Hg from dental amalgam on genome oxidative damage in the saliva and buccal mucosa cells of children exposed to Hg amalgam. A BMCyt assay was used to detect the potential genotoxic and cytotoxic effects of Hg from dental amalgams, while oxidative damage was assessed by quantifying 8-OHdG and MDA in whole saliva.

The hypothesis of this in vivo study was that mercury released from dental amalgam fillings increases oxidative genomic damage in pediatric patients, as evidenced by a higher frequency of NAs in buccal mucosa cells and elevated levels of oxidative stress biomarkers, specifically 8-OHdG and MDA, in saliva.

Materials and methods

Study population

Using Cochran’s sample size formula calculated with R Studio software, with a 95% confidence level and a 90% test power, the required sample size was determined to be 23 individuals per group. The calculation was based on the mean value reported in the study by Ceppi et al. [41] Accounting for a 10% dropout rate, the final number was adjusted to 26 participants per group.

The study was conducted on 120 healthy children, who were referred by the Pediatric Dentistry Clinic of Universidad de Guadalajara (U de G), Guadalajara, Jalisco, México. The study was approved by the medical ethics review committee at the Universidad de Guadalajara, Guadalajara, Jalisco, Mexico. Written informed consent was obtained from the children’s parents before the study began according to the Mexican General Health Law and the NOM-008-SSA2-1993 norm. Medical and dental history was taken from each participant, and the parents also completed a survey to determine if they met the inclusion criteria for the project. None of the children evaluated had been exposed to other known genotoxic agents.

According to the inclusion criteria (Table 1), the participants were classified as follows: Control group (or group 1), strong emphasis was placed on choosing individuals without dental caries and who maintained good oral hygiene. This group included 60 participants (n = 44 females and n = 16 males with a mean age of 7.06 ± 1.72 years) without dental caries, with history of amalgam fillings or any other restorative materials and no clinical signs of gingival inflammation, and good oral health.

Table 1.

Inclusions and exclusion criteria for selection of study sample

Inclusion criteria	Exclusion criteria
Subjects without dental caries (group 1) and with dental caries (group 2)	Subjects with previous any dental amalgam/composite restorations
Both males and females	Subjects with systemic diseases
Subjects aged 4 to 10 years	Subjects who had undergone radiological procedure (> 1 month)

Group 1: control group, group 2: Hg-amalgam exposed group

The Hg amalgam exposed group (or group 2) comprised individuals with dental caries and before and after dental restorations. It included 60 participants (n = 30 females and n = 30 males with a mean age of 6.89 ± 1.70 years) with dental caries involving the enamel or dentin, for whom amalgam fillings were only used in two posterior teeth, and with a no history of amalgam fillings or any other restorative material (Table 1).

Sample preparation

Samples from the buccal mucosa and saliva were collected from all participants. In group 1, two samples were taken: one at the beginning of the study (day 1: baseline sample) and another 30 days later. In Group 2, three samples were collected: one immediately before the placement of Hg containing amalgam fillings (day 1: baseline sample), one days 15 post amalgam, and one 30 days post amalgam restoration. Fillings for each participant were placed in a single session for each participant, and all restorations were performed by a single pediatric dentistry resident (NGML) to ensure consistency in the procedure. Original D Amalgam (Hg, CAS No: 7439-97-6) was used for the restorations, and the material was mixed using an automated amalgamator (Lorma Mod. AM 21). A rubber dam was used to prevent amalgam contact with the soft tissue of the oral cavity.

Buccal micronucleus cytome assay (BMcyt)

A BMcyt assay was used for in vivo determination of the genotoxic (MN, BN and NBs/genotoxicity markers) and cytotoxic (KL, KR, CC and PYC/cytotoxicity and cell death markers) effects in the buccal mucosa cells of Hg containing dental amalgams (Fig. 2).

Fig. 2.

Specific criteria for identifying nuclear abnormalities in buccal mucosa cells. Panel A: Genotoxic markers. a normal cell, b micronuclei (MN), c binucleated cells (BNs), and d nuclear bud (NBs). Panel B: Cytotoxic markers. e karyorrhexis (KR), f abnormally condensed chromatin (CC), g karyolysis (KL) and h pyknotic cell (PYK). Acridine orange stain, objective 60X

Oral mucosal cell samples were collected directly by scraping both cheeks from each participant using microscope slides with rounded edges. The sample was uniformly distributed on duplicate-coded glass slides, allowed to air dry and fixed with 80% methanol for 48 h. The samples were stained with acridine orange (Sigma-Aldrich, St. Louis, Missouri, USA.) a nucleic acid-specific dye, before the analysis of NAs. For the measurement of BMcyt biomarkers, 2,000 cells per individual were counted using an Olympus CX31 microscope equipped with epifluorescence and oil immersion objectives (x60 and x100; Olympus, Tokyo, Japan). Pre-coded slides were examined by one reader, who blindly counted NAs including MN, BN, NBs, KL, KR, CC, and PYK, according to established guidelines (Fig. 1). The results are presented as the number of cells with NAs per 2,000 cells [29, 42]– [43].

Saliva collection

Each participant was instructed on the collection protocol and approximately 4 mL of unstimulated saliva was obtained from each of them in the morning under resting conditions and in a quiet area. The participants were instructed to rinse their mouths with distilled water and saliva was collected into plastic tubes for 5 min. Then, the saliva samples were centrifuged at 10,000 rpm at 4ºC degrees centigrade for 10 min, and the resulting supernatant was transferred to Eppendorf tubes, which were stored at -80ºC until analysis [44].

8-hydroxy-2-deoxyguanosine (8-OHdG) and MDA determination

The levels of 8-OHdG in the saliva samples were measured using a commercially available competitive ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA). The assays were conducted following the instructions provided by the manufacturers, and a colorimetric assessment was carried out using a microplate reader set at a wavelength of 450 nm.

Salivary MDA levels were measured as an index of lipoperoxidation. The quantification of MDA in saliva was carried out using a modified version of the method described by Yaggi in 1998. A 300 µL saliva homogenate was added to 2mL of sulfuric acid (H₂SO₄) N/12, more 0.3 mL of 10% H₃PO₄ and 1mL of 0.6% of TBA. The mixture was heated in a water bath at 95 °C for 1 h and cooled to room temperature, and then 1.3 mL of n-butanol was added. After vigorous mixing, the solution was centrifuged at 3,000 rpm for 15 min. The butanolic phase (upper layer), containing the MDA thiobarbituric acid adducts, was measured using a spectrophotometer at 534 nm [45].

Statistical analysis

The results are expressed as the mean ± SD. All data were tested for normality using the Kolmogorov-Smirnov test. Differences in NAs, 8-OHdG and MDA values were analyzed using the Wilcoxon test for dependent samples and intragroup comparisons and the Mann-Whitney U test with Bonferroni correction for independent samples and intergroup comparisons. A Spearman correlation was performed to test the relationship between NAs and 8-OHdG and MDA. All tests were performed using the medical package of the Statistical Program for the Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows medical pack where a p < 0.05 was considered statistically significant.

Results

General characteristics of the participants

The general characteristics of all participants in terms of gender and age are displayed in Table 2. The frequency of oxidative genomic damage; NAs, 8-OHdG and MDA; and markers was evaluated in the 120 children who participated in our study, of which 74 were females and 46 were males, with a mean age of 6.98 ± 1.71 years. Group 1, who had healthy teeth and did not have a single dental filling (control group/n = 60), comprise mostly females, accounting for 44 participants, and 16 males (Table 2). Group 2, with dental caries and before and after Hg amalgam restorations (Hg exposed group/n = 60), consisted of 30 females and 30 males (Table 2). The mean age of participants was similar between groups. Neither age nor gender were used as a stratification variable because there were not significant differences between groups (Table 2).

Table 2.

General characteristics from participants per group

Variables	Groups
	1: control	2: Hg amalgam exposed
n	60	60
Female	44 (73.3%)	30 (50.0%)
Male	16 (26.6%)	30 (50.0%)
Age, year	7.06 ± 1.72	6.89 ± 1.70

Age is expressed as mean ± SD; n refers to the sample size

Cytogenetic damage analysis: comparison of the frequency of NAs within the study groups

The mean and SD of cells with NAs (MN, BN, NBs, KL, KR, CC, PYK) were measured at different sampling times, and the results of the statistical analysis of these means from both groups are shown in Table 3. The results from intragroup comparisons indicated that in the control group, which included participants without caries and without amalgam or composite restorations, there were no significant increases in the frequency of NAs in buccal mucosa cells were observed at any sampling time (Table 3).

Table 3.

Mercury-containing amalgam effect on NAs number in buccal mucosa cells at the different sampling time: intra and inter group comparisons

		NAs/2000 cells
	Groups	MN	BN	NBs	KL	KR	CC	PYC
Sampling time	1: control
	Basal sampled	0.48 ± 0.53 (0.00)Aa	1.03 ± 0.78 (1.00)Aa	5.31 ± 1.39 (6.00)Aa	0.06 ± 0.25 (0.00)Aa	0.68 ± 0.83 (0.00)Aa	2.50 ± 2.47 (2.00)Aa	0.21 ± 0.52 (0.00)Aa
	day 30	0.46 ± 0.56 (0.00)A	1.05 ± 0.79 (1.00)A	4.80 ± 1.83 (5.00)A	0.11 ± 0.32 (0.00)A	0.68 ± 0.89 (0.00)A	2.20 ± 2.38 (2.00)A	0.13 ± 0.34 (0.00)A
	Intragroup comparisons	NS	NS	NS	NS	NS	NS	NS
	2: exposed
Sampling time	Basal sampled	1.38 ± 0.93 (1.00)A*b**	3.58 ± 1.92 (3.00)A*b**	4.89 ± 2.60 (6.00)A*b**	2.32 ± 5.35 (1.00) A*b**	4.21 ± 4.46 (3.00)A*b**	8.40 ± 7.19 (6.00)A*b**	0.76 ± 1.20 (0.00)A*b**
	day 15	4.94 ± 2.54 (5.00)B*c**	4.94 ± 2.54 (5.00)B*c**	9.54 ± 5.75 (9.00)B*c**	4.45 ± 6.44 (3.00)B*c**	6.00 ± 4.81 (5.00)B*c**	10.43 ± 6.12(10.00)B*c**	1.60 ± 1.25 (1.00)B*c**
	day 30	2.78 ± 1.78 (3.00)C*d**	5.58 ± 3.42 (5.00)C*d**	9.80 ± 5.26 (11.00)C*d**	4.41 ± 5.50 (3.00)C*d**	6.38 ± 4.28 (7.00)C*d**	7.36 ± 4.87 (8.00)Ad**	2.54 ± 2.47 (2.00)C*d**
	Intragroup comparisons	p = 0.0001*	p = 0.0001*	p = 0.0001*	p = 0.0001*	p = 0.0001*	p = 0.003*	p = 0.004*
	Intergroup comparisons	p = 0.0001**	p = 0.0001**	p = 0.0001**	p = 0.0001**	p = 0.0001**	p = 0.0001**	p = 0.0001**

Data are expressed as mean ± SD (medians in parentheses). Differences in NAs values were evaluated as fallow: Intragroup comparisons (vs. basal): Wilcoxon test. Uppercase letters (A, B, C): significant differences (*p < 0.05) and intergroup comparisons (control vs. exposed): Mann-Whitney U test with Bonferroni correction. Lowercase letters (a, b, c, d): significant differences (**p < 0.05). Means within row with different superscript differ significantly. Sampling times: Basal (before amalgam filling), day 15, and day 30 post-filling. BN (binucleated cells), CC (abnormally condensed chromatin), MN (micronuclei), NAs: nuclear abnormalities; NBs (nuclear buds), NS (not significant), KL (karyolysis), KR (karyorrhexis), PYK (pyknotic nucleus)

In group 2, which included participants with dental caries before and after Hg-amalgam restorations, a comparative analysis was conducted to assess the frequency of NAs in the baseline sample, at day 15 and 30 days post restoration. A statistically significant increase was observed in most of genotoxic and cytotoxic markers in oral mucosa cells after the restoration with Hg-containing dental amalgam (Table 3), as described below:

Intragroup comparisons revealed a significant increase (p = 0.0001) in the frequency of MN, BN, and NBs at both 15 and 30 days post amalgam filling compared to the baseline values (Table 3). Furthermore, Hg containing dental amalgam restorations significantly increased (p < 0.05) the frequency of cytotoxic markers (KL, KR, CC, PYK). However, no statistically significant increase was observed in CC frequency at 30 days post-restoration compared to baseline (Table 3).

Comparison of the frequency of NAs between study groups

The results of the intergroup comparisons are as follows (Table 3):

The frequency of MN increased significantly (p < 0.05) in the basal sampled (p = 0.0001) and 30 days (p = 0.0001) after the amalgam filling process compared with the control group (Table 3). BN cells were significantly more frequent, showing increases in the basal sample (p = 0.0001) and at 30 days (p = 0.0001) post amalgam filling compared to the control group (Table 3). NBs increased significantly at 30 days (p = 0.0001) after the amalgam filling process compared to the control group (Table 3). Regarding cytotoxic biomarkers, cells with KL were significantly more numerous in group 2 compared to the controls. The frequency of KL was higher in the basal sample (p = 0.0001) and after 30 days (p = 0.0001), making KL the most frequent NA observed in buccal mucosa cells in this study (Table 3). The cells with KR showed a higher frequency in the group 2 compared with the control group (p < 0.05), and the basal value was six times higher (p = 0.0001) and nine times higher (p = 0.0001) 30 days post amalgam filling compared with the control group (Table 3). The number of cells with CC also increased significantly in the basal sample (p = 0.0001) and 30 days (p = 0.0001) after restoration compared with the controls (Table 3) and the frequency of PYK increased significantly in the basal value (p = 0.0001) and 30 days (p = 0.0001) after amalgam restoration treatment (Table 3). Moreover, most of the NAs values obtained 30 days after the amalgam filling process were significantly higher in the study group as compared with the control group (Table 3).

Oxidative stress analysis

Oxidative damage was evaluated by quantifying the 8-OHdG and MDA in whole saliva samples from all participants.

Comparison of 8-OHdG and MDA levels in the study groups

Intragroup comparisons showed that 8-OHdG and MDA levels did not increase significantly in the control group at any sampling time (Table 4).

Table 4.

Mercury-containing amalgam effect on salivary 8-OHG and MDA levels at the different sampling time: intra and inter group comparisons

		Groups
	8-OHdG (ng/mL)	1: control	2: exposed	Intergroup comparisons
Sampling time	Basal sampled	2.91 ± 0.85 (2.71)Aa	5.46 ± 0.83 (5.25)A*b**	p = 0.0001 **
	day 15		7.54 ± 0.93 (7.45)B*c**
	day 30	2.64 ± 1.02 (2.66)A	7.17 ± 0.56 (7.12)C*d**
	Intragroup comparisons		p = 0.0001*
	MDA (nM/mL)
Sampling time	Basal sampled	3.10 ± 0.65 (3.19)Aa	5.75 ± 1.59 (5.45)A*b**	p = 0.0001 **
	day 15		6.83 ± 1.23 (6.90)B*c**
	day 30	3.35 ± 0.97 (3.43)A	7.38 ± 0.76 (7.31)C*d**
	Intragroup comparisons		p = 0.0001*

Data are expressed as mean ± SD (medians in parentheses). Results are presented as levels of 8-OHdG in ng/mL and MDA in nM/mL. Differences in 8-OHdG and MDA levels were evaluated as fallow: Intragroup comparisons (vs. basal): Wilcoxon test. Uppercase letters (A, B, C): significant differences (*p < 0.05) and intergroup comparisons (control vs. exposed): Mann-Whitney U test with Bonferroni correction. Lowercase letters (a, b, c, d): significant differences (**p < 0.05). Sampling times: Basal (before amalgam filling), day 15, and day 30 post-filling. 8-OHdG: 8-hydroxy-2-deoxyguanosine; MDA: Malondialdehyde

Meanwhile, in the saliva samples from participants with dental caries and 15 and 30 days after restoration with Hg-dental amalgam fillings, 8-OHdG and MDA levels increased significantly (p < 0.05) compared with the basal values (Table 5).

Table 5.

Statistical correlation analysis between genotoxicity and cytotoxicity markers and oxidative stress biomarkers

	Oxidative stress markers
	8-OHdG salivary levels (ng/mL)				MDA salivary levels (nM/mL)
	Sampling time				Sampling time
	Day 15		Day 30		Day 15		Day 30
	r*	p value	r*	p value	r*	p value	r*	p value
Genotoxicity markers
MN	0.989	0.0001	0.980	0.0001	0.661	NS	0.221	NS
BN	0.130	NS	0.178	NS	0.589	NS	0.052	NS
NBs	0.996	0.0001	0.993	0.0001	-0.661	0.0001	0.259	NS
Cytotoxicity markers
KL	0.137	NS	0.077	NS	0.975	0.0001	0.988	0.0001
KR	0.986	0.0001	0.994	0.0001	0.986	0.0001	0.988	0.0001
CC	0.993	0.0001	0.993	0.0001	0.992	0.0001	0.983	0.0001
PYC	0.139	NS	0.114	NS	0.992	0.0001	0.897	0.0001

*Spearman correlation coefficient. Sampling times: day 15 and day 30 days post-amalgam. Statistical significance was considered with a p value < 0.05. 8-OHdG: 8-hydroxy-2-deoxyguanosine; BN (binucleated cells), CC (abnormally condensed chromatin), MN (micronuclei), NAs: nuclear abnormalities; NBs (nuclear buds), KL (karyolysis), KR (karyorrhexis), PYK (pyknotic nucleus); NS, not significant; MDA: Malondialdehyde

In addition, intergroup comparisons were made between the saliva levels of 8-OHdG and MDA from the group 2 (with dental caries before and after the amalgam filling process) and the control group (Table 4).

The basal levels of 8-OHdG and MDA were increased significantly (p = 0.0001) in group 2 compared with the control group (Table 4), and in the samples taken 30 days after the amalgam filling process, 8-OHdG and MDA levels increased significantly (p = 0.0001) compared with the control group (Table 4).

Correlation analysis between genotoxicity and cytotoxicity markers and oxidative stress biomarkers

The correlations among genotoxic (MN, BN, NBs) and cytotoxic (KL, KR, CC, PYC) biomarkers and oxidative stress biomarkers (8-OHdG and MDA) in saliva 15 and 30 days after exposure to Hg containing dental amalgam filling are shown in Fig. 3a and b. Spearman correlation analysis showed strong significant positive correlations at all sampling times among salivary levels of 8-OHdG with MN (p = 0.0001), NBs (p = 0.0001), KR (p = 0.0001) and CC (p = 0.0001) (Table 5). In addition, salivary MDA levels showed strong significant positive correlations at all sampling times with all cytotoxic biomarkers (p = 0.0001) and a negative significant correlation with NBs (p = 0.0001) (Table 5).

Fig. 3.

Spearman correlation heat maps between genotoxicity (a) and cytotoxicity (b) markers and oxidative stress biomarkers. Sampling time: day 15 and day 30 after amalgam filling process. *p = 0.0001. 8-OHdG: 8-hydroxy-2-deoxyguanosine; BN (binucleated cells), CC (abnormally condensed chromatin), MN (micronuclei), NAs: nuclear abnormalities; NBs (nuclear buds), KL (karyolysis), KR (karyorrhexis), PYK (pyknotic nucleus); MDA: Malondialdehyde

Discussion

The present study provides evidence that in vivo exposure to Hg from dental amalgams is associated with increased oxidative genomic damage in children.

To assess the potential cytogenetic damage caused by Hg amalgam fillings, an in in vivo BMCyt assay was applied. We analyzed NAs related to genotoxicity (MNs, NBs), cytokinesis defects (BNs), early apoptosis (CC), apoptotic cell death (KR, KL), and cell degeneration (PYK). Our findings demonstrate a significant increase in both genotoxic and cytotoxic markers in buccal mucosa cells after exposure to Hg-containing amalgams. Moreover, the baseline differences in NAs and oxidative damage markers (8-OHdG and MDA) between caries-free children and those with caries reflect a pre-existing pro-oxidative and genotoxic state in the group with caries [46]. This state arises from microbial dysbiosis, chronic inflammation, and environmental factors [47]. Such oxidative imbalance likely amplifies the genotoxic effects of Hg released from dental amalgams, as evidenced by elevated post-exposure biomarker levels [46]– [47]. These findings underscore the need for preventive strategies targeting oral redox balance modulation, particularly in high caries risk populations. The higher frequency of NAs and the results of this study are consistent with those reported by other authors [11, 17, 48–52]since in this study, a statistically significant difference in the frequency of total NAs was found between subjects with Hg amalgam fillings (38.87 ± 9.52) and the controls (9.45 ± 3.33). This cloud be because Hg has been shown to be involved in various processes that affect cell structure, such as the generation of ROS, which damage macromolecular structures like lipids and DNA [24, 35, 53]. These findings support the reliability of the cytome NAs assay, as it detects unrepaired DNA damage that is transmitted during cell division [48].

Another factor that poses a cytogenotoxic risk is the ability of Hg to bind to the sulfhydryl groups of motor proteins, which affects the formation of the mitotic spindle and disrupts the microtubule network. This interference prevents the binding of guanosine triphosphate to these proteins and blocks the biochemical cascade that is responsible for cellular function [21, 54]. As a result, chromosome distribution is altered, leading to chromosomal loss or missegregation during mitosis due to structural chromosomal damage [45, 55]. This mechanism may explain the increased frequency of MN and NBs, markers of DNA damage, and BN, a marker of cytokinesis defects [27, 49–51].

In the present work, KL was the most frequent NA observed in this study. This suggests that the concentration and exposure time to Hg released by dental amalgams is enough weaken of cellular machinery due to oxidative stress. Both KR and KL are associated with cell death events, such as apoptosis and necrosis, respectively, which can coexist in some pathological processes. When apoptosis is induced, it can transform into necrosis [56]. This induction is supported by the positive correlation observed between cytotoxic biomarkers and elevated salivary MDA levels. MDA, being one of the main secondary oxidation products derived from polyunsaturated fatty acids, indicates lipid peroxidation resulting from oxidative stress [37]. This finding aligns with the results obtained by Pinheiro et al., who demonstrated that chronic exposure to relatively low levels of Hg can inhibit antioxidant enzymatic activity, leading to progressive oxidative stress [55].

Regarding the positive correlation found between increased 8-OHdG levels in saliva and the frequency of MN, BN, and NBs, this may be due to the strong affinity of Hg and its compounds for altering DNA structure. Li et al., demonstrated that Hg species covalently coordinate with the endocyclic and exocyclic nitrogen sites of DNA bases, approximately every three to four base pairs, binding to either organic or inorganic Hg compounds [57]. This finding is consistent with the work of Ogura et al., who also reported an increase in MN and 8-OHdG levels in peripheral blood lymphocytes exposed to low concentrations of organic and inorganic Hg. They attributed this effect to alterations in the mitotic spindle mechanism and the generation of ROS [58].

On the other hand, Al-Saleh et al., associated the presence of Hg in urine with elevated levels of several markers, including 8-OHdG, which showed a positive correlation. They concluded that dental amalgam do not directly increase DNA damage but, rather, reduces the body’s ability to repair it, a phenomenon associated with prolonged exposure to low levels of Hg [59]. This is also consistent with Crespo-López et al., and Asmuss et al., who demonstrated that Hg exerts genotoxic effects by interfering with DNA repair pathways. Specifically, Crespo-López et al., showed Hg induced impairment of base excision repair (BER) systems, while Asmuss et al., documented its disruption of zinc finger domains in repair proteins through cysteine binding and zinc displacement. These effects are further supported by Ajsuvakova et al., who highlighted Hg high affinity for sulfhydryl groups in DNA repair enzymes, leading to deficient damage response [21, 53, 54]. This interference is attributed to its ability to bind to sulfhydryl groups in cysteines and directly interact with the zinc finger domains, leading to deficient or impaired DNA repair [60]– [54].

Compared to other dental restorative materials, Hg containing amalgams exhibits greater genotoxic potential due to its continuous release of Hg vapor, which is readily absorbed by oral tissues and enters the systemic circulation [61]. In contrast, composite resins and glass ionomers, while not entirely risk-free, primarily release organic compounds such as bisphenol A derivatives, which have been associated with endocrine disruption rather than direct DNA damage [62]. Clinical studies demonstrate clear genotoxic differences, beginning with the work of Visalli et al., who found that amalgam bearing patients develop higher MN frequencies in oral mucosa cells. These findings are reinforced by Chen et al., who reported significantly elevated 8-OHdG levels in urine samples from Hg exposed individuals, while Al-Saleh et al., observed increased MDA levels in patients with amalgam restorations [11, 35, 59]. These findings confirm that amalgam fillings pose greater cumulative genotoxic risk over time [63].

The main route of absorption for Hg released from dental amalgams is through the oral mucosa, where it can induce structural changes in basal epithelial cells. As these cells migrate to the corneal epithelium, morphological alterations become evident, indicating cellular and DNA damage [59]. The local oxidative genomic damage caused by Hg in dental amalgams is evidenced in the present work by elevated levels of MDA and 8-OHdG in the saliva of individuals exposed to Hg dental amalgam, as well as an increased frequency of NAs in their oral mucosa of individuals exposed to Hg dental amalgam.

Moreover, Hg is absorbed systemically [11, 59, 64, 65]. For example, Di Pietro et al., detected systemic genotoxic damage in circulating lymphocytes using the comet assay, demonstrating that individuals who have been treated with dental materials experience continuous and prolonged exposure to Hg, both locally and systemically, leading to toxic and genotoxic alterations [11]. Similarly, in a study conducted by Yildiz et al., the results showed that Hg amalgam led to an increase in serum MDA levels, suggesting that prolonged exposure to low levels of Hg can cause DNA damage [65].

Our findings support our initial hypothesis that Hg released from dental amalgam fillings contributes to oxidative genomic damage in children. The observed increase in NAs in buccal mucosa cells, together with elevated salivary levels of 8-OHdG and MDA, which are both established biomarkers of oxidative stress, demonstrate a clear association between Hg exposure and oxidative cellular damage.

Finally, the limitations of this study include the lack of consideration of other environmental or dietary sources of Hg exposure, as well as the fact that we did not account for potential differences in metabolic responses to Hg, which may have influenced the biomarker levels and genotoxic responses. Additionally, we did not control for lifestyle habits, including diet and antioxidant intake, despite using questionnaires to assess these factors. The consumption of certain foods known to increase mercury release, such as hot or hard foods and drinks, was also not accounted for. However, one strength of this study is that it was conducted on children aged 5 to 8 years who neither smoked nor consumed alcohol, thus eliminating these potential confounding factors known to be associated with genetic damage. Another strength is that by analyzing samples before and at multiple time points after amalgam placement, the study provides a clearer understanding of the temporal relationship between Hg exposure and oxidative genomic damage. Additionally, the study’s in vivo design is a strength, as it allows for the direct assessment of tissues exposed to mercury release from dental amalgams. Samples were collected from buccal mucosa cells and saliva, both of which are in direct contact with the oral environment, providing a more accurate evaluation of the potential genotoxic and oxidative effects.

Conclusions

Under the condition of this study, our finding suggest that Hg-containing dental amalgams used for restorations may contribute oxidative genomic damage in children oral mucosa cells and saliva. This conclusion was drawn using the BMcyt assay, which revealed an increase in NAs in oral mucosa cells, as well as elevated levels of oxidative stress biomarkers, specifically 8-OHdG and MDA, in saliva compared to control subjects without dental restorations.

Abbreviations

8-OHdG

8-hydroxy-2’-deoxyguanosine

BMcyt

Buccal micronucleus cytome

BN

Binucleated

CC

Condensed chromatin

Hg

Mercury

Kl

Karyolitic

KR

Karyorrhexis

MDA

Malondialdehyde

MN

Micronucleus

NAs

Nuclear abnormalities

NB

Nuclear bud

PYC

Pyknotic

ROS

Reactive oxygen species

Author contributions

ML, OG, LR and MB: contributed to the design of the work, MV, SR, S Dela R and GV: were involved in conceptualization; data curation; formal analysis. ZG, GM and ZP: drafted the manuscript, project administration; resources and all authors critically revised it.

Funding

This study was supported by a grant from Institute of Dental Research at the University Center for Health Sciences of the University of Guadalajara (Grant No: 276820/1.1.4.8.15).

Data availability

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

Declarations

Ethics approval and consent to participate

This study was conducted in full compliance with the ethical principles set forth in the World Medical Association’s Declaration of Helsinki (adopted in 1964 and revised in 2024). The study protocol was approved by the Research Ethics Committee of the University of Guadalajara, with the ethical register number CI-7908. Written informed consent was obtained from the parents of all pediatric participants prior to their enrollment, and assent was also obtained from the children when appropriate. All participants provided informed consent prior to their inclusion. The study design and procedures ensured the minimization of risks, respect for autonomy, privacy, and confidentiality of personal data, as well as the fair and safe inclusion of minors as a vulnerable population, in accordance with the relevant articles of the Declaration of Helsinki. All methods were carried out in accordance with the relevant guidelines and regulations throughout the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.