Prevalence of Glutathione-S-Transferase T and M Deletion Polymorphisms in Apical Periodontitis: a Two-Center Observational Study

Abstract

Objectives

The primary objective of this study was to examine the potential association between glutathione S-transferases (GSTT1/GSTM1) deletion polymorphisms and the development of apical periodontitis (AP) in a population of patients at two university centers: the Faculty of Medicine at the University of Banja Luka in Bosnia and Herzegovina and the School of Dental Medicine at the University of Belgrade in Serbia.

Materials and Methods

The study involved 200 patients with AP in the experimental and 250 healthy individuals without AP in the control group. As a source of genomic DNA, sterile buccal swabs were taken from each patient. Genotyping of GSTM1 and GSTT1 deletion polymorphisms was conducted using multiplex Polymerase Chain Reaction (PCR). The risk of AP development with regard to the genotypes was evaluated based on odds ratios (ORs) and 95% confidence intervals (CIs) that were calculated via unconditional logistic regression.

Results

There were significant differences in demographic characteristics between the investigated groups (p = 0.446, p = 0.154, respectively). GSTM1 and GSTT1 deletions were associated with a 3.05-fold and 5.69-fold risk (OR = 3.05, 95% CI = 2.07–4.49, OR = 5.69, 95% CI = 3.66–8.86, p < 0.001, p < 0.001, respectively) for the AP development. The co-occurrence of both deletions posed a significantly higher risk for AP development (OR = 52.76. 95% CI = 18.20–152.94, P < 0.001).

Conclusions

The carriers of null GSTT, null GSTM, and double null GSTT/GSTM genotypes are more susceptible to AP development in the populations examined at the two centers.

Introduction

Apical periodontitis (AP) represents a local and systemic inflammatory response of the body to various stimuli that compromise the vitality of the dental pulp, ultimately leading to its necrosis (1). Over the last twenty years, this condition has been alarmingly prevalent worldwide, with a significant increase noted among both untreated and endodontically treated teeth (2, 3). When contextualizing the burden of AP within the broader spectrum of chronic oral conditions, it is important to compare it with marginal periodontitis, a highly prevalent and impactful disease. Recent meta-analysis reports that AP affects approximately 6.3% of teeth in the general adult population, with a prevalence of 41.3% among endodontically treated teeth (2). In contrast, marginal periodontitis has a substantially higher overall prevalence, estimated at 61.6% in dentate adults, with severe forms reaching 23.6% (4). These comparisons underscore the clinical and epidemiological significance of both endodontic and periodontal diseases in global oral health. The widespread occurrence of AP is closely linked to numerous general health impairments and imposes a considerable financial burden on healthcare systems around the globe (5-8). Nevertheless, root canal therapy followed by proper restoration is generally more cost-effective compared to extraction and subsequent prosthetic rehabilitation, such as implants or bridges, which are associated with higher upfront and long-term maintenance costs (9).

The pathogenesis of AP is a complex process characterized by overlapping signaling pathways and inflammatory mediators that drive progressive bone resorption in the periapical region of the affected tooth. Central to this alveolar bone resorption is a definitive bidirectional relationship between multiple etiological factors and the response of the host's immune system (10, 11). It is a well-established that polymicrobial infection affecting the dental pulp that represents the leading cause of apical periodontitis (AP) (12). Furthermore, various factors, including environmental and predisposing elements, have a critical role in inducing and exacerbating the progression of the disease (13).

Numerous investigations have highlighted the influence of heredity on the development of AP (14-17). Recent reviews suggest that specific genotypes of certain genes across various populations are linked to the occurrence and progression of AP, along with its diverse clinical and radiographic manifestations (14-17). However, no definitive genetic marker has been identified that can reliably predict a person’s susceptibility to developing apical periodontitis.

Oxidative stress (OS) arises from a disproportion between the generation of reactive oxygen species (ROS) and the capacity of antioxidant systems to neutralize them, which subsequently leads to cellular damage and inflammation. Previous studies clearly demonstrated the significance of OS in the pathogenesis of apical periodontitis (18). Glutathione S-transferases (GSTs) represent a diverse superfamily of phase II metabolic enzymes that catalyze the conjugation of the tripeptide glutathione (GSH) to a vast array of endogenous and exogenous electrophilic substrates (19, 20). This conjugation process facilitates solubilization, detoxification, and subsequent excretion of the compounds that may be potentially harmful. By mitigating the accumulation of reactive intermediates, GSTs significantly contribute to redox homeostasis and protect cells from oxidative stress, lipid peroxidation, and DNA damage (19, 20). Among the cytosolic classes, GSTM1 (mu), GSTT1 (theta), and GSTP1 (pi) have garnered significant attention in dental research owing to their pronounced polymorphic variability and their implication in the pathophysiology of various inflammatory, neoplastic, and degenerative diseases (19, 20). It has been postulated that disruptions in GST function, due to genetic deletions (e.g., in carriers with GSTM1 or GSTT1 null genotypes), may compromise their protective capacity, thereby contributing to heightened tissue destruction and disease progression (19, 20).

A few studies have previously explored the connection between various GST polymorphisms and the presence of marginal periodontitis (21-26). Their findings highlighted the important role of genetic variations in GST enzymes, particularly GSTM1, GSTT1, and GSTP1, in influencing individual susceptibility to chronic periodontitis. Research has shown that people who have null genotypes of GSTM1 and GSTT1, or those carrying the variant allele (G) at the rs1695 locus of GSTP1, exhibit compromised antioxidant defense, rendering them more susceptible to oxidative stress and inflammatory damage in periodontal tissues (21-26).

Although marginal and apical periodontitis shared similar pathological features, evidence regarding the impact of GST polymorphisms on AP development is lacking. Therefore, the primary objective of this study was to examine a potential association between GSTT/GSTM deletion polymorphisms and the development of apical periodontitis in a population of patients at two university centers: the Faculty of Medicine at the University of Banja Luka in Bosnia and Herzegovina and the School of Dental Medicine at the University of Belgrade in Serbia.

Materials and Methods

The present study represents clinical-laboratory and observational genetic association research. The investigation was performed and reported in alignment with the Strengthening the Reporting of Genetic Association study (STREGA) statement checklist (27) (Supplementary Table 1) and the Preferred Reporting Items for Laboratory and Observational Studies in Endodontology (PRILE, PROBE) guidelines (28, 29).

Ethical considerations

The protocol followed in this two-center investigation was reviewed and approved by both local institutional Research Ethics Committees (protocol numbers: 36/12-2013, and 02-3-4-19-1-6/2022). All study participants or parents/legal guardians provided informed written consent, i.e., an assent document, at both centers. Moreover, the procedures followed were all in accordance with the ethical standards of both the responsible committees on human experimentation (University of Banja Luka, B&H, and University of Belgrade, Serbia) and with the Helsinki Declaration of 1975, as revised in 2002.

Sample description and collection of biological material

The present study involved 200 consecutive individuals exhibiting clinical, radiographic, and histopathological features of AP (30) who sought dental care at the University Dental Clinics in Banja Luka (50 patients) and Belgrade (150 patients) between January 2023 and January 2025. During routine clinical check-ups, information was gathered regarding the patients' general health and habits. The study only included the patients classified as ASA1 and/or 2 following the physical status classification proposed by the American Society of Anesthesiologists. Exclusion criteria were as follows: periodontally involved teeth with probing depths greater than 4 mm or vertical root fractures. In addition, individuals who were immunocompromised or had received antibiotics, antiviral medications, hormone therapy, or immunosuppressive treatments within three months prior to examination were not included in the study. The control group was comprised of 250 DNA samples taken from buccal swabs of healthy patients from the University Dental Clinics in Banja Luka (50 patients) and Belgrade (200 patients) with no history of AP.

As a source of genomic DNA, sterile buccal swabs were taken from each patient. All collected samples were refrigerated at 2–8°C overnight and stored at -70 °C before transport and DNA extraction.

DNA extraction, selection of genetic polymorphisms and genotyping

GSTM1 and GSST1 genes were chosen as the vital components of the cellular antioxidant defense system, and the deletion polymorphisms were included to evaluate their potential role in susceptibility to AP. These variants were chosen based on earlier findings that were able to associate them with the presence of marginal periodontitis (21-26). Homozygous deletions of these genes result in complete loss of enzyme activity, thus leading to impaired detoxification of reactive oxygen species.

All laboratory procedures were performed in scientific laboratories at the Implant-Research Center, School of Dental Medicine, and University of Belgrade, Serbia. The samples collected at the other center were appropriately stored and transported in a liquid nitrogen canister to the laboratory facilities. Genomic DNA that was used for the genotyping analysis was isolated from buccal cells collected using cotton swabs, as adopted in previous evaluations (31). The amount and purity of the DNA was identified by a BioSpec-nano UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan).

Genotyping of GSTM1 and GSTT1 deletion polymorphisms was conducted using multiplex PCR (32). Multiplex PCR mix (total volume 25 µL) was prepared using PCR Master Mix (2X) (Thermo Fisher Scientific) and contained 200 nmol /L of each primer, namely: for GSTT1 5’-TTCCTTACTGGTCCTCACATCTC-3’ and 5’-TCACCGGATCATGGCCAGCA -3’; for GSTM1 5’-GAACTCCCTGAAAAGCTAAAGC-3’ and 5’-GTTGGGCTCAAATATACGGTGG-3’ (32) (Microsynth AG, Balgach, Switzerland) and 0.2 µg of genomic DNA. The amplification products were separated on 8% polyacrylamide (PAA) gels, then stained using ethidium bromide and exposed to UV light for visualization. Amplification of a segment of b-globin gene (110 bp) using primers 5’-ACACAACTGTGTTCAACTAGC-3’ and 5’-CAACTTCATCCACGTTCACC-3’ (33) served as an internal amplification control (IAC) in order to avoid false-negative results. Finally, GSTM1 null genotype (M1–/–) did not exhibit a 215 bp amplicon, while GSTT1 null genotype revealed no 480 bp amplification product.

Statistical analysis

SPSS Statistics for Windows Software, version 22.0 (IBM Corp, Armonk, NY, USA) and R statistical software (R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria) were used to carry out all calculations. In order to assess potential differences in categorical variables, the Pearson chi-square test and Fisher’s exact test were performed. The Student’s t-test or Mann–Whitney U-test were used to determine the statistical significance between numerical data. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) the values of which were then considered in order to estimate the risk of AP development with regard to the genotypes. The statistical analyses were two-sided, and p-values of 0.05 were perceived as statistically significant.

Results

Due to the nature of the genotyping method, it was not possible to determine the allelic distribution for the GSTM1 and GSTT1 deletion polymorphisms.

Among AP patients, 96 were male and 104 female, while the control group consisted of 110 male and 140 female participants. There was no statistical difference in gender distribution (Pearson’s chi-square test, p = 0.446). The mean age of the AP group was 37.72 ± 12.89 years, and the control group was 35.60 ± 12.01 years. No significant difference in age distribution was observed (Student’s t-test, p = 0.154).

The presence of GSTM1 deletion was detected in 131 AP patients (65.5%) and 96 controls (38.4%). Furthermore, this genotype was associated with a 3.05-fold risk increase for developing AP (OR = 3.05, 95% CI = 2.07–4.49, p < 0.001; Table 1).

Table 1. Genotype frequencies of GSTM and GSTT deletion polymorphisms in patients with apical periodontitis and controls.

Polymorphism	AP N = 200 (%)	Controls N = 250 (%)	P value	OR (95% CI)
GSTM deletion
GSTM +	69 (34.5)	154 (61.6)	ref
GSTM -	131 (65.5)	96 (38.4)	<0.001	3.05 (2.07-4.49)
GSTT deletion
GSTT +	99 (49.5)	212 (84.8)	ref
GSTT -	101 (50.5)	38 (15.2)	<0.001	5.69 (3.66-8.86)
GSTM/GSTT deletion
GSTM +/GSTT +	43 (21.5)	121 (48.4)	ref
GSTM +/GSTT -	25 (12.5)	33 (13.2)	0.026	2.13 (1.14-3.98)
GSTM -/GSTT +	57 (28.5)	92 (36.8)	0.031	1.74 (1.08-2.82)
GSTM -/GSTT -	75 (37.5)	4 (1.6)	<0.001	52.76 (18.20-152.94)
AP, apical periodontitis; N, number of patients; OR, odds ratio; CI, confidence interval; GST, glutathione-S-transferases;

GSTT1 deletion was observed in 101 AP patients (50.5%) and 38 controls (13%), leading to a more prominent increase in the risk for the development of AP (OR = 5.69, 95% CI = 3.66–8.86, p < 0.001; Table 1).

In the combined analysis of both polymorphisms, the deletion of GSTT1 in the presence of GSTM1 affected the risk for AP development more notably (OR = 2.13, 95% CI = 1.14–3.98, p = 0.026) than vice versa (OR = 1.74, 95% CI = 1.08–2.82, p = 0.031; Table 1). Strikingly, 75 out of 200 patients with periapical lesions (37.5%) and 4 out of 250 controls (3.3%) exhibited the so-called double null genotype, i.e., deletions of both GSTM1 and GSTT1. The co-occurrence of both deletions posed a significantly greater risk for AP development (OR = 52.76, 95% CI = 18.20–152.94, P < 0.001; Table 1).

Table 1 Genotype frequencies of GSTM and GSTT deletion polymorphisms in patients with apical periodontitis and controls

Discussion

Recent scientific evidence clearly shows that AP should be considered as a multifactorial disease, highlighting the complexity of its underlying causes (1, 12, 13). While the polymicrobial infection of the pulp is undoubtedly the primary factor in its development, various environmental and predisposing factors also can significantly contribute to AP development (1, 12, 13). Exploring this issue, we aimed to assess the frequency of GSTT and GSTM deletion polymorphisms in patients with AP and to compare them with healthy individuals without a history of AP. It has been revealed that carriers of null GSTT, null GSTM, and double null GSTT/GSTM genotypes are significantly more susceptible to developing AP. Given the critical role of GST in maintaining normal tissue homeostasis (19, 20), these results strongly suggest that antioxidant defense is usually compromised in individuals inheriting these specific genotypes. Consequently, they are more vulnerable to oxidative stress and inflammatory damage affecting their periapical tissues.

The obtained findings are in alignment with previously reported results in the field of Periodontology (21-26). Namely, there have been a few studies that focused on investigating the role of different GST polymorphisms and susceptibility to the development of multiple forms of marginal periodontitis (21-26). Kim et al investigated the prevalence of the polymorphisms of genes Cytochrome P450 (CYP) 1A1, CYP2E1, and GSTM1 that have a role in metabolic activation and detoxification of tobacco-derived substances in 115 individuals with periodontitis and 126 controls (22). The authors revealed that individuals with the GSTM allele were at a significant risk of developing periodontitis (OR = 2.1, 95% CI = 1.3–3.6) (22). Concolino et al (23) analyzed the association between GSTT and GSTM deletion polymorphisms and chronic and aggressive periodontitis cases (75 individuals) in a population of Caucasians. They reported there was a significant link between the GSTM-null genotype and both chronic and aggressive forms of periodontitis. In addition, the forms classified as aggressive correlated with the double null combination of GSTM and GSTT genes (23). Similarly, Arshad et al (24) found that the presence of both GSTT and the mutant allele (G) at rs1695 in GSTP significantly correlated with chronic periodontitis in a population of Pakistanis. As opposed to these data, a study conducted in a population of Czech Caucasians was not able to establish a strong link between GST gene variants and an increased risk of developing periodontitis (28). Although the current findings are promising, further research is required in order to confirm the impact of GST polymorphism on the development of various types of periodontal diseases.

Despite the growing number of genetic association studies in Endodontology (34), these specific polymorphisms have not been thoroughly examined. In contrast, recent research has focused on the role of other genes related to oxidative stress imbalance (35-37). By employing logistic regression analysis, Meyfarth et al (35) found that SNPs in nitric oxide synthase (rs2297518 and rs2779249) were not associated with persistent AP and SNP-SNP interactions in a Brazilian population. Da Silva Guimarães et al (36) reported that superoxide dismutase (SOD) gene polymorphisms [SOD2 (rs5746136, rs4880, and rs10370) and SOD3 (rs2855262 and rs13306703)] can influence oral health-related quality of life response in individuals with asymptomatic periapical periodontitis. Finally, Karataş et al (37) evaluated the correlation between polymorphisms in catechol-O-methyl transferase (COMT), opioid receptor Mu 1(OPRM1), and serotonin receptor genes [5-Hydroxytryptamine Receptor 1A (5HT1A), (5HT2A), and (5HTR3B)] and recorded postoperative pain intensity levels after undergoing root canal treatment. It was concluded that the SNPs assessed in relation to COMT, OPRM1, 5HT1A, 5HT2A, and 5HTR3B genes could not be linked to the intensity of postoperative pain. However, additional investigations in this field are essential to establish a definitive conclusion regarding a potential association.

The most prominent limitation of the current study is the restricted number of SNPs that were investigated. Recently, Petty et al (38) were the first to perform a genome-wide association study (GWAS) of apical periodontitis in a population that was large and well-characterized. Namely, the authors provided ample evidence pointing to host-mediated genetic effects on susceptibility to developing apical periodontitis. It has been stated that genome-wide analyses have identified a top-associated variant situated near the RAP1 GTPase-activating protein (RAP1GAP), which functions as an inhibitor of Ras-related protein 1 (RAP1) (38). It is crucial in both physiological and inflammatory pathways, including macrophage-mediated phagocytosis, chemokine-driven cell adhesion, and leukocyte trafficking, as well as the homing of lymphocytes and dendritic cells. Moreover, it also facilitates cellular attachment to extracellular matrix components such as fibronectin, fibrinogen, collagen, and laminin (39). Furthermore, the authors stated that the associations identified by GWAS are not to be explicitly linked to disease causation. Instead, follow-up studies focusing on functional implications should be prioritized in future research (39).

There are also several important strengths of this study. This study was performed and reported according to STREGA guidelines for genetic association studies (27). Moreover, the authors followed all recommendations for primary clinical investigations proposed in the latest umbrella review related to this topic (14). In addition, this is the first two-center study that merged the investigated populations from two university centers (200 individuals with AP and 250 healthy controls, in total). As a result, the primary benefits of this study included enhancing statistical power and increasing the generalizability of the observed results.

Insights into individual genetic variations hold promise for advancing precision-based dental care, thus allowing for more individualized prevention and treatment approaches. Detection of gene polymorphisms linked to exaggerated inflammatory responses may help clinicians estimate a patient's risk of developing or progressing oral diseases. Such genetic insights could also support the customization of therapeutic strategies, thus ensuring better clinical efficacy and a reduced likelihood of disease relapse. Considering the vital role of GSTs in redox balance and tissue health, it is imperative that individuals with compromised antioxidant defenses, particularly those at a heightened risk of cancer, undergo targeted screening for AP. This necessity underscores a significant area for future translational research and preventive strategies.

Conclusion

The carriers of null GSTT, null GSTM, and double null GSTT/GSTM genotypes are more susceptible to the development of disease in the investigated two-center population. Further genetic association studies involving a broader range of relevant gene polymorphisms, their functional implications, and larger population samples are required to validate the significance of the polymorphisms examined in this study.