Are Dental Caries Associated with Oxidative Stress in Saliva in Children and Adolescents? A Systematic Review

This systematic review aimed to assess whether dental caries is associated with oxidative salivary stress. The searches were carried out in electronic databases, including PubMed, Scopus, Web of Science, the Cochrane Library, LILACS, OpenGrey, and Google Scholar, without restrictions on the date of publication and language. The acronym PECO was used, in which the participants (P) were children and adolescents exposed (E) to dental caries compared (C) to those without dental caries, with the outcome (O) of modulation of oxidative biochemical parameters. After the search retrieval, the duplicates were removed, and the articles were evaluated by title and abstract, following the inclusion and exclusion criteria. Then, the papers were read and thoroughly assessed. After selection, the risk of bias assessment and qualitative synthesis were performed using the Newcastle-Ottawa Scale (NOS) for observational studies. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) tool was used to assess the level of evidence. A total of 5790 studies were found, and 30 articles were considered eligible and were included for the qualitative synthesis and the level of evidence assessment. The studies showed an imbalance of the antioxidant and pro-oxidant parameters in individuals with dental caries, with primarily increases in both total antioxidant capacity and lipid peroxidation. Most articles showed a low risk of bias, having comparability as the main issue. When exploring through GRADE, a very low level of evidence was found. It was possible to observe an association between oxidative stress and dental caries, showing a disbalance of antioxidants and pro-oxidants, but the evidence level was still very low.


Introduction
Among oral diseases, the most prevalent chronic disease is dental caries [1]. Caries is a multifactorial disease that affects the hard tissues of teeth through metabolites produced by the microorganisms in the oral flora as a result of the imbalance between the demineralization and remineralization processes. Due to the high production of acids by aciduric/acidogenic bacteria, demineralization is more prevalent through frequent exposure to sugars in the biofilm [2].
Saliva, when constantly bathing the teeth and oral mucosa, works as a cleaning solution, having lubricating and buffering actions as well as acting as a reservoir of calcium and phosphate. These minerals are essential ions for the remineralization of the initial carious lesions through the process of remineralization and demineralization of dental enamel that occurs in the oral cavity (DES-RE process). The biochemical composition of of publication bias were the contemplated aspects to rate the overall certainty of evide [19,20].

Selection and Characteristics of the Studies
A total of 5790 records were identified from the searches of the databases, and 2 duplicates were found and removed. The remaining 3136 records were evaluated by and abstract according to the eligibility criteria, and as a result, 3096 studies were exclu at this stage.
The remaining studies (n = 40) were assessed by reading the full text, and ten stu were excluded due to the following causes: three didn't evaluate oxidative stress and ies, one didn't evaluate caries in children and adolescents, five didn't have caries free caries active groups, and one is an in vitro study, conflicting with the previously es lished eligibility criteria. Finally, 30 studies were selected in this systematic review acco ing to the eligibility criteria [10,[13][14][15]. Results are presented in Figure 1.
Only eight studies [13,24,25,35,39,40,44,45] evaluated LPO; six [13,25,35,39,40,44] found an increase in the exposed group; one [24] found that the LPO level was low in all groups; and one found no difference between groups [45]. In 1996, Corvalán et al., showed that the levels of xanthine oxidase, GSH-px, GSH, vitamin C, CAT, and SOD were higher in the control group. On the other hand, Silva et al., Jurczak et al., 2017 showed that levels of SOD, UA, GSH, and GSSG were higher in the exposed group.
Due to the significant methodological heterogeneity of the studies, mainly concerning the method of analysis of the biochemical parameters observed in Table 1 in the analysis tab, it was impossible to perform the quantitative research through a meta-analysis.

Certainty of Evidence
The narrative syntheses for the modulation of oxidative biochemical parameters showed a very low certainty of evidence. The synthesized results on the TAC were affected mainly by the inconsistency of the effect sizes of the studies assessed and the absence of overlap among their confidence intervals. The products on the MDA and NO levels were also affected by the inconsistency of the effect sizes and by the impression due to the limited number of participants evaluated (GRADE recommended rule of thumb threshold: sample sizes larger than 400). This last criterion used to downgrade the evidence's certainty was also applied to all other outcomes, including a single study (GSH, GSSG, GSH/GSSG, SOD, UA).
It is essential to mention that, although it was considered that the syntheses were not affected by indirectness, these results do not provide direct evidence for a specific age group since the included studies evaluated individuals of different ages. Publication bias was unsuspected for all the outcomes. Table 4 shows the certainty assessment of the most relevant results. There was no statistical difference between the exposed and control groups regarding the TAC levels. The LPO levels were higher in the exposed group (active caries) when compared to those in the control group (caries-free; p = 0.001). Considering the comparison between genders, the LPO levels were higher in male participants (p = 0.02) when compared to female participants. The mean salivary MDA in the ECC group (4.8 ± 0.6) was significantly higher than that in the caries-free group (2.9 ± 0.5) (p = 0.01). Total protein levels were higher in the extensive caries groups when compared to those in the other groups (p < 0.001). Moreover, there was a moderate positive correlation between protein levels and caries severity (Spearman's r = 0: 7084, p < 0.0001); The LPO levels were lower in the extensive caries group when compared to those in the other groups (p < 0.0001). Moreover, there was a strong negative correlation between LPO levels and caries severity (Spearman's r = −0.8570, p < 0.0001). The TAC levels were higher in the extensive caries group when compared to those in the other groups (p < 0.001), and there was a strong positive correlation between caries severity and TAC levels (Spearman's r = 0.8.425, p < 0.0001); The SOD activity was higher in the extensive caries group when compared to those in the other groups (p < 0.001), and there was a strong and positive correlation between caries severity and SOD activity (Spearman's r = 0.7320, p < 0.0001); The salivary uric acid levels were higher in the extensive caries group compared to those in the other groups (p < 0.0001). Also, there was a weak and positive correlation between uric acid levels (corrected by protein levels) and caries severity (Spearman's r = 0.4659, p < 0.0001). Griess reaction method (wavelength 540 nm).
The mean concentration of nitrites and nitrates was lower in the exposed group in both conditions: early childhood caries and severe early childhood caries. TAC Assessed by thiobarbituric reactive species production inhibition.
In both situations, the exposed groups, early childhood caries and rampant caries, had higher TAC levels than in the control groups (p < 0.05). The salivary antioxidant status in the exposed group was lower than the levels found in the control group. The salivary TAC levels in the exposed group (active caries) were higher than those in the control group (caries-free). Moreover, the TAC level was higher in younger participants. The salivary TAC levels in the exposed group (active caries) were higher than those in the control group (caries-free). The salivary TAC levels were higher in the exposed group (active caries) when compared to those in the control group (caries-free; p = 0.025). Moreover, the salivary protein levels were higher in the exposed group (p = 0.033). The salivary TAC levels in the exposed group (active caries) were higher than those in the control group (caries-free). Moreover, the total protein levels were higher than in the control group. The salivary TAC and total protein levels increased significantly in the exposed group when compared to those in the control group. The TAC and MDA levels increased in children with active caries when compared to caries-free controls (p < 0.05). Moreover, the total protein levels also increased in the active caries group when compared to those in the control group (p = 0.017). The TAC was significantly higher in the active caries group when compared to that in the control group (p < 0.05). LPO, SOD, and uric acid levels were more elevated in the active caries group when compared to those in the control group (p < 0.05).   The symbol * was adopted to refer to the number of points/stars attributed to each category.   ⊕ VERY LOW a . The certainty of the evidence is downgraded by one level due to the variation in the effect size. There is no overlap between the confidence intervals. b . The certainty of the evidence is downgraded by one level because the total number of individuals included in the synthesis is limited (GRADE recommended rule of thumb threshold: sample sizes larger than 400). The score to GRADE certainty vary from High to very low, ranked as 1-4. ⊕ symbol represents a full score (equivalent to one) and represents a zero score.

Discussion
In this systematic review, 30 articles were found. All of them showed an imbalance in pro-oxidants and antioxidants in children or adolescents with caries, suggesting an association of caries with salivary oxidative stress. The main parameter evaluated was the TAC, which was increased in the group with caries in 16 of the selected articles. Regarding the methodological quality of the studies, 29 of them scored in all domains; however, the level of evidence was very low, indicating that the association of oxidative stress with caries cannot be determined with certainty.
Saliva proved to be a fluid with a high capacity to detect molecules that can act as biomarkers of several oral diseases, such as periodontitis and dental caries [7]. It also serves as a potent means of diagnosing oxidative stress in saliva since we find markers of oxidative stress in saliva, which causes saliva to show a reflection of the changes that occur both in the oral cavity and in the entire organism. It is also related to the balance of both pH and antioxidants in the oral cavity [7]. In this systematic review, these articles investigate the parameters associated with the modulation of antioxidant defenses (TAC, GSH, vitamin C, SOD, UA, and CAT) and pro-oxidants (LPO and NO) in the saliva of patients with dental caries. Of these parameters, those that were shown to be altered in these articles were mainly TAC, LPO, SOD, GSH, and UA, which were increased in the groups with active decay, and the levels of nitrates and nitrites were lower in the group with dental decay. These changes show us that dental caries are associated with antioxidant defense responses and an imbalance in the oxidative process.
Among the pro-oxidant factors evaluated, the chosen studies evaluated LPO and nitrates/nitrites. Some studies included in this review showed that there was an increase in lipid peroxidation (LPO) when they analyzed malondialdehyde (MDA) [13,25,35]. MDA is the final product of LPO and is related to salivary oxidative stress, thus showing whether there is an imbalance of the pro-oxidant and antioxidant systems [48].
Interestingly, no evaluation has been carried out to analyze the oxidative damage of proteins and DNA, although the latter can lead to even more deleterious consequences than damage to lipids. The sulfur-containing amino acids cysteine and methionine are particularly susceptible to ROS, and the oxidative damage to proteins can be measured by protein carbonylation [49]. DNA damage by oxidative stress includes base modifications, basic sites, and strand breaks. While guanine usually pairs with cytosine, oxidized guanosines (8-hydroxy-2'-deoxyguanosine -8-OHdG-, and 8-oxo-7,8-dihydro-2'-deoxyguanosine -8-oxodG-), which are the most frequent type of oxidative base damage, may cause mispairing with adenine through a conformational change. This is a classical route to induced mutations that is also used to evaluate oxidative DNA damage by quantitation of 8-OHdG and 8-oxodG as resulting byproducts [50]. It is somewhat surprising that no study has evaluated the possible oxidative genotoxicity of caries, considering that salivary DNA damage has been used as a marker in other studies [51][52][53]. Furthermore, considering the vulnerable population being analyzed (children and adolescents), it is urgent to obtain reliable results about the possible genotoxic consequences of caries, because this population usually has a long lifespan and, consequently, high probability of accumulating mutations that eventually lead to carcinogenesis.
Nitric oxide is a free radical and is one of the smallest and simplest biosynthesized molecules [54]. NO is synthesized through the oxidation of one of the two guanine nitrogen bases of L-arginine (an essential amino acid for many functions in our body), which is converted into L-citrulline. This reaction is catalyzed by the enzyme NO-synthase (NOS) [55,56]. There are several isoforms of NO-synthase. In the oral cavity, the inducible NOS (i-NOS) that performs NO synthesis in the oral cavity is produced by macrophages and other cells activated by cytokines, and this enzyme is expressed in the salivary glands [56,57]. The increase in NO is related to individuals who have poor hygiene and dental caries [58]. Studies that analyzed NO showed that levels were low in groups with dental caries [27,37]. Nitric oxide participates in the defense of the oral cavity against bacterial multiplication. It is observed that the increase in NO levels in saliva can be a defense mechanism when there is neglect of oral hygiene and an increase in dental caries in individuals [15].
Among the antioxidant factors evaluated, the chosen studies evaluated TAC, GSH, vitamin C, SOD, UA, and CAT. The articles selected in this study that analyzed the TAC showed an increase in these antioxidants in individuals with dental caries [13,14,22,23,[25][26][27][29][30][31][32][33][34][35]38,46]. TAC evaluation is one of the fastest, cheapest, and most accessible methods, thus facilitating the general observation of all antioxidants [59]. The TAC shows the combined effect of antioxidants, mainly non-enzymatic, present in the plasma and body fluids, such as saliva, since they all work together [59]. However, the analysis of TAC has some limitations, mainly because it provides limited information on specific mechanisms of free radical scavenging and therefore cannot provide the contribution of individual antioxidant species to the pathology of specific diseases [59]. In addition, the different systems used to measure TAC appear to be sensitive to different antioxidants, and the oxidative damage index used to define the free radical-induced oxidation process is also different. So, the data between experiments may not be comparable [59]. Thus, the increase in TAC shows an imbalance between antioxidants and pro-oxidants in the oral cavity.
Another analysis that was made in the studies was that of UA. The studies that performed this analysis observed an increase in this antioxidant [15,25]. UA is another non-enzymatic antioxidant. UA is very efficient in eliminating ROS in both the plasma and saliva, which contributes to minimizing the damage caused by the possible oxidative imbalance caused by dental caries [60].
To observe the enzymatic antioxidant system, we performed an analysis of superoxide dismutase (SOD) [61]. SOD catalyzes the dismutation of the superoxide anion into oxygen and hydrogen peroxide so that the superoxide anion causes a decrease in the bioavailability of nitric oxide (NO) [61][62][63]. The articles that analyzed SOD showed an increase in the group with dental caries, which are related to the stage at which dental caries are found. Higher levels of SOD are found in the most severe cases of caries, thereby illustrating an attempt to restore an oxidative balance [15].
Glutathione (GSH) is the most critical low molecular weight antioxidant synthesized in animal cells and is found mainly in the cytosol [64]. Due to the cysteine residues, GSH ends up being oxidized non-enzymatically to GSSG by free radicals, which causes a loss of GSH within the cells. So, GSH/GSSG is the leading redox pair that determines the antioxidant capacities of cells and fluid [65]. GSH is responsible for directly or indirectly neutralizing free radicals through the reaction catalyzed by GPx peroxidase and other peroxidases, thus neutralizing H2O2 and nitric oxide [65]. The levels of GSH are altered in the studies in which it was analyzed, also showing an increase in the group with caries, indicating the body's need to try to fight and neutralize free radicals [14].
It is worth pointing out that the antioxidant capacity measured in the various studies elected may suffer modulation by supplementation with antioxidants and even by diet. Although the articles do not report this, nor was it the object of study in their investigations, these are points that deserve highlighting. Vijayavel et al., 2006 showed that, ascorbic acid and α-tocopherol supplementation reduced lipid peroxidation and increased enzymatic (Superoxide dismutase, Catalase, and Glutathione peroxidase) and nonenzymatic (Glutathione) antioxidants, indicating a possible activity against free radicals [66]. Antioxidants such as ascorbic acid and α-tocopherol can undergo oxidation and thereby provide electrons that will be used by the oxidized glutathione through the action of glutathione reductase. In the same way, fruits and vegetables provides supplementation of vitamins C and E, carotenoids, and flavonoids, which are nutrients with high antioxidant capacity and can act together with endogenous antioxidants against free radicals generated by physiological conditions or by exposure to free radical generating agents [67,68].
As shown by Araujo et al., 2020, antioxidants are altered depending on the stage of caries. Early diagnosis of dental caries is crucial, making the treatment less invasive and more productive [69]. Thus, based on the International Caries Detection and Assessment System (ICDAS) and the International Caries Classification and Management System (ICCMS ™), which were the methods found in the included articles, it allows dentists to have a guide to measure the risk of caries accordingly. Clinical practice is more effective when information is shared with other professionals [48]. The level of caries aggression is directly linked to the imbalance of the pro-oxidant and antioxidant systems; children with severe caries had a higher level of TAC, SOD, UA, MDA, and GSH and lower levels of NO, thus showing a direct relationship between the severity of caries and the activity index of the antioxidant system [13,15,26,37]. There is then an increase in total antioxidant levels to minimize the oxidative damage caused [15].
The levels of antioxidants and pro-oxidants are also altered depending on the age of the individual, which is also related to the severity of this pathology. The study by Araujo et al., 2020 showed that TAC levels could change depending on the individual's age; children have a greater TAC when compared to adolescents. Children with caries in the early stages have more cariogenic bacteria, such as Streptococcus mutans, which have high acidogenic activity and are not found in adolescents with caries [45].
The degree of association of the biochemical oxidative parameters is altered according to age [46]. Salman et al., 2021 conducted the analysis by observing groups of children and adolescents. When they analyzed children from 3 to 12 years, there was no statistical difference that showed an association between the markers of oxidative stress and dental caries. This may be because the immune system is not yet fully formed, which makes the inflammatory process incomplete. When observed in adolescents aged 13 to 18 years, there was a significant decrease in the total antioxidant capacity in the group with caries, which may possibly be associated with the excess of free radicals [46].
The articles included in this systematic review obtained scores in almost all the domains evaluated but presented comparability problems. Age directly influences the antioxidant capacity of saliva; another factor that is directly related to antioxidant capacity is eating habits, in which a healthy diet composed of probiotics and antioxidants derived from fruits and vegetables increases these defenses against oxidative stress. Bad habits, such as smoking, drinking, and eating fast food, are related to a decrease in these defenses, in which it was observed that adolescents had worse habits [70].
The level of evidence of studies carried out jointly by GRADE was considered very low. This tool assesses whether the evidence from the study selection is strong enough to conclude the association of oxidative stress with dental caries. GRADE parameters consider less than 400 participants as a qualifier for severe imprecision. The variation in effect sizes also contributed to the fact that the level of evidence was very low. Larger sample sizes and a combination of more sensitive biochemical analyses are essential to properly observe whether there is a direct relationship between caries and oxidative stress.
When all studies were analyzed, it was possible to observe that there was an agreement between the studies, showing that there is an association between oxidative stress and caries activity in children and adolescents, with an increase in the biochemical parameters evaluated in individuals with caries being also observed, especially those who had caries in a more advanced stage.
However, this systematic review showed that the biochemical modulations between pro-oxidants and antioxidants that occur in saliva are associated with the presence of dental caries. Despite this association, most of the studies do not subdivide into ages, which may affect the results obtained, due to the relationship between age and salivary pro-oxidant and antioxidant levels. Another limitation of this review is that many of the studies included, only evaluated the total antioxidant capacity, which does not give us a complete picture of the biochemical modulations of saliva.
Despite the limitations, this systematic review has shown that with possible advances in the analysis of oxidative biochemical parameters in saliva, we can suggest possible carious formation at an early stage and thus prevent the disease from progressing and showed the biochemical modulation of saliva against carious disease at different ages. More studies with more parameters such as GSH, SOD, LPO, UA, and NO together to give us more details about the relationship between dental caries and oxidative stress in saliva are needed.

Conclusions
It was possible to observe a biochemical modulation linked to caries and to the prooxidant and antioxidant systems. The articles showed a high level of antioxidant response by increasing mainly TAC in the caries group, but we also saw an increase in lipid damage by the LPO parameter in the same group, showing a disbalance of antioxidants and prooxidants. We could see a very low level of evidence. Future studies with more combined analyses will give a clearer picture of the association of caries with oxidative stress.