Effect of Dental Local Anesthetics on Reactive Oxygen Species: An In Vitro Study

Introduction Oxidative stress, an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, plays an important role in various dental diseases. Local anesthetics are frequently used in dentistry. The potential antioxidant activity of dental local anesthetics can contribute to dental practice. Therefore, this study aimed to investigate the ROS-scavenging activities of three commonly used dental local anesthetics, lidocaine, prilocaine, and articaine, focusing on their effects on hydroxyl radicals (HO•) and superoxide anions (O2•−). Materials and methods The electron spin resonance (ESR) spin-trapping technique was employed to specifically measure the ROS-scavenging activities of these local anesthetics at varying concentrations. Results Lidocaine, prilocaine, and articaine exhibited concentration-dependent HO•-scavenging activities, with IC50 values of 0.029%, 0.019%, and 0.014%, respectively. Lidocaine and prilocaine showed concentration-dependent O2•−-scavenging activity, with IC50 values of 0.033% and 0.057%, respectively. However, articaine did not scavenge O2•−. Conclusions The proactive use of dental local anesthetics may mitigate oxidative injury and inflammatory damage through direct ROS scavenging. However, further research is needed to elucidate the specific mechanisms underlying the antioxidant effects of these dental local anesthetics and their potential impact on the dental diseases associated with oxidative stress.


Introduction
Oxidative stress, an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, plays an important role in various dental diseases.Local anesthetics are frequently used in dentistry.The potential antioxidant activity of dental local anesthetics can contribute to dental practice.Therefore, this study aimed to investigate the ROS-scavenging activities of three commonly used dental local anesthetics, lidocaine, prilocaine, and articaine, focusing on their effects on hydroxyl radicals (HO • ) and superoxide anions (O 2 •− ).

Materials and methods
The electron spin resonance (ESR) spin-trapping technique was employed to specifically measure the ROSscavenging activities of these local anesthetics at varying concentrations.

Introduction
Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the ability of the body to neutralize them through antioxidant defenses.Oxidative stress plays a significant role in various aspects of medicine as it is involved in the pathogenesis of various diseases and the aging process [1,2].Notably, oxidative stress has been implicated in the pathogenesis of diseases, such as cardiovascular and neurodegenerative diseases, metabolic disorders, chronic inflammatory diseases, cancers, aging, and age-related diseases [1][2][3].Oxidative stress also influences several pathologies in dentistry and oral maxillofacial surgery.Increased oxidative stress markers have been observed in patients with periodontitis, suggesting a link between ROS and rapid destruction of periodontal tissue [4].ROS damage the DNA and promote cell proliferation and the development of oral squamous cell carcinoma and other types of oral cancers [5,6].Therefore, oxidative stress may play a role in medication-related jaw osteonecrosis by inhibiting bone healing and promoting inflammation [7,8].Additionally, oxidative stress is involved in dental caries, oral lichen planus, oral submucous fibrosis, and aphthous stomatitis [9][10][11][12].
ROS are a group of independently existing molecules that possess at least one oxygen atom and one or more unpaired electrons.ROS includes various oxygen-containing free radicals, such as hydroxyl radical (HO • ), superoxide anion (O 2 •− ), hydrogen peroxide, peroxyl radicals, hydroperoxides, and alkoxyl radicals [13].HO • is one of the most powerful oxidizing agents among free radicals and can cause significant cellular damage by inducing lipid peroxidation, protein oxidation, and DNA strand break [14][15][16].O

Statistical analysis
To establish a baseline, the spectral intensity after the addition of distilled water was designated as I 0 .The

HO • -scavenging activities of dental local anesthetics
To determine the antioxidant properties of dental local anesthetics, we examined the HO • -scavenging activities of lidocaine, prilocaine, and articaine.Notably, distilled water exhibited strong HO • generation (Figure 2A-2C).However, HO • generation was significantly inhibited by lidocaine (Figure 2A), prilocaine (Figure 2B), and articaine (Figure 2C) in a concentration-dependent manner, with IC 50 values of 0.029, 0.019, and 0.014 w/v%, respectively (Figure 2D).Although several studies have shown the antioxidant effects of local anesthetics, reports on the direct capture of ROS are limited.For example, studies using the ESR spin-trapping technique showed that lidocaine scavenged HO • in a concentration-dependent manner, with IC 50 of approximately 80 µM [26,27].
Considering that the molecular weight of lidocaine is 288.81, its IC 50 (80 µM) corresponded to 0.0023%.
Overall, the results of the present study are consistent with the previous reports (Figure 2A, 2D) [26,27].
However, the concentrations at which the antioxidant effect against HO • was detected were lower than those typically used in clinical practice (0.5-2%).In dental practice, 2% lidocaine containing epinephrine (1:80,000) prepared in dedicated cartridges is typically used.Notably, the concentration of lidocaine in the oral mucosa was diluted to approximately 360-120 μg/g after 10-60 min of injection with 0.5 mL of the local anesthetic [28].Considering that the IC 50 of lidocaine against HO • in the present study was approximately 0.029% (Figure 2D), may sufficiently scavenge HO • even when injected locally.In contrast, lidocaine did not reportedly scavenge O  3D), thereby confirming its limited scavenging activity.
Notably, we demonstrated the direct ROS scavenging activities of prilocaine and articaine.A study on lipid peroxidation using a liposome membrane system showed that prilocaine has considerably lower antioxidant activity than other local anesthetics [30].Additionally, Hattori M et al. showed that O 2 •− inhibitory effect of prilocaine in neutrophils was as low as that of lidocaine [29].Moreover, a study using the xanthine-xanthine oxidase-induced chemiluminescence showed that prilocaine inhibited O 2 •− levels in a concentrationdependent manner but did not inhibit HO • [31].In this study, similar to lidocaine, prilocaine showed high HO • -and O 2 •− -scavenging activity (Figures 2, 3).Additionally, articaine effectively scavenged HO • in a concentration-dependent manner (Figure 2C, 2D), but did not scavenge O 2 •− (Figure 3C, 3D).Articaine differs from other amide-based local anesthetics as it contains an ester bond and a thiophene ring [32].Therefore, articaine has higher lipid solubility compared with other local dental anesthetics and undergoes hydrolysis by nonspecific cholinesterase in the body [32].In a study using the xanthine-xanthine oxidaseinduced chemiluminescence, high articaine concentrations showed reactivity against O 2 •− [31].Although whether articaine can effectively scavenge O 2 •− is debatable, the differences in chemical structure may stiff affect its O 2 •− -scavenging ability.
Local anesthetics are used to manage local pain in clinical settings and dental practice.Lidocaine eliminates ROS through direct scavenging, ROS-generating enzyme inhibition, mitochondrial protection, inflammatory pathway modulation, and antioxidant defense upregulation [14][15][16][17][18][33][34][35].Therefore, these multiple mechanisms may have contributed to the antioxidant properties of prilocaine and articaine in the present study, and the proactive use of dental local anesthetics may potentially mitigate oxidative injury and inflammatory damage.Recently, a study using lipid raft model membranes reported that local anesthetic-induced lipid raft disruption may indirectly affect the activity of raft-associated proteins, leading to anesthetic action [36].Lipid peroxidation, which leads to ROS production, may be one of the mechanisms underlying lipid raft disruption.
Despite the promising findings, this study had several limitations.First, we only focused on the chemical reaction of ROS-scavenging activities of local anesthetics.Second, although the antioxidant mechanism of lidocaine has been explored, the mechanisms of action of prilocaine and articaine remain speculative.Third, we did not investigate the potential clinical implications of the observed ROS-scavenging activities, and further research is needed to determine whether these properties translate into beneficial clinical outcomes.Finally, considering that this study is in vitro, the results may not be directly applicable to the complex environment of the human body [22].Despite these limitations, the present study provides valuable insights into the potential antioxidant properties of lidocaine, prilocaine, and articaine.Further research is needed to fully understand the mechanisms and clinical implications of these findings.

Conclusions
We investigated the ROS-scavenging activities of lidocaine, prilocaine, and articaine, focusing on their effects on HO • and O 2 •− .These activities were concentration-dependent, except for articaine's O 2 •−scavenging effect.Notably, antioxidant effects were observed at sub-clinical concentrations.While currently used solely for pain relief, our findings suggest these dental local anesthetics may have potential applications in treating oxidative stress-associated oral diseases, expanding their utility beyond pain management.
5 mT, field modulation width of 0.079 mT, sweep time of 1 min, and time constant of 0.03 s.Data acquisition was achieved using WIN RAD ESR Data Analyzer software ver.1.30(JEOL).The gain was set to 320, and the software recorded 4096 data points.HO • and O 2 •− were generated a minimum of six times.
spectral intensity corresponding to each local anesthetic was labeled I.The relative spectral intensity (%Intensity of I/I 0 ) was assessed by calculating the I:I 0 ratio.The concentration dependence of the local anesthetic reaction was determined by fitting the data to the following equation: A=Amax/[1+ (IC 50 /[X] 0 ) h ]+Amin, where IC 50 is the half-maximal inhibitory concentration for each local anesthetic with a Hill coefficient (h) of 1. Amax and Amin are the maximal and minimal responses, respectively[24,25]. [X] 0 indicates the concentration of local anesthetic applied.Data are expressed as the mean ± standard deviation (SD) of n observations, where n represents the number of separate experiments.Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparisons.Statistical significance was set at p<0.05.All statistical analyses were performed using GraphPad Prism 7.05 (Graph Pad Software, La Jolla, CA, USA).

FIGURE 2 :
FIGURE 2: Dose-dependent relationships between hydroxyl radical signal intensities and dental local anesthetics(A-C) Representative typical ESR spectra of HO • in response to various concentrations of lidocaine (A), prilocaine (B), and articaine (C).Signal intensities (in grey dotted boxes) were inhibited by each local anesthetic in a concentration-dependent manner.(D) Concentration-response relationships of different local anesthetics.Data points illustrate I/I 0 as functions of each local anesthetic concentration.Grey circles represent lidocaine; red circles represent prilocaine; and blue circles represent articaine.Curves (solid lines) were fitted according to the equation described in the text.Each data point represents the mean ± SD of data from six separate experiments.Significant differences between data points are indicated using asterisks.*p<0.05.D.W., distilled water; IC50, the 50% inhibitory concentration; ESR, electron spin resonance; SD, standard deviation

FIGURE 3 : 9 Discussion
FIGURE 3: Dose-dependent relationships between superoxide anion signal intensities and dental local anesthetics (A-C) Representative typical ESR spectra of O 2 •− in response to different concentrations of lidocaine (A), prilocaine (B), and articaine (C).Signal intensities (in grey dotted boxes) of D.W. were inhibited by lidocaine (A) and prilocaine (B) in a concentration-dependent manner.(D) Concentration-response relationships of different local anesthetics.Data points illustrate I/I 0 as functions of each local anesthetic concentration.Grey circles represent lidocaine; red circles represent prilocaine; and blue circles represent articaine.Curves (solid lines) were fitted according to the equation described in the text, except for the articaine.Each data point represents the mean ± SD of data from six separate experiments.Significant differences between data points are indicated using asterisks.*p<0.05.D.W., distilled water; IC50, the 50% inhibitory concentration; ESR, electron spin resonance; SD, standard deviation 2 [17]re primarily generated as byproducts of cellular respiration in the mitochondrial electron transport chain or produced by other enzymes, such as NADPH oxidases and xanthine oxidase[17].Importantly, high levels of O 2