DL-Buthionine-Sulfoximine

Melatonin is an effective protector of gingival cells damaged by the cytotoxic effect of glutamate and DL-buthionine sulfoximine

Verónica M. Sola1,2 | Juan J. Aguilar3,4 | Ana P. Vazquez Mosquera2,4 | Agata R. Carpentieri1,2
1 Cátedra “B” de Química Biológica, Facultad de Odontología, Universidad Nacional de Córdoba, Córdoba, Argentina
2 INICSA/UNC-CONICET, Enrique Barros esquina Enfermera Gordillo, Ciudad Universitaria, Córdoba, Argentina
3 Instituto Dr. José M.Vanella, Facultad de Ciencias Médicas, UNC, Córdoba, Argentina
4 Cátedra “B” de Introducción a la Física y Química Biológica, Facultad de Odontología, UNC, Córdoba, Argentina

Abstract

Background and Objective: Cellular damage related to oxidative stress (OS) is impli- cated in periodontal diseases (PD). Melatonin (MEL) has multiple functions, and it has been described as a potential treatment for PD. We aim at evaluating the protective effects of MEL on an in vitro model of cellular damage triggered by glutamate (GLUT) and DL-buthionine sulfoximine (BSO), on gingival cells (GCs) in culture.
Material and Methods: A primary culture of GCs from Wistar rats was developed in order to test the protective property of MEL; BSO and GLUT were administered alone as well as in combination with MEL. The viability and apoptosis were measured with MTT assay and TUNEL, respectively, and the concentration of superoxide anion (O2) was measured with the NBT method.
Results: The combination of BSO and GLUT treatment resulted in a decreased viability of GCs. This was evidenced by the increase in both the production of superoxide anion and apoptosis. After MEL administration, the oxidant and pro-apoptotic effects of BSO and GLUT were totally counteracted.
Conclusions: These findings demonstrated that MEL has an effective protective role on GCs subjected to cellular damage in a model of OS and cytotoxicity triggered by BSO and GLUT. Consequently, MEL could be used as a therapeutic agent in PD which begin with a significative loss of GCs.

K E Y WO R D S
cellular damage model, gingival cell culture, melatonin, periodontal disease

1 | INTRODUC TION

In healthy organisms, there are mechanisms and molecules that guarantee that reactive oxygen species (ROS) remain in low or moderate concentrations. The lack of relation between ROS and their neutralization leads to oxidative stress (OS).1 Cellular im- pairments generated by abundant ROS may alter physiological processes such as immune response and inflammation.2 Therefore, OS causes damage to different biomolecules leading to various diseases such as neurodegeneration, diabetes, cancer, and peri- odontitis, among others.3 Periodontitis is one of the most com- mon oral inflammatory conditions among humans. It involves the destruction of the tissues supporting due to the immune re- sponse displayed by the of dysbiotic biofilm.4 Periodontitis is now considered an inflam- matory disease triggered by periodontopathic bacteria, which is necessary but not sufficient for causing the tissue and cellular damage observed clinically.5 Polymorphonuclear neutrophils are the most abundant cells in the periodontal environment; they are the “guardians” in a homeostatic gingival sulcus, where they mi- grate among the epithelium through chemotaxis. Moreover, they are abundant in the dysbiosis, where periodontitis is established, producing a huge amount of ROS.6
However, there is also an endogenous OS enhanced during peri- odontitis when depletion of Glutathione (GSH), a key intracellular antioxidant, occurs. There is a report of a dose-dependent reduction of GSH in periodontal ligament as a result of smoking; furthermore, GSH has been shown to protect against the cytotoxic actions of nic- otine and BSO which was used to cause a synergic effect to study the reduction of GSH.7 It has also been shown that fibroblasts re- lease ROS during periodontal disease (PD) as well.8,9
In the model of periodontitis proposed by Meyle and Chapple,4 cellular damage related to OS was indicated as a critical factor for the initiation and progression of PD. For this reason, a model to study what happens in an environment of endogenous generated ROS related to the depletion of GSH can allow us to identify possible agents that can prevent or stop the progression of cellular damage.
Melatonin (MEL), a ubiquitous hormone that has receptors in most of the cells of the organism, is mainly synthesized and secreted by the pineal gland, and it is recently known to be generated and released by other tissues such as the retina, intestine,10 salivary glands,11 and gingival tissue.12 In addition to its synchronizing, it is remarkable that MEL not only has antioxidant effects due to its direct scavenging of ROS but also indirect effects by stimulating antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase.13,14 Furthermore, MEL demonstrated antia- poptotic effects in different cells of the organism, such as intestinal cells,15 retinal cells,16 and immune cells.17
The beneficial effects of MEL in other tissues10,18-20 led us to hypothesize that MEL could be advantageous both as a preventive agent and as a therapeutic agent in the treatment of PD.21
Primary culture of gingival cells (GCs) is a useful tool to study the outcomes of cellular damage due to OS on the periodontal en- vironment and to test possible drugs that may prevent or diminish cellular injury.22
We can then propose a model of GCs damage, induced by the combination of DL-buthionine sulfoximine (BSO) and glutamate (GLUT), which was previously tested on neuronal cells in our labo- ratory.13 On the one hand, DL-buthionine sulfoximine (BSO), a drug used as a chemotherapy coadjuvant,23 inhibits glutamate cysteine ligase, which is an enzyme that synthesizes glutathione, a key anti- oxidant molecule for the organism.
On the other hand, GLUT is an abundant aminoacidic excit- atory neurotransmitter in the brain.24 There are few reports about its presence and influence on periodontal tissues and in PD.25 However, GLUT is industrially used as a flavor enhancer in the form of monosodium glutamate. Thus, periodontal tissues, particularly the gingiva, are often exposed to this aminoacid. Moreover, Shredah et al26 showed its toxicity on periodontal cells in vivo. Proteolysis caused by periodontopathic bacteria in the periodontal pocket has been found to lead to an increase in GLUT in the periodontal en- vironment.27 As a result, it would be interesting to try GLUT as an OS inducer in GCs.
Through GSH depletion and cytotoxicity, the combination of BSO and GLUT was able to produce OS in ganglionar cells13 and in an immortalized retinal cell line.28
Taking into account these findings, we aim at replicating the oxi- dative model induced by BSO and GLUT in GCs, as well as evaluating the potentially protective effect of MEL in this model.

2 | MATERIAL AND METHODS

2.1 | Chemicals

All chemicals were purchased from Sigma-Aldrich Co unless otherwise stated. Weighed according to the doses indicated below, MEL was freshly prepared by dissolving it with 0.1% ethanol in Minimum Essential Media (MEM, Invitrogen) solution. DL-buthionine sulfoximine and gluta- mate were dissolved only with MEM solutions according to each dose.

2.2 | Cell culture

Gingival tissue for primary GCs culture was obtained from six fe- male rats (150-200 g. body weight). The protocol was approved by the Commission for Care and Use of Laboratory Animals, Faculty of Medical Sciences, National University of Cordoba, in accord- ance with the guidelines laid down by the National Institute of Health in the USA regarding the care and use of animals for ex- perimental procedures and in accordance with local laws and regu- lations. We chose to work with GCs of rats. Primary cell culture of GCs was performed according to Angelopoulos et al29 protocol with some modifications. In short, after the sacrifice of the rats with isoflurane (Baxter, Healthcare Puerto Rico, 00784 USA), the gingival tissue was resected from vestibular gums of the maxil- lary bone and placed in phosphate-buffered saline (PBS). The tis- sue samples were washed and disinfected with ethanol 70% for 2-3 minutes and then washed in PBS. Subsequently, gingiva was cut into pieces, approximately 1 mm2 in size, and placed in a petri capsule with a solution of trypsin 0.25% (Gibco, Thermo Fisher scientific) and in a mechanical agitation at room temperature for 2 hours. Next, the solution containing the cells was spun in a cen- trifuge, and then, the pellet of cells was translocated to a bottle of 25 cm2 with MEM (Gibco) supplemented with 10% fetal calf serum (Natocor), 1 µg/mL gentamicin (Klonal) to prevent growth of microorganisms. The culture plate was incubated at 37°C in a hu- midified atmosphere of 95% air and 5% CO2, and the old medium was replaced with a fresh medium once per week. Cells were used from the second passages through eight passages.

2.3 | Cell viability assay

Cell viability was determined by performing an MTT (3-(4, 5-dimethyl- 2-thiazolyl)-2, 5-di-phenyl-tetrazolium bromide) assay.30 This assay relies on the activity of a mitochondrial dehydrogenase to convert MTT into a purple-colored insoluble formazan product. GCs were cul- tured in a 48- or 96-well plate for 48 hours, and then, the cells were treated. After culturing GCs under the conditions specified for each experiment, the cells were washed with PBS and treated with MTT solution 1.2 M for 2 hours. The formed formazan was dissolved in dimethyl sulfoxide (DMSO), and its absorbance at 570 nm was meas- ured using a microplate reader (BioTek ELx800). The total amount of formazan produced is directly proportional to the number of viable cells in the culture.31

2.4 | Superoxide anion measurements

Cells were washed twice with PBS and incubated with nitroblue tetrazolium (NBT) 6.1 M at 37°C for 1 hours. The formazan precipi- tates formed were dissolved in DMSO and KOH 2 M and quanti- fied at 570 nm using a microplate reader (BioTek ELx800). OD values were considered as direct indicators of ⋅O2 concentration in the samples.15

2.5 | Apoptosis determination

In order to estimate apoptosis, TUNEL (Terminal deoxynucleoti- dyl transferase-mediated dUTP-biotin Nick End Labeling) assay was performed with the In Situ Cell Death Detection Kit, POD (Roche Diagnostics GmbH), according to the manufacturer’s instructions.
Briefly, cells were fixed with paraformaldehyde 4% in PBS. After rins- ing with PBS solution for 5 minutes, the GCs culture was incubated with 20 μg/mL proteinase K for 15 minutes. Afterward, it was rinsed with distilled water and incubated in a solution of 3% hydrogen per- oxide (H2O2) and PBS for 5 minutes at room temperature, in order to prevent enzyme activity. Then, after rinsing the cells twice with PBS for 5 minutes, they were placed inside a darkened humidified chamber and incubated with the TUNEL reaction mix, at 37°C for 60 minutes, to allow the end-labeling reactions to occur. A colored reaction was activated by the addition of diaminobenzidine (DAB) and allowed to elapse for 3-6 minutes. The samples were counterstained with 0.5% methyl green solution, prior to being analyzed by light microscopy (Olympus Motorized Inverted Research IX81. Imaging Software: Cell M). Under light microscopy, the apoptotic nuclei were colored black, whereas non-apoptotic cells were light gray. The TUNEL-positive cells were quantified in relation to the total number of cells and expressed as the percentage of apoptotic index. This was evaluated at a 20× mag- nification with the assistance of ImageJ software.13

2.6 | Statistical evaluation

All data are expressed as means ± SD. The results were evaluated by one-way analysis of variance (ANOVA) and the Tukey’s test as a post hoc analysis. Differences were considered statistically signifi- cant at P < 0.05. All assays were performed in three independent ex- periments done in sextuplicate. All the analyses were carried out by using GraphPad Prism version 6 (GraphPad Software). 3 | RESULTS 3.1 | Melatonin is not toxic in low and high concentrations for GCs In order to corroborate that MEL is innocuous for these cells, we seeded 3 × 105 cells in a 48 well plate with different concentrations of MEL from 32 nmol/L to 1 mM for 48 hours, and viability was eval- uated with MTT assay. MEL was not toxic at all concentrations tested (Figure 1). For this reason and taking into account our previous experiments with neu- ronal cells, MEL concentration of 0.5 mM was chosen for the rest of the experiments, in order to study its antioxidant and antiapoptotic effects.13 3.2 | Determination of minimum inhibitory concentration of BSO and GLUT needed to induce cell damage The minimum inhibitory concentrations (MICs) of BSO or GLUT for producing cell damage were used, based on the microdilution method of the CLSI (formerly NCCLS) (M27-A) 28 using 96-well microtiter plates with some modifications. Each well contained GCs at a final concentration of 2.5 × 103 cells/mL and the tested drugs. The final concentrations of GLUT ranged from 0.3 to 6 mM whereas that of BSO ranged from 0.1 mM to 1.3 mM. Cells without tested agents were included as control. All plates were incubated at 37°C for 48 hours, and viability was determined by spectropho- tometry at 570 nm in a microplate reader (BioTek ELx800). MIC was defined as the lowest concentration of tested agent demon- strating more than 50% of growth inhibition compared with that of the agent-free growth control. The data are reported as the median of at least three independent assays. We used the MIC values of BSO or GLUT to determine the fractional inhibitory con- centration (FIC); that is, the ratio between the MIC of the com- bination of the drugs and the MIC of the agent used alone. The FIC index (S FIC, the sum of individual FICs) was calculated using the formula: ∑FIC = MIC(Acombo)/MIC(Aa-lone) + MIC(Bcombo)/ MIC(Balone). PFIC < 0.5 indicates synergy; 0.5-4.0, indifference; and >4, antagonism.32
The results showed a synergic effect at BSO 0.3 mM and GLUT 0.3 mM, significant decrease in the viability of the GCs when they were treated with GLUT alone in concentrations superior to 0.8 mM (Figure 2A). Nevertheless, low doses of GLUT such as 0.3 mM com- bined with 0.3 mM of BSO demonstrated the best synergic effect on cell damage (∑FIC = 0.075, Figure 2B). The microphotography under light microscope of GCs dyed with crystal violet demon- strated that the damage was consistent with the results of MTT assay. For this reason, concentrations of BSO and GLUT both at 0.3 mM concentration were selected for further experiments.

3.3 | Melatonin protects GCs against the deleterious effects of the combination of BSO and GLUT

With the purpose of understanding the effects of the addition of MEL to BSO and GLUT observed in the experiments shown above, GCs were treated with MEL 0.5 mM and its viability was evaluated after 48 hours. Consistently, GCs viability dramatically decreased with BSO and GLUT treatment. MEL administration totally reversed this effect, restoring GCs viability to a control-like status (Figure 3).
To evaluate whether the decreased cell viability with BSO and GLUT was due to increased superoxide anion radical, a NBT assay was performed. In the groups of GCs treated with these drugs, a significant increase of superoxide anion was observed compared to controls. The addition of MEL restored the superoxide anion to con- trol levels. The results demonstrated a clear protective effect of MEL on GCs (Figure 4).
To investigate whether OS cell death was due to apoptosis, the TUNEL immunohistochemical technique was used: We found that the GCs treated with BSO and GLUT have close to an 80% apoptotic index, while MEL treatment rescued them from the apoptotic effect. The results demonstrated an evident antiapoptotic effect of MEL in GCs (Figure 5).
All these experiments demonstrated a notable protective effect of MEL through antiapoptotic and antioxidant effects on GCs.

4 | DISCUSSION

According to our results, MEL was able to reduce superoxide anion levels and apoptosis index previously increased by the cell-damaging combination of BSO and GLUT. These findings clearly demonstrate that MEL preserves the cell vitality of GCs against oxidative damage in our in vitro model.
The design of an experimental model, capable of mimicking the events that occur when the cells are under endogenous OS related to the GSH depletion, could be useful to correlate with what occurs in vivo, since OS has a key role on PD.33 In this study, we implemented in GCs a novel model of OS which has been previously tested on neurons in order to evaluate the possible protective effect of MEL against the endogen OS.
In our previous study, GLUT was administered in 20 mM concen- trations13 while in the current study, GLUT was toxic for GCs from 0.8 mM (Figure 2B). Yu et al34 found that GLUT receptor NMDA is present in human periodontal ligament fibroblasts. This fact could explain the higher sensitivity of the GCs to GLUT.
Chang et al used BSO in combination with nicotine to show a synergic cellular damage7; however, they used more concentration of BSO than we did in our study (0.5 mM). In the current study, we demonstrate for the first time that the use of the combination of BSO and GLUT is effective in primary cultures of GCs.
Some studies have investigated the effect of MEL as an antia- poptotic agent in different cell lines. We have previously shown that the treatment with MEL protects retinal ganglion cells (RGC) against the damage triggered by BSO and GLUT.13 Nevertheless, in oral cells there are no studies describing the effect of MEL in the OS model using BSO and GLUT.
MEL and its metabolites act as antioxidants by neutralizing ROS. In addition, MEL stimulates antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, catalase, and glutathione reduc- tase10; it also suppresses pro-oxidant enzymes such as nitric oxide synthase.35 MEL is possibly able to activate the enzyme responsible for GSH synthesis, making it more effective for the antioxidant pro- cess. Moreover, other processes involved in such mechanisms could be induced, counteracting in both cases the oxidant effect of BSO.
In addition, MEL has been observed to protect mitochondria against oxidative injuries and to enhance respiratory function, both in vivo and in vitro.36 This effect could explain MEL antiapoptotic action ob- served in the GCs in vitro.
Due to the low toxicity of MEL, its protective effects and its capacity to enhance cell differentiation, this indoleamine could be useful not only in the treatment of PD when the condition is installed but also to prevent the initiation of this inflammatory condition trig- gered by OS.18 In this sense, Renn et al37 demonstrated that MEL was effective as a prophylactic drug to this target. In our study, we found that MEL reversed the OS, by diminishing the superoxide anion.
It is not the first time that the protective properties of MEL in cells of the oral environment have been studied. Proksch et al found that MEL counteracts the toxicity of chlorhexidine digluconate in an osteoblast cell line. They showed that chlorhexidine digluconate, at lower doses than those used clinically for periodontal pocket irriga- tion, resulted toxic to these cells. The treatment with MEL inhibited the apoptosis in this cell line. The authors concluded that melatonin is a “promising tool” for periodontal therapies.38 In this research, GCs primary cultures were used and the damage was characterized by it intensity and duration. It is noteworthy that we found that MEL was non-toxic for GCs in a wide range of doses (Figure 1). In the cellular damage model proposed in this study, MEL was supplied at a much higher concentration than that used by Proksch et al to produce cel- lular protection. This difference in dose may be due to the fact that cells from cell lines are much more resistant to cell damage than cells from a primary culture. Moreover, GCs being undifferentiated are more sensitive to cellular damage, which would indicate that higher doses of MEL should be used. This is useful since a primary cell cul- ture mimics better the cells that gave rise to it and the results can be more reliably transposed.39 Consequently, it is reasonable to think it could be safe to apply MEL on gingival tissue during periodontal treatment.
We demonstrated that in a primary GCs culture, the cell dam- age could be caused mainly by the increase of superoxide anion and apoptosis. We previously found that treatment with MEL protects RGC against the damage triggered by BSO and GLUT.13 Other stud- ies have investigated the effect of MEL as an antiapoptotic agent in various cell lines.40 Although MEL is well known for its synchronizing effects,41 in the last decade there has been an increasing body of ev- idence documenting the antioxidant and antiapoptotic effects of the molecule.2,36,42 Moreover, there are valuable studies in vivo which support these effects. Kara et al43 evaluated the administration of MEL to rats with ligature-induced PD, showing an improvement in the clinical, inflammatory, and oxidant parameters. Kose et al44 demonstrated that melatonin improved not only periodontal status but also hyperglycemia in diabetic rats. In a similar study, Virto et al45 observed that MEL was effective in obese rats as adjunctive ther- apy for reducing alveolar bone destruction and bleeding on probing. Moreover, the levels of biomarkers in the gingival tissue and plasma, such as inflammatory cytokines, insulin, leptin, osteocalcin, osteo- pontin, among others, were restored after the administration of MEL. Balci et al found that adjunctive MEL administration restored clinical periodontal parameters in diabetic rats and it was able to decrease osteoclast numbers.46 MEL was also tested in irradiated rats.47 Radiotherapy increased OS and PD. MEL was able to pre- vent the OS and negative periodontal outcomes like alveolar bone destruction and attachment level. In a study, Balaji et al found that levels of MEL were higher in gingival tissue of individuals with PD.48 In a study of diabetic patients by Cutando et al,49 the topical applica- tion of MEL was found to show improvements to the gingival index and pocket depth, reducing RANKL salivary concentrations and increasing osteoprotegerin salivary concentrations. These findings indicate that topically applied MEL can reduce alveolar bone loss, improving alveolar bone quality and helping to slow the progression of PD. All this evidence together with the findings obtained in the current study could explain the protective function of the hormone on gingival tissue since it is the first immunological barrier periodon- tal pathogens must deal with.
Despite the well-documented effects of MEL on PD and co-morbid diseases, it is not yet used in daily clinical therapeutic procedures. Our study contributes to elucidating the mechanisms by which GCs are damaged during an OS-related cell injury, and it provides new evidence that confirms the protective effects of MEL derived from its antioxidant and antiapoptotic properties. These findings support that the application of MEL may be useful in the treatment of periodontitis together with non-surgical peri- odontal therapy.

5 | CONCLUSIONS

In this study, we demonstrated that MEL has an effective protective role in GCs with cellular damage related to OS. This was evidenced by the reduction of the concentrations of superoxide anion and the decrease of apoptosis triggered by BSO and GLUT model. Our re- search provides new evidence that supports the effective protective role of MEL on GCs and its possible use as a therapeutic agent in PD, which begins with the destruction of the GCs.

R EFER EN CE S

1. Tripathi P, Blaggana V, Upadhyay P, Jindal M, Gupta S, Nishat S. Antioxidant therapy (lycopene and green tea extract) in periodontal disease: a promising paradigm. J Indian Soc Periodontol. 2019;23:25-30.
2. Galano A, Tan DX, Reiter RJ. Melatonin: a versatile protector against oxidative DNA damage. Molecules. 2018;23:530.
3. Rehman K, Akash MH. Mechanism of generation of oxidative stress and pathophysiology of type 2 diabetes mellitus: how are they in- terlinked? J Cell Biochem. 2017;118:3577-3585.
4. Meyle J, Chapple I. Molecular aspects of the pathogenesis of peri- odontitis. Periodontol 2000. 2015;69(1):7–17.
5. Bartold PM, Van Dyke TE. Host modulation: controlling the inflammation to control the infection. Periodontol 2000. 2017;75(1):317–329.
6. Silva LM, Brenchley L, Moutsopoulos N. Primary immunodeficien- cies reveal the essential role of tissue neutrophils in periodontitis. Immunol Rev. 2019;287:226-235.
7. Chang YC, Hsieh YS, Lii CK, Huang FM, Tai KW, Chou MY. Induction of c-fos expression by nicotine in human periodontal ligament fi- broblasts is related to cellular thiol levels. J Periodontal Res. 2003;38:44-50.
8. Skaleric U, Manthey CM, Mergenhagen E, Gaspirc B, Wahl M. Superoxide release and superoxide dismutase expression by human gingival fibroblasts. Eur J Oral Sci. 2000;108:130-135.
9. Murrell GA, Francis MJ, Bromley L. Modulation of fibroblast prolif- eration by oxygen free radicals. Biochem J. 1990;265:659-665.
10. Carpentieri A, Díaz de Barboza G, Areco V, Peralta López M, Tolosa de Talamoni N. New perspectives in melatonin uses. Pharmacol Res. 2012;65:437-444.
11. Shimozuma M, Tokuyama R, Tatehara S, et al. Expression and cellu- lar localization of melatonin-synthesizing enzymes in rat and human salivary glands. Histochem Cell Biol. 2011;135:389-396.
12. Madapusi BT, Rao SR. Preliminary evaluation of human gingiva as an extrapineal site of melatonin biosynthesis in states of periodon- tal health and disease. J Clin Diagn Res. 2018;89:321-346.
13. Del Valle BC, Fajreldines HD, De Barboza G, et al. Protective role of melatonin on retinal ganglionar cell: in vitro an in vivo evidences. Life Sci. 2015;218:233-240.
14. Rodríguez Lozano FJ, García Bernal D, Ros Roca M, AlgueróC OS, Oñate Sánchez RE, Camacho Alonso F. Cytoprotective effects of melatonin on zoledronic acid-treated human mesenchymal stem cells in vitro. J Craniomaxillofac Surg. 2015;43:855–862.
15. Areco V, Rodriguez V, Marchionatti A, Carpentieri A, Tolosa de Talamoni N. Melatonin not only restores but also prevents the inhibition of the intestinal Ca(2+) absorption caused by glutathi- one depleting drugs. Comp Biochem Physiol A: Mol Integr Physiol. 2016;197:16-22.
16. Blasiak J, Reiter RJ, Kaarniranta K. Melatonin in retinal physiology and pathology: the case of age-related macular degeneration. Oxid Med Cell Longev. 2016;9:681-736.
17. Carrascal L, Nunez-Abades P, Ayala A, Cano M. Role of melatonin in the inflammatory process and its therapeutic potential. Curr Pharm Des. 2018;24:1563-1588.
18. Reiter RJ, Rosales Corral SA, Liu XY, Acuna Castroviejo D, Escames G, Tan DX. Melatonin in the oral cavity: physiological and patholog- ical implications. J Periodontal Res. 2015;50:9-17.
19. Roohbakhsh A, Shamsizadeh A, Hayes AW, Reiter RJ, Karimi G. Melatonin as an endogenous regulator of diseases: the role of auto- phagy. Pharmacol Res. 2018;133:265-276.
20. Wongprayoon P, Govitrapong P. Melatonin as a mitochon- drial protector in neurodegenerative diseases. Cell Mol Life Sci. 2017;21:3999-4014.
21. Carpentieri A, Peralta Lopez E, Aguilar J, Solá VM. Melatonin and periodontal tissues: molecular and clinical perspectives. Pharmacol Res. 2017;125:224-231.
22. Klingbeil FG, Herson MR, Cristo EB, dos Santos PD, Yoshito D, Mathor MB. Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes. Cell Tissue Banking. 2009;10:197-204.
23. Ongaro A, Pellati A, Mattei MD, Terlizzi FD, Rossi C, Campana L. Enhancement of melphalan activity by buthionine sulfoxi- mine and electroporation in melanoma cells. Anticancer Drugs. 2015;26:284-292.
24. Ting SM, Zhao X, Zheng X, Aronowski J. Excitatory pathway en- gaging glutamate, calcineurin, and NFAT upregulates IL-4 in isch- emic neurons to polarize microglia. J Cereb Blood Flow Metab. 2020;40:513-527.
25. Haas HS, Linecker A, Pfragner R, Sadjak A. Peripheral glutamate signaling in head and neck areas. Head Neck. 2010;32:1554-1572.
26. Shredah M. Molecular study to the effect of monosodium gluta- mate on rat gingiva. Tanta Dent J. 2017;14:155.
27. Téllez N, Aguilera N, Quiñónez B, Silva E, González L. Arginine and glutamate levels in the gingival crevicular fluid from patients with chronic periodontitis. Braz Dent J. 2008;19:318-322.
28. Maher P, Hanneken A. The molecular basis of oxidative stress-in- duced cell death in an immortalized retinal ganglion cell line. Invest Ophthalmol Vis Sci. 2005;46:749-757.
29. Angelopoulos I, Brizuela C, Khoury M. Gingival mesenchymal stem cells outperform haploidentical dental pulp-derived mesenchymal stem cells in proliferation rate, migration ability, and angiogenic po- tential. Cell Transplant. 2018;27:967-978.
30. Lim SW, Loh HS, Ting KN, Bradshaw TD, Allaudin ZN. Reduction of MTT to purple formazan by vitamin E isomers in the absence of cells. Trop Life Sci Res. 2015;26:111-120.
31. Kim SR, Park MJ, Lee MK, et al. Flavonoids of Inula britannica pro- tect cultured cortical cells from necrotic cell death induced by glu- tamate. Free Radic Biol Med. 2002;32:596-604.
32. Wei G-X, Bobek LA. In vitro synergic antifungal effect of MUC7 12- mer with histatin-5 2-mer or miconazole. J Antimicrob Chemother. 2004;53:750-758.
33. Sulijaya B, Takahashi N, Yamazaki K. Host modulation therapy using anti-inflammatory and antioxidant agents in periodontitis: a review to a clinical translation. Arch Oral Biol. 2019;105:72-80.
34. Yu H, Lee P, Kim I, Jang J. Identification of N-methyl-D- aspartate receptor subunit in human periodontal ligament fibro- blasts: potential role in regulating differentiation. J Periodontol. 2009;20:338-346.
35. Fischer TW, Kleszczyński K, Hardkop LH, Kruse N, Zillikens D. Melatonin enhances antioxidative enzyme gene expression (CAT, GPx, SOD), prevents their UVR-induced depletion, and protects against the formation of DNA damage (8-hydroxy-2’-deoxyguano- sine) in ex vivo human skin. J Pineal Res. 2013;54:303-312.
36. Acuña Castroviejo D, López C, Escames G, López A, García A, Reiter J. Melatonin-mitochondria interplay in health and disease. Curr Top Med Chem. 2011;11:221-240.
37. Renn TY, Huang YK, Feng SW, et al. Prophylactic supplement with melatonin successfully suppresses the pathogenesis of periodon- titis through normalizing RANKL/OPG ratio and depressing the TLR4/MyD88 signaling pathway. J Pineal Res. 2018;64:e12464.
38. Proksch S, Strobel SL, Vach K, et al. Melatonin as a candidate ther- apeutic drug for protecting bone cells from chlorhexidine-induced damage. J Periodontol. 2014;85:79-89.
39. Kaur G, Dufour JM. Cell lines. Spermatogenesis. 2012;2:1-5.
40. Sifat M, Samsonraj RM, Munmun F, et al. Biological effects of mel- atonin on osteoblast/osteoclast cocultures, bone, and quality of life: Implications of a role for MT2 melatonin receptors, MEK1/2, and MEK5 in melatonin-mediated osteoblastogenesis. J Pineal Res. 2018;64:e12465.
41. Carpentieri A, Oliva C, Díez A, Cambras T. Melatonin administra- tion DL-Buthionine-Sulfoximine modifies circadian motor activity under constant light de- pending on the lighting conditions during suckling. Chronobiol Int. 2015;32:994-1004.
42. Carpentieri A, Díaz de Barboza G, Areco AV, Peralta Lópeza M, Tolosa de Talamoni N. New perspectives in melatonin uses. Pharmacol Res. 2012;65:437-444.
43. Kara A, Akman S, Ozkanlar S, et al. Immune modulatory and antiox- idant effects of melatonin in experimental periodontitis in rats. Free Radic Biol Med. 2013;55:21-26.
44. Kose O, Arabaci T, Kara A, et al. Effects of melatonin on oxidative stress index and alveolar bone loss in diabetic rats with periodonti- tis. J Periodontol. 2016;87:82-90.
45. Virto L, Cano P, Jiménez-Ortega V, et al. Melatonin as adjunctive therapy in the treatment of periodontitis associated with obesity. J Clin Periodontol. 2018;45:1336-1346.
46. Balci Yuce H, Karatas O, Aydemir Turkal H, et al. The effect of mela- tonin on bone loss, diabetic control, and apoptosis in rats with diabe- tes with ligature-induced periodontitis. J Periodontol. 2016;87:35-43.
47. Köse O, Arabaci T, Kizildag A, et al. Melatonin prevents radia- tion-induced oxidative stress and periodontal tissue breakdown in irradiated rats with experimental periodontitis. J Periodontal Res. 2017;52:438-446.
48. Balaji TM, Vasanthi HR, Rao SR. Gingival, plasma and salivary levels of melatonin in periodontally healthy individuals and chronic peri- odontitis patients: a pilot study. J Clin Diagn Res. 2015;9:23-25.
49. Cutando A, López-Valverde A, de Diego RG, et al. Effect of topical application of melatonin to the gingiva on salivary osteoprotegerin, RANKL and melatonin levels in patients with diabetes and peri- odontal disease. Odontology. 2014;102:290-296.