The Influence of Some Synthesis Compounds on Healthy and Tumoral Oral Cell Lines: PGK, HGF and SCC4

This present study aimed to assess the influence of different chemical compounds which are commonly found in the composition of products used for oral hygiene on primary gingival keratinocyte, human gingival fibroblast and tongue squamous cell carcinoma behaviour. Chlorhexidine (Chx), aspartame (Asp), xylitol (Xyl) and sodium bicarbonate (NaB) were evaluated in an in vitroresearch regarding cells morphology and cytotoxicity. Primary gingival keratinocytes (PGK)and fibroblasts (HGF)treated with different concentration of the tested compounds did not show significant changes, nor related to morphology nor related to viability at the lowest concentrations used (< 25μM). In contrast, the tumor cells were affected by the test compounds, especially xylitol and sodium bicarbonate, both in terms of morphology and cytotoxicity, at concentrations over 5μM.

This present study aimed to assess the influence of different chemical compounds which are commonly found in the composition of products used for oral hygiene on primary gingival keratinocyte, human gingival fibroblast and tongue squamous cell carcinoma behaviour. Chlorhexidine (Chx), aspartame (Asp), xylitol (Xyl) and sodium bicarbonate (NaB) were evaluated in an in vitroresearch regarding cells morphology and cytotoxicity. Primary gingival keratinocytes (PGK)and fibroblasts (HGF)treated with different concentration of the tested compounds did not show significant changes, nor related to morphology nor related to viability at the lowest concentrations used (< 25µM). In contrast, the tumor cells were affected by the test compounds, especially xylitol and sodium bicarbonate, both in terms of morphology and cytotoxicity, at concentrations over 5µM.
Oral care products (for teeth and mouth care) are available as toothpaste, mouthwash, tooth whitening products, denture care products etc. Before placing on the market, they pass through standard safety assessments and are approved by regulatory bodies. However, in various situations, side effects have been reported. Specialty studies that treat biocompatibility and adverse reactions due to these types of products are in minority compared to those that treat their efficacy. The main reasons are: (i) the passive oral exposure of tissues to these products due to the short contact period between them and the preferential sponsorship of research which aim to evaluate the positive regards of oral care products in terms of efficacy and clinical performance compared to research which aim to assess adverse effects of these products [1].
Aspartame (E951), was discovered in 1965 and is synthetized through the combination of amino acids, namely L-phenylalanine, L-aspartic acid, and connected through methyl ester bonds being considered a source of phenylalanine (which must be considered for people with phenylketonuria) [5]. In literature, are data which describe the toxicity and hepatocellular alteration in long-term exposure to it [6].Currently is investigated for its safety, even if wasgenerally recognized as safe. Some pathologies are linked with his nephrotoxicity, hepatotoxicity, and damage to nerves, cancer, and even type 2 diabetes [7][8][9][10].
Xylitol (E967), a five-Cpolyol, synthesized by hydrogenation of xylose (for the first time in 1891) presents around 95% sweetness of sucrose being the sweetest compound from its class, contributing with 2.4 kcal/g. Can be also obtained by different extraction methods from birch and other woods, almond husks, corncobs, straw and paper production surplus. Can be found in many fruits and vegetables [1,10].
Sodium bicarbonate exerts an antibacterial effect because presents cleansing action by loosening debris and dissolving mucus. Due to the increase values of the pH in the oral cavity after administration of the compound, can be prevented theovergrowth of acidophilic bacteria. Some studies pointed out that mouthwash withNaHCO 3 1% causes less irritation to the oral mucosa in a group of sensitive cancer patients as compared with other similar products [1].
The present study was proposed to assess the activity on normal and tumoral oral cell lines of four different chemical compounds found in oral care products: chlorhexidine (Chx), aspartame (Asp), xylitol (Xyl) and sodium bicarbonate (NaB).

Experimental part Materials and methods Chemicals and reagents
All chemical reagents and standard substances used were of analytical reagent grade and obtained from Sigma-Aldrich and Merck (Germany). The chemical compounds stock solutions used were initially dissolved in complete medium, Dulbecco's Modified Eagles Medium(DMEM) at concentrations of 10 mM for chlorhexidine and sodium bicarbonate, and 1 mM for aspartame and xylitol, respectively. After preparation, the stock solutions were filtered through a 0.22mm filter and diluted with medium in order to obtain the finalworking concentrations (adjusted to pH 7.4) used within the tests.

Cells culture
The cell lines utilized in the current experiments were primary gingival keratinocytes and fibroblasts (normal/ healthy cells -PGK and HGF) and tongue carcinoma (tumor squamous cells -SCC4) purchased from American Type cell Collection, commercial forms -frozen vials. The specific medium for cells culture and kit, like: Dermal Cell Basal Medium (DMEM), Fibroblast Basal Medium (FBM) and Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F-12 (DME:F12), Keratinocyte Growth Kit and MTT were supplied from Sigma Aldrich and ATCC. Other characteristic reagents among whichfetal bovine serum (FBS), phosphate saline buffer (PSB), tr ypsin, ethylenediaminetetraacetic acid (EDTA), penicillin and streptomycin mixture, Trypan Blue,hydrocortisone were purchased from Sigma Aldrich, Merck and ATCC. The cells exposed to various concentrations of Chx, Asp, Xyl, NaB (1, 5, 10, 25 and 50 µM) over three time periods (24 hours) were examined under a phase-contrast microscope for any changes in their attachment to the culture flask, size, shape, integrity of their cell characteristics.
The MTT cell viability assay was used and the protocol was adapted from the literature [11]. In brief, the cells were seeded in 96-well cell culture plates and exposed to the test compounds as mentioned above. After incubation period (24 hours), the MTT solution was added to each well and the culture plates were incubated at 37°C for 4hours and after removal of the liquids from the wells DMSO was added in order to dissolve the formazin crystals and the absorbance was read at 540nm with a reference wavelength of 690nm. Results were expressed as % cell viability.

Statistical analysis
The statistical and software programs applied in the present study were GraphPad Prism 7. Data wereprocessed using One-way Anova followed by Tukey's post-tests, in order to establish the statistical difference groups; *, **, *** and **** indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively.

Results and discussions
Cells morphology.
The cells were observed to have an intact morphology and a similar size and shape as the untreated controls up to a concentration of chemical compounds of approximately 25 µM. Cells presented clear morphological changes when observed under the phase-contrast microscope after exposure to test compounds in excess of 50 µM, after 24 h post-stimulation. They lose their welldefined form as can be observed in the figures 1-3.

Cells viability.
The effect on viability of PGK,HGF and SCC4 cells treated with different concentrations of the fourchemical compounds chlorhexidine, aspartame, xylitol andsodium bicarbonate, over 24 h was assessed by using MTT method. All the compounds caused a decrease in cell viability of the tumor cells in a concentration and timedependent manner as compared to controls, however to varying degrees. A slightly higher cell viability, as compared to untreated cells, was observed for healthy all cells-PGK  and HGF-when treated with low concentrations ofsodium bicarbonate (1-10 µM), however exposure to chemical compoundsapplied in higher concentrations caused decreased cell viability in a dosedependent manner. In case of PGK after post-stimulation with the compounds at 50 µM the viability was as follows: ~ 84% for Chx, ~ 71% for Asp, ~ 83% for Xyl and ~ 92% for NaB while regarding HGF the percentage were: ~ 79% for Chx, ~ 81% for Asp, 8 7% for Xyl and ~ 88% for NaB After 24 h of incubation, no statistically significant differences could be detected between the following compounds, chlorhexidine, aspartame and xylitolat 5 and 10µM concentrations. The tests compounds concentrations starting at 10 µM caused a decrease in cell viability in a dose-dependent manner for the SCC4 cells, and as can be seen in the figure 4, this cell line seemed to be more sensitive to all the compounds except xylitol compared to the healthy cell lines. For the SCC4 cell line the greatest decrease in cell viability was observed with chlorhexidine (~ 68% at 25µM and ~ 52% at 50 µM) and sodium bicarbonate (~ 51% at 25µM and ~ 33% at 50 µM).
Aspartame at low doses can be considered a potential angiogenic compound that can produce regenerative cytokine production. It enhances the specific markers and receptors and finally can be formed new blood vessels, which develop proper conditions that in vivo may result in the promotion of specific pathologies such as: diabetic retinopathies, rheumatoid arthritis and tumor cell invasion and spreading of metastases [12]. Some authors, like Park and collaborators stated that xylitol inhibited the proliferation of cancer cells such as A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2 cells, but had less effect on the cell viability of HGF cells and also induces cell morphological changes and autophagy in lung cancer cells [13].
All these preliminar y studies may have clinical relevance only after correlating with other in vivo detailed studies due to the fact that the utilized cells do not have the salivary pellicle layer, andcannot reproduce the immunologic issues in vivo or the protective barriers at the tissue level [14].

Conclusions
Chemical compounds, like disinfectants and artificial sweeteners are currently classified as safe for consumption in various products by many international regulatory bodies, while some of them are still classified to be used 'with caution' in some countries. The results obtained in this study offer important data regarding the influence of Chx, Asp, Xyl and NaB on cell lines behaviour but they must be extrapolated to the in vivo conditions. The present study serves to provide further information on the possible effect these agents have on mammalian cells, however further studies will have to be performed to clarify what exactly these effects might be.