Background
Tooth color contributes significantly to a person’s aesthetic appearance. Normal tooth color is determined by the optical and chromatic properties of dentine and enamel: hue, value, chroma, thickness, texture, and translucency [
1]. Both intrinsic and extrinsic factors in dentin or enamel, or in both, can cause tooth discoloration [
1‐
3]. Nightguard vital bleaching (at-home-bleaching) was first introduced in 1989 to reduce tooth discolorations [
4]. The 10% carbamide peroxide used in a custom-fitted nightguard was converted by saliva in the oral cavity to 3% H
2O
2 (~ 0.9 M) and 7% urea [
3‐
5]. H
2O
2 diffuses into the enamel-dentine junction and dentine, and using a redox reaction, decomposes chromogens, which whitens teeth [
2,
6]. The most noteworthy side effects associated with at-home-bleaching are tooth sensitivity and oral mucosa irritation. Some studies [
5,
7,
8], however, report that these side effects are transient and can be recovered from after treatment. Safety for exposure to a long-term H
2O
2 bleaching agent has also been a concern in more recent studies [
9,
10]. H
2O
2 generates free radicals, especially hydroxyl radical, in oxidative reactions; free radicals are considered genotoxic and carcinogenic [
11,
12]. Long-term exposure to high-dose H
2O
2 can damage soft and hard oral tissue [
4,
6]. The inappropriate application or abuse of H
2O
2 during cleaning treatments has other potential adverse effects [
13].
We explored the pathological effects of H
2O
2 on gingival mucosa under different concentration doses and exposure durations using primary cultured normal human oral keratinocytes (NHOKs) as a model. Gingival mucosa is composed of highly-regenerative keratinized stratified squamous epithelium and submucosal connective tissues. The NHOKs in the epithelium form a barrier to defend basal cells with regenerative capability from chemical and physical insults of oral environment including the reactive oxygen species generated during tooth bleaching [
14]. Through previous oral transmucosal bioavailability studies, various factors were known to reduce the chemical exposure to basal cells thus reducing the actual H
2O
2 concentration at this level from tooth bleaching [
15]. The keratinized gingival mucosa is known to harbor a lower permeability, intermediate residence time, and high blood flow as compared to the buccal and palatal muocosa [
16,
17]. H
2O
2 gel also effectively reduced risk of high dose exposure to oral mucosa compared to the liquid form [
18]. Besides, the EU Commission established in the cosmetics directive that the concentration of hydrogen peroxide in consumer oral hygiene products should be limited to 0.1% [
19]. Therefore, we tested the proposed equivalent exposure dose at basal level in the range of 0.01~100 mM (0.000033% ~ 0.33%) in this study. The mechanism of H
2O
2-induced cell death was further explored by assessing mitochondrial membrane potential- and apoptosis-related pathways. The antioxidant defense system was also investigated for its role in the short- and the long-term exposure.
Discussion
After we exposed NHOKs to H
2O
2, we comprehensively evaluated the effects on cell viability, DNA damage, cellular defense response to oxidative damage, and apoptosis in the estimated basal cell level exposure dose range of 0.01~100 mM, which is about 1/10 that of the H
2O
2 in the bleaching gel. From the dose- and duration-dependent decrease in cell viability, we confirmed that exposing NHOKs to H
2O
2 induced significant cellular damage, especially when the dose exceeded 5 mM. The study also revealed that the degree of cell proliferation inhibition was dose- and duration-dependent. This finding is consistent with O’Toole et al., who used a similar dose range: 2, 4, and 7 mM [
25]. They reported that a low dose of H
2O
2 have no effect on NHOKs viability at doses ≤700 μM for even after exposure for 24 h. We found that > 90% cell survival could be maintained using doses < 1 mM, even after 8 h of exposure, which is the exposure time to Nightguard vital bleaching.
Dental bleaching is based on the ability of H
2O
2 to penetrate through tooth structure and produce free radicals to oxidize the colored organic molecules. There are many reports investigated the effect of dose and exposure time of H
2O
2 to the pulpal tissues as the bleaching agents penetrated through the tooth structure [
26‐
28]. However, only few addressed the effect of acute or chronic exposure of the leaked bleaching agents to oral mucosal keratinocytes. Although 3% H
2O
2 was most commonly used for tooth bleaching, the actual exposure dose of to the basal cell layer of oral mucosa that regenerate the mucosal barrier was affected by multiple factors. At one end, the peroxide releases into saliva from home bleaching systems require to diffuse through the carrier while being diluted and degraded by the saliva. It has been reported that gel formulation significantly reduced the peroxide concentration in the saliva more than that of the liquid form [
27]. The leaked peroxide was rapidly degraded by the saliva to 52 and 24% of the original concentrations at 2 and 6 h after exposure, respectively [
29]. The remaining salivary H
2O
2 needs to penetrate deep into the basal layer and subgingival tissues through epithelium barrier to exert pathological effects [
30]. According to EU recommend oral hygiene products, the tissue exposure of H
2O
2 concentration should be limited to 0.1% or 29.4 mM. In this study, the direct exposure at dose range of 0.01 mM to 100 mM was applied using primary cultured gingival oral kerayinocyte as the model. The pathological effects of both short (1 h) and long (8 h) exposures were analyzed according to the common clinical practices
.
The safety of H
2O
2 tooth bleaching is still controversial: its genotoxicity and carcinogenicity are under active discussion [
9,
10]. Diaz-Llera et al. showed that 0.34–1.35 μM of H
2O
2 induced hypoxanthine guanine phosphoribosyltransferase (HPRT) mutation both in vitro and in vivo [
31]. High-dose H
2O
2 was reported to be mildly carcinogenic for the duodenum of catalase-deficient mice [
9]. In another report, 1% H
2O
2 (~ 0.3 M) in drinking water induced forestomach tumors in rats [
10]. These reports showed that exposure to high-dose H
2O
2 for a sustained period induces oxidative stress that leads to DNA damage in mammalian cells.
In this study, we used primary cultured NHOKs isolated from the basal layer of oral gingival epithelium that is closely related to the most susceptible cell types in clinical practice of tooth bleaching (gingival mucosa). The primary cultured NHOKs presented similar properties of basal layer keratinocytes that maintained certain replication and differentiation potential suitable for study the peroxide induced DNA damage and subsequent pathogenic signaling. Unlike nDNA, mtDNA lacks histone protections and sophisticated DNA repair mechanisms to shelter it from oxidative attacks [
32,
33]. Free radical attacks normally cause mtDNA mutation, deletion, or other types of damage and can be preserved in the cells in heteroplasmic format. One common type of mtDNA damage is the 4977-bp deletion (
nt8469–13,447) that is related to aging [
34,
35] and to different diseases, such as Kearns-Sayre syndrome [
36]. A comet assay confirmed that H
2O
2 induced nDNA damage. In the tested dose range of 0.01 to 200 mM, we found that the number of nDNA single strand breaks was dose-dependent. Interestingly, only the difference in nDNA damage between 1 and 8 h of exposure to each dose of H
2O
2 was significant. However, we found no plateau dose in the treatment range.
The comet assay showed only nDNA damage but not mtDNA damage [
37]. We thus used qPCR to quantify the ratio of mtDNA damage. For the control NHOKs, mtDNA
4977 deletion showed only 0.81% baseline deletion in vitro. During the first hour of exposure to H
2O
2, mtDNA
4977 deletion dose-dependently increased. Despite a significant drop in the mtDNA deletion ratio at a dose of 100 mM H
2O
2, we suspect that it can be attributed to direct massive cellular damage, which was supported by the MTT assay. After the NHOKs had been exposed to H
2O
2 for 8 h, all groups but the 100-mM treatment group were restored to their approximate baseline levels. This confirmed that H
2O
2 can be genotoxic to both nDNA and mtDNA in NHOKs. Moreover, in keratinocytes treated with < 10 mM of H
2O
2 for 8 h, mtDNA
4977 deletion, but not nDNA, returned to normal. This is consistent with Ballinger et al., who used two treatment doses (0.1 and 0.5 mM) for 1 h [
32].
Croteau and Bohr [
38] reported that mitochondria are more efficient at DNA repair than nDNA, and that mitochondria are able to repair 65% of the lesions within 4 h. However, nDNA has a more sophisticated defense and repair mechanism than does mtDNA because it includes histone protection and nucleotide excision repair. Both nDNA and mtDNA damage contribute to carcinogenesis [
39]. Therefore, the genotoxicity of both mtDNA and nDNA should be a concern for people who use at-home-bleaching over the long term.
Unlike nDNA damage, mtDNA damage can be preserved in a heteroplasmic state, thus allowing cells to survive and proliferate when the damage is repairable [
40]. Both normal and damaged mtDNA copies can be amplified and passage in the organelles. A large-scale deletion would affect the supply of essential proteins in the electron transport chain, thus interfering with the normal function of mitochondria; this can be considered a loss of ΔΨ
m [
41]. In the present study, ΔΨ
m was low at 8 h, as well as it was after 1 h, which was consistent with the treatment-time-associated alterations in the ratio of mtDNA
4977 deletion. A dramatic (~ 407-fold) increase in ΔΨ
m loss occurred within 1 h after NHOKs had been treated with doses < 1 mM. For longer exposure (8 h), the degree of ΔΨ
m loss was attenuated to between 10- and 30-fold, and it occurred with doses as low as 0.01 mM. Thus, mtDNA
4977 deletion seemed to be more sensitive to oxidative attacks than was ΔΨ
m to the dose and duration of exposure to H
2O
2, which is conceivable because replacing a protein complex in damaged DNA takes time through transcription and translation. The same reason applied for the observation that after 8 h, ΔΨ
m loss occurred even at doses as low as 0.01 mM, while the ratio of mtDNA
4977 deletion had already been restored at doses < 100 mM.
Oxidative attacks induce various types of cell death: apoptosis, autophagy, and necrosis [
42‐
44]. These cell death mechanisms are in fact not completely disadvantageous: they prevent severely damaged cells from contributing to the development of cancer. After 1 h of exposure to H
2O
2, the number of apoptotic cells dose-dependently increased, and the apoptotic fraction dropped significantly at doses > 200 mM. The number of necrotic cells increased at doses > 5 mM. At the highest dose (200 mM), a large number of NHOKs became necrotic instead of apoptotic, as evidenced by a large PI-positive population. After 8 h of exposure, apoptosis was also dose-dependent, but the apoptotic cell population rapidly fell when treatment doses were > 50 mM. The necrotic population grew as the apoptotic population declined.
At lower levels of oxidative attack, pro-apoptotic Bcl-2 family proteins increase mitochondrial membrane permeability and stimulate voltage-dependent anion channel (VDAC) activity which reduces H
+ and ΔΨ
m in the inner membrane, as shown in our ΔΨ
m assay. When the matrix expands to rupture the outer membrane, cytochrome
c and apoptosis-inducing factor are released to the cytoplasm to induce cell apoptosis [
45‐
47]. The observed timing and dose response in our experiments support this model. In addition, a drastic decline of cell viability at high doses of H
2O
2 should be a primary consequence of direct chemically induced necrosis, but apoptosis was activated at low doses of H
2O
2.
In addition to DNA repair and various types of cell death, activation of antioxidant molecular mechanisms is important for cellular defense against at-home-bleaching-induced health hazards. It is known that catalase and GSH are essential for the cellular antioxidant defense system, and that they detoxify H
2O
2-induced damage in vivo [
48]. GSH is one of the most important nonenzymatic antioxidants that exist in large amounts within cells, including in mitochondria [
22]. Thus, measuring GSH content in response to different doses of H
2O
2 should provide important mechanistic insights. After the first hour of treatment, GSH levels were higher than at baseline when H
2O
2 doses were between 0.01 mM and 5 mM. This was followed by a dose-dependent decline from 86 to 16% of baseline in the dose range of 10 mM to 200 mM, which implies that at lower doses, a protective increase in GSH was activated. At a high dose, however, the endogenous GSH was rapidly consumed to protect cells against oxidative attacks. After the NHOKs had been exposed to H
2O
2 for 8 h, excess GSH was detected when the dose was < 0.1 mM, but the levels were significantly lower than they had been. At higher doses, dose-dependent decreases in GSH were detected, but they were consistently lower than they had been at the same doses. These findings implied that a negative balance between GSH generation and consumption occurred during continuous exposure to H
2O
2.