Ex vivo study of molecular changes of stained teeth following hydrogen peroxide and peroxymonosulfate treatments

Abstract

White teeth can give confidence and tend to be associated with a healthier lifestyle in modern society. Therefore, tooth-bleaching strategies have been developed, including the use of hydrogen peroxide. Recently, peroxymonosulfate has been introduced as an alternative bleaching method to hydrogen peroxide. Although both chemicals are oxidizing agents, their effects on the molecular composition of the stained teeth are yet unknown. In this study, the molecular profiles of teeth bleached with hydrogen peroxide and peroxymonosulfate were compared using Liquid Chromatography-Tandem Mass Spectrometry. Statistical analyses were used to assess the samples. In addition, reference spectral libraries and in silico tools were used to perform metabolite annotation. Overall, principal component analysis showed a strong separation between control and hydrogen peroxide and peroxymonosulfate samples (p < 0.001). The analysis of molecular changes revealed amino acids and dipeptides in stained teeth samples after hydrogen peroxide and peroxymonosulfate treatments. Noteworthy, the two bleaching methods led to distinct molecular profiles. For example, diterpenoids were more prevalent after peroxymonosulfate treatment, while a greater abundance of alkaloids was detected after hydrogen peroxide treatment. Whereas non-bleached samples (controls) showed mainly lipids. Therefore, this study shows how two different tooth-whitening peroxides could affect the molecular profiles of human teeth.

Figure 1

Bleaching methods significantly alter teeth' biochemical profiles. (a) Boxplots of non-normalized whiteness index (WIO) show a significant difference between Control and H₂O₂/MPS bleaching methods. This is a whiteness index based on human perception studies, − WIO are darker teeth and + WIO are whiter teeth. Analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) Test. ***p < 0.05. (b) PCA shows a clear separation between control and bleaching methods according to the Permutational Analysis of Variance (PERMANOVA), R2 = 0.27, p < 0.001. (c) Heatmap of the top 47 features with VIP scores > 1.00 extracted from pairwise PLS-DA models. The asterisks in the scores plots represent group centroids.

Figure 2

Univariate analyses and annotated metabolites in H₂O₂ and MPS treated samples. (a) Volcano plot between H₂O₂ and MPS treated samples and nine examples (7–15) among the higher VIP score metabolites related to each group of samples. (b) Molecular networking displaying log2 fold change of metabolite relative abundances: blue indicates metabolites present in high abundance after MPS treatment while red after H₂O₂ treatment, and numbers with green backgrounds (1–5) represent the highlighted networks in (c) and (d). (c) Chemical information on metabolites considered important in MPS post-bleaching predicted using a systematic classification of unknown metabolites (CANOPUS). (d) Information on metabolites considered important in H₂O₂ post-bleaching predicted via CANOPUS.

Figure 3

Overview teeth metabolome investigation. (a) The roots of the teeth were removed; (b) the crown was separated into three pieces; and (c) the samples randomized as the cut tooth sections were randomly added to bins A, B, and C, prior to destaining; (d) samples from groups A and B were bleached until their whiteness index (WIO) gave the same delta; (e) the samples were powdered in a TissueLyser and then, extracted using 96-deep-well plates; (f) LC–MS/MS obtained the molecular profiles for groups A, B, and control, and to understand the differences in the molecules detected, GNPS library search and in silico predictions via SIRIUS were used.