In vivo effect of fluoride combined with amoxicillin on enamel development in rats

Abstract Some evidence in vitro suggested that amoxicillin and fluoride could disturb the enamel mineralization. Objective: To assess the effect of amoxicillin and of the combination of amoxicillin and fluoride on enamel mineralization in rats. Methodology: In total, 40 rats were randomly assigned to four groups: control group (CG); amoxicillin group (AG - amoxicillin (500 mg/kg/day), fluoride group (FG - fluoridated water (100 ppm -221 mg F/L), and amoxicillin + fluoride group (AFG). After 60 days, the samples were collected from plasma and tibiae and analyzed for fluoride (F) concentration. The incisors were also collected to determine the severity of fluorosis using the Dental Fluorosis by Image Analysis (DFIA) software, concentration of F, measurements of enamel thickness, and hardness. The data were analyzed by ANOVA, Tukey’s post-hoc test, or Games-Howell post-hoc test (α=0.05). Results: Enamel thickness of the incisors did not differ statistically among the groups (p=0.228). Groups exposed to fluoride (AFG and FG) have higher F concentrations in plasma, bone and teeth than those not exposed to fluoride (CG and AG). The groups showed a similar behavior in the DFIA and hardness test, with the FG and AFG groups showing more severe fluorosis defects and significant lower hardness when compared with the AG and CG groups, with no difference from each other. Conclusion: The rats exposed to fluoride or fluoride + amoxicillin developed dental fluorosis, while exposure to amoxicillin alone did not lead to enamel defects.

assessed in controlled studies in vivo. Therefore, this in vivo study aimed to investigate the effect of amoxicillin and its combination with fluoride on amelogenesis in rats.

Methodology
Study groups and experimental design After a 5-day adaptation period, the rats were randomly assigned to four groups of 10 rats each, with administration of the following substances every The substances were administered for 60 days, period during which incisors could fully develop again. 33 Amoxicillin was prepared by diluting the content of the flask in deionized water at the concentration of 500 mg in 5 mL and that administration was done as described by Mihalaş, et al. 34 (2016) and Souza, et

Photographic analysis
In total, 10 lower incisors from each group were photographed and analyzed by the quantitative method known as Dental Fluorosis by Image Analysis

Analysis of enamel thickness
In total, 10 upper incisors from each group were transversely sectioned in three regions (incisal, medial, and apical) using a diamond bur at high speed. Each region was fixed on an acrylic resin block, with the cross-sectional tooth surface parallel to the horizontal plane. Three enamel thickness measurements were made in each region using 10× magnification on a microhardness tester (Future Tech, FM-7 model, Kawasaki, Japan) coupled to FM-ARS software.

Hardness analysis
In total, ten upper incisors from each group were embedded on acrylic resin discs with their long axes parallel to the disc surface (Arotec EMB-30, Arotec

Results
A total of 40 rats (10 per group) were assessed. No statistical difference (ANOVA: p>0.05) was observed between the group means for water intake (42.1±5.6 mL) and for body mass (309.8±64.8 mg) among the rats included in the study. Table 1 shows the means and standard deviations of enamel thickness (in μm) and of the DFIA index, which determine the severity of fluorosis, according to the experimental groups assessed. No significant differences were found between the means obtained for enamel thickness between the groups. On the other hand, the means of DFIA for the groups exposed to fluoride (FG and AFG) presented significantly higher values than control (CG) or amoxicillin (AG) groups.
Exposure to amoxicillin (AG) alone or with fluoride (AFG) did not influence the DFIA values. Figure 1 shows the diagram of DFIA index for quantification of fluorosis through the differences between the dark and light areas. Table 2 shows the means and standard deviations of Knoop hardness, angular coefficients, and correlation coefficients of two linear regressions for depths of 10 to 30 μm. The groups exposed to fluoride (AFG and FG) had significantly lower mean hardness than that of non-exposed groups (AG and CG) at all depths. We can observe that the fluorosis was more severe on the outer enamel surface (10 μm), since the results are the same and greater for depths from 20 to 70 μm. Regarding exposure to amoxicillin, the enamel hardness values from rats only exposed to amoxicillin (AG) did not differ from the control group (CG), and are similar to the groups exposed to fluoride and amoxicillin (AFG) and only fluoride (FG).
The regressions show significant linear increase in hardness at approximately 10 μm to 30 μm in groups not exposed to fluoride (CG and AG), with an increase by 7.9 and 7.1 KHN (Knoop hardness number) per μm, compared to an increase by 4.0 and 5.4 KHN per μm in the FG and AFG, respectively. Figure 2 shows the area of the three indentations that were performed, namely: incisal, medial, and apical regions. Inc -Lower incisor, indicating the area demarcated for analysis. Black arrow -indicating the highest pixel intensity. White arrowindicating the lowest pixel intensity. In the image of the rat tooth with dental fluorosis showing striated white bands distributed in the orange enamel. In the gray scale image, the black area corresponds to the original orange bands and the white area corresponds to the original white bands. For each segmented area, the method computes the average of the pixel intensities in the input image concentrations in plasma, bone, and teeth. Groups exposed to fluoride (AFG and FG) have higher concentrations than those not exposed to fluoride (CG and FG) with no effect of amoxicillin on F concentrations. can exert some influence on fluorosis, 24,28 this study showed no differences between the control group and the group exposed to amoxicillin, despite the high doses of amoxicillin. One possible explanation could be that the rat strain used in this study is not sensitive to 500 mg amoxicillin/kg. The rats exposed to fluoride presented severe fluorosis in the image analysis (DFIA index) and in surface hardness test, as expected, since the model for the induction of dental fluorosis in rodents used in this study is already known. 35 The severity of fluorosis can be quantified and correlated with the level of injury caused by fluoride based on its plasma concentration. 36 In this study, the analysis of fluoride on plasma and bone was performed to demonstrate the experiment's validity. Bone tissue presents a highly dynamic behavior, with a constant rate of remodeling, and, therefore, fluoride exposure can affect the bone composition, its three-dimensional structure, and bone cells. * Group means in the column followed by identical letters did not show any differences in the Games-Howell test (p>0.05). The total daily dose was ministered once a day, conveniently, since the effects on tooth development seem similar when the total daily dose was divided into 3 times a day 10 or in a single dose. 38

Discussion
The amoxicillin did not influence on the enamel hardness and thickness in this study. No significant difference was observed between the groups exposed to fluoride + amoxicillin (AFG) and the fluoride group (FG) on the DFIA and hardness, as well as no difference between the AG and CG groups. Therefore, we suggest that exposure to amoxicillin did not exhibited interaction on the effect of fluoride on in vivo dental enamel. These data are not in accordance with other previous published data.
In animals' study, Gottberg, et al. 11 (2014) observed that the side effect caused by amoxicillin on dental enamel was dose-dependent in rodents. Souza, et al. 26 (2016) have evidenced in a histological analysis that, during the secretory stage of the enamel matrix (seven days), exposure to amoxicillin reduced enamel thickness, but in the subsequent stage, at the end of secretion and at the outset of mineralization (12 days), this reduction in thickness was not observed between the exposed and non-exposed groups, thus suggesting a one-off effect of amoxicillin or a possible change in the secretion rate of the enamel matrix, which was later restored. Our results do not show statistically significant effects of amoxicillin on dental fluorosis in Holtzman rats, in accordance with de Souza,et al. 25 (2020) who used the same rat strain and observed none effect of amoxicillin on the enamel mineralization.
Regarding enamel thickness, no difference among the groups was identified. The exposure to fluoride leads to changes in enamel mineralization, causing enamel fluorosis without morphological changes in the secretory stage, which is in line with the assumptions about the pathogenesis of dental fluorosis. 30,41,42 However, in vitro 28 and in vivo 24,26 studies have shown that exposure to amoxicillin, to fluoride, or to both can affect enamel thickness during the initial secretory stage of enamel matrix formation. These controversial results should be analyzed in future studies that should also analyze the bioavailability of amoxicillin in the organism of the animal and the possible intestinal environment of absorption of the drug, as previously suggested by Chesa-Jiménez, et al. 43 (1994).
Previous studies also demonstrate that some endogenous factors 15 and exogenous molecules 21 may modulate fluoride effects on dental enamel underlying the necessity for further studies with dose-response effects on various strains with careful considerations to external conditions. In rodents, the association between fluoride and bisphenol-A seems to increase severity of fluorosis in rats. 19

Conclusion
We conclude that chronic exposure to fluoride caused significant changes in dental enamel, compatible with the plasma concentrations of fluoride and with the reduction of enamel hardness, especially at the first 10 μm. Amoxicillin associated to fluoride did not influence the severity of fluorosis. Moreover, the exposure to amoxicillin alone has not affected amelogenesis in our rat model.

Declarations of interest
The authors declare no competing interests.