The Effect of Acidified Drinking Water on Molar Tooth Enamel in C57BL/6 Mice
The use of hydrochloric acid treatment of drinking water in many academic research rodent colonies and commercial vendors prompted us to investigate its effect on tooth enamel health in mice. Drinks with a low pH such as fruit juices and soft drinks have been demonstrated to cause demineralization of tooth enamel in humans and rodents. This study explored the hypothesis that acidified drinking water at the recommended range of 2.5 to 3.0 pH can lead to enamel erosion and compromised tooth integrity in mice. Specifically, we sought to quantify the effects of pH 2.5 or 3.0 daily drinking water exposure on molar enamel and bone mineral density over 1 and 3 mo. Methylene blue was used to quantify enamel erosion of the molar teeth, while dual-energy X-ray absorptiometry was used to quantify the bone mineral density of the mandible. After 1 mo of drinking water exposure, no statistically significant difference between the groups in enamel erosion or bone mineral density was observed. However, after 3 mo, a significant difference in enamel erosion for the pH 2.5 group compared with the other groups was identified, suggesting a potentially destructive process. There were no differences in bone mineral density between groups at any time point. These findings indicate that acidified drinking water of pH 2.5 may have deleterious effects on the enamel integrity of molar teeth in mice; however, drinking water of pH 3.0 seems safe for tooth enamel in mice during a short-term exposure of 3 mo. As this study only had a 1- to 3-mo exposure period, further study is needed to determine the effects of longer-term use of acidified drinking water at pH 3.0 in mice.
Schematic representation of the experimental timeline for 42 C57BL/6 mice (18 males [M] and 18 females [F] in the experimental groups, plus 6 mice in the baseline group). After a 3-d acclimation period, the baseline group (3 males and 3 females) were euthanized without HCl-treated drinking water intervention. The remaining 36 mice were divided into an acute group (18 mice) and a chronic group (18 mice). Each group was further allocated into 3 subgroups receiving HCl-treated drinking water at pH 2.5 or 3.0 or neutral water at pH 7.0 (3 males and 3 females per subgroup). The acute group was euthanized at 1 mo for data collection, while the chronic group continued HCl-treated water until 3 mo, at which point they were euthanized for data collection. Out of the pH 7.0 3-mo time point group, 1 male mouse died and was excluded from data collection. This design allowed for both short- and long-term assessment of acidified water exposure on tooth enamel and mandibular bone density. Created in BioRender. Coleman J. (2025) https://BioRender.com/q39g064.
Representative gross anatomic and processed images of a C57BL/6 mouse mandible exposed to 3 mo of pH 2.5 acidified water or nonacidified purified water. (A and D) Gross anatomic view of the mandibles before methylene blue staining. (B and E) Gross anatomic view of the mandibles 24 h after methylene blue staining, highlighting significant uptake of the dye in areas of enamel erosion, primarily on the molars in mandibles exposed to acidified water. (C and F) ImageJ-processed view of the mandibles, illustrating the detection and quantification of blue or white variations in molar teeth. The area of unstained white compared with stained blue areas of enamel was quantified (magnification factor: 10×).
Effect of pH levels on the ratio of white-to-teeth area over time. The ratio of white-to-teeth area for 3 pH levels (2.5, 3.0, and 7.0) at 3 time points: baseline (black), month 1 (light gray), and month 3 (dark gray). The baseline data was not subjected to the pH conditions. For pH 2.5, significant differences were observed between baseline and month 1 (P = 0.0127) and month 1 and month 3 (P = 0.0022). For pH 3.0, significant differences were observed between baseline and month 1 (P = 0.0016) and baseline and month 3 (P = 0.0053). For pH 7.0, significant differences were observed between baseline and both month 1 and month 3. Cross-group comparisons show significant differences between pH 2.5 month 3 and pH 3.0 month 3 (P = 0.0012), as well as between pH 2.5 mo 3 and pH 7.0 mo 3 (P = 0.0009). Error bars represent SE. *, P < 0.05, statistical difference.
Effect of pH levels on bone mineral density (BMD) over time. The BMD (measured in g/cm2) across 3 pH levels (2.5, 3.0, and 7.0) at 3 time points: baseline (black), month 1 (light gray), and month 3 (dark gray). The baseline data were not subjected to the pH conditions. Significant differences were observed for pH 2.5 between baseline and month 1 (P = 0.0221) and between month 1 and month 3 (P = 0.0020). For pH 7.0, a significant difference was noted between month 1 and month 3 (P = 0.0011). Error bars represent SE. *, P < 0.05, statistical difference.
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