Reagent

Sodium fluoride (NaF) Cat. S299-100 was obtained from Fisher Scientific (Pittsburgh, PA, USA).

Animals

Systemic administration of fluoride to the mice was performed following a previously established protocol (Suzuki et al. 2017). C57BL/6J mice (Strain #000664) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) and bred in-house. To ensure consistency with our previous fluoride studies, we selected C57BL/6J mice, a widely used and genetically well-characterized strain that supports reproducibility and relevance in biomedical research. To reduce variability potentially introduced by hormonal factors, only male mice were evaluated in this study. The ages for adolescent and mature mice were determined based on the Jackson Laboratory’s life history stages in C57BL/6J mice and previous studies (Fox 2007; JacksonLaboratory 2023; Mytidou et al. 2021). 5- to 9-week-old male mice were designated as the adolescent group, and 16- to 20-week-old male mice were assigned as the mature group. Each adolescent and mature group was randomly divided into four sub-groups (4–6 mice per sub-group) and provided drinking water containing either 0, 50, 100, or 125 ppm fluoride as NaF for 6 weeks as previously reported (Okamoto et al. 2025; Suzuki et al. 2015). Throughout the experiment, the animals were fed a fluoride-free chow (F1515, rodent standard diet, AIN-76A) from Bio-Sev (Frenchtown, New Jersey, USA), beginning one week prior to fluoride water treatment until the end of the fluoride treatment period. During the fluoride treatment period, the body weight of each mouse was recorded once a week. No significant differences in body weight change among sub-groups of the same age group were detected (Supplementary Fig. S1). After 6 weeks of systemic fluoride treatment, the animals were euthanized. Mandibular and maxillary incisors were extracted for quantitative light-induced fluorescence (QLF), Vickers microhardness, Micro-Computed Tomography (micro-CT), Scanning Electron Microscopy (SEM), Scanning Electron Microscopy with Energy-Dispersive X-ray Spectroscopy (SEM–EDX) and histological analysis. Serum and urine were extracted for measurement of fluoride concentration. All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals approved by the Institutional Animal Care Use Committee (IACUC) at Augusta University (where the corresponding author carried out experiments until January 2023. Registration number: 2019-0989) and at Nova Southeastern University (Registration number: 2023.02.MSuz1) which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). A certificate of approval is available upon request.

Photography of mouse incisors

After euthanasia, photographs of the maxillary and mandibular incisors were taken using a Nikon digital camera D7500 with AF-S DX Micro MIKKOR 85 mm f/3.5G ED VR lens (Nikon, Tokyo, Japan). Representative images from three mice per group are shown.

Quantitative light-induced fluorescence assay (QLF) of mandibular incisors

QLF has previously been used to assess the severity of fluorosis in mice (Carvalho et al. 2009; Everett et al. 2002; Okamoto et al. 2025; Sharma et al. 2011; Suzuki et al. 2017, 2014b; Vieira et al. 2005a; Yan et al. 2011). The mandibular incisors were dissected in pairs and subjected to QLF using a Nikon epifluorescence micro-camera (Nikon, Tokyo, Japan) equipped with a Chroma Gold 11006v2 set cube (exciter D360/40x, dichroic 400DCLP, and emitter E515LPv2). Fluorescence images of the teeth were converted to 8-bit grayscale values 0 (black) to 255 (white), and their intensities were analyzed using the ImageJ software (http://imagej.nih.gov/ij/). Average 8-bit grayscale intensities were taken from 10 sites on the enamel surfaces from both incisors and used as the overall value for each animal.

Vickers microhardness testing of mouse incisor enamel

Resin-embedded and well-polished specimens were prepared as previously described (Okamoto et al. 2024). A mirror-finish surface specimen of the mandibular incisor was measured using a microhardness tester (ALPHA-MHT-1000Z) from PACE Technologies (Tucson, Arizona, USA). Indentation testing was performed with a load of 25 g for 10 s using a Vickers tip. The tip region of the enamel layers served as the evaluation site for microhardness testing. There was a total of twelve indentations in each incisor, and the average value of twelve indentations was calculated as enamel microhardness (HV) of each mouse.

Micro-CT analysis

Micro-CT images of mandibular incisors were acquired using a Skyscan 1176 micro-CT scanner (Bruker, Billerica, MA, USA) at a resolution of 18 μm. Scans were performed at 50 kV and 500 μA, Al 0.5 mm Filter, 270-ms integration time, Camera binning 2 × 2, Camera pixel size 11.95 µm, total degrees of rotation 360 degrees, Rotation step 0.5 degrees, Frame averaging OFF. After scanning, images were reconstructed using NRecon software (Bruker). CTAn (Bruker) was used for evaluating enamel mineral density during enamel formation. The reference line between the first and second molars was defined as the approximate transition point from the secretory stage to the mature stage. Furthermore, the enamel regions selected for measurement were divided into the following three areas; (1) Eruption area: Incisors that have erupted and are not surrounded by the alveolar bone. (2) Maturation area: Incisors completely covered by the alveolar bone, extending from the proximal root of the first molar in the mesial direction. (3) Secretory area: Incisors located between the mesial and distal roots of the second molar, surrounded by the alveolar bone. Each region of the maturation and secretory stages of mandibular incisor enamel was defined based on a previous report (Jalali et al. 2015; Suzuki and Bartlett 2019). Enamel mineral density (EMD) was measured in adolescent mice and mature mice in the same regions. Pseudo-colors were rendered onto the 3-D structures using CTvox 3.3.0 (Bruker) to maximize the visualization of the teeth and the enamel.

Scanning electron microscopy (SEM) with energy-dispersive X-ray (SEM–EDX) and elemental line analysis of enamel

Scanning electron microscopy (SEM) and Energy-dispersive X-ray spectroscopy (EDX) analysis of the enamel in mice were performed according to a previously established protocol (Okamoto et al. 2025). Maxillary incisors were embedded in epoxy resin (Epofix cold setting embedding resin) from Electron Microscopy Sciences (Hatfield, PA, USA). Resin-embedded samples were evaluated sagittally, and the specimen surfaces were polished using diamond abrasive sheets (30–0.3 μm grain size) from Maruto Instruments (Tokyo, Japan). The sagittally exposed and polished surfaces were ultrasonically cleaned to remove debris, dried for over 24 h, and then subjected to elemental mapping using SEM–EDX (Xplore 30 EDS), with Aztec Live platform/software (Oxford Instruments, Abingdon, UK) to determine the constituent elements of the maxillary incisor and their distribution. The analyzed elements included C, O, F, Na, Mg, P, Ca, Fe, and Sr. The region of interest was analyzed by mapping the enamel in the tip aspect. The specimens were subsequently decalcified using 40% phosphoric acid for 40 s. After decalcification, they were thoroughly rinsed under running water, cleaned using an ultrasonic device, and then dried. The dried specimens were gold-coated, and SEM images were obtained using a Quanta 200 SEM (FEI Company, Hillsboro, OR, USA) as secondary electron images.

Histological analysis

Mouse incisors were extracted after fluoride treatment for 6 weeks and fixed in 4% paraformaldehyde for 24 h, demineralized with 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks, and embedded in paraffin. Five μm sagittal sections of the maxillary incisor were made for hematoxylin–eosin (H&E) staining or immunohistochemical (IHC) analysis after deparaffinization. H&E staining was performed with Harris hematoxylin and Eosin-Y Solution (Thermo Fisher Scientific, Waltham, MA, USA) after deparaffinization. For IHC, the sections were deparaffinized, hydrated, and heated overnight in 0.01 M citrate buffer at 60 °C to unmask antigenic sites. Sections were incubated with primary antibody: rabbit anti-KLK4 antibody (1:500) (Abcam, Waltham, MA, USA) or rabbit mAb IgG XP® Isotype negative control (DA1E) (1:200) (Cell signaling Technology, Danvers, MA, USA) followed by incubation with a peroxidase-conjugated secondary antibody, Vectastain® Elite® ABC-HRP Kit (PK-6101, Vector Laboratories, Newark, CA, USA), and ImmPACT® VIP substrate kit, HRP (SK-4605, Vector Laboratories). Sections were counterstained with methyl green for 4 min at 60 °C. Sections were analyzed using light microscopy (Revolve, ECHO, San Diego, CA, USA). Sections from at least three mice per group were assessed. Histological sections were independently assessed by two investigators who were blinded to the treatment groups, using a semi-quantitative scoring approach. To evaluate pathological changes in ameloblasts, each slide was examined for the presence of abnormal structures, defined as either disruptions in the ameloblast layer or the formation of ectopic cyst-like structures. Mice were classified as positive if at least one abnormal structure was identified, and negative if none were detected. The number of positive and negative cases was recorded for each experimental group. Representative images are shown in the figure (Fig. 6 and Supplementary Fig. 6S).

Measurement of fluoride in serum and urine

After six weeks of fluoride treatment, urine and serum samples were collected and stored at − 20 °C. Samples were thawed immediately prior to preparation for fluoride measurement.

Urine Samples: 20–50 µL of urine were first diluted 1:1 with ultrapure water, followed by a second 1:1 dilution with total ionic strength adjustment buffer (TISAB). This step adjusted the pH to 5–6, maintained constant ionic strength (Whitford 2017b) and ensured a minimum final volume of 75 µL required for fluoride analysis. The diluted samples were pipetted between the bottom of a Petri dish and the sensing surface of a double-channel fluoride ion-selective electrode (Orion 9609BNWP, Thermo Fisher Scientific) connected to an ion concentration meter (Orion Dual Star pH/ISE Meter, Thermo Fisher Scientific).

Serum Samples: Diffusion samples were prepared as previously described (WHitford 2017a). Briefly, 75 µL of serum was added to 3 mL of ultrapure water in the bottom of a 60 × 15 mm Petri dish. A 2 mm hole was made in the lid, and the inner rim was coated with petrolatum. Fifty µL of fluoride-trapping solution (0.05 N NaOH) was dispensed in three drops onto the inside surface of the lid. The lid was sealed to the dish, and a hexamethyldisiloxane (HMDS)-saturated 3 N sulfuric acid solution was injected through the hole. The hole was immediately sealed with petrolatum. HMDS-facilitated diffusion (Taves 1968; Whitford 1994, 1996) was carried out for 4–5 h at room temperature on a variable rocker. After diffusion, the lid was removed, flipped upside down, and 20 µL of 0.2 N acetic acid was added to the NaOH trap to adjust the pH to 5–6. The solution was aspirated with a pipette and adjusted to 75 µL with ultrapure water inside the pipette tip. The 75 µL sample was then carefully pipetted between the bottom of a Petri dish and the sensing surface of a double-channel fluoride ion-selective electrode (Orion 9609BNWP, Thermo Fisher Scientific) connected to an ion concentration meter (Orion Dual Star pH/ISE Meter, Thermo Fisher Scientific).

Calibration and Detection Limits: Fluoride calibration standards were prepared and treated identically to urine and serum samples. An eight-point calibration (0.049–200 ppm) for urine, and a five-point calibration (0.06–1.27 ppm) for serum was performed before the recording of each sample batch. Fluoride concentrations below 0.02 ppm were recorded as below the limit of detection (LOD).

Measurement of ALP activity in serum

Serum was collected after 6 weeks of fluoride treatment. Alkaline Phosphatase (ALP) activity was measured using the Alkaline Phosphatase Assay kit (Colorimetric) (Abcam) according to the manufacturer’s protocol. Briefly, serum was reacted with the ALP reaction solution for 60 min at room temperature under protection from light. The absorbance was then measured at 405 nm with a BioTek Synergy LX Multimode Reader (Agilent, Santa Clara, CA, USA).

Statistical analysis

Fluoride dose effects within each age group were analyzed using one-way ANOVA. To assess the differential effects of fluoride dose and age group (adolescent vs. mature), a two-way ANOVA was performed, followed by Tukey’s multiple comparisons test for post hoc analysis. Student’s t-test was used for direct comparisons between two groups where appropriate. All statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, Boston, MA, USA). Statistical significance was defined as p < 0.05.

Graphical abstract

The graphical abstract was created using BioRender (https://BioRender.com) (Toronto, Canada).