5 mL Eppendorf tube containing 300 μL HNO3 at 1.8% (v/v). The labial face of the incisal third of the lower incisor was maintained in contact with the acid for 20 s (the tube was inclined at 35°). A dentine fragment obtained from the lingual aspect of the incisor root was completely digested in 500 μL HNO3 at 50% (v/v).

The mass of bone, dentine, and enamel of each acid extract was calculated on the basis of its phosphorus content.16 All the samples were assayed in triplicate. The mass (g) of enamel, dentine, and bone was determined assuming phosphorus contents of 17.0%, 15.97%, and 13.5% in enamel,17 dentine,18 and bone,19 respectively. For fluoride analysis, 100 μL of the acid extract were mixed with 900 μL deionized water buffered with 100 μL TISAB II (1.0 M of acetate buffer, pH 5.0 with 1.0 M NaCl and 0.4% cyclohexanediaminetetraacetic JAK2 inhibitor drug acid).19 Fluoride was determined in

an ion-specific electrode, calibrated with standard fluoride solutions (0.5–5.0 μg/mL). Whole Antiinfection Compound Library high throughput blood and calcified tissues were collected for determination of Pb levels. Blood samples were withdrawn using metal-free syringes with lyophilized heparin. A detailed description of the applied technique can be found in our previous report.13 Pb levels were obtained as μg of Pb/dL of whole blood or as μg of Pb/g of calcified tissue. Enamel, dentine, and bone lead and fluoride concentrations were compared by ANOVA followed by Bonferronís Multiple Comparison Test. Fluorosis scores were compared by Kruskal–Wallis test. Differences were considered statistically significant at P < 0.0083 (5% significance level divided by 6 comparisons). This study aimed to compare the enamel characteristics in the different groups. In order to do that, a fluorosis, or better, an enamel defect index comprising 5 categories of defects was proposed. Representative pictures of the 5 scores suggested for this index are shown in Fig. 1, and a detailed description of each score is displayed in

Table 1. From a histopathological viewpoint, all the normal and fluorotic teeth presented positive birefringence in water and negative birefringence in Thoulet́s 1.62. Sharp changes in enamel birefringence were detected with increasing fluorosis scores, and these alterations consisted of enhanced positive Lck birefringence in water and decreased (less negative) negative birefringence in Thoulet́s 1.62. The most remarkable contrast between white and pigmented bands was found upon water immersion and with the target area at the position of maximum birefringence, using the Red I plate. Normal enamel displayed low positive birefringence in water (Fig. 2a) and a homogeneous mineralization in the microradiograph (Fig. 3a). White bands exhibited higher positive birefringence, seen as blue bands (Fig. 2b), and lower radiopacity (Fig. 3b) compared with pigmented bands.