Prenatal Manganese Exposure Assessment

Mothers were asked to bring in at the time of the 7-year assessment a tooth the child had shed. In the present study, we used incisors that were free of obvious defects (caries, hypoplasias, fluorosis, cracks, extensive attrition) and prepared ~100–150 μm sections in an axial labio-lingual plane. Using light microscopy, we identified the neonatal line (NL), a histological feature formed in deciduous teeth at birth (see Supporting Information, Figure S1). With the NL as a reference point, we measured Mn levels (as ⁵⁵Mn:⁴³Ca) in mantle dentine immediately adjacent to the enamel-dentine junction (EDJ) with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) (Figure 1, Supporting Information Table S1, and Arora et al. 2011 [17 (link)]). We normalized Mn measurements to ⁴³Ca to account for intra- and inter-sample variations in mineralization. We confirmed that our sampling points were in mantle dentine by examining sections in polarized light where mantle dentine is clearly demarcated from circumpulpal dentine (Figure 1d).
We sampled mantle dentine at 30 equally spaced points adjacent to the EDJ from cusp tip to the cemento-enamel junction and calculated the area under the curve (AUC) of Mn levels across all the sampling points in prenatally formed mantle dentine to estimate cumulative Mn exposure in the prenatal period. Because floor dust and blood samples were collected during the second trimester, we also undertook analyses restricted to Mn levels in sampling points located in mantle dentine formed in the second trimester.