Overview of the dentine Pb biomarker. ( a ) Schematic of a deciduous human incisor. During tooth formation the deposition of the enamel (E) and primary dentine (PD) matrix commences at the enamel-dentine junction (dashed line). At birth the neonatal line (red line) is formed in enamel and dentine providing a landmark to distinguish prenatally formed parts of teeth from those formed postnatally. In our analysis, prenatally formed primary dentine adjacent to the enamel-dentine junction is sampled to obtain prenatal Pb exposure information. Sampling points in dentine at the neonatal line measure fetal Pb exposure at the time of birth. Secondary dentine (SD) formation starts after the completion of the tooth root and proceeds at a slower rate as long at the tooth remains vital. Measurements in this region are used to estimate cumulative long-term Pb exposure from root completion to the time tooth was shed. Pulp chamber (P) and cervical margin (C) between the tooth crown and root are also indicated. ( b ) Lead distribution and developmental timing of primary dentine sampled in a deciduous molar. It is our hypothesis that Pb levels at each dentine sampling point represent Pb exposure experienced when that part of dentine was being mineralized. In this individual dentine Pb levels showed a marked rise prior to birth. Key time points for change in dentine Pb levels are shown; other points on the graph can be similarly dated. doi:10.1371/journal.pone.0097805.g001 

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... to environmental toxicants is heightened during prenatal and early childhood periods when many systems are developing and vulnerable to the disruptive effects of chemicals [1]. However, accurate objective assessment of exposure timing, especially during fetal development, remains a major challenge in environmental epidemiologic research. This primarily arises from the absence of direct fetal biomarkers of exposure that can be safely used in large study populations. In earlier work, we used naturally shed deciduous tooth dentine to uncover early life exposure to metals including Pb, Mn, Ba and Sr [2–6]. We combined micro-spatial elemental analysis of teeth with detailed histological techniques to construct a detailed temporal ‘map’ of exposure during the prenatal and early childhood periods. Figure 1 depicts the basic structure of a tooth crown and provides an example of our approach to estimating exposure timing on a fine scale over the prenatal and early postnatal periods. A brief description of dental anatomical terminology used in this manuscript is given in Table S1 (see Information S1). Although the use of teeth to obtain cumulative metal exposure information has been proposed for many decades [7–10], the validity of obtaining exposure timing from dentine remains to be adequately tested. There exists a need to validate the temporal Pb exposure information obtained from dentine against Pb concentrations in other established biomarkers including maternal blood and bone, cord blood and serial childhood blood Pb levels. Furthermore, comparing the cumulative Pb exposure information from secondary dentine with another measure of integrated long- term Pb exposure such as the cumulative blood Pb index (CBLI) [11],[12] would provide evidence that life-long exposure can also be estimated from dentine. In the study presented here, we evaluate if the dentine biomarker accurately measures (i) the intensity and timing of fetal exposure, (ii) the intensity and timing of early childhood exposure, and (iii) cumulative long-term exposure. We undertook this study in a prospective mother-child cohort where we measured Pb in umbilical cord blood collected at birth, and in serial venous blood samples collected at approximately 6- to 12-monthly intervals from the age of 3 months to 6 years, and then measured blood Pb again between the ages of 7 to 11 years. We also measured maternal blood Pb in each trimester of pregnancy and at multiple time points after the birth of their child. Importantly, we have measured Pb in the bones of the mothers which allowed us to explore the association of maternal Pb body burden with our tooth Pb biomarker. Mother–child pairs in this study were drawn from the longitudinal birth cohort studies in Mexico City that comprise the Early Life Exposures in MExico and NeuroToxicology (ELEMENT) project. Subjects were originally recruited between 1994 and 2003 to investigate the long-term consequences of prenatal environmental factors on child development [13–15]. Detailed information on the study design and data collection procedures has been published previously [13,15,16]. Mothers were recruited during pregnancy and maternal venous blood was sampled once each during the first, second and third trimesters. Anthropometric data from the mother and newborn, and umbilical cord and maternal venous blood samples were gathered within 12 hr of delivery. Information on estimated gestational age, based on the date of last menstrual period, and characteristics of the birth and newborn period were extracted from the medical records. Exclusion criteria included factors that could interfere with maternal calcium metabolism; medical conditions that could cause low birth weight; prematurity ( , 37 weeks) or an infant with Apgar score at 5 min of # 6, a condition requiring treatment in neonatal intensive care unit; or serious birth defects; psychiatric illness, seizures, or kidney or cardiac disease; preeclampsia, systolic BP . 140 mmHg or diastolic BP . 90 mmHg; gestational diabetes; consumption of alcoholic beverages; addiction to illegal drugs; and continuous use of corticosteroids. The mother–child pairs were contacted and recalled for the follow-up assessment between 2008 and 2010 when the children were 7–15 years of age, and tooth collection was incorporated into this visit. Only one child for each mother was included in this study, regardless of birth order. Medical history, physical examination, and venous blood samples were collected from or performed on mothers and children. All participating mothers signed a written consent form, received a detailed explanation of the study intent and research procedures, as well as counseling on how to reduce environmental lead exposure. In addition, the children signed a written assent of minor form, easy to understand for them, and also received a detailed explanation of the study. All participants were encouraged to ask questions about the study in order to ensure their understanding. The research protocol was approved by the human subjects committees of the National Institute of Public Health of Mexico, the Harvard School of Public Health, Michigan School of Public Health, National Institute of Perinatology, and the American British Cowdray Medical Center (ABC Hospital). The sampling times of various biological media used in this study are shown in Table 1, and the distribution of Pb levels in these media is detailed in Table 2. Both median (range) and mean (SD) are provided to allow comparison with other studies in this cohort. Maternal tibia (cortical) and patella (trabecular) bone lead levels were measured within 1 month of delivery using a spot- source 109 Cd K-shell X-ray fluorescence (K-XRF) instrument (ABIOMED, Danvers, MA, USA) [17]. Umbilical ...