Determining Prenatal, Early Childhood and Cumulative Long-Term Lead Exposure Using Micro-Spatial Deciduous Dentine Levels

Abstract

The aim of this study was to assess the validity of micro-spatial dentine lead (Pb) levels as a biomarker for accurately estimating exposure timing over the prenatal and early childhood periods and long-term cumulative exposure to Pb. In a prospective pregnancy cohort sub-sample of 85 subjects, we compared dentine Pb levels measured using laser ablation-inductively coupled plasma mass spectrometry with Pb concentrations in maternal blood collected in the second and third trimesters, maternal bone, umbilical cord blood, and childhood serial blood samples collected from the ages of 3 months to ≥6 years. We found that Pb levels (as 208Pb:43Ca) in dentine formed at birth were significantly associated with cord blood Pb (Spearman ρ = 0.69; n = 27; p<0.0001). The association of prenatal dentine Pb with maternal patella Pb (Spearman ρ = 0.48; n = 59; p<0.0001) was stronger than that observed for tibia Pb levels (Spearman ρ = 0.35; n = 41; p<0.03). When assessing postnatal exposure, we found that Pb levels in dentine formed at 3 months were significantly associated with Pb concentrations in children's blood collected concurrently (Spearman ρ = 0.64; n = 55; p<0.0001). We also found that mean Pb concentrations in secondary dentine (that is formed from root completion to tooth shedding) correlated positively with cumulative blood lead index (Spearman ρ = 0.38; n = 75; p<0.0007). Overall, our results support that micro-spatial measurements of Pb in dentine can be reliably used to reconstruct Pb exposure timing over the prenatal and early childhood periods, and secondary dentine holds the potential to estimate long-term exposure up to the time the tooth is shed.

Full text

Determining Prenatal, Early Childhood and Cumulative
Long-Term Lead Exposure Using Micro-Spatial
Deciduous Dentine Levels
Manish Arora
1,2
*, Christine Austin
1,2
, Babak Sarrafpour
2
, Mauricio Herna
´ndez-A
´vila
3
, Howard Hu
4
,
Robert O. Wright
1
, Martha Maria Tellez-Rojo
5
1Department of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America, 2Faculty of Dentistry, University of Sydney,
Sydney, Australia, 3Office of the Director, National Institute of Public Health, Cuernavaca, Morelos, Me
´xico, 4Dalla Lana School of Public Health, University of Toronto,
Toronto, Ontario, Canada, 5Division of Statistics, Center for Evaluation Research and Surveys, National Institute of Public Health, Cuernavaca, Morelos, Me
´xico
Abstract
The aim of this study was to assess the validity of micro-spatial dentine lead (Pb) levels as a biomarker for accurately
estimating exposure timing over the prenatal and early childhood periods and long-term cumulative exposure to Pb. In a
prospective pregnancy cohort sub-sample of 85 subjects, we compared dentine Pb levels measured using laser ablation-
inductively coupled plasma mass spectrometry with Pb concentrations in maternal blood collected in the second and third
trimesters, maternal bone, umbilical cord blood, and childhood serial blood samples collected from the ages of 3 months to
$6 years. We found that Pb levels (as
208
Pb:
43
Ca) in dentine formed at birth were significantly associated with cord blood Pb
(Spearman r= 0.69; n = 27; p,0.0001). The association of prenatal dentine Pb with maternal patella Pb (Spearman r= 0.48;
n = 59; p,0.0001) was stronger than that observed for tibia Pb levels (Spearman r= 0.35; n = 41; p,0.03). When assessing
postnatal exposure, we found that Pb levels in dentine formed at 3 months were significantly associated with Pb
concentrations in children’s blood collected concurrently (Spearman r= 0.64; n = 55; p,0.0001). We also found that mean
Pb concentrations in secondary dentine (that is formed from root completion to tooth shedding) correlated positively with
cumulative blood lead index (Spearman r= 0.38; n = 75; p,0.0007). Overall, our results support that micro-spatial
measurements of Pb in dentine can be reliably used to reconstruct Pb exposure timing over the prenatal and early
childhood periods, and secondary dentine holds the potential to estimate long-term exposure up to the time the tooth is
shed.
Citation: Arora M, Austin C, Sarrafpour B, Herna
´ndez-A
´vila M, Hu H, et al. (2014) Determining Prenatal, Early Childhood and Cumulative Long-Term Lead Exposure
Using Micro-Spatial Deciduous Dentine Levels. PLoS ONE 9(5): e97805. doi:10.1371/journal.pone.0097805
Editor: Jaymie Meliker, Stony Brook University, Graduate Program in Public Health, United States of America
Received December 20, 2013; Accepted April 23, 2014; Published May 19, 2014
Copyright: ß2014 Arora et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by funding from NIEHS grants R00ES019597 (Arora), P42ES05947 (Hu), R01ES07821 (Hu), P42ES00000 (Hu), R01ES07821 (Hu,
Wright) R01ES013744 (Wright), R01ES014930 (Wright), and Australian NHMRC grant APP1028372 (Arora). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: manish.arora@mssm.edu
Introduction
Susceptibility 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 concen-
trations 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
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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.
Methods
Study Population
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.
Ethics Statement
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).
Collection and Analysis of Child and Maternal Samples
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 cord blood lead
was analyzed using an atomic absorption spectrometry instrument
(model 3000; PerkinElmer, Chelmsford, MA, USA). Child blood
Pb was analyzed with the finger prick method using the LeadCare
System (Magellan Biosciences, Chelmsford, MA, USA) at 3, 6, 18
and 30 months. At other ages venous blood Pb was measured
Figure 1. 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.
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using inductively coupled plasma mass spectrometry (Elan 6100;
PerkinElmer, Norwalk, CT, USA). External blinded quality
control samples (concentrations ranging from 2 to 88 mg/dL)
were analyzed and provided by the Maternal and Child Health
Bureau and the Wisconsin State Laboratory of Hygiene Cooper-
ative Blood Lead Proficiency Testing Program. These analyses
demonstrated good precision and accuracy with quality control
specimens (Pearson r.0.98; mean difference ,1mg/dL). Addi-
tional details have been published previously [13,15,16].
Mothers were asked to provide their child’s naturally shed
deciduous teeth. At the time of this study, we had 34 incisors, 25
canines and 26 molars that could be analyzed for metal
concentrations. The teeth were stored dry at room temperature
in packets provided by the research team, and were collected in
person by a member of the field team. Information on the reason
the tooth was shed, age at shedding, and if tooth was previously
stored in any liquid was also collected from the mothers. Teeth
were examined by a dentist for any visible defects including caries,
attrition, cracks and discoloration. In the present study we use data
from the 85 participants who donated deciduous teeth.
Measurement of Pb in Teeth by LA-ICP-MS
Our approach to measuring metals in teeth using laser ablation-
inductively coupled plasma mass spectrometry (LA-ICP-MS) and
assigning developmental times has been detailed elsewhere (see
Arora et al.[2], Hare et al.[6] and Austin et al.[5]). Briefly, we used
the neonatal line (a histological feature formed in enamel and
dentine at birth) and daily growth incremental markings to assign
temporal information to sampling points.
The laser ablation unit used was a New Wave Research NWR-
193 system (Kennelec Technologies, Mitcham, Victoria, Australia)
equipped with a Nd:YAG laser emitting a nanosecond laser pulse
in the fifth harmonic with a wavelength of 193 nm. The standard
ablation cell was replaced with a Large Format Cell (LFC). The
LFC has a large volume chamber capable of holding samples up to
15.2 cm
2
in area. The x-y-z stage of the LFC employs a small
volume ‘roving’ sampling cup that traverses the sample while the
laser beam remains stationary. An approximately 40 cm length of
TygonHtubing (i.d. 3 mm) connected the laser ablation unit to an
Agilent Technologies 7700cx (Agilent Technologies Australia,
Forrest Hill, Victoria, Australia) ICP-MS. The instrument was
fitted with a ‘cs’ lens system for enhanced sensitivity. The system
was tuned daily for sensitivity using NIST SRM 612 (trace
elements in glass). Polyatomic oxide interference was evaluated
and minimized by monitoring the Th
+
/ThO
+
(m/z 232/248)
ratio. Typical oxide formation was consistently under 0.3%.
Using the laser we sampled 50 sampling points of 35 mm
diameter in enamel and primary dentine adjacent to the enamel-
dentine junction, and seven sampling points of 100 mm diameter
in secondary dentine. Data were analyzed as
208
Pb:
43
Ca ratios to
control for any variations in mineral content within a tooth and
between samples.
Statistical Analysis
To test our hypothesis that dentine captures the timing and
intensity of prenatal Pb exposure we undertook four comparisons.
First, we studied the association of Pb levels in dentine sampling
points at the neonatal line (a measure of fetal Pb exposure at birth
according to our hypothesis) with Pb concentrations in umbilical
cord blood, another marker of fetal Pb exposure. Second, we
compared Pb levels in dentine formed at birth with blood Pb levels
measured at different ages in childhood. If dentine indeed captures
the timing of Pb exposure as we propose, the association of birth
dentine Pb should be strongest with cord blood Pb and
Table 1. Timing of collection of Pb biomarkers used in the present study
a
.
Prenatal trimester Birth Postnatal months
1
st
2
nd
3
rd
1361218243036486084
+
Metal Biomarkers
Fetal/cord blood X
Infant/child blood XXXXXXXXXX
Maternal blood X X X
K-XRF bone Pb X
Teeth X
a
additional measures are available in ELEMENT but not relevant for analyses presented in this study.
doi:10.1371/journal.pone.0097805.t001
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progressively weaker with Pb in blood samples collected at older
ages. Third, we compared Pb levels in dentine formed during the
second and third trimesters with maternal blood Pb levels collected
at corresponding times during pregnancy. We hypothesized that
prenatal dentine Pb should also be positively associated with
maternal blood Pb during pregnancy because Pb levels in
maternal and fetal circulation are strongly associated [18]. Fourth,
we considered maternal bone Pb measurements at 1 mo
postpartum which represent cumulative Pb exposure to the
mother and a source of fetal exposure [14,17,19]. Because bone
Pb represents integrated long-term exposure, we compared this
maternal measure to the area under the curve (AUC) of Pb levels
in all sampling points in prenatally formed dentine. K X-ray
fluorescence provides a continuous, unbiased point estimate of the
true bone lead measurement. However negative values are
sometimes produced when the true values are below the detection
limit of the instrument (in this study, 8 patella and 11 tibia Pb
estimations, respectively, were below this limit). To avoid bias due
to these negative values, we analyzed mean tibia and patella lead
levels using both standard summary statistics that include the
negative values and an estimation using interval regression, which
simulates a normal distribution between 0 and the detection limit
(2 mg Pb/g bone mineral), an approach we have used previously in
this cohort [14]. To assess the potential influence of these negative
values, we performed statistical analyses following two approaches:
1) including the negative estimates and 2) replacing them with new
simulated values randomly generated with a uniform distribution
between 0 and the lower limit of 2 mg Pb/g bone mineral. Since
the results obtained were very similar (,5 percent difference in the
estimated coefficients), we present the results only from the former
approach.
We also tested whether dentine captures early childhood Pb
exposure. We compared Pb levels in dentine formed at 3 months
postnatally with Pb concentrations in blood collected at this age. In
our sample of incisors (teeth for which the coronal primary dentine
completes at the earliest age) the most cervical dentine was formed
between the ages of 1.2 to 3 months based on our histological
analysis. Consequently, in some cases, the timing of Pb exposure
represented by the dentine sampling points is not exactly
concurrent with the 3-month blood Pb concentrations. However,
it is important to consider that this disparity between the incisor
dentine and blood measurements is likely to reduce the observed
association between the two biomarkers and would not result in an
inflated correlation (i.e. a bias towards the null). This issue was not
of concern for canines and molars, where primary coronal dentine
continues to form for 6 months or longer, and we were readily able
to compare Pb levels in dentine formed at 3 months postnatally
with 3-month blood Pb concentrations. We also extended this
analysis by comparing Pb levels in these dentine sampling points
with blood Pb levels measured at older ages in childhood.
Finally, we wanted to test the utility of secondary coronal
dentine as a marker of cumulative long-term Pb exposure. This
compartment of coronal dentine is formed from approximately
1.5 to 2.0 yrs for incisors and 2.5 to 3.0 yrs for canines and molars
to the time of tooth shedding [20]. To test this hypothesis we
compared the average Pb levels of seven sampling points in
secondary dentine with CBLI. The CBLI is calculated by
integrating multiple blood Pb measurements at different ages,
and gives a single composite score that describes long-term Pb
exposure (details on calculating CBLI and its interpretation have
been presented by others [11,12]). We excluded any participants
who had less than 3 valid measurements of blood Pb from the ages
of 1.5 years to the last included measurement which was 5 to 6
years for incisors and 7 to 11 years for canines and molars.
Spearman’s correlation analysis was used to measure the
association of
208
Pb:
43
Ca in teeth with blood and bone Pb
measurements. When displaying bivariate associations as scatter
plots with regression lines, we log
e
transformed our data to achieve
normally distributed variables. In some of our data analyses, we
multiplied our dentine
208
Pb:
43
Ca measurements with 10 or 10
4
to
avoid negative values during log
e
transformation (and have
indicated in the results when this was done). Results were
considered statistically significant at p,0.05. All analyses were
undertaken in STATA 10.0 (StataCorp, College Station, Texas).
Results
When assessing the intensity and timing of prenatal exposure
from dentine Pb, we found that Pb levels (as
208
Pb:
43
Ca) in
sampling points at the neonatal line (which represented approx-
imately 12–15 days of dentine deposition around birth) were
significantly associated with cord blood Pb levels (Spearman
r= 0.69; n = 27; p,0.0001; Fig. 2a). Additional comparisons of Pb
levels in these sampling points in dentine formed at birth with
blood Pb concentrations measured later in life showed that the
association continued to decline with increasing age at blood
sampling (Fig. 2b.). Birth dentine Pb levels were not significantly
associated with the child’s blood Pb sampled after 2 years of age.
Because Pb deposits in maternal bone are mobilized during
pregnancy and serve as a major source of Pb exposure to the fetus
[18], we compared Pb levels in prenatal dentine with maternal
bone Pb measured at 1 month postpartum. Prenatal dentine Pb
was significantly associated with both maternal tibia and patella Pb
concentrations (Fig. 3a, b). However, the association with maternal
patella Pb (Spearman r= 0.48; n = 59; p,0.0001) was stronger
than that observed for tibia Pb levels (Spearman r= 0.35; n = 41;
p,0.03). When we compared the Pb levels in dentine formed
during the second and third trimesters with Pb concentrations in
Table 2. Lead levels in biological media.
Matrix n Median Pb (range)
Prenatal dentine
a
82 0.31 (0.06 to 3.99)
Postnatal dentine
a
83 0.27 (0.06 to 2.72)
Secondary dentine
b
84 0.01 (0.00 to 0.12)
Cord blood
c
28 3.95 (0.90 to 14.7)
d
Child blood at 3 mo
c
55 3.50 (0.20 to 11.8)
Maternal blood during pregnancy
c
1
st
trimester 51 5.70 (0.9 to 26.3)
2
nd
trimester 52 4.70 (0.8 to 17.1)
3
rd
trimester 49 5.20 (0.90 to 16.0)
Maternal bone
e
Tibia 43 6.45 (0.09 to 33.4)
f
Patella 61 11.32 (0.01 to 42.9)
g
a
area under curve (AUC) of Pb levels in all sampling points within a region of
dentine formed over a selected developmental period. Because the number of
sampling points varies, AUC is adjusted for number of sampling points within
that region.
b
average of 7 measurements (as
208
Pb:
43
Ca).
c
mg/dL.
d
mean (SD) = 4.70 (3.72).
e
mg Pb/g bone mineral.
f
adjusted for negative values, mean (SD) = 8.95 (8.03).
g
adjusted for negative values, mean (SD) = 12.9 1 (11.21).
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maternal blood collected concurrently, we also found significant
positive associations (Second trimester: Spearman r= 0.60; n = 36;
p,0.0001; Fig. 3c. Third trimester: Spearman r= 0.64; n = 44;
p,0.0001; Fig. 3d).
To assess exposure timing in early childhood we compared Pb
levels in dentine formed at approximately 3 months postnatally
with Pb concentrations in the child’s blood sampled at 3 months of
age. We found a significant positive association (Spearman
r= 0.64; n = 55; p,0.0001). Furthermore, when we compared
the association of Pb levels in these dentine sampling points (which
were formed at approximately 3 months of age) with blood Pb
concentrations measured at later ages we observed a progressively
weaker association (Fig. 4).
To test the hypothesis that secondary coronal dentine captures
long-term Pb exposure we compared the average of 7 sampling
points in secondary coronal dentine of each tooth with CBLI
which represents the integrated exposure in childhood (see
Methods for additional details). We found that mean Pb
concentrations (as
208
Pb:
43
Ca) in secondary dentine correlated
positively with CBLI (Spearman r= 0.38; n = 75; p,0.0007).
Furthermore, when secondary dentine Pb levels were compared to
Pb concentrations in maternal bone and maternal blood during
pregnancy the correlations were weaker and statistically insignif-
icant, supporting our hypothesis that secondary dentine Pb is a
measure of cumulative postnatal exposure Pb exposure and not
affected by prenatal Pb exposure (Table 3).
Discussion
Measuring exposure timing, especially during fetal develop-
ment, is a major challenge in environmental epidemiology. Even
when maternal biomarkers are available during pregnancy, they
are not always a reliable measure for fetal exposure. The use of
deciduous teeth provides a unique opportunity to directly measure
fetal exposure. Furthermore, the ability to retrospectively reconstruct
prenatal and early childhood exposure would permit temporal
exposure information to be ascertained in case-control studies.
This, in our opinion, is likely to improve efficiency in the study of
diseases that occur at lower frequency and manifest during
childhood and adolescence (autism spectrum disorders, for
example).
Whole teeth and fragments of dentine have been successfully
used in many studies to estimate cumulative Pb exposure
[9,10,21]. Needleman and colleagues [9] observed that children
who lived in environments with greater risk of Pb exposure had
higher dentine Pb levels than children who lived in areas with
lower Pb exposure. Further validation was undertaken by
Rabinowitz and colleagues who showed that dentine Pb measure-
ments and blood Pb concentrations were significantly correlated
[10]. Subsequently, dentine and whole tooth Pb levels were used in
studies of childhood neurodevelopment and this Pb biomarker was
found to be associated with several developmental parameters
including IQ [8,21]. Gulson and colleagues advanced these
methods by means of stable Pb isotope analysis that allowed
identification of source of Pb exposure [7]. With the advent of
micro-spatial analytical methods, including laser ablation and
proton- and synchrotron-induced X-ray emission methods, it is
now possible to take advantage of the incremental structure of
teeth to reconstruct the timing of Pb exposure. However, before
micro-spatial dentine Pb analysis can be applied to the study of
health outcomes, it is important that this method is validated
against other exposure biomarkers.
Much work has been undertaken on the kinetics of Pb in blood
which is why it has been the marker of choice to explore the
adverse health effects of Pb exposure in most epidemiologic
studies. The application of K-XRF analytical methods to the
in vivo measurement of Pb levels in bone provided a way to study
cumulative Pb exposure, which cannot be obtained from single
blood Pb measurements due to the short half-life of Pb in blood
[17]. Therefore, to validate dentine Pb as a biomarker of the
timing and intensity of Pb exposure, we compared dentine Pb with
these established biomarkers.
When using the dentine-Pb biomarker to uncover prenatal
exposure, we found that Pb levels in sampling points in dentine at
the neonatal line were positively associated with cord blood Pb
concentrations. Our laser sampling points are 35 mm in diameter
which represent exposure over approximately 12–15 days around
birth. Because cord blood Pb represents levels in fetal circulation
in the latter part of the third trimester, a strong positive correlation
between these two matrices supports our hypothesis that dentine
Pb is a suitable marker of prenatal exposure. However, to confirm
that sampling points in dentine capture the timing of prenatal
Pb exposure we undertook additional analyses. We compared Pb
levels in dentine sampling points at the neonatal line with Pb
concentrations in blood collected at different ages in childhood. It
Figure 2. Association of Pb levels in dentine formed at birth with blood Pb concentrations at birth and childhood. (a) Significant
positive association of Pb levels (as log
e
[
208
Pb:
43
Ca10
4
]) in dentine at the neonatal line (representing fetal Pb exposure at birth) with umbilical cord
blood Pb concentrations (as log
e
[Pb610 mg/dL]). (b) Spearman’s r(Y-axis; vertical lines show 95% CI) of Pb levels in dentine formed at birth with Pb
concentrations in blood collected at different ages (X-axis; in months). The association of birth dentine Pb is strongest with umbilical cord blood Pb
and is progressively weaker with blood sampled at later ages. Sample size at different time points: birth = 27, 3 m = 55; 6 m = 43; 12 m = 61;
18 m = 63; 24 m = 68; 30 m = 64; 36 m = 63; 48 m = 67; 60 m = 48.
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was our hypothesis that if dentine indeed captures the timing of Pb
exposure, then measurements in dentine formed at birth would
show the strongest correlation with cord blood Pb and a
progressively weaker association with circulating Pb levels at older
ages. Our observations (Fig. 2b) confirmed this hypothesis.
Beyond the analysis of dentine formed at the time of birth, we
wanted also to check the utility of our biomarker in predicting
cumulative prenatal exposure. Lead stored in maternal bones is
mobilized to maternal circulation during pregnancy and lactation
and serves as an important source of fetal Pb exposure [18]. Bone
Pb measurements were undertaken at 1 month postpartum in our
study and have been applied effectively to study prenatal Pb
exposure and childhood health in this cohort [19]. When we
compared Pb levels in prenatal dentine (as area under curve of all
prenatal sampling points), we found a significant positive
association with both patella and tibia Pb measurements. The
stronger association with patella Pb is not surprising because Pb
deposits in trabecular bone (predominant component of patella)
are comparatively more mobile and exchange more readily with
plasma than Pb stored in cortical bone (predominant component
of tibia) [17,18]. Similarly, we found that Pb levels in dentine
formed during the second or third trimesters were significantly
associated with Pb concentrations in maternal blood collected
concurrently.
An important feature of secondary dentine is that it continues to
accrue Pb and other elements as long as the tooth remains vital. It
is, therefore, a potential target to measure integrated life-time
Figure 3. Association of Pb levels in prenatal dentine with maternal bone and blood Pb concentrations. Prenatal dentine Pb (as log
e
[AUC
208
Pb:
43
Ca610
4
]) was positively associated with Pb concentrations (as mg Pb/g bone mineral) in a) tibia (Spearman r= 0.35; n = 41; p,0.03) and
b) patella (Spearman r= 0.48; n = 59; p,0.0001). Relevance of negative bone Pb concentrations is discussed in detail by Hu et al.[17] Significant
positive associations were observed between Pb concentrations in maternal blood and prenatal dentine formed during the c)2
nd
trimester
(Spearman r= 0.60; n = 36; p,0.0001) and d)3
rd
trimester (Spearman r= 0.64; n = 44; p,0.0001).
doi:10.1371/journal.pone.0097805.g003
Figure 4. Timing of postnatal Pb exposure estimated from
dentine. Spearman’s correlation coefficients (and 95% CI) for Pb levels
in dentine formed at 3 months postnatally and child blood Pb also
measured at 3 months. When compared to Pb levels in blood collected
at older ages in childhood, the association was progressively weaker
supporting the hypothesis that dentine retains the timing of postnatal
Pb exposure.
doi:10.1371/journal.pone.0097805.g004
Dentine Lead Biomarker
PLOS ONE | www.plosone.org 6 May 2014 | Volume 9 | Issue 5 | e97805

exposure up to the time the tooth is shed (or no longer depositing
dentine). We examined secondary dentine in our tooth sections,
avoiding tertiary dentine deposits which may corrupt our analysis,
and measured average Pb concentrations over seven sampling
points. We compared this measure to the CBLI which integrates
blood Pb concentrations over different ages and is an established
measure of cumulative Pb exposure [11,12]. In this analysis we
used data from children who had blood Pb measurements from
the ages of 1.5 to 6 years, making this one of the most detailed
analyses of secondary dentine Pb with serial blood Pb measure-
ments undertaken to date. We observed a significant positive
association between the two measures suggesting that average Pb
concentrations in secondary dentine do provide a measure of long-
term cumulative exposure.
The main limitation of our study is that we did not have a
complete set of biomarkers on all 85 participants due to
incomplete follow-up and sample collection. However, our sample
size was sufficient to test our hypotheses and uncover statistically
significant associations between respective biomarkers. Secondly,
we have used data from the first 85 participants who donated
teeth. To examine differences in exposure profile of our
participants from the wider ELEMENT cohort, we compared
the Pb levels in maternal blood and bone to those reported from
earlier work on the ELEMENT participants. We only observed
minor differences (compare data in Table 2 with data in Tellez-
Rojo et al. [14] and Hu et al. [22]). Another limitation of our study
is that we do not have multiple teeth from each participant.
Consequently, we are unable to explore if tooth type influences
dentine Pb concentrations. Ideally, within each individual, we
should compare Pb levels in multiple teeth in dentine formed at
the same time (at birth, for example), thereby parsing out the
influence of tooth type from differences in exposure.
Metal concentrations in teeth have served as exposure
biomarker in many studies. With modern techniques that allow
micro-spatial measurement of multiple metals in teeth, we now
have the opportunity to reconstruct past exposure history not only
in terms of cumulative exposure but also for the timing of
exposure. This will allow us to examine critical windows of
susceptibility even when those windows occur prenatally, which
would be a major advance in environmental epidemiology
considering that maternal exposure biomarkers do not always
represent fetal exposure adequately. A barrier in the application of
this biomarker to the study of health outcomes has been the lack of
appropriate validation of this methodology. In the present study,
we have shown that measurements of Pb in discrete regions of
dentine can be reliably used to reconstruct Pb exposure history
over the prenatal and early childhood periods and secondary
dentine holds the potential to estimate long-term exposure up to
the age of tooth shedding.
Supporting Information
Table S1 Brief description of anatomical terms used in this
manuscript.
(DOCX)
Acknowledgments
The authors acknowledge Maritsa Solano and Katherine Svensson for
assistance with data management.
Author Contributions
Conceived and designed the experiments: MA CA BS MH-A HH ROW
MMT-R. Performed the experiments: MA CA BS. Analyzed the data: MA
CA HH ROW MMT-R. Contributed reagents/materials/analysis tools:
MA CA BS MH-A HH ROW MMT-R. Wrote the paper: MA CA BS
MH-A HH ROW MMT-R.
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