TOOTH ENAMEL BIOMARKER FOR HEAVY METAL EXPOSURE ASSESSMENT
J.E. Ericson*, A. Rinderknect (Department of Environmental
Analysis & Design, University of California, Irvine, CA 92697‑7070,
USA); M.T. Kleinman (Department of Community & Environmental Medicine,
University of California, Irvine, CA 92697‑1825, USA): jeericso@uci.edu
ABSTRACT
Measurements
of lead (Pb), manganese (Mn), and calcium (Ca) were made in transects of tooth
enamel and used as biomarkers for assessing heavy metal exposure continuously
during prenatal and neonatal development. This technique was applied to
histological cross‑sections of rat tooth and deciduous tooth enamel of
three human subjects, using ion mass spectrometry (IMS). The findings suggest
that this method may be applied to cross‑sectional and longitudinal
studies to assess health effects of ambient heavy metal exposure on prenatal
and postnatal CNS development.
INTRODUCTION
The
accurate measurement of prenatal exposure, given the exogenous and endogenous
factors in maternal transfer, short-term variations in nutrient/Pb ratios, and
complexity of fetal neurological development provide limitations for assessing
the relationships between Pb exposure and biological effects (Goyer, 1996). It
is not surprising that two decades of prospective research conducted in Mexico,
the former Yugoslavia, Port-Pirie, Australia, Cincinnati, Boston, Cleveland and
Glasgow, using conventional methods do not appear to demonstrate relationships
between low level prenatal Pb exposure and long-term deficits in IQ. Standard
techniques of exposure assessment of Pb including serial maternal blood Pb
(Rothenberg et al., 1995), cord blood Pb, circumpulpal dentine (Rabinowitz et
al., 1993), cortical bone tissue Pb measurement (Bellinger et al., 1994), and
more recently sectioning of tooth samples (Gulson and Wilson, 1994), have been
used to assess prenatal and postnatal exposure and neurotoxic effects. However,
these surrogate measures, including maternal and placental blood Pb
concentrations are imprecise estimators of fetal Pb exposure and do not provide
useful metrics of when exposure occurred during development. Therefore, there
is a well‑recognized need to define temporal specificity of dose‑effect
when addressing issues of CNS development.
Another toxic metal for
which temporally relevant exposure assessments are needed is the neurotoxin Mn.
The ban on the use of the gasoline additive methyl tertiary butyl ether (MTBE)
by executive order in March 2000 could lead to the combustion of
methylcyclopentadienyl manganese tricarbonyl or MMT, a controversial octane‑boosting
fuel additive, which will increase environmental Mn. The emitted Mn can be
absorbed through respiration, dermal contact, and the gastrointestinal tract. Epidemiological
studies and laboratory evidence support the neurotoxicity of high Mn exposure,
particularly for fetuses and young children (Lonnerdal et al., 1983, Wagner,
2000). Determining the time course of Mn exposure by examining patterns of Mn
deposition in deciduous teeth can assist in assessing the potential role of Mn
in the etiology of CNS dysfunctions.
METHODS
It is demonstrated here for the
first time measurements of Pb, Mn and Ca in human tooth enamel by ion mass
spectrometry within a clean room environment. The ion (microprobe) mass
spectrometer provides simultaneous, high resolution, high sensitivity analysis
of four to six elements or isotopes at spatial resolutions of 10 mm at
the 30ppb Pb level in cross‑sections of tooth enamel. Pb, Mn, and Ca were
measured in polished, carbon-coated, longitudinal cross sections of enamel of
shed, deciduous mandibular central incisors.
Three shed deciduous mandibular
central incisors were selected for this initial experiment. The deciduous
mandibular central incisors in humans, used in this method, are the first teeth
to mature and erupt at six to eight months of age that are subsequently shed at
six to seven years of age (McDonald 1974). The first formed enamel of these
teeth occurs at 4.5 months (18th week) from conception and is
located at the cusp tip at the enamel‑dentine junction (Deutsch and
Pe’er, 1982). The last formed enamel at nine to ten months’ gestation (Deutsch
and Pe’er 1982) occurs at the cervical cementum‑enamel junction (CCEJ)
and matures by 2.5 months after birth or 50 weeks gestation. The
average enamel rate of growth is 21mm/day from cusp tip to cementum‑enamel junction
(Bernard, 1997). Once matured, tooth enamel is a metabolic isolate and does not
undergo metabolic mineral exchange (Ericson et al., 1979), although surface
enamel can absorb Pb from biological fluids of the oral cavity. Beginning at
the cusp tip, the probe followed the approximately 4.5 mm transect at a
distance of 0.3 mm from the labial enamel-dentine junction to the cervical
junction.
USA1 was a 1986 shed tooth from a six-year-old, Los
Angeles, California boy, born and raised in that urban environment. Sample 1562
and sample 1720 were 1998 shed teeth from two six-year-old Mexican children
living in Tijuana although born in rural Sinaloa and urban Tijuana,
respectively. The blood Pb levels of the subjects of the Tijuana Childhood Pb
Project, analyzed in 1998, were 11 µ/dl and 7 µ/dl, respectively. Blood level
of USA1 is unknown. In 1998, subject 1562 reported eating three types of known
Pb‑contaminated candies, eating frijoles from a low-fired Pb glazed
ceramic, received additional Pb exposure from father's occupation and storage
of Pb automobile batteries at his residence. Neither USA1 nor subject 1720
reported any specific exposure sources.
To test the temporal relevance of the method, we
injected pregnant Sprague Dawley rats with stable Pb isotopes at various times
during prenatal and postnatal development. The animals were housed in an AALAAC‑accredited
facility and all procedures were reviewed and approved by the Institutional
Animal Care and Use Committee. 204Pb was administered by i.p.
injection to the dams on the 12th day of gestation for the prenatal
dose and 206Pb was administered to the pups on day 7. The pups were euthanized
by a lethal injection of sodium pentobarbital (65 mg/kg) at 14 days of age
and the teeth were reserved for IMS analysis.
The IMS analyses were performed using the
CAMECA IMS 1270, were analyzed using the large-radius triple focusing mass
spectrometer with primary ion sources capable of producing mass-filtered ion
beams. The primary beam (160‑), approximately 6nA
was focused to a spot size approximately 46 mm in diameter and mass resolution was 3400, which is
sufficient to resolve the known molecular interferences. Apatite from Durango,
Mexico (Young et al., 1969) was used for Pb, Mn and Ca calibration of unknown
samples. Five minutes of auguring by IMS at each spot removed surface
contamination and then an electromagnetic "window" was placed over the
spot. Pb concentration, 206Pb, 207Pb, 208Pb, 55Mn
and 48Ca were analyzed
simultaneously by beam switching at each spot. The 208Pb and 55Mn
results were normalized relative to 48Ca in the sample and
calibrated by using 208Pb normalized relative to 48Ca in
the Durango apatite standard. The normalization to 48Ca acts as a
control for density variations within the sample and standard. If body burden
Pb can be associated with common Pb coming from the environment, then 208Pb
multiplied by a factor of 1.9 (208Pb represents 52.4% total Pb), can
be used to determine total Pb exposure accumulated during prenatal and
postnatal development.
RESULTS AND DISCUSSION
Multiple
measurements of Pb concentrations in enamel histology were made, shown in
Figure 1. The US child has the lowest enamel Pb of the group,
consistent with the US 1975 phase-out of lead in gasoline. The phase‑out
of Pb in gasoline in Mexico commenced in 1992 although still available in
Tijuana until 1997. A double dissociation between prenatal tooth enamel Pb and
postnatal (at age six years) blood Pb level between the two Mexican children is
noted. The prenatal Pb exposure of the Sinaloa born child is lower than the
Tijuana born child. The Sinaloa child reflects low prenatal Pb exposure in a
rural environment with subsequent exposure to excessive Pb as a six year-old
living in Tijuana via his four exposure variables. Within each concentration
profile, we observe an increase in Pb, near the cervical cementum-enamel
junction (40th to 50th week along the transect), which is
believed coeval with postnatal Pb exposure.
Figures
2a and b indicate an inverse relationship between Mn and Ca along the enamel
transect of each incisor. Cross‑sampling and longitudinal data can be
shown within these figures. In Figures 2a and b, subject USA1 has relatively
lower 55Mn accumulation than the Tijuana‑born subject 1720,
while having slightly elevated 48Ca levels relative to the other
subjects.
Figure
3 shows prenatal and postnatal histological exposure to 206Pb and 204Pb,
respectively, within the enamel of the rat mandibular incisor. Data points were
taken along a transect in which p1 represents the earliest formed enamel laid
down during prenatal development and p6 documents a data point near the
cementum‑enamel junction, indicating a section of enamel that was laid
down during postnatal development. These experimental data document metabolic
transfer of blood Pb to the tooth during enamel development.
We
have developed a method to determine the distribution of Pb and Mn within the
histological structure of deciduous tooth enamel and have established
techniques such that these measurements may serve as a biological indicator
(biomarker) of exposure providing a continuous record through prenatal and
perinatal time periods. The method can be extended to later periods of
perinatal early childhood exposure by using eruption sequences of deciduous
teeth. Changes in micronutrients can also be evaluated (such as deficiencies of
Ca, Fe, Zn, Mg and P) to elucidate synchronous co‑variational effects
with Pb or Mn. Likewise, the source or change of Pb exposure can be determined
retrospectively through isotopic characterization of the enamel histology.
Additional work, especially with respect to characterizing the temporal
relationships between location in the tooth matrix and heavy metal
concentrations is needed.
REFERENCES
Bellinger,
D., Hu, H., Titlebaum, E., and H.L. Needleman. Arch. Env. Health. 49(2)
98‑105 (1994).
Bernard,
G.W. (1997). Encyclopedia of Human Biology, 2nd Edition;
Academic Press. 3:2033‑215.
Deutsch,
D., and E. Pe'er (1982). J. Dent. Res. (Sp.Iss): 1543-1551.
Ericson, J.E., Shirahata, H., and
C.C. Patterson (1979). New England Journal of Medicine, 300, 946‑951.
Goyer,
R.A. (1996). Env. Health Perspectives. 104(10): 1050-1054.
Gulson,
B.L., and D. Wilson (1994). Arch. Env. Health, 49:279-283.
Lonnerdal B., Keen C.L., Ohtake M., Tamura T. Am J Dis Child, 1983;
137:433-437
McDonald,
R.E. (1974). Dentistry for the Child & Adolescents. The C.V. Mosby
Co., St. Louis, MO.
Rabinowitz,
M.B., A. Leviton and D. Bellinger (1993). Calcif Tissues Int. 53:338‑341.
Rothenberg,
S.J., Karchmer, S., Schnaas, L., Perroni, E., Zea, F., Alba, J.F. (1995). Env.
Health Perspectives. 102:876-880.
Wagner J.R. The toxicity of the fuel
additive MMT in the dopaminergic PC12 cell line, UMI, 2000.
Young,
E.J., Myers, A.T., Munson, E.L., and Conklin, N.M. (1969). U.S. Geological
Survey Prof. Paper 650-D, D84-93.
