Manish Arora*ab and
Dominic J. Hareacd
aSenator Frank R. Lautenberg Environmental Health Sciences Laboratory, Department of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, USA. E-mail: manish.arora@mssm.edu
bFaculty of Dentistry, University of Sydney, Sydney, New South Wales, Australia
cElemental Bio-imaging Facility, University of Technology Sydney, Broadway, New South Wales, Australia
dThe Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia
First published on 29th July 2015
Accurate assessment of early life lead exposure requires an accessible and reliable biomarker. Blood lead levels alone are not a suitable measure of chronic lead exposure, due to the rapid turnover and proportionally smaller amount compared to calcified tissues, such as bone and teeth. To further validate and standardise tooth lead levels as an appropriate biomarker of exposure, we designed a series of experiments where Wistar rats were fed, through drinking water, a regime of lead dosage that included stable, increasing and decreasing exposures, as well as prenatal exposure via the mother. At 15 days of age the animals were culled and blood, kidney, liver, brain, bone and teeth lead levels were assessed using inductively coupled plasma-mass spectrometry. Bone and tooth lead levels were by far the highest, and were reflective of the dosing parameters used. Brain lead levels were the lowest, suggesting that the blood–brain barrier provided some protection against lead uptake. These results give further support to the use of tooth lead levels as a biomarker of environmental lead exposure, particularly during early life periods.
Exposure to lead during the neo- and postnatal periods represents a critical window that can result in significant adverse effects on neurodevelopmental outcomes. The precise mechanism by which lead imparts neurotoxicity during this period is unclear, though animal studies have shown that it disrupts neurotransmitter synthesis,6 impedes calcium-dependent processes including nitric oxide synthesis7 and increases the rate of apoptosis through elevated oxidative stress in hippocampal neurons.8 Long-term adverse health outcomes of children exposed to high levels of environmental lead include decreased intelligence quotient (IQ), poorer academic performance and delinquent behaviour.9 Studies in bacteria have shown that chronic lead exposure has significantly greater adverse effects on cell viability than acute toxicity,10 thus the persistency of lead in the environment is a pressing health concern.
The concept of critical windows of susceptibility to lead toxicity thus requires a better understanding of the differential lead body burden that is dependent on exposure timing. For instance, fetal exposure to lead during the first and second trimesters is associated with a higher risk of premature birth,11 and prenatal lead exposure appears to increase the production of β-amyloid into young adulthood.12 Recent evidence has also shown that coexposure to lead with other potentially toxic metals, such as manganese during the second year of life can potentiate lead neurotoxicity.13
The highly variable half-lives of lead stores in the body makes direct association of the timing and magnitude of exposure difficult to ascertain. Historically, much of our understanding of lead body burden comes from work performed in the mid-1900s, prior to the development of many of the more accurate analytical methods available today.
We have previously taken advantage of such methods, such as imaging by laser ablation-inductively coupled plasma-mass spectrometry to assess the metal levels within teeth.14–16 Teeth are a particularly attractive biomarker of past chemical exposure, as they are able to exchange metal ions into the hydroxyapatite crystal structure and do not readily remobilise metals,17 as is the case with bone lead. Additionally, children's deciduous teeth can be collected with relative ease and in a non-invasive manner, and in combination with histological analysis; spatial information can be directly correlate with specific time periods of exposure.
In this work, we examined the distribution of lead in Wistar rats following pre- and postnatal lead exposure using inductively coupled plasma-mass spectrometry, with a particular focus on the uptake in teeth. The aim of these experiments was to investigate how differential timing of lead exposure during early life affected the distribution of the metal throughout the body, and if tooth lead concentrations provided an appropriate measure of lead body burden, to further support our efforts to develop a tooth-based biomarker of early life metal exposure.
A total of 41 animals were raised for these experiments. Two rat pups belonging to Group 2 appeared to be rejected by the mother and showed physical signs of distress in the first week after birth, including low body temperature, wrinkled skin and low body weight. These animals were immediately culled. Body weights of all animals were within the reported normal range (5.0–6.4 g at birth)18 and there were no significant differences in body weights between rat pups exposed to lead and those in the control group (p > 0.05; two-tailed Student's t-test). A total of 39 Wistar rat pups were included in the study (N per group: Group 1 = 11; Group 2 = 5; Group 3 = 7, Group 4 = 7; Group 5 = 9).
Details of the dose and timing of lead nitrate administration are given in Table 1. Group 1 served as a control and was not exposed to any lead nitrate. Group 2 received lead only during the prenatal period, while Groups 3 and 4 were given lead in an increasing and decreasing pattern respectively. Group 5 were given a fixed daily dose of lead during the experimental periods. These different patterns of lead exposure were used to mimic possible lead exposure events in a typical human environment. Drinking water bottles containing the ultrafiltered deionised water or lead nitrate were changed daily. The volume of water consumed was measured to determine the lead intake by the mother in each cage.
Time period | Concentration of lead (mg L−1) | ||||
---|---|---|---|---|---|
Group 1 no lead | Group 2 prenatal dose | Group 3 increasing dose | Group 4 decreasing dose | Group 5 steady dose | |
Prenatal | |||||
Day-5 to birth | 0.00 | 40.00 | 10.00 | 40.00 | 40.00 |
Postnatal | |||||
Day 1 to day 6 | 0.00 | 0.00 | 20.00 | 20.00 | 40.00 |
Day 7 to day 11 | 0.00 | 0.00 | 40.00 | 10.00 | 40.00 |
Exposure to lead nitrate occurred over three time periods, which included the prenatal period and two postnatal periods. The prenatal period of exposure started approximately five days before the birth of the pups, anticipating the commencement of crown formation in the first mandibular molar teeth approximately two days before birth.19 The margin of approximately three days was given to allow transplacental transfer of lead from the mother to the developing fetus. The postnatal period of exposure extended to 11 days after the birth of the pups, as the crown of the first mandibular molar tooth is completely formed at that time.20 The long postnatal period of tooth development provided the opportunity to administer two varying doses of lead nitrate. The postnatal period of lead exposure was divided into two time periods that extended from day-1 to day-6 and from day-7 to day-11 respectively.
All the experimental animals were culled four days after the second postnatal period, on day-15. The designated doses of lead nitrate are not considered toxic and are substantially lower than the established fatal dose.21 Previous studies that have used similar or higher doses did not report any observable signs of acute toxicity.22,23
Following blood collection, the unerupted left and right mandibular first molar teeth, the liver, right kidney, brain and the right femoral bone were removed from each rat pup, using clean, sterile surgical instruments in a biological safety cabinet (Email Westinghouse Ltd NSW, Australia) to avoid any contamination from the laboratory environment. Organs were stored in sterile metal-free tubes (EZ Micro Test Tubes, Bio-Rad Laboratories, USA) at −20 °C. Analysis was restricted to first mandibular molar teeth, as these are the only molar teeth in Wistar rats that form enamel and dentine prenatally and can therefore be used to assess prenatal exposure to lead.14
Molars were preferred over incisors in the present study because of the greater anatomical similarity between rodent and human molar teeth. Rat incisors lack enamel on the lingual aspect and do not have a root.19 In addition, an iron pigment is present on the surface of rat incisors,19 which may potentially affect the lead deposition in the enamel. Finally, the orientation of enamel rods in rat molar enamel is similar to that in humans, which is not the case in rat incisor enamel.24
The analysis of the bone samples (right femurs) was essentially identical. Soft tissue was removed mechanically from the external surface of the bone sample. The samples were placed in an ultrasonic bath of 5% hydrogen peroxide for 5 minutes followed by cleaning in an ultrasonic bath of deionised water for 10 minutes. The samples were dried at 60 °C for 2 hours and dissolved in 2 mL of 65% nitric acid. The digest was subsequently diluted to 10 mL with ultrafiltered deionised water and analysed by ICP-MS. NIST SRM-1486 (Bone Meal) was used as the external reference standard for bone-lead analysis.
More than 95% of the lead in blood is contained in the red blood cells, but it is the lead in plasma that exchanges with the different biological compartments.26 Lead concentrations in whole blood show strong positive correlations with plasma-lead levels.27 Whole blood was analysed as this allowed for the measurement of the total lead content in all fractions of blood and also facilitated easier detection of lead by making a larger concentration of lead available for measurement by ICP-MS.
Method blanks were prepared by diluting 2 mL of 65% nitric acid with deionised water to a final volume of 10 mL. Method blanks were analysed in every run with five blanks for every 15–20 samples analysed. Matrix matched survey validated standard reference materials for blood, QC 410448 and QC02407 (Quality Control Technologies Pty. Ltd Australia) were analysed in each run. The standards were prepared in an identical manner to the blood samples.
Method blanks were prepared by diluting 2 mL 65% nitric acid and 2 mL 30% hydrogen peroxide with deionised water to final volumes of 10 mL. Method blanks were analysed in every run with approximately five blanks for every 15–20 samples analysed. The standard reference material NIST SRM-1577b (bovine liver) was used as an external standard. 2 mL each of nitric acid and hydrogen peroxide were added to 1 g of SRM-1577b. The solution was processed as for organs describe above. Two standard solutions were placed in each analytical run.
QC410448 (blood) | QC02407 (blood) | SRM-1577b (liver) | SRM-1486 (bone meal) | ||||
---|---|---|---|---|---|---|---|
Measured concentrationa | % recovery | Measured concentrationa | % recovery | Measured concentrationb | % recovery | Measured concentrationc | % recovery |
a n = 5.b n = 10.c n = 11. | |||||||
0.0480 ± 0.0045 | 96.53 ± 1.61 | 0.4180 ± 0.0084 | 102.6 ± 2.3 | 0.1230 ± 0.0067 | 98.47 ± 4.80 | 1.333 ± 0.051 | 100.2 ± 3.8 |
Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | |
---|---|---|---|---|---|
Total maternal dose (mg) | 0.00 | 1.68 ± 0.28 | 12.50 ± 1.83 | 7.41 ± 0.41 | 15.79 ± 1.76 |
Maternal blood-lead (μg g−1) | 0.01 ± 0.02 | 0.19 ± 0.04 | 0.36 ± 0.03 | 0.32 ± 0.07 | 0.61 ± 0.08 |
Fig. 1 shows mean lead levels in whole teeth, blood, bone and key organs (liver, kidney and brain) of the Wistar rat pups in Groups 1–5, measured by ICP-MS. Notably, teeth and bone had the highest concentrations of lead, while brain samples showed the lowest lead levels in every group.
Mean tooth-lead levels of the pups were significantly different between all five groups (p < 0.05) and showed the same pattern as the mean maternal blood-lead levels. Pups in Group 5 had the highest mean tooth-lead levels, followed by pups in Groups 3, 4 and 2 in decreasing order of mean tooth-lead concentrations. Additionally, the mean tooth-lead levels were generally ranked in the same order as the mean lead levels in the key organs in the exposure groups, with the one exception of the lead levels in livers of the pups in Group 3 which were lower than those of Group 2. Mean blood-lead levels of the pups also mirrored the maternal mean blood-lead levels, with only Groups 1 and 2; 2 and 4; and 3 and 5 showing no significant variation between experiments (p > 0.05). As expected, Groups 1 and 3 and 1 and 5 showed the most significant difference between mean blood lead levels (p < 0.001).
The Pearson's correlation coefficient analysis of lead levels in the kidney, liver, brain and bone with lead concentrations in teeth and blood are given in Table 4. When compared with blood-lead, tooth-lead levels correlated more strongly with lead levels in each of the organs studied and this difference was statistically significant for liver, brain and bone (p < 0.05). The Bland–Altman analysis further allowed a quantitative relationship to be established between the tooth-lead levels and lead concentrations in the different organs studied (Table 5). With the exception of bone, lead levels in teeth were substantially higher than the lead concentrations of the other organs.
Organ | Tooth-lead | Blood-lead | p value |
---|---|---|---|
Kidney (n = 38) | 0.82 | 0.73 | 0.35 |
Liver (n = 37) | 0.90 | 0.38 | 0.001 |
Brain (n = 34) | 0.91 | 0.60 | 0.001 |
Bone (n = 38) | 0.89 | 0.56 | 0.001 |
Tooth-lead vs. organ-lead | Mean | 95% confidence interval |
---|---|---|
a For the Bland–Altman analysis, logarithmic transformations were applied when comparing lead concentrations in teeth, kidney, liver and brain. Consequently, results are reported as ratios of lead concentration in teeth and the respective organ. For comparisons between tooth-lead and bone-lead levels, logarithmic transformations were not required and results are reported as difference in mean lead concentration (μg g−1).28 | ||
Tooth-lead:kidney-lead | 14.60 | 11.81, 18.04 |
Tooth-lead:liver-lead | 22.81 | 18.36, 28.34 |
Tooth-lead:brain-lead | 79.60 | 63.82, 99.30 |
Tooth-lead minus bone-leada (μg g−1) | −2.25 | −5.40, 0.88 |
Maternal blood-lead levels were highest in Group 5 followed by Groups 3, 4 and 2 in decreasing order of lead concentration. The tooth-lead and blood-lead levels of the pups closely reflected this pattern in maternal blood-lead levels, taken at the time of culling, and the total maternal intake of lead (Table 4). Notably, blood-lead levels of the pups were not significantly different between every group, while, the tooth-lead levels showed statistically significant differences between all groups. This outcome indicates that dental lead levels more accurately reflect the intensity of exposure which is most likely a consequence of the greater affinity of lead for calcified dental tissues coupled with a significantly longer period of retention of lead in teeth30 as opposed to the relatively short half-life of lead in blood.4 Furthermore, the correlation analysis revealed that tooth-lead levels were significantly better predictors of lead levels in the bone, liver and brain than blood-lead levels in the present model. Both these facts support the proposition that dental lead levels are a reliable marker of exposure and body burden of lead, and that assessment of tooth-lead levels could be beneficial in childhood lead exposure studies, where blood data alone would be insufficient to accurately identify individuals at a health risk.
The results of the Bland–Altman analysis allowed quantitation of the relationship between tooth-lead levels and lead levels in key organs, which lends further support for the applicability of tooth-lead levels in the prediction of the body burden of lead. With further development of methods using prospective human studies, such as spatial analysis using laser ablation ICP-MS,15,31,32 it may be possible to predict the concentration of lead in the key organs using tooth-lead levels.
Small amounts of lead were detected in samples from the control group (Group 1). Although the exact source of lead cannot be identified, it is believed that lead stores present in the maternal rats prior to commencement of the experiment may have been mobilised to blood during gestation and lactation, which is known to happen in humans,5,33 and this may explain the levels of lead found in the teeth, blood, bone and organs of the control rat pups. The levels of lead observed were, however, negligible and would not significantly influence the results of the present study.
Teeth have significant potential for use as a biomarker of early-life lead exposure. Though not as readily available as blood, saliva, or urine, they provide a retrospective assessment of temporal, rather than cumulative exposure.14 Nails and hair also provide some temporal information, though the nature of primary tooth formation and development presents a broader window extending from the second trimester to several years post-birth.34 When implemented into large-scale cohort studies,16 deciduous teeth provide a non-invasive and easily collected resource for measuring neo, peri and postnatal metal exposure35 that requires minimal handling and simple storage conditions. Discrete sampling using methods such as laser ablation-ICP-MS15,36 also permit repeated measurements of the sample with minimal destruction or sample disruption. Additionally, comparative measures of multiple deciduous teeth from the sample donor can be used to cross-validate and determine intra-sample variability with a high degree of accuracy and precision, which is not restricted to lead; complementary studies to the one presented here can also help further validate teeth as a biomarker of multiple metal exposures.
This journal is © The Royal Society of Chemistry 2015 |