Christine Austin

Barium distributions in teeth reveal early-life dietary transitions in primates

Early-life dietary transitions reflect fundamental aspects of primate evolution and are important determinants of health in contemporary human populations. Weaning is critical to developmental and reproductive rates; early weaning can have detrimental health effects but enables shorter inter-birth intervals, which influences population growth. Uncovering early-life dietary history in fossils is hampered by the absence of prospectively validated biomarkers that are not modified during fossilization. Here we show that large dietary shifts in early life manifest as compositional variations in dental tissues. Teeth from human children and captive macaques, with prospectively recorded diet histories, demonstrate that barium (Ba) distributions accurately reflect dietary transitions from the introduction of mother’s milk through the weaning process. We also document dietary transitions in a Middle Palaeolithic juvenile Neanderthal, which shows a pattern of exclusive breastfeeding for seven months, followed by seven months of supplementation. After this point, Ba levels in enamel returned to baseline prenatal levels, indicating an abrupt cessation of breastfeeding at 1.2 years of age. Integration of Ba spatial distributions and histological mapping of tooth formation enables novel studies of the evolution of human life history, dietary ontogeny in wild primates, and human health investigations through accurate reconstructions of breastfeeding history.

Elemental bio-imaging of trace elements in teeth using laser ablation-inductively coupled plasma-mass spectrometry

OBJECTIVES In this study we present the application of a novel laboratory method that employs laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to construct two-dimensional maps of trace elements in teeth. METHODS Teeth were sectioned longitudinally, embedded in resin and polished to a smooth surface. Data were generated by laser ablating the entire sectioned tooth surface. Elemental images were constructed using custom-built software. RESULTS Quantified images of (66)Zn, (88)Sr, (111)Cd and (208)Pb, with a spatial resolution of 30 μm(2), were generated from three teeth. Concentrations were determined by single-point calibration against NIST SRM 1486 (bone meal). Zn and Sr concentrations were determined in the μg g(-1) range and Cd and Pb in the ng g(-1) range. Concentrations of Pb, Zn and Cd were higher in dentine particularly in regions adjacent the pulp. CONCLUSIONS Elemental bio-imaging employing LA-ICP-MS is a novel method for constructing μm-scale maps of trace elements in teeth. This simple imaging method displays the heterogeneity of trace elements throughout the tooth structure that correspond to specific structural and developmental features of teeth. As a preliminary study, this work demonstrates the capabilities of LA-ICP-MS imaging in dental research.

Factors affecting internal standard selection for quantitative elemental bio-imaging of soft tissues by LA-ICP-MS

Element response variations under different laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) operating conditions were investigated to identify important factors for selecting an internal standard (IS) for quantitative elemental bio-imaging. Analytes covering a range of atomic masses and first ionisation potentials (FIP) were selected to investigate the signal response variation with changes in laser spot diameter, mass bias and cell sampling position. In all cases, an IS improved experimental precision regardless of a close match in element mass or FIP but optimal analyte/IS combinations depended on the difference in masses of the analyte and IS. Particular attention was paid to 13C as this isotope is typically used as an IS in elemental bio-imaging applications. Despite its non-ideal IS characteristics (often different mass and FIP to many analytes), possibility of abundance sensitivity effects and poor signal-to-background ratio, 13C was a suitable IS candidate exhibiting a linear response with respect to the mass ablated, apparent independence from the high abundance of the adjacent 14N mass peak and effective analyte normalisation after background subtraction as long as the 13C signal from the sample was at least 6% of the gross signal.

Spatial distribution of manganese in enamel and coronal dentine of human primary teeth.

Emerging evidence indicates that excessive exposure to manganese (Mn) during the prenatal period and early childhood may result in neurodevelopmental deficits. However, accurate exposure biomarkers are not well established, limiting our understanding of exposure-response relationships over these susceptible periods of development. Naturally shed deciduous teeth are potentially a useful biomarker of environmental exposure to Mn. However, the uptake and distribution of Mn in human teeth has not been studied in detail. Mn distribution was measured at high resolution (~20 μm) in eight human primary teeth using laser ablation-inductively coupled plasma-mass spectrometry. A bio-imaging methodology was applied to construct detailed elemental maps of three incisors, and bone meal (NIST SRM 1486) was used to validate the analyses. The distribution of Mn in enamel and coronal dentine showed a distinct and reproducible pattern. In enamel, the ⁵⁵Mn:⁴³Ca ratio was highest at the outer edge of enamel (range=0.57 to 4.74) for approximately 20-40 μm but was substantially lower in deeper layers (range=0.005 to 0.013). The highest levels of Mn were observed in dentine immediately adjacent the pulpal margin (⁵⁵Mn:⁴³Ca range=2.27 to 6.95). Importantly, a clearly demarcated high Mn zone was observed in dentine at the incisal end of the teeth. Using confocal laser scanning microscopy to visualize the neonatal line, this region was identified as being in the prenatally formed dentine. The high-resolution map of the spatial distribution of Mn in human primary teeth highlighted specific reproducible patterns of Mn distribution in enamel and coronal dentine.

Quantification method for elemental bio-imaging by LA-ICP-MS using metal spiked PMMA films

A method for quantitative analysis of biological soft tissues by laser ablation-inductively coupled plasma-mass spectrometry has been developed. Polymer film standards were produced by spin coating spiked solutions of polymethylmethacrylate onto quartz substrates. Calibration curves throughout the range of 0–400 μg g−1 yielded correlation coefficients better than 0.999 for 66Zn and 63Cu. Spiked, homogenised soft tissue standards were quantified by LA-ICP-MS against the thin film standards. The results agreed with values calculated from solution nebulisation ICP-MS. A procedure for internal standardisation by employing ruthenium or yttrium in the underlying thin film was also assessed.

Ruthenium Phthalocyanine-Bipyridyl Dyads as Sensitizers for Dye-Sensitized Solar Cells : Dye Coverage versus Molecular Efficiency

The application of ruthenium phthalocyanine complexes as sensitizing dyes in dye-sensitized solar cells (DSCs) is explored. Four monomeric complexes are reported which vary in peripheral substitution and axial ligand anchoring groups. Sensitizing dyes containing two ruthenium centers are also presented. These dyads, which contain ruthenium phthalocyanine and bipyridyl chromophores, were prepared using a protection/deprotection strategy that allows for convenient purification. DSCs fabricated using the phthalocyanine complexes and dyads were less efficient than those incorporating a standard DSC dye. However, on the basis of the number of molecules bound to the TiO(2) electrode surfaces, several of the new complexes were more efficient at photocurrent generation. The results highlight the importance of molecular size, and thus the dye coverage of the electrode surface in the design of new sensitizing dyes.

Determining Fetal Manganese Exposure from Mantle Dentine of Deciduous Teeth

Studies addressing health effects of manganese (Mn) excess or deficiency during prenatal development are hampered by a lack of biomarkers that can reconstruct fetal exposure. We propose a method using the neonatal line, a histological feature in deciduous teeth, to identify regions of mantle dentine formed at different prenatal periods. Micromeasurements of Mn in these regions may be used to reconstruct exposure at specific times in fetal development. To test our hypothesis, we recruited pregnant women before 20 weeks gestation from a cohort of farmworkers exposed to Mn-containing pesticides. We collected house floor dust samples and mothers blood during the second trimester; umbilical cord blood at birth; and shed deciduous incisors when the child was ∼7 years of age. Mn levels in mantle dentine formed during the second trimester (as (55)Mn:(43)Ca area under curve) were significantly associated with floor dust Mn loading (r(spearman) = 0.40; p = 0.0005; n = 72). Furthermore, (55)Mn:(43)Ca in sampling points immediately adjacent the neonatal line were significantly associated to Mn concentrations in cord blood (r(spearman) = 0.70; p = 0.003; n = 16). Our results support that Mn levels in mantle dentine are useful in discerning perinatal Mn exposure, offering a potentially important biomarker for the study of health effects due to environmental Mn exposure.

Elemental bio-imaging of thorium, uranium and plutonium in tissues from occupationally exposed former nuclear workers

Internal exposure from naturally occurring radionuclides (including the inhaled long-lived actinides (232)Th and (238)U) is a component of the ubiquitous background radiation dose (National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States; NCRP Report No. 160; NCRP: Bethesda, MD, 2009). It is of interest to compare the concentration distribution of these natural alpha-emitters in the lungs and respiratory lymph nodes with those resulting from occupational exposure, including exposure to anthropogenic plutonium and depleted and enriched uranium. This study examines the application of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) to quantifying and visualizing the mass distribution of uranium and thorium isotopes from both occupational and natural background exposure in human respiratory tissues and, for the first time, extends this application to the direct imaging of plutonium isotopes. Sections of lymphatic and lung tissues taken from deceased former nuclear workers with a known history of occupational exposure to specific actinide elements (uranium, plutonium, or americium) were analyzed by LA-ICPMS. Using a previously developed LA-ICPMS protocol for elemental bio-imaging of trace elements in human tissue and a new software tool, we generated images of thorium ((232)Th), uranium ((235)U and (238)U), and plutonium ((239)Pu and (240)Pu) mass distributions in sections of tissue. We used a laboratory-produced matrix-matched standard to quantify the (232)Th, (235)U, and (238)U concentrations. The plutonium isotopes (239)Pu and (240)Pu were detected by LA-ICPMS in 65 mum diameter localized regions of both a paratracheal lymph node and a sample of lung tissue from a person who was occupationally exposed to refractory plutonium (plutonium dioxide). The average (overall) (239)Pu concentration in the lymph node was 39.2 ng/g, measured by high purity germanium (HPGe) gamma-spectrometry (Lynch, T. P.; Tolmachev, S. Y.; James, A. C. Radiat. Prot. Dosim. 2009, 134, 94-101). Localized mass concentrations of thorium ((232)Th) and uranium ((238)U) in lymph node tissue from a person not occupationally exposed to these elements (chronic natural background inhalation exposure) ranged up to 400 and 375 ng/g, respectively. In lung samples of occupationally nonexposed to thorium and uranium workers, (232)Th and (238)U concentrations ranged up to 200 and 170 ng/g, respectively. In a person occupationally exposed to air-oxidized uranium metal (Adley, F. E.; Gill, W. E.; Scott, R. H. Study of atmospheric contaminiation in the melt plant buiding. HW-23352(Rev.); United States Atomic Energy Commission: Oakridge, TN, 1952, p 1-97), the maximum (235)U and (238)U isotopic mass concentrations in a lymph node, measured at higher resolution (with a 30 mum laser spot diameter), were 70 and 8500 ng/g, respectively. The ratio of these simultaneously measured mass concentrations signifies natural uranium. The current technique was not sufficiently sensitive, even with a 65 mum laser spot diameter, to detect (241)Am (at an overall tissue concentration of 0.024 ng/g, i.e., 3 Bq/g).

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

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.

Teeth as a biomarker of past chemical exposure.

Purpose of review Accurate prenatal exposure assessment is one of the major challenges in environmental epidemiologic studies. Variations in placental transport make maternal biospecimens unreliable for many chemicals and fetal specimens collected at birth do not provide information on exposure timing over the prenatal period. Recent findings The skeletal compartment is an important chemical repository, making calcified tissues important for measuring exposure. For decades teeth have been used to estimate long-term cumulative exposure to metals and some organic chemicals. Recently developed methodologies that combine sophisticated histological and chemical analysis to precisely sample tooth layers that correspond to specific life stages have the potential to reconstruct exposure in the second and third trimesters of prenatal development and during early childhood. Summary Such a retrospective biomarker that precisely measures exposure intensity and timing during prenatal development would substantially aid epidemiologic investigations, particularly case–control studies of rare health outcomes.