The tooth exposome in children’s health research

Abstract

Purpose of review

The exposome concept proposes a comprehensive assessment of environmental exposures from the prenatal period onwards. However, determining exposure timing especially over the prenatal period is a major challenge in environmental epidemiologic studies.

Recent findings

For decades teeth have been used to estimate long-term cumulative exposure to metals. Recently developed high-dimensional analytical methods that combine sophisticated histological and chemical analysis to precisely sample tooth layers that correspond to specific life stages have the potential to reconstruct the exposome in the second and third trimesters of prenatal development and during early childhood.

Summary

A retrospective temporal exposomic approach that precisely measures exposure intensity and timing during prenatal and early childhood development would substantially aid epidemiologic investigations, particularly case–control studies of rare health outcomes.

Introduction

The ‘Exposome’ concept was introduced in 2005 to address the disparity between the genomic sciences, where rapid technological advances provided an expanse of high-precision analyses, and the environmental exposure sciences that measured a small fraction of the thousands of environmental exposures individuals experience [1]. The exposome concept encompasses lifecourse environmental exposures (including lifestyle factors), from the prenatal period onwards [1]. It is important to consider that the exposome includes not only external exposures but also internal factors (e.g. inflammation, infection, and the microbiome) [2]. While the definition of the exposome evolves (see Miller and Jones [3]), the fundamental concept of the exposome continues to gain momentum internationally. Notably, in the US, the Human Exposome Project supported by the National Institutes of Health and, in Europe, the HELIX, HEALS and EXPOsOMICS projects, as well as projects at various academic institutions are examining specific aspects of the exposome.

Challenges to Uncovering the Fetal Exposome

There are many challenges that must be overcome before the exposome concept can be taken from a theoretical foundation to wide-spread practical application, as was the case with the genomic sciences [2]. Foremost, the exposome, unlike the genomic sequence, is highly variable and dynamic, and continues to evolve throughout the individual’s lifetime [1, 4]. Longitudinal birth cohort studies that collect biomarkers of environmental chemical exposure during pregnancy and then follow offspring into later life would provide the strongest evidence to assess the impact of exposures during key developmental windows in humans. However, the expense and time required for such studies are major barriers to investigating lower frequency conditions with long latency periods. Another important barrier to studying the fetal exposome is that maternal biomarkers do not necessarily provide accurate measures of fetal exposure for all chemicals. Reliance on maternal biomarkers of fetal exposure fails to account for variability in placental transport and metabolism, potentially overlooking the significant interplay at the maternal-fetal interface [5–7]. Umbilical cord blood has been successfully collected at birth in epidemiologic studies and has provided valuable exposure information [8–11]. However, for compounds with a short half-life in blood, cord blood levels can only provide information on the latter part of the third trimester.

The Tooth Exposome

To overcome the need of large sample sizes and have a direct measure of the fetal environment, we propose that the exposome biomarkers would benefit from two attributes - be retrospective and incorporate temporal signatures. This has recently been referred to as the ‘retrospective temporal exposome’ [12]. For health outcomes that occur at lower frequencies, this biomarker would be applied in population-based case-control designs. Unlike contemporary approaches that are cross-sectional, such biomarkers would provide time-series exposure data similar to that obtained from a longitudinal study, whilst doing so retrospectively.

In this overview, we propose the use of teeth as a matrix that provides an opportunity to retrospectively reconstruct the dynamic exposome. We also identify the limitations of the use of teeth, which future work will hopefully address. Key aspects of the well-defined incremental formation of teeth and its relevance to exposure assessment have been detailed previously [13 15].

Components of the Exposome that are Measurable in Tooth Matrix Biomarkers

i. Metallomics

Metals have been measured in teeth for many decades, with lead being the most studied toxicant in teeth [16–18]. Because many metal toxicants accumulate preferentially in bone, early studies considered teeth as a useful biomarker for measuring long-term exposure [16–18]. Most notable are studies on lead, as the skeletal compartment comprises the major depository of total body burden and is also a potential source of internal exposure due to release of lead during bone remodeling, such as occurs in pregnancy or osteoporosis [19]. Several studies have shown that children living in lead contaminated locations have higher lead levels in their deciduous teeth than children from lower exposure environments [17, 18, 20–22]. The suitability of teeth as exposure biomarkers for other metals was also explored (cadmium, for example [23]).

Over the last two decades, microspatial sampling combined with sophisticated histological analysis has provided a means to uncover the timing of metal uptake, including prenatal exposure, from teeth biomarkers [24–29]. However, detailed validation against environmental samples and other biomatrices has only been performed in the last five years. For validation of Mn, there was a significant positive association of levels in parts of dentine formed in the second trimester with Mn loading in floor dust sampled during the second trimester of pregnancy [30]. That study also showed that Mn levels in dentine adjacent the neonatal line was strongly associated with cord blood Mn concentrations, both biomarkers reflecting Mn uptake close to the time of birth. Another study undertook detailed validation of tooth Pb measurements against maternal pregnancy blood levels and also bone lead levels postpartum [13]. Of note for metals analysis is the application of laser ablation-based mass spectrometry that allows measurement of multiple metal targets in the same scan as shown in Figure 1a.

Figure 1.

Components of the exposome measureable using tooth matrix biomarkers. (a) Metallomics: elemental bio-imaging of teeth using laser ablation-based mass spectrometry provides detailed spatial distribution of multiple metals across enamel and dentine. (b) Dietary transitions: recently developed biomarkers using barium signatures distinguish breast milk intake (between dashed lines) from introduction of infant formula (below black dashed line). (c) Targeted and untargeted organics analysis in teeth reveals exposure to multi-class organic chemicals within (2^nd versus 3^rd trimester) and between (Child A versus Child B) children. QTOFMS was operated in a dual electrospray ionization mode (positive and negative modes). Study design and results are presented in Andra et al [12]. Abbreviations for the chemicals on Y-axis: BPA, bisphenol A; MMP, mono-methyl phthalate; MEP, mono-ethyl phthalate; MBP, mono-nbutyl phthalate; MBzP, mono-benzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; NIC, nicotine; COT, cotinine; HCOT, hydroxycotinine; BAP; bisphenol AP; BPF, bisphenol F; MP, methyl paraben; PP, propyl paraben; BP, butyl paraben; PFOA, pentadecafluorooctanoic acid; PFOS, perfluorooctanesulfonic acid; and DMP, dimethylphosphate. (d) Uncovering historical exposure to external stressors: Raman spectroscopic analysis of macaque tooth dentine shows signatures that correspond to various medical events.

ii. Dietary components and essential nutrients

Trace element and stable isotope signatures in teeth (and bone) have been used for several decades to reconstruct major diet transitions in past populations, such as terrestrial versus marine resource exploitation [64]. Typically, the ratio of a non-essential element to a chemically similar essential element is used to determine the trophic steps up a food chain [65]. The most common ratios used to assess diet in past populations are Sr/Ca and Ba/Ca. Due to the process known as biopurification, Sr/Ca and Ba/Ca ratios decrease during metabolic processes that involve Ca leading to a decrease in these ratios in consumers relative to diet [66]. The ratio decrease between diet and tooth values are relatively constant so that the diet of past populations can be compared against known herbivores and carnivores to identify the relative importance of plant and animal products in the diet [67]. Similarly, stable isotope ratios of light elements carbon and nitrogen also show partitioning through metabolic processes used to identify trophic level [68]. Carbon and nitrogen isotopes are typically measured from dentine collagen and reflect the major protein sources within a diet [69]. Recently, oxygen isotope values measured from the skeletal remains of Richard III were interpreted as an increase in wine intake [70]. Due to the sensitivity and sample preparation restrictions of the techniques used for stable isotope analysis, the temporal resolution is limited.

Stable isotopes have been used to reconstruct weaning practices based on the child being one trophic level higher than mother [71–74], however several drawbacks have been identified in relation to assumptions of trophic offsets [68, 72, 75]. The study of trace elements rather than isotopes offers the advantage of better temporal resolution. Barium was recently identified as a sensitive biomarker in teeth to infant diet transitions from exclusive breastfeeding to the introduction of infant formula and process of weaning [76]. In children’s teeth, Ba levels in dentine rose after birth and remained relatively steady for the duration of exclusive breastfeeding, rising again at the introduction of infant formula (Figure 1b). The distinction in diet is made possible due to differences in Ba dietary exposure. A similar pattern was observed in teeth from captured macaques that showed a decrease in Ba over the process of weaning. Strontium has also been used to determine weaning patterns in past populations and nonhuman primates [77, 78] but Ba is considered a more sensitive marker of diet [74, 76, 79].

The isotopic partitioning of other elements (Mg, Fe, Cu and Zn) from diet to tissue is also under investigation with the promise that the use of multiple elemental and isotopic systems will provide more robust results [65]. The isotopic composition of teeth (and bones) can also provide information on migration and habitat conditions [64]. The precise determination of diet components in mixed diets remains a challenge via stable isotope ratio analysis [64].

iii. Environmental organics: Targeted exposome analysis

Current prenatal exposome approaches do not allow proper characterization of timing of exposure to an organic contaminant or mixtures and association with health effects at a later life stage [31]. Correlations between targeted organic contaminant concentrations in maternal and fetal matrices collected at various stages of pregnancy and child birth suggest inconsistent associations and mixed outcomes in assessing prenatal exposures [9]. It is well acknowledged that a unifying bio-matrix to assess perinatal exposome for xenobiotics is needed [32]. Teeth offer a unique advantage of accurate fetal organic chemical exposure assessment on a temporal scale [33]. This is not true for conventional biomarkers such as maternal bio-matrices due to variations in placental transport or for cord blood due to short half-lives of many chemicals [34, 35].

Teeth analysis was explored for the presence and quantification of various organic chemicals, contaminants and metabolites such as analgesics, pesticides and plastics additives [36], anesthetics [37], antibiotics [38, 39], illegal drugs [37, 40], metabolites of alcohol [41] and tobacco [42–44], and organochlorines [45–48]. The major limitation of the methodologies employed in these studies was to grind and analyze whole teeth (that constitutes the tissue and blood vessels within the pulp chamber) leading to exposure misclassification because of differential deposition of organic chemicals and contaminants in different tooth compartments. Andra and colleagues [12, 33] have demonstrated micro-spatial organic chemical measurements of specific growth rings in dentine that correspond to trimester-specific fetal developmental windows (Figure 1c). For example, mono-benzyl phthalate was quantified in dentine layers formed during the second and third trimester using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) targeted analysis [33]. This work needs to be expanded to encompass a comprehensive validation of the dentine-phthalate biomarkers against data available from conventional bio-matrices such as maternal urine during pregnancy and at birth, and newborn and childhood urine.

iv. Environmental organics: Untargeted exposome profiling

Analyzing bio-matrices for measuring the totality of exposures to organic pollutants is (a) to assess a fraction of the vast and complex internal chemical milieu made of exogenous sources and endogenous responses, [49] and (b) a component of the top-down approach for scaling human exposome [50]. Advances in high-resolution mass spectrometers (MS), such as fouriertransform MS, [51] hybrid ion trap-orbitrap MS, [52] quadrupole time-of-flight MS, [53, 54] allow increased metabolic detection [55] and capture a wider, untargeted chemical space in the exposome [56, 57]. The power of measuring environmental organics exposome as a tool to evaluate health risks is gaining attention spanning several scientific domains [1, 4, 58, 59]. The blood exposome was the first effort directed towards incorporating literature data for about 1600 exo- and endogenous chemicals into identifying associated metabolic pathways and disease etiologies [60]. Other emerging exposome approaches that consider measuring organics with distinct features are (a) tooth exposome that utilizes a novel bio-matrix, [12] (b) volatolomics that use a specific physical fraction (viz., exhaled breath or volatile organic compounds pool), [61, 62], and (c) pregnancy exposome that relies on collective data from multiple matrices and multiple sampling points during prenatal and child birth phase [63].

Recently, we reconstructed the prenatal and early childhood exposure to multiple organic chemical classes using teeth [12]. We performed global screening of small molecules in trimester-specific formed dentine layers from deciduous teeth using liquid chromatography coupled quadrupole time-of-flight mass spectrometry (QTOF-LC/MS) metabolomics approach. QTOF-LC/MS analyses show unique and differential chemical signatures of environmental exposure that are individual and development-stage dependent. The results of this study (a) revealed more than 12,000 unique chemical signatures in trimester-specific dentine layers, (b) indicate high inter- and intra-child variability in screened chemical profiles, (c) show novel ‘known unknowns’ and ‘suspected unknowns’ compounds, (d) demonstrate exposure misclassification error that can cause misleading inferences about causality, and (e) most importantly, the reconstruction of exposure was done 7 to 10 years after prenatal and early childhood exposure. An example to demonstrate inter- and intra-individual differences in chemical fingerprints on a temporal prenatal scale is shown in Figure 1c. In future, we will develop a hybrid approach for tooth exposome-wide measures for reconstructing fetal exposures. First, we will apply discovery methods that employ QTOF-MS detection after liquid and/or gas chromatography separation to generate large datasets, and full mass spectra scan plus MS/MS fragmentation of organic compounds and biomolecules. Second, we will perform targeted analysis of the relevant biomarkers after accurate mass identification of compounds from above, and library searching combined with advanced chemometrics and bioinformatics data mining tools. Finally, the findings will be validated in multiple matrices and exposure cases from an exposome perspective.

v. Stress signatures

Formation of the neonatal line (NL) is thought to be due to disturbances in the cells which lay down the tooth matrix for mineralization [80] and it’s width has been related to the difficulties in delivery [81]. Other accentuated growth lines have been observed, largely in enamel, which are similarly believed to be due to the disruption of tooth matrix deposition due to external stressors, both physical and social [82–84]. Several studies have compared the timing of these accentuated lines with clinical events such as injuries, bouts of dehydration/diarrhea, and hospitalizations [85–87] and other events such as weaning [88, 89], and separation from dam [90]. These accentuated lines are typically identified and aged using light microscopy. However, the response to external stressors involves complex mechanisms and this technique cannot identify the biological systems or pathways impacted. Additionally, light microscopy methods are subjective, and highly dependent on operator expertise, quality of sample preparation and microscopy technique.

Recently a novel multi-tiered approach was presented that enables the identification of specific stress impacted systems using objective techniques to identify different signals in teeth and overlaying these with temporal mapping [90]. Firstly, elemental signals in teeth observed through laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging indicated disruptions specific to bone remodeling. Second, it was demonstrated that molecular markers of specific homeostatic pathways activated in response to external stressors could be targeted by direct antibody labelling on thin sections of teeth. The final approach used Raman spectroscopy to chemically image teeth at high resolution that revealed both fine-scale regular rhythms and accentuated lines that corresponded to medical events (Figure 1d). Though this study was performed on a small biomedical animal model, it demonstrates the potential of these methods to measure an individual’s stress response to external stimuli across a population experiencing the same challenges.

Conclusion

Tooth matrix biomarkers provide an opportunity to incorporate the intensity and timing of exposure in environmental health studies. Recent advances in technology allow high dimension analyses of a large range of targets in a single scan, which takes us closer to the ideal of the exposome concept of capturing the entirety of exposures over a life-time. In-depth validation and recognition of the limitations of dental tissues, including missing information during the first trimester, are important considerations for future development of tooth matrix biomarkers.

Key Points.

• Fetal and early postnatal development comprise critical developmental periods when environmental stressors may disrupt life-long health trajectories

• Reconstructing the exposome during this time period is a major challenge in epidemiologic research due to the need for large sample sizes and long follow-ups

• Tooth matrix biomarkers incorporate the intensity and timing of exposure can overcome some of these challenges