Science of the Total Environment 409 (2011) 1315–1319

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Spatial distribution of manganese in enamel and coronal dentine ofhuman primary teeth

Manish Arora a,b,c,d,⁎, Dominic Hare c, Christine Austin c, Donald R. Smith d, Philip Doble c

a Institute of Dental Research, Westmead Centre for Oral Health, Westmead Hospital, Westmead, Australiab Oral Pathology and Oral Medicine, Faculty of Dentistry, University of Sydney, Sydney, Australiac Elemental Bio-imaging Facility, Department of Chemistry and Forensic Science, University of Technology, Sydney, Australiad Department of Environmental Toxicology, University of California, Santa Cruz, CA, USA

⁎ Corresponding author. Institute of Dental ResearcHealth, Westmead Hospital, Westmead, NSW, 2145, Aufax: +61 2 9845 7599.

E-mail address: manish.arora@sydney.edu.au (M. Ar

0048-9697/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.scitotenv.2010.12.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 October 2010Received in revised form 4 December 2010Accepted 6 December 2010Available online 5 January 2011

Keywords:Bio-imagingDentineEnamelLaser ablationManganese

Emerging evidence indicates that excessive exposure to manganese (Mn) during the prenatal period and earlychildhood may result in neurodevelopmental deficits. However, accurate exposure biomarkers are not wellestablished, limiting our understanding of exposure–response relationships over these susceptible periods ofdevelopment. Naturally shed deciduous teeth are potentially a useful biomarker of environmental exposure toMn. 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 detailedelemental 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 μmbutwas substantially lower in deeper layers (range=0.005 to 0.013). The highest levels ofMnwere observed indentine immediately adjacent the pulpal margin (55Mn:43Ca range=2.27 to 6.95). Importantly, a clearlydemarcated high Mn zone was observed in dentine at the incisal end of the teeth. Using confocal laser scanningmicroscopy 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 specificreproducible patterns of Mn distribution in enamel and coronal dentine.

h, Westmead Centre for Oralstralia. Tel.: +61 2 9845 8772;

ora).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Manganese (Mn) is an essential nutrient required for manyphysiological processes including bone mineralization, protein andenergy metabolism, and protection of cells from free radical species(Agency for Toxic Substances and Disease Registry (ATSDR), 2008).While occupational exposures to Mn have been linked with permanentneurologic damage, emerging evidence indicates that chronic exposureto lower levels of Mn may also be harmful and may result indevelopmental deficits if exposure occurs during early life (Santamariaand Sulsky, 2010). Inverse associations have been reported betweenMnbiomarkers and measures of cognition, behavior, and motor function inchildren (Wasserman et al., 2006; Wright et al., 2006; Bouchard et al.,2007). Ina recent studyan invertedU-shaped relationshipwasobservedbetween blood Mn levels at 12 months of age and childhood mentaldevelopment scores (Henn et al., 2010).

Uptake ofMn in calcified tissues (bones and teeth)may be related toexposure from environmental sources (Ericson et al., 2001; Pejović-Milić et al., 2009), as has been well-demonstrated for lead (Needlemanet al., 1974; Bellinger et al., 1994; Hu et al., 1998). Furthermore, Mn2+,

the predominant form of Mn in the body, is a biologic analog to Ca2+

(da Silva and Williams, 2001). It is, therefore, possible that tooth Mnconcentrations may serve as a useful biomarker of environmentalexposure to Mn during crucial developmental windows including theprenatal and early childhood periods. However, the limited informationon the uptake of Mn in teeth in general, and in different toothcompartments (enamel, dentine and cementum) in particular, is amajor barrier to the use of teeth as a reliable Mn biomarker.

In the present study, we aim to establish, at approximately 20 μmresolution, the spatial distribution of Mn in enamel and coronal dentineof naturally exfoliated human primary teeth using laser ablationinductively coupled plasma mass spectrometry (LA–ICP–MS), a sensi-tive analytical technique that offers detection limits suitable for theanalysis of trace elements in teeth. Briefly, a focused laser beam with aμm-range diameter is directed onto the surface of a sample. Material isvaporized, and the ejected particles are then carried to the ICP–MSwhere thematerial is ionized andseparatedonabasis ofmass-to-charge

Table 1Typical operating conditions for LA–ICP–MS system.

Agilent 7500ce ICP–MS New Wave UP213 Laser Ablation

RF Power 1250 W Wavelength 213 nmPlasma gas flow rate 15 L min−1 Repetition frequency 20 HzCarrier gas flow rate 1.15–1.25 L min−1 Laser energy density 1 J cm−2

Sample depth 4.0 mm Spot size 20 μmQP Bias −5 V Scan rate 20 μm s−1

OctP Bias −8 V Line spacing 20 μmScan mode Peak hopping Carrier gas ArDwell time 0.1 s per m/zMeasured m/z 13,31,66,88,111,208Extracts 1, 2 6.8, −126 V

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ratios. Imaging using LA–ICP–MS can be achieved through the assemblyof single lines of ablation into two-dimensional images (Austin et al.,2009; Hare et al., 2009). Using this novel bio-imagingmethodology, wealso aim to construct elementalmaps to visualize the distribution of Mnwithin different anatomical regions of teeth.

2. Methods

Eight naturally shed deciduous teeth, collected from eight childrenliving in a rural community of Australia, were analyzed. Children camefrom backgrounds of no known elevated environmental or dietaryexposure to Mn. Written informed consent was obtained from theparents/guardians of the participants. Ethics approval for this studywasobtained from the Human Research Ethics Committee of the FarWestern Area Health Service, New South Wales, Australia. Teeth withdental caries and gross attrition, abrasion or erosion of dental hardtissues, as well as teeth with any large dental restorations ordevelopmental dental defects were excluded from the study. Overall,three incisors, three molars and two canines were analysed.

2.1. Sample preparation

Teeth were washed in distilled water by the dentist post-extraction,air-dried and individual teeth were placed in sterile plastic specimencontainers (Sarstedt, Australia). The participant's identification number,type of tooth, and date of extraction were noted on the container andforwarded to the laboratory. Teeth were subsequently washed in anultrasonic bath of ultrapure water (Milli-Q), dried and embedded inresin at 70 °C for 10 h (Taab Laboratories Equipment Inc. Berkshire, UK).Embedded teeth were sectioned using a rotary stainless-steel blade(Leitz-1600, Germany) in a vertical plane that passed through the cusptip. Incisors and canineswere sectioned in the labio-lingual plane, whilemolars were sectioned in the bucco-lingual plane. Following sectioningandprior toanalysis, sampleswere again cleaned in anultrasonic bath ofultrapure water and dried in an oven at 60 °C for 2 h.

2.2. Laser ablation-inductively coupled plasma-mass spectrometry

Teeth were analyzed using an Agilent Technologies 7500cx (AgilentTechnologies Australia, Forrest Hill, Victoria, Australia) ICP-MS, fittedwith a New Wave Research UP-213 laser ablation system (KennelecTechnologies, Mitcham, Victoria, Australia). The laser ablation systemwas equippedwith a Nd:YAG laser emitting a nanosecond laser pulse inthe fifth harmonic with a wavelength of 213 nm. The standard ablationcell was replaced with a Large Format Cell (LFC). The LFC has a largevolume chamber capable of holding samples up to 15.2 cm2 in area. Thex-y-z stage of the LFC employs a small volume ‘roving’ sampling cup thattraverses the sample while the laser beam remains stationary. Thedimensions of the LFC are large enough to enable analysis of all samplesin a single experiment. An approximately 40 cm length of Tygon®tubing (i.d. 3 mm) connected the laser ablation unit to the ICP–MS. TheICP–MS was fitted with a ‘cs’ lens system for enhanced sensitivity. Thesystem was tuned daily for sensitivity using NIST SRM 612 Traceelements in glass. Polyatomic oxide interference was evaluated andminimized by monitoring the Th+/ThO+ (m/z 232/248) ratio. Typicaloxide formation was consistently under 0.3%. Operating conditions forthe optimized LA–ICP–MS system are given in Table 1.

For all eight teeth included in this study,55Mn:43Ca levels weredetermined by rastering/scanning the laser beam across the sectionedtooth surface along a straight line. Three such line scans wereundertaken; one each in the occlusal/incisal, middle and cervical 1/3rdof every sample tooth. A spot diameter of 20 μm was used in theseanalyses. In addition to these analyses, elemental bio-images wereconstructed for three incisors. To achieve this, the entire surface of thethree incisors was rastered and the elemental data were converted toelemental images as described in the following section.

2.3. Image processing

Each line of ablation produced a single data file in comma separatedvalue (.csv) format. Data were processed using Interactive SpectralImaging Data Analysis Software (ISIDAS), a custom-built software toolwritten using Python programming language. ISIDAS reduces all .csvfiles into a single, exportable visualization toolkit (.vtk) file format.Images were produced by exporting .vtk files into MayaVi2 (EnthoughtInc., Austin, Texas, USA), an open source data visualization application.

2.4. Standard reference material (SRM) analysis and Mn quantitation

Approximately 1 g of NIST SRM 1486 bone meal was pressed into ahigh-density pellet using a KBr press. Prior to the analysis of each toothsample, theNIST SRM1486pelletwas analyzedusing seven separate laserablation lines. Identical ablation conditions were used for the SRM 1486standard and tooth sample analysis. Quantitative data recorded by theablation of NIST SRM1486was used to convert the observed toothMn:Cacounts per second to concentration (μg/g) values. NIST SRM 1486 wasselected as an appropriate standard for the quantitation of trace elementsin teeth due to the major component being crude hydroxyapatite.Suitability of NIST SRM 1486matrix was confirmed by direct comparisonofmean 44Ca/31P ratios in both the standard (1.14) and each tooth sample(range: 1.05–1.25). NIST provides certified concentrations (μg/g) for Caand P in bone meal as 26.58±0.24 and 12.30±0.19 respectively, and anon-certified concentration ofMn as 1 μg/g. However,Mn concentrationsmeasured in NIST 1486 have been reported in the literature with closeagreement: Saiki et al., 2009, 1.08±0.12 μg/g; ZaichickandZaichick, 2010,0.98±0.08 μg/g.

3. Results

The line scans of all eight teeth revealed a similar pattern of Mndistribution which was further confirmed by detailed analyses andelemental bio-image construction of the three incisors. TheMn bio-image(Fig. 1a) and the 55Mn:43Ca plot (Fig. 1b) reveal a distinct pattern thatwasobserved in all three incisors. The distribution of Mn in molars andcanines, as determined by three line scans per tooth, was also similar tothat seen in Fig. 1b. In enamel, the highest 55Mn:43Ca levels wereconsistently seen on the outer edge of enamel although this regionshowed large variability in 55Mn:43Ca ratios (range=0.57 to 4.74). Thewidth of this high Mn zone was estimated as being approximately20–40 μmfrom the surface of enamel, but the exact dimensions could notbe determined as the laser spot size of 20 μm allowed only one or twomeasurements in this zone. Deeper into enamel the 55Mn:43Ca countswere low (range=0.005 to 0.013) and in two of eight teeth were belowthe Mn background (100 to 300 cps). With the exception of surfaceenamel, Mn levels in enamel were generally lower than in dentine.

Infive of the eight teeth analyzed, therewas a discernable increase inthe 43Ca-normalized Mn counts (range=0.015 to 0.145) at thedentine–enamel junction (DEJ), and thesehigherMn:Ca levels extendedfor approximately 80±26 μm into dentine from the DEJ. Closer to the

Fig. 1. Spatial distribution of Mn in enamel and coronal dentine. Panel A shows a background corrected elemental bio-image of Mn distribution in the crown of a primary incisor.The 55Mn:43Ca levels are represented using a relative concentration index. Variations in Mn levels can be seen between enamel (E), dentine (D) and the dentine–enamel junction (DEJ).A clearly demarcated zone of highMn is evident in the incisal end of dentine (indicated by solid black arrow). Panel B shows the distribution of 55Mn:43Ca counts in the region selected inPanel A. Arrow indicates an increase in Mn levels at the DEJ.

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pulp chamber, there was again a sharp increase in Mn:Ca levels(range=2.27 to 6.95). This highMnzone,whichwasobserved in all 8 ofthe teeth analyzed, appears yellow to green in Fig. 1a. Within this zone,theMn distributionwas not homogenous but showed localized spots ofmarkedly higher Mn:Ca levels that appear red in Fig. 1a. Fig. 1b showsthe typical Mn:Ca distribution from one transect of the tooth imaged inFig. 1a, and a spike inMn:Ca levels at theDEJ is indicatedbyagrey arrow.A similar distribution of Mn can be seen in another deciduous incisorshown in Fig. 2.

An interesting feature to emerge from the elemental maps was theclearly higherMn:Ca levels in the region of dentinenear the incisal tip ofteeth (indicated by black arrow in Figs. 1a and 2b). This was observed in

Fig. 2. Mn distribution in enamel and coronal dentine of a human primary incisor. Backgrouregion of the prenatally formed dentine (indicated by arrow). This region is shown in hiconcentration index.

all 3 teeth that were bio-imaged. In Figs. 1a and 2b, this clearlydemarcated region of higherMn:Ca levels corresponds to the prenatallyformed dentine in incisors. To further confirm this, confocal microscopywasundertaken to image theneonatal line andwewere able to visualizethe intersection of the neonatal linewith theDEJ in these teeth (data notshown). In both teeth, the zone of higher Mn:Ca levels lay within thatregion of dentine bounded by the neonatal line, confirming that Mn:Calevels in prenatally formed dentinewere higher than in dentine formedafter birth.

Beyond measuring 55Mn:43Ca counts, we also adopted a simpleapproach to quantifying the Mn concentrations in sample teeth usingbone meal NIST SRM 1486 as a standard reference material. The

nd corrected 55Mn:43Ca map (Panel A) shows high Mn levels near the incisal tip in thegher magnification in Panel B. The 55Mn:43Ca levels are represented using a relative

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concentrations of Mn ranged from 0.1 to 0.2 μg/g in inner enamel(excluding the surface enamel) to 0.5 to 0.7 μg/g in the dentine adjacentthe pulp. The highest concentrations, observed in discreet spots near thepulp chamber were of the order of 1.0–2.0 μg/g (shown in red inFigs. 1a and 2).

4. Discussion

The results of this study provide a detailed high-resolution (20 μm)elemental map of Mn in human primary teeth. High Mn levels(i.e., 55Mn:43Ca) on the surface of enamel and lower levels in innerenamel are consistent with the distribution of other metals including Pbthathavepreviouslybeendetailed inhumanandanimal teeth (Buddet al.,1998; Arora et al., 2005). The higher abundance of Mn on the surface ofenamelmay result during thematuration phase of enamelmineralizationwhen the surface becomes hypermineralized relative to the inner aspectsof enamel (Smith, 1998). Alternatively, it is possible that, like Ca, P and Fions, the enamel surface exchanges Mn ions with saliva and, over time,accumulates a higher concentration of Mn. However, this cannot beconfirmed as there are limited data on the stability of Mn in enamelsurface and sub-surface layers.While it is estimated that thehighMnzoneextended for approximately 20–40 μm from the enamel surface, thereliability of thismeasurement is limitedby the fact that a20 μmlaser spotsize was used, allowing only one or two measurements in this region ofenamel.

Higher 55Mn: 43Ca counts were seen at the DEJ. The DEJ is a complexstructure and higher metal concentrations have been reported here inearlier studies (Arora et al., 2005). In a previous investigation of ratmolar teeth, we detected higher levels of Pb in dentine in the vicinity ofthe DEJ using proton-induced X-ray emission (Arora et al., 2005). Thehigh levels of Mn determined here extended beyond the DEJ intodentine for approximately 80 μm, but this was observed in only five ofthe eight teeth analyzed. This region of dentine corresponds withmantle dentine, an outer layer of dentine characterized by profusedbranching of the dentinal tubules (Berkovitz et al., 2009). Some reportshave found the mineral content of mantle dentine to differ from otherparts of circumpulpal dentine (Herr et al., 1986) and it is, therefore,possible that the unique mineralization pattern of mantle dentine mayresult in the higher Mn levels in this region.

Ca–normalized Mn counts were higher in dentine than enamel, andof all regions of enamel and coronal dentine analyzed in this study, thedentine immediately adjacent to the pulp chamber showed the highestMn levels. This highMnzone extended as a bandalong the dentine-pulpmargin (shown in yellow/green in Fig. 1a)with localized regions of veryhighMn (shown in red in Fig. 1a). The higherMn levels in the vicinity ofthe pulp may be due to the high density of blood vessels facilitating theexchange ofMn in the newly formed dentine and pre-dentinematrix. Itis also possible that the biological processes at play during rootresorption may result in localized increase in Mn along the dentine-pulp margin. Indeed, in one study higher levels of mitochondrialMn-superoxide dismutase activity were observed in the vicinity ofresorbing bovine tooth roots (Arai et al., 1989). Alternatively, the slowerrate of dentine deposition after crown completion may allow for alonger time period for Mn from blood to be incorporated in the newlyformed dentine adjacent the pulp. Additional work is needed toadequately address these hypotheses.

An important observation,made fromtheelemental bio-imagemapsof three teeth,was the presence of a clearly demarcatedhighMn zone indentine at the incisal end of the crown (indicated by black arrow inFigs. 1a and 2a,b). This zone appeared to be in the prenatally formeddentine and to confirm this we undertook confocal laser scanningmicroscopy to visualize the neonatal line. The neonatal line is ahistological feature in all primary human teeth (and some firstpermanent molars) that demarcates the prenatally formed regions ofteeth from those formed after birth (Schour, 1936). Results of these

analyses confirmed that in all three teeth this high Mn zone waslocalized within the prenatally formed dentine.

It is known that the developing fetus is supplied with Mn frommaternal sources and that Mn may be actively transported across theplacenta (Yoon et al., 2009). Earlier reports have also shown that Mnconcentrations inmaternal blood and breastmilk are lower than in cordblood indicating that Mn exposure during the prenatal period issubstantially higher than in early childhood (Ljung et al., 2009; Zotaet al., 2009). The fact that a clearly demarcated high Mn zone could beobserved in the prenatally formed dentine of all three teeth suggeststhat deciduous human teeth may be a potentially useful biomarker Mnexposure during tooth development. Whether high levels of Mn inprenatally formed dentine reflect higher fetal exposure or greaterefficiency in Mn absorption during prenatal development requiresfurther research.

Mn levels observed in the sample teeth were quantified using NISTSRM 1486 (bone meal). The Mn concentrations in enamel were in therange of 0.1 to 0.2 μg/g, and in dentine were 0.5 to 1.0 μg/g. Theseconcentrations of Mn are similar to Mn concentrations of caries-freeprimary teeth reported by Shashikiran et al. (2007) but lower than Mnconcentrations of approximately 3.5 μg/g in roots of permanent teethreported in another study (Malara et al., 2006). It is possible that toothtype and environmental factors are responsible for the varyingconcentrations of Mn in teeth reported in different studies.

The detailedmap of the spatial distribution of Mn in human primaryteeth highlighted distinct patterns with key differences in enamel anddentine. The elemental bio-imaging approachundertaken in thepresentstudy offers a number of advantages over more commonly usedmethods of measuring metal concentrations in tooth fragments or ofusing line scans to analyze discrete regions of teeth. Most notably, byimaging the spatial distribution of Mn in coronal dentine we have beenable to uncover two novel findings; firstly, that localized regions of veryhigh Mn levels are seen at the pulp-dentine junction. Secondly, thereexists a clearly demarcated high Mn zone in prenatally formed dentine.

Acknowledgments

The LA–ICP–MS analyses were supported by research grants fromthe Australian Dental Research Foundation Inc. and the Dental Board ofNSW, Australia.

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