The application of synchrotron radiation induced X-rayemission in the measurement of zinc and lead in Wistarrat ameloblasts

Manish Arora a,*, Brendan J. Kennedy b, Chris G. Ryan c, Ross A. Boadle d,D. Murray Walker a, Cathy L. Harland e, Barry Lai f, Zhonghou Cai f,Stefan Vogt f, Hans Zoellner a, Sheena W.Y. Chan a

aCellular and Molecular Pathology Research Unit, Department of Oral Pathology, Faculty of Dentistry, University of Sydney,

Westmead Centre for Oral Health, Westmead Hospital, Westmead, NSW 2145, Australiab School of Chemistry, University of Sydney, NSW 2006, AustraliacCSIRO Exploration and Mining, Geosciences, Building 28E, Monash University, Clayton, VIC. 3168, AustraliadElectron Microscope Unit, ICPMR, Westmead Hospital, Westmead, NSW 2145, AustraliaeAustralian Synchrotron Research Program, Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, AustraliafAdvanced Photon Source, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 9 3 8 – 9 4 4

a r t i c l e i n f o

Article history:

Accepted 6 April 2007

Keywords:

Synchrotron radiation

X-ray emission

Ameloblasts

Wistar rats

Trace elements

Zinc

Lead

a b s t r a c t

The development of analytical techniques for the measurement of trace elements in cellular

compartments of developing teeth remains an important methodological issue in dental

research. Recent advances in third generation synchrotron facilities have provided high

brilliance X-ray sources that can be effectively used to study trace element distributions in

small spatial regions with low detection limits. The present study describes for the first time

the application of synchrotron radiation induced X-ray emission (SRIXE) in measuring the

distribution of zinc and lead in the ameloblasts of developing Wistar rat teeth. Wistar rats

were fed a standard rat diet, containing the normal dietary requirements of zinc, ad libitum

and exposed to 100 ppm of lead in drinking water. Resin embedded sections of first

mandibular molars were analysed using a 13.3 keV incident monochromatic X-ray beam

focussed to a 0.2 mm spot. Characteristic X-rays arising from the entire thickness of the

sample were measured using an energy dispersive detector for quantitative analysis of

elemental concentrations. The results showed that intranuclear concentrations of zinc were

greater than levels in the cytoplasm. Furthermore, nuclear and cytoplasmic concentrations

of zinc in the maturation stage (742 � 27 and 424 � 25 ppm, respectively) were significantly

higher than the zinc levels observed in the nucleus and cytoplasm of presecretory stage

ameloblasts (132 � 10 and 109 � 10 ppm, respectively) ( p < 0.05). A clear lead signal above

the background was not detected in the ameloblasts and lead concentrations could only be

reliably measured in the developing enamel. Overall, SRIXE was an effective method of

studying the spatial distribution of zinc in the cells of developing teeth and offered a unique

combination of sub-micron spatial resolution and parts-per-million detection limits (0.8–1

and 0.6–1 ppm for zinc and lead, respectively).

# 2007 Elsevier Ltd. All rights reserved.

avai lable at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journals /arob

* Corresponding author. Tel.: +61 2 9845 7892.E-mail addresses: marora@mail.usyd.edu.au (M. Arora), b.kennedy@chem.usyd.edu.au (B.J. Kennedy), Chris.Ryan@csiro.au

(C.G. Ryan), rossb@icpmr.wsahs.nsw.gov.au (R.A. Boadle), charland@aps.anl.gov (C.L. Harland), BLAI@aps.anl.gov (B. Lai), CAI@aps.anl.gov(Z. Cai), VOGT@aps.anl.gov (S. Vogt), hzoellne@mail.usyd.edu.au (H. Zoellner).

0003–9969/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2007.04.003

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 9 3 8 – 9 4 4 939

1. Introduction

Zinc is an essential dietary element and serves numerous

biological functions including protein synthesis, maintenance

of neuro-sensory functions, cell-mediated immunity, thyroid

function as well as bone formation and mineralisation.1–3

There is also an increasing body of evidence supporting the

role of zinc in the development and maintenance of dental

tissues. Data from animal models shows that restriction of

dietary zinc increases the incidence of dental caries,4 reduces

the lipid content of developing enamel, and causes morpho-

logical alterations in ameloblasts.5 In addition, a number of

zinc-finger transcription factors have recently been identified

in developing mouse tooth germs and current evidence

suggests that they serve an important role in cytodifferentia-

tion of enamel epithelium and amelogenesis.6–9 In contrast to

zinc, lead exerts an adverse effect on developing teeth, with

lead exposed rats showing an increased incidence of dental

caries, disruption of salivary gland function, as well as delays

in enamel mineralisation and tooth eruption.10–12 The positive

association of lead exposure to dental caries has also been

confirmed in epidemiological studies.13–14

In addition to the individual effects of lead and zinc on

oral and systemic health, a number of studies suggest an

interaction between these elements. Co-administration of

zinc is known to reduce tissue and organ lead concentrations

in animal models.15–17 Furthermore, lead may displace zinc

from a number of zinc-binding sites including d-aminolevu-

linic acid dehydratase (ALAD), metallothioneins and some

transcription factors.18–20

An important methodological issue in understanding the

oral health effects of zinc, lead and other metal toxins is

the development of suitable analytical techniques to measure

the spatiotemporal distribution of theseelements in the cellular

compartments of developing tooth germs. While bulk chemical

methods of analysis including inductively coupled plasma-

mass spectrometry (ICP-MS), inductively coupled plasma-

optical emission spectrometry (ICP-OES) and atomic absorption

spectrometry (AAS), are capable of providing detection limits in

the parts-per-million (ppm) or parts-per-billion (ppb) range,

these methods are not suitable for determining the spatial

distribution of trace elements. Techniques such as energy

dispersive X-ray analysis (EDAX), electron probe analysis (EP)

and nuclear microprobe analysis (NMP) have proven useful in

measuring the intracellular concentrations of trace elements in

various cell types.21–23 These methods are, however, limited by

either high detection limits or poor spatial resolution. Over the

past two decades, developments in third generation synchro-

tron sources have provided high intensity X-ray beams that can

be effectively used to study trace element distribution in small

spatial regions with low detection limits.

In synchrotron radiation induced X-ray emission (SRIXE)

analysis, the high-energy X-rays generated by an undulator

source are focussed into a sub-micron spot on the sample,

where they create vacancies in the inner shells of the target

atoms by photoelectric absorption. When these vacancies are

filled by electrons from outer shells, characteristic X-rays are

emitted that are measured in an energy dispersive detector for

quantitative analysis of elemental concentrations.24 For the

purposes of trace element analysis, SRIXE offers a unique

combination of sub-micron resolution and detection limits in

the ppm range or better. While this technology is rapidly

gaining acceptance in many areas of science, its application in

the measurement of trace element concentrations in dental

soft tissues remains largely unexplored. The primary aim of

this study was to measure the change in intracellular

distribution of lead and zinc during ameloblast differentiation

from presecretory to early maturation stage. To our knowledge

this paper describes for the first time the application of SRIXE

in measuring the intracellular distribution of zinc and lead in

ameloblasts of developing Wistar rat teeth. The advantages

and limitations of this technique are also discussed.

2. Materials and methods

2.1. Animal handling

Three female Wistar rats, aged 12–14 weeks, of normal weight

were mated with similarly aged male Wistar rats of normal

weight.25 The animals received 100 ppm of lead nitrate (Asia

Pacific Speciality Chemicals, NSW, Australia) in the drinking

water and were fed a standard rodent diet containing 40 mg/kg

of zinc (AIN93G, Glen Forrest Stockfeeders, Western Australia)

ad libitum. Lead exposure was commenced 5 days before the

expected birth of the pups. The pups were housed with their

mothers, fed only by lactation and had no access to the

drinking water.

Groups of three rat pups were sacrificed by decapitation

from each of the litters at birth and on the third and seventh

day after birth. These specific time points were chosen

because the presecretory and secretory stages of ameloblast

development can be observed in a rat first molar tooth germ at

birth and 3 days after birth, while the early maturation stage of

ameloblast development can be visualised on day-7.26 The

continuously erupting rat incisor has been widely used as a

model for tooth development. Rat molars were used in the

present analyses, however, to permit the measurement of

trace elements in radicular tissues, which was the aim of a

parallel-running project. All procedures in the present study

were approved by the Animal Ethics Committee, Westmead

Hospital, NSW, Australia (Ethics clearance no. 124.06).

2.2. Preparation of tissue sections

Immediately after culling, the mandibles were removed using

sterile surgical instruments. The samples were processed in a

manner similar to that reported by Dillon et al.27 for SRIXE

analysis of hamster lung cells. First mandibular molar tooth

germs were removed under a dissecting microscope and fixed

overnight at 4 8C in Karnovsky’s fixative, followed by washing

in MOPS buffer (3-(N-morpholino)-propane-sulphonic acid).

The samples were dehydrated with graded ethanols (50–100%)

(Selby Biolab, Australia) for 15 min each at room temperature.

Following dehydration, tooth germs were placed in a 50:50

mixture of low viscosity Spurr’s resin (Polysciences Inc., USA)

and ethanol for 1 h at room temperature. The tooth germs

were then transferred to a 90:10 mixture of Spurr’s resin and

ethanol overnight. The final infiltration was in Spurr’s resin at

70 8C for 10 min, and this was repeated three times. For

Fig. 1 – Zinc concentrations in the nucleus and cytoplasm of

presecretory, secretory and early maturation stage

ameloblasts measured using synchrotron radiation

induced X-ray emission. From two tooth germs, 6–8 cells

were analysed per stage. Nuclear concentrations of zinc

were significantly different between all three

developmental stages ( p < 0.05). However, cytoplasmic

concentrations of zinc only showed a significant increase

in the early maturation stage ( p < 0.05).

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 9 3 8 – 9 4 4940

embedding, tooth germs were placed individually in large

embedding moulds (Leica Microsystems, Austria) and orien-

tated to ensure that the buccal surfaces of respective tooth

germs were parallel to the cutting faces of the resin blocks.

Spurr’s resin was then placed in the mould using a dropper

and care was taken to ensure that orientation of the tooth

germs remained unchanged. The moulds were placed in a

sealed container and the resin was polymerised at 70 8C

for 10 h.

Serial sections (1.0–1.5 mm) of the embedded tooth germ

samples were cut using an ultramicrotome (Reichert-Jung

Ultracut E, Austria). Adjacent sections were either stained with

methylene blue or mounted on an aluminium sample holder

prepared at the Advanced Photon Source, Argonne National

Laboratory, Illinois, USA. The aluminium mounts were 3.5 cm

long, 0.5 cm wide and two holes, 2 mm in diameter, were

present at one end of the mount. Gold finder grids (Prositech,

Australia) were placed on one hole to serve as reference points.

A thin coating of 0.3% (w/v) Formvar 15/95E (Monsanto

Chemicals, USA) was placed over the second hole to provide

support to the resin-embedded tissue section. Photomicro-

graphs of methylene blue-stained sections were used to

identify ameloblasts for SRIXE analysis. Using these photo-

micrographs as guides, tissue sections on aluminium mounts

were imaged under optical phase contrast microscopy and co-

ordinates for regions of interest were recorded. The finder

grids on each aluminium mount served as fixed reference

points. The co-ordinates recorded under phase contrast

microscopy were then transferred to the motorised SRIXE

sample stage, permitting accurate positioning of the samples.

To further confirm the location of the ameloblasts, coarse

SRIXE scans were conducted prior to high-resolution scans

(see following section on SRIXE Analysis). To avoid sample

contamination, tooth germ sections were mounted on the

aluminium holders only 5 days prior to the scheduled SRIXE

analysis. Mounted samples were stored in sterile tissue

culture dishes (Becton Dickinson, NJ, USA) and transported

to the Advanced Photon Source, Argonne National Laboratory,

Illinois, USA.

2.3. SRIXE analysis

SRIXE experiments were performed on the 2-ID-D beamline at

the Advanced Photon Source. All experiments were conducted

under a helium atmosphere to eliminate the argon K-shell

fluorescence signal and to reduce absorption of low-Z

fluorescence by air, and thus improve the signal-to-noise

ratio of the acquired data. Fluorescence lines were collected

using a Canberra germanium detector (Canberra, Meriden, CT,

USA). Full spectra were acquired at every scan position and the

K and L shell fluorescence signals were used for zinc and lead,

respectively.

A 13.3 keV incident monochromatic X-ray beam was

focussed to a 0.2 mm spot. The regions of interest were located

using the coordinates determined by light microscopy. Coarse

scans, with step sizes of 1–5 mm, were conducted to locate

individual cells in the X-ray microprobe. Detailed maps

were then collected using 0.2–0.5 mm steps and 2–10 s dwell

times per pixel. Mapped areas varied in dimensions from

15 mm � 20 mm to 80 mm � 80 mm. Because the tissue sections

analysed in the present study were 1.0–1.5 mm in thickness,

the emitted radiation was derived from the entire thickness of

the samples. The standard reference material (SRM) 1832

(National Institute of Standards and Technology, USA) was

used during the calibration process. SRM 1832 consists of a

silica-based glass film that has been deposited onto a

polycarbonate filter and is primarily used as a standard in

X-ray fluorescence spectrometry. Data analysis was con-

ducted using GeoPIXE II software (Commonwealth Scientific

and Industrial Research Organisation, Australia) and MS Excel

2000 (Microsoft Corporation, USA).

2.4. Statistical analysis

For each developmental stage the data from the two tooth

germs analysed were pooled and differences in element

concentrations between the different stages were analysed

using Kruskal–Wallis test at p < 0.05 using Stata (Intercooled

Stata 9.0; Stata Corporation, College Station, Texas, USA)

3. Results

Results of the SRIXE analysis of the developing rat tooth germs

are presented in relation to the stages of ameloblast devel-

opment in Figs. 1–3. The secretory and maturation stage

ameloblasts were analysed in detail with fine scans. However,

due to time restrictions, presecretory ameloblasts could only

be analysed using a coarse scan. A total of six tooth germ

sections from six different animals were analysed. The

method detection limits were 0.8–1 ppm for zinc and 0.6–

1 ppm for lead.

Mean zinc concentrations in nuclei and the cytoplasm

immediately adjacent to the nuclei in ameloblasts during the

Fig. 2 – (a and b) Spatial distribution of zinc and lead in secretory stage ameloblasts. Synchrotron radiation induced X-ray

emission was used to map the distribution of zinc and lead in a section of a resin-embedded first mandibular molar tooth

germ from a 3-day old Wistar rat pup whose mother received 100 ppm of lead via drinking water. In each case, Panel A

shows a coarse image of the trace element content of secretory ameloblasts (As), stratum intermedium (SI) and enamel (E)

generated using a step size of 1 mm and a 2 s dwell time. Panel B is a high resolution map, with a step size 0.3 mm and a

dwell time of 10 s, of the area highlighted in Panel A. Panel C is a high resolution map of the nuclei of two ameloblasts (as

indicated by the arrow in panel B), with a step size of 0.2 mm and a dwell time of 10 s. Light colours indicate high

concentrations and dark colours represent low concentrations as described by the relative concentration index. Note: the

maximum concentration for each element in a scan is used to determine the relative concentration index for the colour

image corresponding to that particular element. Consequently, similar colours in different elemental maps may not

indicate similar concentrations and image colour alone cannot be used to compare different elemental maps. (For

interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 9 3 8 – 9 4 4 941

Fig. 3 – Spatial distribution of zinc in secretory stage

ameloblasts. Synchrotron radiation induced X-ray

emission was used to map the distribution of zinc in a

section of a resin-embedded first mandibular molar tooth

germ from a 3-day old Wistar rat. The secretory stage

ameloblasts (As) and cells of the stratum intermedium (SI)

are indicated. Light colours indicate high concentrations

and dark colours represent low concentrations as

described by the relative concentration index. (For

interpretation of the references to colour in this figure

legend, the reader is referred to the web version of the

article.)

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 9 3 8 – 9 4 4942

presecretory, secretory and maturation stages are shown in

Fig. 1. At every developmental stage, zinc concentrations were

higher in the nucleus than the cytoplasm. Furthermore, in the

nucleus, the zinc concentrations showed a statistically

significant increase from the presecretory to the secretory

and early maturation stages (p < 0.05). In the cytoplasm, the

increase in zinc concentrations was mainly evident from the

secretory to the early maturation stage (p < 0.05).

Fig. 2a shows the typical intracellular distribution of zinc in

secretory stage ameloblasts. Lead concentrations, however,

could only be measured in regions of the mineralizing enamel,

and no lead was detected above the background signal in the

region of the ameloblasts (Fig. 2b). In enamel, zinc levels (0.1–

0.5%) were markedly higher than lead (34–117 ppm). The zinc

maps provided the clearest distinction between the nuclear

and cytoplasmic regions of the imaged cells as shown in Fig. 3,

which is a high-resolution map of secretory stage ameloblasts

and cells of the stratum intermedium. The terminal ends of

the ameloblasts and enamel were not imaged in this scan.

4. Discussion

SRIXE has been used successfully in measuring the intracel-

lular distribution of trace elements in a number of cell

types27–30 and in dental hard tissues.31 To the best of our

knowledge the intracellular measurements of zinc and lead in

ameloblasts of developing tooth germs using SRIXE have not

previously been reported. In the present study, SRIXE was

successfully applied in the measurement of zinc in the

presecretory, secretory and early maturation stages of

ameloblast differentiation. Lead concentrations in the cellular

strata were, however, below the background signal, despite

the use of tooth germs from lead-supplemented rats.

The concentration of zinc in the nuclei and cytoplasm of

ameloblasts increased with progression of the ameloblast

life cycle from the presecretory to the early maturation

stage. Zinc is normally present in calcified tissues including

enamel, and its entry into the calcifying enamel matrix may

be regulated by ameloblasts in a manner similar to calcium.

The increase in zinc concentrations with advancement of

ameloblasts from presecretory to the secretory and early

maturation stages agrees with the data on calcium reported

by Engel and Hilding32 and Engel,23 who measured calcium

levels at different stages of mouse molar tooth development

using EDAX. In those studies, maturation stage amelo-

blasts at the age of 14 days had calcium levels of

1671 � 687 counts/100 s, which were significantly higher

than the calcium levels of 220 � 90 counts/100 s observed

in ameloblasts from 5-day old animals. Studies on the

incorporation of zinc in osteoblasts lend further support to

our observations. Kanno et al.,33 by means of fluorescent

microscopic analysis using Zinquin, observed an increase in

zinc concentrations in cultured osteoblasts in response to

mineralisation induced by the addition of b-glyceropho-

sphate to the culture medium.

Zinc binds to alkaline phosphatase (ALP) and is important

for the activity of this enzyme.34 Because, ALP is essentially

absent from presecretory and secretory ameloblasts, but is

present in the maturation stage ameloblasts,35,36 ALP asso-

ciated increases in zinc concentrations may partially account

for the higher zinc levels in maturation stage ameloblasts.

Further, it has been recently shown that there is an increase in

several matrix metalloproteinases, including enamelysin37

and transcription factors such as Krox-25 and Krox-26 in the

secretory ameloblasts.6,7 The presence of zinc-finger domains

in these and other molecules may further contribute to

the increase in the intracellular zinc concentrations in

ameloblasts.

The mean concentrations of zinc were higher in the

nuclei of ameloblasts as compared with concentrations

measured in the cytoplasm immediately adjacent to the

nuclei. While data on the intracellular concentrations of zinc

in ameloblasts has not previously been reported, higher

levels of zinc in the nucleus compared with the cytoplasm

have been observed in a number of cell types.27,38 In relation

to the data on calcium levels in ameloblasts, Boyde and

Reith39 observed no difference in calcium concentrations

between the nuclear and non-nuclear regions of rat

secretory ameloblasts using EDAX. Studies on hamster lung

cells and ovarian cancer cells using SRIXE, however, have

shown that calcium concentrations in the nucleus are

indeed higher than in the cytoplasm.28,40

While lead could not be detected in the ameloblasts, a peak

in lead concentration was observed on the edge of the

mineralizing enamel (Fig. 2b). A number of studies have

reported similarly high concentrations of lead on the enamel

surface of primary human teeth,41–43 and some have attrib-

uted this finding to the absorption of lead from saliva.44 In the

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 9 3 8 – 9 4 4 943

present study, however, uptake of lead from saliva is unlikely

as the tooth germs had not erupted into the oral cavity. High

levels of lead in the superficial layers of enamel in unerupted

rat molars, measured using a nuclear microprobe, have been

previously reported.45 It is possible that the presence of lead in

the outer layers of enamel is due to ionic exchange with

calcium in the apatite lattice, similar to the exchange of

fluoride ions.46 Lead uptake in enamel may also be influenced

by considerable changes in the enamel organ during early

maturation, which is known to coincide with variations in the

uptake of calcium.47,48 However, the inability of the present

study to reliably detect the intracellular distribution of lead in

the ameloblasts precludes further analysis of the cause of high

concentrations of lead found at the mineralizing front of

enamel using this methodology. One limitation to the

approach used is that sample preparation requires fixation,

dehydration and resin infiltration. Each step may affect

concentrations of unbound trace elements, potentially con-

founding the data. However, zinc concentrations are unlikely

to be affected by this, as intracellular zinc is largely in bound

form.49

Despite these limitations, the present study demon-

strates the value of SRIXE as an effective and high-resolution

method for the measurement of intracellular trace elements

in developing rodent tooth germs. In comparison to EDAX,

EP and NMP, SRIXE offers a unique combination of low

detection limits and sub-micron resolution.50 It is also a non-

destructive technique and shows limited susceptibility to

matrix effects when analysing thin tissue sections as used in

the present study. The main limitations of SRIXE include the

long data collection times, limited access to synchrotron

facilities, higher detection limits when compared with

neutron activation analysis, and laser-ablation inductively

coupled plasma-mass spectrometry. SRIXE, however, offers

significantly better spatial resolution than these other

techniques.

In summary, the results presented here have shown that

SRIXE is a powerful technique for the determination of trace

elements in the cellular compartment of developing tooth

germs. Concentrations of zinc in the ameloblasts of the Wistar

rat tooth germs increased from the presecretory to early

maturation stage, and nuclear concentrations of this element

were higher than levels in the cytoplasm.

Acknowledgements

The authors thank Dr. Ross Mathews and the technical staff at

the Westmead Animal Holding, Westmead Hospital, Sydney,

for their help with animal handling, and the staff of the

Electron Microscope Unit, Westmead Hospital, Sydney, for

their assistance in sample preparation. Dr. Arora was

supported by the Early Career Development Award from the

College of Health Sciences, University of Sydney. This work

was supported by the Australian Synchrotron Research

Program, which is funded by the Commonwealth of Australia

under the Major National Research Facilities Program. Use of

the Advanced Photon Source was supported by the US

Department of Energy, Office of Science, Basic Energy

Sciences, under Contract no. W-31-109-Eng-38.

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