mechanically induced epr signals in tooth enamel

Applied Radiation and Isotopes 55 (2001) 375–382

Mechanically induced EPR signals in tooth enamel

D. Aragno, P. Fattibene*, S. Onori

Istituto Superiore di Sanit "a, Physics Laboratory, Viale Regina Elena 299, I-00161 Rome, Italy

Received 2 January 2001; accepted 13 February 2001

Abstract

Sample preparation of tooth enamel for electron paramagnetic resonance (EPR) dosimetry usually involves

mechanical operations. The present study shows that mechanical operations performed without water cooling generatea paramagnetic center inducing a stable isotropic EPR signal with g-value of 2.00320 and linewidth of about 0.1mT.Using EPR spectrum simulation, the similarity between the mechanically induced signal and the signal generated when

the enamel is heated in air at a temperature above 6008C was investigated. Results indicate that the mechanicallyinduced signal is related to sample temperature increase during mechanical friction. # 2001 Elsevier Science Ltd. Allrights reserved.

Keywords: EPR signals induced mechanically; Retrospective dosimetry; Tooth enamel

1. Introduction

The electron paramagnetic resonance (EPR) dosereconstruction with tooth enamel is based on themeasurement of paramagnetic centers induced by

ionizing radiation in tooth enamel hydroxyapatite(Voight and Paretzke, 1996; European Commission,1996). Mostly, the radiation induced free radical has

been identified as CO2� (Callens et al., 1987; Vugman

et al., 1995). The EPR dose reconstruction with toothenamel has now been established as a valid biologicalindividual dosimetric method for retrospective dosime-

try in case of nuclear accidents. Many of the researchgroups active in this field have developed samplepreparation protocols (Wieser et al., 2000). Even if at

different extents, all the sample preparation proceduresmake use of mechanical operations, like sawing forseparation of the root from the crown, drilling for

removal of the dentine and grinding for powdering theenamel. These operations may introduce defects andchanges in the morphology and ultra-structure of the

enamel, with the consequent induction of EPR signals

which may affect the CO2� dosimetric signal evaluation.

The possibility of inducing free radicals by mechanical

operations in bone like tissues has been reported a longtime ago (Marino and Becker, 1968). More recently,some authors have reported the presence of EPR signals

in enamel correlated with the different mechanicaloperations included in the sample preparation. Inparticular, the effect of grinding has been studied by

various authors (Polyakov et al., 1995; Sholom et al.,1998; Fattibene et al., 1998), and the effect of sawing hasbeen shown by Desrosiers et al. (1989). Aldrich et al.(1992) have studied the effect of drilling and showed that

drilling on tooth enamel produces a range of radicalswhose EPR signals appear similar to those produced byheating. In many of the quoted papers the mechanical

operations were stressed to a level such that themechanically induced signal was clearly detectable inorder to obtain a non-ambiguous cause-effect relation.

In particular, Desrosiers et al. (1989) have shown thatthe use of a high-speed diamond abrasive wheel inducesthe formation of a signal with spectroscopic features

very similar to those of the dosimetric CO2� signal. It

was also reported that vigorous mechanical operationscould heat the sample up to temperatures as high as10008C (Ikeya, 1993). Recently, Fattibene et al. (2000)reported the formation of different paramagnetic centers

*Corresponding author. Tel.: +39-06-4990-2248; fax: +39-

06-4938-7075.

E-mail address: [email protected] (P. Fattibene).

0969-8043/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 6 9 - 8 0 4 3 ( 0 1 ) 0 0 0 7 8 - 1

when heating tooth enamel above 3508C. For tempera-tures in the 600–10008C range the EPR spectrum

simplifies and appears dominated by a symmetric singleline signal with g ¼ 2:00320 and a linewidth of about0.11mT.

Aim of the present paper was to investigate if sawingand drilling induced radicals are originated by a localoverheating of the enamel. Human teeth were mechani-cally treated with and without water cooling during the

mechanical operations. The experiment was performedwith teeth not irradiated in laboratory to keep the CO2

�

radical signal to a minimum value, in order to reduce its

interference with the mechanically and thermally in-duced signals. Similarity of the mechanically inducedsignals to thermally induced signals was investigated

with the help of spectrum simulation.

2. Materials and methods

2.1. Sample preparation

Non-irradiated healthy molars of adult people anddeciduous teeth from the local population were used.The tooth crown was separated from the root and then

cut in two halves using a power driven low speeddiamond saw. Dentine was removed using a high speeddental drill. Both the saw and the drill could be

optionally used with or without water cooling. Sixenamel samples (A, MT, B, HMT, C and D) wereprepared using five teeth (three adult molar teeth and

two position 6 deciduous teeth). The samples A and MT(mechanically treated) were obtained, respectively, fromthe two halves of an adult tooth. The samples B andHMT (heavily mechanically treated) were prepared

using two deciduous teeth. Sample B was obtainedpooling the powder obtained from one half of eachtooth and HMT was obtained pooling the powder

obtained from the other two halves. Samples A and Bwere the controls for samples MT and HMT, respec-tively. The other two adult teeth were used for the

preparation of samples C and D, respectively. SamplesA, B, C and D were prepared using water cooling duringdrilling according to the ISS protocol (Onori et al.,

2000). Samples MT and HMT were prepared withoutwater cooling during drilling. Moreover, for the sampleHMT, the drilling operation was stressed using moreintense strength and applying the drill for longer time

than in the case of the sample MT. After drilling, all thesamples were gently ground by hand with mortar andpestle, to 0.5–1mm grain size, without cooling. For

samples B, C, MT and HMT no etching was applied.Each sample had a mass of about 150mg.Sample C was heated in air in a ventilated oven at

10008C for 6min. After heating, the sample was cooledin air at room temperature for about 20min. Then the

sample was inserted in a quartz tube that was closedwith a plastic cup and measured immediately (Fattibene

et al., 2000). Sample D was irradiated at 400mGy with a6MV photon beam produced by a Varian 2100C linearaccelerator and measured after a few weeks to zeroing

possible transient signals (Sholom et al., 1998).

2.2. EPR measurements

The EPR measurements were carried out with aBRUKER ESP 300 spectrometer, operating in X band,with a TM cylindrical cavity. The microwave frequency

was 9.74GHz. According to the ISS protocol (Onoriet al., 2000), the EPR acquisition was performed at0.2mT of modulation amplitude and 25mW of micro-

wave power. Moreover, to improve the spectrumresolution of both thermally and mechanically inducedsignals, some EPR measurements were performed at

0.05mT of modulation amplitude using different levelsof microwave power in the range 1–200mW. Figurecaptions will report the experimental parameters of eachacquisition. The other EPR acquisition parameters

(sweep field, time constant, conversion time) were setin order to assure a correct recording of the signals. Themicrowave frequency was monitored during the EPR

acquisition with a frequency counter (53150A, HewlettPackard, Santa Clara, California) controlled by apersonal computer. A MgO/Mn2+ powder sample was

used as a reference sample for g-value and for signalintensity normalization. It was inserted at the bottom ofthe cavity and its position was held fixed for each set of

EPR acquisitions, as described by Aragno et al. (2000).The enamel powder samples were inserted in a 3mminternal diameter quartz tube and centered in the cavity.Each sample was measured three times. Between

measurements the sample was extracted out of thecavity, shaken and repositioned in the cavity.

2.3. Spectrum simulation

The signal intensity evaluation was based on simula-

tion of the enamel EPR spectrum and on the best fit ofthe simulated spectrum to the experimental spectrum.Both the simulation and the best fit of all the

experimental spectra acquired were performed with theprogram POWFIT, based on the SIMPLEX algorithm(the program is part of the public EPR Software Tools(PEST) developed at NIEHS/NIH, software is available

at http://epr.niehs.nih.gov/). Following the ISS proto-col, three signals were used for the simulation of theenamel spectrum: an isotropic signal for the native

background signal, an axial signal for the radiationinduced signal (CO2

� signal) and a sextet isotropic signalfor the Mn2+ standard. The Hamiltonian parameters

used for the simulation of the three signals are reportedin Table 1. Details of spectrum simulation are given in

D. Aragno et al. / Applied Radiation and Isotopes 55 (2001) 375–382376

Onori et al. (2000) and in Aragno et al. (2000). For the

simulation of the tooth enamel spectra recorded at0.05mT modulation amplitude, the linewidth of thesextet isotropic signal of the Mn2+ standard was

reduced to 0.07mT to account for the effect of themodulation amplitude on the signal linewidth.

3. Results

The spectrum of the mechanically treated sample MT,

along with the spectra of its control sample A and of thesample D irradiated at 400mGy, is shown in Fig. 1. TheEPR acquisition parameters were 0.2mT modulation

amplitude and 25mW microwave power. Also, thefigure shows (dotted lines) the spectra simulated usingthe Hamiltonian parameters reported in Table 1. In the

spectrum of the MT sample a signal induced by themechanical treatment is visible at about 349mT. The

spectrum simulated with only the radiation induced

CO2� and native background signal does not match the

experimental spectrum.In order to obtain a higher resolution of the

mechanically induced signal with respect to the nativebackground and to the CO2

� native signals, themeasurement was repeated with the HMT sample usingthe following EPR acquisition parameters: 0.05mT

modulation amplitude, 1mW microwave power. Thespectra of the HMT sample and of its control sample B,acquired with these parameters, are shown in Fig. 2

along with the subtracted spectrum. Under theseacquisition conditions the main visible difference be-tween the HMT sample and the control sample spectra

was the presence of a signal at 349.1mT field value.The mechanically induced signal was stable in time:

no significant signal amplitude decrease was observedover 6 month storage at 188C and 30–40% relative

humidity.

Table 1

Spectral parameters used for the simulation of the tooth enamel spectrum according to the ISS protocol (acquisition parameters:

0.2mT modulation amplitude, 25mW microwave power)

Radical species g Linewidth (mT) Line shape

Radiation induced (CO2� signal) 2.00322(?), 1.99835(k) 0.326(?), 0.340(k) Gaussian (axial)

Background 2.00534 0.654 Lorentzian (isotropic)

Mn2+ 2.00639a 0.080 Lorentzian (isotropic)

aThis Mn2+ g-value corresponds to the effective center field of the 3rd and 4th line of the Mn2+ signal, calculated using the isotropic

g-value 2.00101 (Abragam and Bleaney, 1970) and accounting for the relative positions in the cavity of the enamel sample and of the

MgO/Mn2+ samples as reported in Aragno et al. (2000).

Fig. 1. Experimental (solid lines) and simulated (dotted lines) EPR spectra of three enamel samples. From top to bottom: spectrum of

a sample prepared with accurate water cooling during the mechanical operations (control sample A); spectrum of a sample prepared

without water cooling during the sample preparation mechanical operations (MT sample); spectrum of a sample irradiated in the

laboratory at 400mGy (sample D). All the experimental spectra were detected with the same EPR parameters. Acquisition parameters

were 0.2mT modulation amplitude, 25mW microwave power. The simulated spectra were calculated using signals reported in Table 1.

Baseline offset on the ordinate has been introduced for better reading.

D. Aragno et al. / Applied Radiation and Isotopes 55 (2001) 375–382 377

In Fig. 3 the experimental (continuous line) andsimulated (dotted line) EPR spectra of the enamelsample C heated at 10008C are shown. Acquisition

parameters were 0.05mT modulation amplitude and1mW microwave power. Spectrum simulation led to thefollowing Hamiltonian parameters: Lorentzian line

shape, g-value 2.00320, linewidth 0.108mT. The spec-trum was then recorded at different microwave powerlevels. In Table 2 the linewidths resulting from the bestfit of the simulated spectra to the experimental spectra

are reported.

Fig. 2. From top to bottom: experimental EPR spectrum of the control sample B; experimental EPR spectrum of the sample prepared

with heavy mechanical treatment and without water cooling (HMT sample); spectrum obtained by subtraction of B from HMT

spectra. Acquisition parameters were 0.05mT modulation amplitude, 1mW microwave power. Baseline offset on the ordinate has been

introduced for better reading.

Fig. 3. Experimental (solid line) and simulated (dotted line) EPR spectra of a sample heated in air atmosphere at 10008C. Acquisitionparameters were 0.05mT modulation amplitude, 1mW microwave power.

Table 2

Signal linewidth (calculated with spectrum simulation) of the

sample heated at 10008C as a function of the microwave power

Microwave power (mW) Linewidth (mT)

1 0.108

4 0.108

10 0.110

25 0.111

100 0.121


Similarly, the spectrum of the HMT sample wasrecorded at different microwave power levels and at0.05mT modulation amplitude. Fig. 4 shows the varia-

tion of the signal amplitude with the microwave powerfor the HMT and 10008C samples. For comparison, thefigure shows also the microwave power dependence of

signal amplitude for the 6008C sample as reported in thepaper of Fattibene et al. (2000).Fig. 5 shows experimental and simulated spectra of

the HMT sample at different microwave power levels.

The experimental spectra were simulated with four

signals: three were those used in the ISS protocol andreported in Table 1, the fourth was an isotropic signal tosimulate the mechanically induced signal. For the native

background and for the radiation induced signals theHamiltonian parameters reported in Table 1 were used.The linewidth of the sextet isotropic signal for the Mn2+

standard was reduced to 0.07mT, as explained in theMaterials and methods Section. The Hamiltonianparameters used for the simulation of the mechanicallyinduced signal were those obtained for the simulation of

the 10008C signal (Lorentzian line shape, 2.00320 g

Fig. 4. Relative amplitude of the mechanically induced signal in the HMT sample, and of the signals of the 6008C and 10008C heatedsamples as a function of microwave power. The amplitude values were normalized to the relative amplitudes at 1mW. Modulation

amplitude was 0.05mT. Solid lines are only for eye-guide.

Fig. 5. Experimental EPR spectra (solid lines) of the HMT sample detected with different microwave power levels and the respective

simulated spectra (dotted lines). Modulation amplitude was 0.05mT. Baseline offset on the ordinate has been introduced for better

reading.


value, linewidths reported in Table 2). The agreementbetween experimental and simulated spectra is quitesatisfactory.

Simulation of the MT sample spectrum shown inFig. 1 was repeated with the same signals used for thesimulation of the HMT sample spectrum. Hamiltonian

parameters were those reported in Table 1 to take intoaccount the microwave power and modulation ampli-tude (25mW, 0.2mT) used for the MT sample spectrumacquisition. Experimental and simulated spectra are

reported in Fig. 6. Comparison with Fig. 1 clearly showsan improvement in the simulation of the MT spectrum.

4. Discussion

Mechanical treatment of enamel samples, performedwith saw and drill, induced a narrow EPR signal in theg-value region of the radiation induced signal (Figs. 1

and 2). These signals are generated when samples wereprepared without the use of water cooling during themechanical operations, but the signal intensity was

much stronger for the HMT sample with respect to theMT sample. Indeed, for the same acquisition para-meters, the peak-to-peak amplitude of the mechanicallyinduced signal was about twice the peak-to-peak

amplitude of the native background signal in the caseof the HMT sample, while it was about half in the caseof the MT sample.

The spectra of the MT sample and of the irradiatedsample shown in Fig. 1 appear, at a first sight, similar. Infact, with respect to the control sample, both show a

signal overlapped to the native background signal, butnothing can be said about the line shape and the g-value,

because they are partly masked by the native back-ground signal. The only difference that can be noted isthat the peak minimum of the signal induced by the

mechanical treatment is shifted to lower field values withrespect to the peak minimum of the CO2

� signal inirradiated samples, suggesting that the mechanically

induced signal is probably different from the radiationinduced CO2

� signal. The difference between themechanically induced and the radiation induced signalswere put in evidence by spectrum simulation (shown in

Fig. 1 as dotted lines). While there is a good agreementbetween experimental and simulated spectra for theirradiated sample, the agreement was not satisfactory in

the case of the spectrum observed for the MT sample.This suggests that the mechanically induced signal in theMT sample is to be ascribed to free radicals different

from the radiation induced CO2� radicals. Also, Fig. 2

clearly shows that the signal induced by mechanicaltreatment in the HMT sample was very different from

the radiation induced CO2� signal. Indeed, as it can be

seen from the spectrum obtained by subtracting thecontrol sample spectrum from the HMT samplespectrum, it is a single line signal with about 0.1mT

linewidth.The mechanically induced signal shown in Fig. 2

reminds a signal found when enamel samples were

heated in air atmosphere at temperatures above 6008C(Fattibene et al., 2000). It is known that vigorousmechanical stress can heat the sample up to tempera-

tures as high as 10008C (Ikeya, 1993). Evidence that thesample reached locally high temperature values camefrom the formation of grey spots on the enamel samplesurface when the tooth was treated mechanically with-

out water cooling. The same color change in enamel

Fig. 6. Experimental (solid line) and simulated (dotted line) EPR spectra of the MT sample. Acquisition parameters were 0.2mT

modulation amplitude, 25mW microwave power. Simulation was performed adding a 10008C like signal to the signals used for thesimulation shown in Fig. 1.


samples was found in samples heated around 5008C inO2-free atmosphere (Holcomb et al., 1980), and at

temperatures up to 10008C in air atmosphere (Fattibeneet al., 2000). The change of the enamel color from whiteto grey has been related to partial decomposition of

organic matter in the enamel (Holcomb et al., 1980).Therefore, the present study investigated the similarityin the spectral characteristics of the signal induced insamples heated at 10008C and of the signal induced bymechanical treatment. The temperature of 10008C waschosen on the basis of the data presented in the paper byFattibene et al. (2000), where a phenomenological

picture of the tooth enamel heated at 350, 400, 450,600 and 10008C was reported. Spectra of samples heatedat a temperature below 4508C were quite complex, whilethey changed to very simple (a single spectral line ofabout 0.1mT linewidth) above 6008C heating. The EPRsignals of the samples heated at 6008C and 10008C werevery similar in terms of Hamiltonian parameters buttheir dependence on microwave power was different.This difference was attributed to some signals stillpresent in the spectrum of the 6008C heated sample,that could affect the microwave power dependence, evenif their intensity was likely too weak to be observed.Therefore, in the present paper a sample heated at

10008C, where it is likely that only one species gives riseto the signal under study, was used to verify thehypothesis of similarity between thermally and mechani-

cally induced signals. The first step was to determine bysimulation the spectroscopic parameters of the 10008Csignal. Then, these parameters were used to simulate theHMT sample signal. Even though the agreement

between experimental and simulated signals of theHMT sample was satisfactory (Fig. 5), the dependenceof the HMT signal intensity on microwave power

(Fig. 4) did not fit adequately the microwave powerdependence of the signal intensity neither of the 10008Cheated sample nor of the 6008C heated sample. Thissuggests that the mechanically induced signal could be aconvolution of different signals, which are dominated bythe 10008C signal. In other words, radicals generated bymechanical operations that heat the enamel at hightemperatures (around 10008C) are mostly present in thesample, but at the same time it is likely that otherradicals, as those generated at lower temperatures, might

be present too. Also, the modalities of sample heatingare different in the case of an oven heated sample(uniform heating of sample volume) with respect to the

mechanically treated sample (discontinuous surfaceheating process). Of course, similar considerations canbe given for the MT sample, even if in this case the

temperature increase could have been lower. Anyway,Fig. 6 demonstrates the need to add a 10008C like signalin the MT sample simulation.

Many papers studied free radical induction bymechanical treatment in enamel and in bone-like tissues.

In two previous papers, it was reported the induction ofan EPR signal centered at about g ¼ 2:002 when thetooth samples were powdered with diamond saw(Desrosiers et al., 1989) or when the enamel was sawedor the dentine drilled out (Aldrich et al., 1992).

Unfortunately, the comparison of the mechanicallyinduced signal shown in the present work with thoseof the quoted papers is difficult since Aldrich et al.(1992) used laboratory irradiated teeth and Desrosiers

et al. (1989) did not report the EPR acquisitionparameter values. However, it should be noted that thesignals shown in those papers might be different from

the signal shown in the present work since mechanicallyinduced radicals could be different in the differentmechanical procedures applied. Indeed, different me-

chanical procedures might induce locally different sur-face temperature increase.

5. Conclusions

The use of saw and drill without water cooling in the

preparation of tooth enamel samples for individual dosereconstruction with EPR induces an EPR signal in theg ¼ 2:003 region of the enamel spectrum. The mechani-cally induced signal is near to the dosimetric CO2

� signal,but spectrum simulation showed that, actually, it is notradiation induced CO2

�. The origin of the signal was

related to the enamel temperature increase caused bymechanical friction. Spectrum simulation showed simi-larity of the mechanically induced signal with the signalinduced by enamel heating at 10008C, even if thepresence of other radicals generated at lower tempera-tures cannot be excluded. Care should be paid to controlthe temperature increase during sample preparation

operations, since the presence of a mechanically inducedsignal, which overlaps to the radiation induced CO2

�

signal, may lead to a non-reliable dose assessment.

Acknowledgements

The authors are grateful to E. Petetti for technicalsupport. This work was partially supported by theCommission of the European Community, Contract

FI4P-CT95-0011, under Framework IV, Nuclear Fis-sion Safety, Project E1.4, Dose Reconstruction.

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mechanically induced epr signals in tooth enamel

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