Can in vivo surface dental enamelmicrobiopsies be used to measure ...

Environmental Science and Pollution Research https://doi.org/10.1007/s11356-017-0988-9

RESEARCH ARTICLE

Can in vivo surface dental enamelmicrobiopsies be used to measure remote lead exposure? Kelly Polido Kaneshiro Olympio 1 & Manuel Fernando Gonzalez Huila 2 & Cristiane de Almeida Baldini Cardoso 3 & Ana Paula Sacone da Silva Ferreira 1 & Adrielly Garcia Ortiz 4 & Henrique Eisi Toma 2 & Ricardo Henrique Alves da Silva 4 & Maciel Santos Luz 5 & Maria Regina Alves Cardoso 6 & Gislayne Aparecida Rodrigues Kelmer 2 & Pedro Vitoriano de Oliveira 2 & Etelvino José Henriques Bechara 2 & Wanda Maria Risso Günhter 1 & Marília Afonso Rabelo Buzalaf 7 Received: 10 April 2017 / Accepted: 10 December 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract Measuring lead in the surface dental enamel (SDE) using biopsies is a rapid, safe, and painless procedure. The dental enamel lead levels (DELLs) decrease from the outermost superficial layer to the inner layer of dental enamel, which becomes crucial for the biopsy depth (BD) measurement. However, whether the origin of lead found in SDE is fully endogenous is not yet established. There is also controversy about the biopsy protocol. The aims of this study were to investigate if DELLs are altered by extrinsic contamination (A) and to evaluate the real geometric figure formed by the erosion provoked by biopsy procedure and the respective BD in SDE (B). To accomplish the aim A, lead from 90 bovine incisor crowns lead was determined by graphite furnace atomic absorption spectrometer as a function of exposure time and lead concentration. Two biopsies were performed in each tooth, before and after lead exposure. Six 15-tooth groups differed by exposure time (1 or 30 min) and lead concentrations (A. 0 mg/L—placebo, B. 0.01 mg/L—standard for drinking water, or C. 0.06 mg/L—concentration found in contaminated groundwater). Phosphorus was determined by an inductively coupled plasm optical emission spectrometer to quantify the enamel removed. To compare intakes/losses of lead in SDE among the groups, values of DELL differences between before and after lead exposure were compared by ANOVA (p < 0.05). To attain the objective B, one extracted human permanent tooth was studied by confocal Raman microscopy. Lead measurements and the surface profile were determined. There was no difference in DELL among the groups (p = 0.964). The biopsy bottom surface area, analyzed by microscopy, showed an irregular area, with regions of peaks and valleys, where areas with depth ranging from 0.2 (peaks) to 1.8 μm (valleys) (± 0.1 μm) could be found. BD carried out in vivo is commonly calculated using the cylinder height formula. The real BD was shown to be very similar to already published data. In conclusion, the SDE of erupted teeth does not seem to be susceptible to environmental lead intake, being thus reliable to measure remote exposures to lead. Keywords Lead exposure . Dental enamel . Tooth microbiopsies . Tooth Raman spectroscopy . Biopsy protocol . Biomarker

Introduction According to the World Health Organization, lead exposure accounts for 0.6% of the global burden of lead poisoning principally in developing countries. About 600,000 new cases of children with intellectual disabilities are yearly attributed to Responsible editor: Philippe Garrigues * Kelly Polido Kaneshiro Olympio [email protected] Extended author information available on the last page of the article

lead exposure. Legal restrictions on the use of lead in gas, paint, plumbing, and solder have resulted in substantial reductions in blood lead levels. However, significant sources of exposure are still reason of concern, mainly the so-called hot spots in developing regions (WHO 2016). Blood is the biomarker most commonly used to evaluate recent lead exposures (Rabinowitz 1995); however, remote lead exposure must be evaluated as well (Olympio et al. 2010a, b, c). Remote lead exposure has been associated with human dental tissue evaluation by archeological sciences (Budd et al. 1998, 2000, 2004). In vivo early lead exposure can be verified by surface dental enamel sampling as a

Environ Sci Pollut Res

biomarker for remote or chronic lead exposure (Brudevold et al. 1975; Olympio et al. 2010a, b, c). Lead determination in biopsies of the surface dental enamel (SDE) is a rapid, safe, and painless procedure. However, whether the lead found in SDE is fully endogenous has not yet been clarified (Budd et al. 1998). Different protocols to evaluate the dental enamel lead levels (DELLs) in microbiopsies revealed significant differences in previous studies (Brudevold et al. 1977; Cleymaet et al. 1991a; Olympio et al. 2010a, b, c). Confocal laser scanning microscopy is a nondestructive 3D technique widely used to perform surface and depth measurements on mineral matrices, and it has been applied to assess the amount of tissue loss of eroded enamel (Mann et al. 2014; Heurich et al. 2010). Importantly, the lead levels in dental enamel decrease from the outermost superficial layer to the inner layer of dental enamel, and applying CLSM, which allowed the incremental line visualization in human teeth (Renz et al. 1997; Dean and Scandrett 1996), these data led us to conduct the present DELL study in order to verify possible occurrence of extrinsic contamination and the effect of biopsy geometry and depth for DELL once these features are crucial to a reliable evaluation of lead concentration in the dental enamel.

circular perforation was placed on the labial surface of the tooth, delineating a window for the biopsy site. This window was etched according to the following procedure: 10 μL of 1.6 mol/L HCl in 70% (v/v) glycerol was applied to the area for 35 s (Brudevold et al. 1975). The biopsy solution was then transferred to a centrifuge tube (Axygen Scientific, Inc., Union City, CA) containing 200 μL Millipore Alpha Q water. The surface was then rinsed twice for 10 s with 10 μL Millipore Alpha Q water, which was then transferred to a centrifuge tube, making a final volume of 230 μL. Two biopsies were made from each bovine tooth before and after lead exposure to proceed the experiments described in “In vitro evaluation of the post-eruptive lead incorporation onto the superficial dental enamel” section. The human tooth biopsied for the Raman microscopy study had a smaller biopsy area aiming to preserve the highest amount of dental enamel. The biopsy employed the adhesive tape with a 2-mm-diameter circular perforation placed on the labial surface of the tooth to limit the biopsy site. This window was etched using 5 μL of the same acidulated glycerol solution applied to the area for 35 s. A shorter exposure time does not promote a homogenous eroded area, which is needed to estimate the real biopsy depth (Olympio et al. 2010a, b, c). In vitro evaluation of the post-eruptive lead incorporation onto the superficial dental enamel

Materials and methods The Institutional Review Board of Bauru Dental School of the University of São Paulo reviewed and approved the protocol adopted to carry out the present work (Protocol 26/2010).

Experimental design Preparation of bovine and human teeth Recently extracted sound bovine incisors were stored in sodium azide 0.2% in a refrigerator at 4 °C and submitted to visual analysis for detection of possible stains and cracks. If visible, they were excluded from the set of samples. The cleaning of the teeth consisted of removing any residual and gingival tissue adhered to the surface with the aid of a periodontal curette (Duflex. Ind. Bras.). Ninety sound bovine incisors were selected for measurements. One sound permanent tooth extracted because of periodontal disease from a volunteer and donated to this study was cleaned and stored by the same procedure. In vitro bovine enamel and human permanent biopsy procedures The bovine teeth were previously cleaned with a rotary brush and pumice slurry, washed, and dried. An adhesive tape (Magic Tape, 810 Scotch 3M) containing a 4-mm-diameter

The 90 sound bovine incisor crowns underwent two biopsies in each tooth, before and after lead exposure. Six 15-tooth groups differed by exposure time (1 or 30 min) and lead concentrations (A. 0 mg/L—placebo, B. 0.01 mg/L—standard for drinking water, or C. 0.06 mg/L—concentration found in contaminated groundwater) (Fig. 1). These concentrations were chosen based on Regulation 518, March 25, 2004 (Brazil 2004), which establishes the procedures and responsibilities for the control and surveillance of water quality for human consumption and potability standards and other parameters, beyond the reports on analyses of groundwater contaminated area by the Brazilian Environmental Agency (CETESB), in 2002. All glasswares were previously decontaminated to avoid external lead, as described in the next section (Chemical analysis). Chemical analysis Lead and phosphorus determinations To avoid contamination, all polypropylene and glass flasks used in the preparation and storage of solutions were cleaned with a detergent solution, rinsed in 10% (v/v) HNO3 (Emsure®, Merck, Darmstadt, Germany) overnight, rinsed with deionized water 18.2 MΩ cm at 25 °C, dried, and stored in a closed polypropylene container. High-purity water produced by a Milli-Q


Fig. 1 Experimental design for the in vitro evaluation of the post-eruptive lead incorporation onto the superficial permanent dental enamel

water purification system (Millipore, Bedford, MA) and doubly distillated chloride acid (Emsure®, Merck) were used throughout the experiments. All the reagents were of analytical grade. The sample manipulations were conducted in a class 100 laminar flow bench (Veco, Campinas, SP, Brazil) to avoid airborne contamination. The contamination processes were monitored through blank solution analysis. Lead was determined in a graphite furnace atomic absorption spectrometer (GF AAS) (Perkin-Elmer, Shelton, CT, USA), model SIMAA-6000, equipped with a longitudinal Zeeman-effect background correction system, Echelle optical arrangement, solid-state detector, end-capped transverse-heated graphite tubes (EC-THGA) with integrated pyrolytically coated platforms (Perkin-Elmer, Norwalk, CT), and a hollow cathode lamp. Solutions were delivered into the graphite tube by means of an AS-72 autosampler. Aliquots of 10 μL of samples or analytical solutions were introduced into the graphite furnace with 10 μL of a chemical modifier (5 μg Pd and 3 μL Mg). This chemical modifier was prepared using Suprapur solutions of 10 g/L Pd in 15% (v/v) HNO3 and 10 g/ L Mg, prepared from Pd(NO3)2 and Mg(NO3)2 inorganic salts (Merck, Darmstadt, Germany), respectively. The calibration curve (2–40 μg/L) was prepared in the autosampler vials using an analytical-grade Titrisol® solution containing 1000 mg/L of lead (Pb(NO 3 ) 2 ) from Merk diluted in 0.06 mol/L HCl in 7% (v/v) glycerol. The samples were analyzed without previous treatment. Samples with higher concentrations of Pb (> 40 μg/L) were diluted in 0.06 mol/L HCl in 7% (v/v) glycerol (two to five times). The analytical signals of each sample were recorded in triplicate. The accuracy of the analytical procedure was checked by analyzing a standard reference material of animal bone (H-5, IAEA from Austria). Phosphorus was determined using an inductively coupled plasm optical emission spectrometer (ICP-OES) (Thermo Fisher Scientific, Cambridge, England), model iCAP 6300, equipped with a duo view plasma torch. The calibration curve was obtained using an analytical-grade Titrisol® solution containing 1000 mg/L of phosphorus (H3PO4) from Merck after

appropriate dilution in water. After lead determination in the samples (known volume), the resulting solutions were diluted up to 1.75 mL with deionized water. The analytical range was 0.5–10 mg/L. The analytical signals of each sample were recorded in triplicate. The limits of detection (LoD) were estimated considering the variability of ten consecutive measurements of blank solution, according to 3 sblk/b (sblk = standard deviation of the blank and b = calibration curve slope) and the limits of quantification (LoQ) were estimated as 3.33 × LoD. In the special case of ICP-OES, the background equivalent concentration (BEC) was also considered, which is defined as the concentration of analyte that produces a signal equal to the background emission intensity at the spectral line of interest. The results in mass over mass were calculated, considering 20 μg of tooth enamel. Calculation of biopsy depth by a chemical method The biopsy depth calculation described by Cleymaet et al. (1991c) is based on the assumption that 17.4% of the dental enamel weight corresponds to phosphorus and that the mean density of the dental enamel is 2.95 g/cm3. Biopsy depths were estimated according to the following equation: Biopsy depth ¼ enamel mass ðμgÞ = 2:95 biopsy area mm2 :

Measurement of the biopsy depth by confocal Raman depth profilometry One sound human permanent incisor, extracted due to periodontal disease, was biopsied as detailed in “In vitro bovine enamel and human permanent biopsy procedures” section. Confocal Raman measurements were acquired with a microscope WITec® alpha 300R, with a frequency-doubled Nd:YAG laser (λ = 532 nm) coupled through a single-


channel fiber. The scattered light was captured through a multi-channel fiber coupled to a UHTS 300 spectrophotometer equipped with an electron multiplier-CCD detector. Crosssectional scans (in an xz plane) of a tooth surface positioned horizontally (xy plane) were obtained with a piezo-driven xyz feedback-controlled scan stage and a spectrum was collected at every pixel. All measurements were made with a laser power of 40 mW cm−2 (measured at laser output), a 100×/0.8NA Nikon® objective, and a 25-μm pinhole. The Raman spectrum of dental enamel is basically composed by hydroxyapatite peaks (Fig. 2). The integral peak intensities of a 959-cm−1 hydroxyapatite phosphate peak were corrected from the background spectra using the WITec® Project 2.06 software to construct the Raman depth profile images. The scan geometry (with x depth) was 100 μm × 20 μm and used 200 × 200 pixels to ensure a high vertical image resolution with a step of 0.1 μm. The integration time for each spectrum was 0.045 s and the entire scan took c.a. half an hour (40,000 spectra).

Statistical analysis Intakes or losses of lead in SDE by the tooth groups before and after lead exposure were compared by ANOVA (p < 0.05) using the STATA statistical software version 9.1.

Results and discussion Most studies evaluating dental enamel lead levels in vitro used flattened and polished bovine enamel blocks to perform the experiments. This procedure removes the enamel in deep and impairs the simulation of the real conditions under which the enamel biopsy is carried out. In the present study, we intended to simulate the real conditions without flattening and polishing the dental enamel specimens. The data presented here show that the superficial dental enamel does not incorporate

exogenous lead in vitro once there was no difference in dental enamel lead levels among the analyzed groups (p = 0.964). Means and standard deviations values for the biopsies 1 and 2 and the differences between them are presented in the Table 1. Some lead concentrations were found below the detection limits of the method. The LoD and LoQ in tooth enamel for lead determination by a GF AAS were 12 and 40 μg/g, respectively, and those for phosphorus determination by an ICP-OES were 130 and 433 μg/g, respectively. The differences between the enamel lead levels found before and after the lead exposure for diverse exposure times were calculated and the mean was − 34.58 ± 116.67 μg/g, showing that sometimes the lead concentration found in the first biopsy was higher than lead concentration found after the experiment, in the second biopsy. The median for the first biopsy was 104.64 μg/g and that for the second biopsy was 82.62 μg/g. This result corroborates the findings by Budd et al. (1998) that the lead levels in both current and archeological samples of human teeth exhibit similar patterns expected from lead levels in immediate surface enamel tissues of biogenic origin that results from the natural process of tissue generation. Molina et al. concluded that if lead is present in the oral environment, it might deposit in the enamel during the caries process in primary teeth. However, these authors used very high lead concentrations in their study. The lead concentration found in contaminated groundwater was 0.06 mg/L and the authors used 0.3 mg/L (Molina et al. 2011). Observing the results we got using Raman microscopy, the biopsy depth was found to be around 1.5 μm in the real biopsy conditions. This very small depth cannot be compared with 50-, 100-, 150-, and 200-μm depths measured in the Molina et al.’s study. Moreover, it is needed to keep in mind that the polishing of the enamel removes around 200 μm even before the experiment is performed (Levy et al. 2014; Souza et al. 2015). In a previous study, Olympio et al. (2010a, b, c) claimed that the surface dental enamel provides a reliable biomarker for lead exposure as attested by the fact that biopsy depth in homologous teeth from the same individual examined by a single methodology yielded very close dental enamel lead levels. The use of different procedures rendered significantly distinct biopsy depths in homologous teeth. Biopsy depths depend both on the pH of the extraction solution and on its residence time on the surface dental enamel. When the surface dental enamel is exposed to acid for shorter periods, the layer removed is Table 1 Means and standard deviations for the biopsies 1 and 2 and their differences Variable

Fig. 2 Raman spectrum of dental enamel. The most intense peak at 959 cm−1 is assigned to hydroxyapatite phosphate

Number Mean

Biopsy 1 89 Biopsy 2 89 Differences (biopsy 1 and 2) 89

Standard deviation

121.7427 129.4587 87.1580 80.6597 − 34.5847 116.6758

Environ Sci Pollut Res Fig. 3 Tooth photographs from different angles (a–c) and surface micrographs taken with 20× and 100× objectives (d and e respectively) at the attacked region. The circle in red (front view) and red semicircle depicting a front tooth attacked region view by the acid

expected to be shallower. Olympio et al. (2010a, b, c) showed that when the biopsy depth values were determined using protocols I (4 mm of diameter, application of 10 μL 1.6 mol/L HCl in 70% (v/v) glycerol to the area for 35 s) and II (1.6 mm of diameter, application of 5 μL 1.6 mol/L HCl in 70% (v/v) glycerol to the area for 20 s), by the cylinder formula, the layer removed after 20-s exposure to acid was shallower. Accordingly, the individual dental enamel lead level values were higher in the Protocol II compared to Protocol I. This finding is biologically unexpected as deeper biopsies contain less lead than superficial ones, since the surface enamel has a steep lead gradient (Brudevold et al. 1977). Hence, the Protocol II yielded not reliable results for dental enamel lead levels because the biopsy depth is critical for the accuracy of this kind of measurement. A profilometry test was then conducted on bovine enamel crowns to elucidate these divergent results. It was

found that when surface dental enamel was exposed to acid for a shorter time, a more superficial layer was removed. This explains the observation that shorter exposure time results in higher dental enamel lead levels. In the present study, Raman microscopy was used to elucidate the real geometric shape of the eroded area in dental enamel biopsies. Photographs of the tooth used in this study are shown in Fig. 3. In the top, the attacked region (around 2-mm diameter) is indicated by a red circle (Fig. 3(a)). The lateral view indicates that the acid attack removes only a little fraction of dental enamel as the apparent hole created is in the micrometer scale (Fig. 3(b)). The frontal view of tooth also shows that the surface has both a flattened part and a cylindrical part (Fig. 3(c)). Therefore, one can conclude that the attacked region is irregular and the extracted enamel volume resembles approximately a spherical cap (a hemisphere) or metaphorically an “orange cover portion.”

Fig. 4 Sketch of the tooth in 3D (a) illustrating the eroded region (red) and the cross section scanned with the confocal Raman microscope. Surface profile extracted from Raman mapping of the 959-cm−1 hydroxyapatite phosphate peak (b), where the black line defines the

surface of the tooth (gray) and the eroded region is the geometrical area in red. Image analysis of profile in Image J software let us to estimate the average height of approximately 0.83 μm


Fig. 5 Raman spectra of dental enamel at different plane levels (a) being the most intense just at the surface (red spectrum) and less intense out of the surface (magenta spectrum). Original image of Raman mapping of the

959-cm−1 peak (b) and silhouette mask (c) of the same mapping with calibrated height bars (pixel aspect ratio of 0.0662 μm/pixel) used to calculate the average depth

Besides the geometrical alterations, the edge near the flattened region was chosen in order to estimate the roughness of attacked area and the depth of hole created. Figure 3(d) shows a dental enamel region as a bright area and the attacked region appears darker. A closer inspection of the attacked region reveals micrometric holes on the surface that referred to here as “microerosions” (Fig. 3(e)). At this point, the depth of the entire attacked region can be actually defined by the average height of microerosions, and the cylinder was viewed with a high diameter-to-depth aspect ratio. The tooth was mounted on a scan stage and a highly focused laser beam used to acquire the Raman spectrum of a micrometric point (Fig. 4(a)). The scanning principle is based on moving the sample and taking the spectra point by point until they construct an entire image. It can create a height profile of the enamel surface since the Raman signal is at maximum just at the surface of the sample. Figure 4(b) shows the height profile of the constructed enamel surface using the 959-cm−1 Raman peak (Fig. 5).

The profile was strategically traced at the edge of the attacked region for the untreated surface can act as the zero plain level; therefore, the average depth of the erosions could be calculated as approximately 0.83 μm and the maximum peak to valley depth of the microerosions as 1.5 μm (see the supporting information for more details, Fig. 5). These data support the chemical method to estimate the biopsy depth. The Raman spectra of the attacked and not attacked regions by acid were equal, which may indicate that the composition of the tooth after the acid attack has no further change than just a change in roughness. In Fig. 5, it could also visualize that the acid treatment removes only a minimal fraction of the surface compared to its entire extent. The difference in the thickness of hydroxyapatite between these regions is due only to a physical effect that the penetration power of the Raman signal is favored in regions with a surface smoother as it is in the non-attacked region. The maximum possible confocal determination depth was 40 μm but this is only the


maximum geometry that the piezoelectric scanner allowed. The Raman signal is sensitive almost exclusively to the surface and it is only possible to penetrate surfaces since they are surfaces of low roughness. It is not possible to measure lead by the Raman spectrum because it has no peaks in the region of the studied spectrum that was 200 to 3600 cm−1. A limitation of our study was that we have analyzed one tooth by Raman microscopy. As Raman microscopy is not mainly idealized to perform profilometric measures, consequently, the demanded time to do this is very long. However, it was an innovation that we have gotten the depth measures by this technique. An additional limitation is that we have not proceeded with pH-cycling regimen simulating caries developing. Our study only can be considered in environmental oral that excluded the cariogenic challenge, which is frequent depending on the bacterial infection, food, host, and time (Fejerskov et al. 2003). In this way, it would be useful to test our hypothesis in a pH-cycling protocol.

Conclusions In vivo biopsy depths have been commonly calculated by the cylinder height formula. The measurements of lead concentrations in tooth enamel as a function of the biopsy depths using chemical and physical methods converge to values similar to those published elsewhere that used the conventional cylinder approach. Importantly, SDE of erupted permanent teeth does not seem to be susceptible to environmental lead intake, which justifies its use as a remote lead exposure biomarker. Accordingly, a statistically significant association between lead exposure and antisocial behavior of adolescents has been reproduced by longitudinal and cross-sectional studies independently upon the biological samples employed—blood, bones, or tooth enamel (Dietrich et al. 2001; Needleman et al. 2002; Wright et al. 2008, 2009; Olympio et al. 2009, 2010a, b, c; Arbuckle et al. 2016)—and the cultural environment of the population cohorts examined. Financial support Petrobrás, FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo—grants 01/09641-1 and 06/56530-4); CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico); and the Projeto Milênio Redoxoma. KPKO was recipient of a fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

Compliance with ethical standards

The Institutional Review Board of Bauru Dental School of the University of São Paulo reviewed and approved the protocol adopted to carry out the present work (Protocol 26/2010). Conflict of interest The authors declare that they have no conflict of interest.

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Affiliations Kelly Polido Kaneshiro Olympio 1 & Manuel Fernando Gonzalez Huila 2 & Cristiane de Almeida Baldini Cardoso 3 & Ana Paula Sacone da Silva Ferreira 1 & Adrielly Garcia Ortiz 4 & Henrique Eisi Toma 2 & Ricardo Henrique Alves da Silva 4 & Maciel Santos Luz 5 & Maria Regina Alves Cardoso 6 & Gislayne Aparecida Rodrigues Kelmer 2 & Pedro Vitoriano de Oliveira 2 & Etelvino José Henriques Bechara 2 & Wanda Maria Risso Günhter 1 & Marília Afonso Rabelo Buzalaf 7 1

Departamento de Saúde Ambiental, Faculdade de Saúde Pública, Universidade de São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, São Paulo, SP CEP 01246-904, Brazil

2

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, Cidade Universitária, Butantã, São Paulo, SP CEP 05508-000, Brazil

3

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Programa de Pós-Graduação em Odontologia, área de concentração Odontopediatria, Universidade Cruzeiro do Sul (UNICSUL), Rua Galvão Bueno, 868, Liberdade, São Paulo, SP CEP 01506-000, Brazil Departamento de Estomatologia, Saúde Coletiva e Odontologia Legal, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Av do Café, s/n, Ribeirão Preto, SP CEP 14040-904, Brazil

5

Centro de Tecnologia em Metalurgia e Materiais, Laboratório de Processos Metalúrgicos (CTMM/LPM), Instituto de Pesquisas Tecnológicas do Estado de São Paulo (IPT), Av. Prof. Almeida Prado, 532, Cidade Universitária, Butantã, São Paulo, SP CEP 05508-901, Brazil

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Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, São Paulo, SP CEP 01246-904, Brazil

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Departamento de Ciências Biológicas, Faculdade de Odontologia de Bauru, Universidade de São Paulo, Alameda Otávio Pinheiro Brisolla, 9-75, Vila Nova Cidade Universitária, Bauru, SP CEP 17012-901, Brazil