A new mixed model of periodontitis-induced preeclampsia: A pilot study.

OBJECTIVES/BACKGROUND: Recent studies have shown that periodontal disease is strongly related to gestational complications such as preeclampsia (PE). PE is responsible for 42% of maternal deaths worldwide and kills approximately 76 000 women a year. In addition, children born under PE conditions are at increased risk of hospitalization due to metabolic disorders, epilepsy, and other complications. Numerous reviews and clinical studies on PE have been published, but the mechanisms underlying the relationship between periodontal disease and PE and the way periodontopathogens alter vascular response in pregnant women remain unclear. METHODS: This study aims to verify whether periodontal disease induces PE by using the association of two periodontitis (PD) models: ligature and oral Porphyromonas gingivalis (P. gingivalis) W83 inoculation in Wistar rats. At gestational day 5, the ligature was placed on each mandibular first molar, which was followed by daily oral P. gingivalis inoculation for 15 days. At gestational day 19, urine was collected, and invasive arterial pressure was measured. The animals were euthanized, and plasma and tissues were collected. RESULTS: After 15 days of the association of ligature and P. gingivalis inoculation, the animals presented the characteristic symptoms of PE: altered blood pressure, proteinuria, and change in litter size (number of pups) and pup weight when compared to the control group (p < .005). The PE animals also presented greater bone porosity, trabecular separation, and reduced bone volume in the hemimandibles, as well as altered inflammatory response. The level of cytokine IL-6 was higher in the PE group than in the control group (p < .005). CONCLUSION: The association of two PD models effectively induced PE. To our knowledge, this is the first study on the oral use of P. gingivalis for PE induction. Our results support the importance of PD as a possible cause for PE development, opening an important new avenue to study cause and consequence relationships in inflammation and PE due to exposure to periodontal infection.

Sex determination of human remains from peptides in tooth enamel.

The assignment of biological sex to archaeological human skeletons is a fundamental requirement for the reconstruction of the human past. It is conventionally and routinely performed on adults using metric analysis and morphological traits arising from postpubertal sexual dimorphism. A maximum accuracy of ∼95% is possible if both the cranium and os coxae are present and intact, but this is seldom achievable for all skeletons. Furthermore, for infants and juveniles, there are no reliable morphological methods for sex determination without resorting to DNA analysis, which requires good DNA survival and is time-consuming. Consequently, sex determination of juvenile remains is rarely undertaken, and a dependable and expedient method that can correctly assign biological sex to human remains of any age is highly desirable. Here we present a method for sex determination of human remains by means of a minimally destructive surface acid etching of tooth enamel and subsequent identification of sex chromosome-linked isoforms of amelogenin, an enamel-forming protein, by nanoflow liquid chromatography mass spectrometry. Tooth enamel is the hardest tissue in the human body and survives burial exceptionally well, even when the rest of the skeleton or DNA in the organic fraction has decayed. Our method can reliably determine the biological sex of humans of any age using a body tissue that is difficult to cross-contaminate and is most likely to survive. The application of this method will make sex determination of adults and, for the first time, juveniles a reliable and routine activity in future bioarcheological and medico-legal science contexts.

Genes Involved in the Enamel Development Are Associated with Calcium and Phosphorus Level in Saliva.

Saliva components play a crucial role in the integrity of the dental enamel and in caries susceptibility. The saliva characteristics are controlled by many factors, including genetic factors. Therefore, this study aimed to evaluate the association between the genetic variations in genes expressed in enamel development with calcium and phosphorus levels in saliva. We collected 276 unrelated 12-year-old children from private and public schools. Saliva was collected for DNA extraction from oral cells and for measurement of calcium and phosphorus. Inductively coupled plasma-mass spectrometry determined calcium and phosphorus levels in whole saliva. Fifteen genetic variations in 9 genes were analyzed. The genotype was determined by real-time polymerase chain reactions. Data were analyzed using Plink with an alpha of 5%. Genetic variations in AMELX, AMNB and ESRRB were associated with the calcium level in saliva (p < 0.05). A borderline association was observed in ENAM allele distribution shown with phosphate level in saliva (p = 0.049). In conclusion, our results are the first to report that genetic variations contribute to calcium and phosphorus levels in saliva.

Lead concentrations in whole blood, serum, saliva and house dust in samples collected at two time points (12 months apart) in Santo Amaro, BA, Brazil.

UNLABELLED: Whole Blood Lead Level (BLL) is the main marker used to verify lead contamination. The present study explores how BLL is associated with lead concentrations in serum, saliva and house dust. Samples were collected twice from Santo Amaro, BA, Brazil, a region that was contaminated by a lead smelter in the past; a time interval of 12 months was allowed between the two collections. It is noteworthy that the following measures have recently been taken to diminish exposure of the population to lead: streets have been paved with asphalt, and educational campaigns have been launched to reduce exposure to contaminated dust. RESULTS: Compared with the first time point, all the samples collected at the second time point contained lower lead concentration (p<0.05), which suggested that the adopted measures effectively reduced exposure of the population to lead present in contaminated soil and dust. Statistically significant correlations only existed between lead in blood collected in the first year and lead in blood collected in the second year (Spearman's r=0.55; p<0.0001; n=62), and lead in house dust collected in the first year and lead in house dust collected in the second year (Spearman's r=0.5; p<0.0001; n=59). CONCLUSIONS: Results support the validity of lead determination in blood and in house dust to assess lead exposure over time. However, lead in blood and lead in dust did not correlate with lead in serum or lead in saliva.

Arterial hypertension perpetuates alveolar bone loss.

Few studies have focused on the impact of hypertension on the progression of periodontitis (PD). The purpose of this study was to evaluate whether hypertension affects PD by enhancing bone loss even after the stimulus for PD induction is removed. Ligature-induced PD was created on the first mandibular molars of spontaneously hypertensive rats (SHR) and normotensive rats (Wistar Kyoto-WKY). The animals were assigned to non-ligated controls (C) and PD groups: WKY-C, WKY-PD, SHR-C, and SHR-PD. After 10 days, five animals of each group were killed and the ligatures of the other animals were removed. On the 21st day (11 days without PD induced), the remaining animals were killed. The jaws were defleshed and the amount of bone loss was measured. After 10 days, the PD groups showed more bone loss than its controls (P < .05); SHR-PD = 0.72 ± 0.05 mm, SHR-C = 0.39 ± 0.04 mm, WKY-PD = 0.75 ± 0.04 mm, and WKY-C = 0.56 ± 0.04 mm. The cumulative bone loss on day 21 (0.94 ± 0.13 mm) was significantly worse than on day 10 only in SHR-PD group (P < .05). The final bone loss differences between PD and C groups accounted for 102% (SHR) and 26% (WKY) increase in comparison with the initial control levels. Hypertension is associated with progressive alveolar bone loss even when the stimulus for PD induction is removed and it may be speculated that host condition perpetuates alveolar bone loss.

Matrix Metalloproteinase 2 as a Pharmacological Target in Heart Failure.

Heart failure (HF) is an acute or chronic clinical syndrome that results in a decrease in cardiac output and an increase in intracardiac pressure at rest or upon exertion. The pathophysiology of HF is heterogeneous and results from an initial harmful event in the heart that promotes neurohormonal changes such as autonomic dysfunction and activation of the renin-angiotensin-aldosterone system, endothelial dysfunction, and inflammation. Cardiac remodeling occurs, which is associated with degradation and disorganized synthesis of extracellular matrix (ECM) components that are controlled by ECM metalloproteinases (MMPs). MMP-2 is part of this group of proteases, which are classified as gelatinases and are constituents of the heart. MMP-2 is considered a biomarker of patients with HF with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF). The role of MMP-2 in the development of cardiac injury and dysfunction has clearly been demonstrated in animal models of cardiac ischemia, transgenic models that overexpress MMP-2, and knockout models for this protease. New research to minimize cardiac structural and functional alterations using non-selective and selective inhibitors for MMP-2 demonstrates that this protease could be used as a possible pharmacological target in the treatment of HF.

BmooMPα-I, a Metalloproteinase Isolated from Bothrops moojeni Venom, Reduces Blood Pressure, Reverses Left Ventricular Remodeling and Improves Cardiac Electrical Conduction in Rats with Renovascular Hypertension.

BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.

Structure and Chemical Composition of ca. 10-Million-Year-Old (Late Miocene of Western Amazon) and Present-Day Teeth of Related Species.

Molecular information has been gathered from fossilized dental enamel, the best-preserved tissue of vertebrates. However, the association of morphological features with the possible mineral and organic information of this tissue is still poorly understood in the context of the emerging area of paleoproteomics. This study aims to compare the morphological features and chemical composition of dental enamel of extinct and extant terrestrial vertebrates of Crocodylia: Purussaurus sp. (extinct) and Melanosuchus niger (extant), and Rodentia: Neoepiblema sp. (extinct) and Hydrochoerus hydrochaeris (extant). To obtain structural and chemical data, superficial and internal enamel were analyzed by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (SEM-EDS). Organic, mineral, and water content were obtained using polarizing microscopy and microradiography on ground sections of four teeth, resulting in a higher organic volume than previously expected (up to 49%). It is observed that both modern and fossil tooth enamel exhibit the same major constituents: 36.7% Ca, 17.2% P, and 41% O, characteristic of hydroxyapatite. Additionally, 27 other elements were measured from superficial enamel by inductively coupled mass spectrometry (ICP-MS). Zinc was the most abundant microelement detected, followed by Pb, Fe, Mg, and Al. Morphological features observed include enamel rods in the rodent teeth, while incremental lines and semiprismatic enamel were observed in the alligator species. The fossil enamel was in an excellent state for microscopic analyses. Results show that all major dental enamel's physical, chemical, and morphological features are present both in extant and extinct fossil tooth enamel (>8.5 Ma) in both taxa.

Ancient enamel peptides recovered from the South American Pleistocene species Notiomastodon platensis and Myocastor cf. coypus.

We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. SIGNIFICANCE: This study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.

Matrix metalloproteinase inhibition improves cardiac dysfunction and remodeling in 2-kidney, 1-clip hypertension.

BACKGROUND: Enhanced cardiac matrix metalloproteinase activity (MMPs) has been associated with ventricular remodeling and cardiac dysfunction. It is unknown whether MMPs contribute to systolic/diastolic dysfunction and compensatory remodeling in 2-kidney, 1-clip (2K1C) hypertensive rats. To test this hypothesis, we used 2K1C rats after 2 weeks of surgery treated or not with a nonspecific inhibitor of MMPs (doxycycline). METHODS AND RESULTS: We found that blood pressure and +/- dP/dt increased in 2K1C rats compared with sham groups, and these parameters were attenuated by doxycycline treatment (P < .05). Doxycycline also reversed cardiac hypertrophy observed in 2K1C rats (P < .05). Hypertensive rats showed increased MMP-2 levels in zymograms and in the tissue by immunofluorescence (P < .05) compared with sham groups. Increased total gelatinolytic activity was observed in untreated 2K1C rats when compared with sham groups (P < .05). Doxycycline decreased total gelatinolytic activity in 2K1C rats to control levels (P < .05). CONCLUSION: An imbalance in gelatinolytic activity, with increased MMP-2 levels and activity underlies the development of morphological and functional alterations found in the compensatory hypertrophy observed in 2K1C hearts. Because function and structure were restored by doxycycline, the inhibition of MMPs or their modulation may provide beneficial effects for therapeutic intervention in cardiac hypertrophy.

Variation in mineral, organic, and water volumes at the neonatal line and in pre- and postnatal enamel.

OBJETIVES: The neonatal line (NNL) in enamel is hypomineralized, but quantitative data on the enamel component volumes of the NNL are lacking. This study aimed at quantifying the variation in the mineral, organic, and water volumes at the NNL and in pre- and postnatal enamel. MATERIALS AND METHODS: In buccal enamel longitudinal ground sections of exfoliated primary incisors (upper and lower; n = 17), the enamel component volumes were quantified at five histological sites (located at 40 µm intervals along a transversal line): the NNL, two sites in prenatal enamel, and two sites in postnatal enamel. Mineral volume was quantified using microradiography, and non-mineral volumes were quantified using polarizing microscopy. RESULTS: Differences in component volumes between the NNL and pre- and postnatal enamel had high effect sizes (Hedge's G ranging from 0.89, for the water volume, to 1.88, for the mineral volume; power > 90 %). The distance from the NNL correlated with the normalized component volume: r = 0.459, 95 % CI = 0.274/0.612 (mineral); r = -0.504; 95 % CI= -0.328/-0.647 (organic), and r = -0.294; 95 % CI= -0.087/-0.476 (water). Approaching the NNL from postnatal enamel, the percentage differences in component volumes were: -1.93 to -3.22 % for the mineral volume, +21.26 to +35.42 % for the organic volume, and +3.86 to +6.03 % for the water volume. Towards postnatal enamel, the percentage differences had the opposite trend. CONCLUSIONS: The enamel NNL is slightly hypomineralized with an increased organic volume one order of magnitude higher than the percentage differences in both mineral and water volumes.

Calcium and Phosphorus Levels in Saliva are Influenced by Genetic Polymorphisms in Estrogen Receptor Alpha and Microrna17.

Homeostasis between salivary calcium and phosphorus is important for maintaining oral health. The aim of this study was to evaluate if polymorphisms in ESR1 (Estrogen Receptor Alpha), ESR2 (Estrogen Receptor Beta) and miRNA17 (microRNA17) are associated with calcium and phosphorus levels in saliva. Saliva from 276 12-year-old children were collected by masticatory stimulation and calcium and phosphorus levels were determined by Mass Spectrometry. Genomic DNA was extracted from remaining saliva and genetic polymorphisms in ESR1 (rs12154178, rs1884051, rs9340799 and rs2234693), in ESR2 (rs4986938 and rs1256049) and in miRNA17 (rs4284505) were genotyped using TaqMan chemistry and a real-time PCR equipment. Statistical differences in genotype and allele distributions between 'low' and 'high' calcium and phosphorus levels were determined using chi-square or Fisher´s exact tests. The analysis was also adjusted by sex (alpha of 5%). ESR1 rs9340799 had the less common genotype associated with higher calcium levels (p=0.03). The less common allele of ESR1 rs1884051 was associated with lower phosphorus levels (p=0.005) and there was an excess of heterozygotes for miRNA17 rs4284505 among individuals with lower calcium levels (p=0.002), both adjusted by sex. This study provides evidence that genetic polymorphisms in ESR1 and miRNA17 are involved in determining salivary calcium and phosphorus levels.

Lead in saliva from lead-exposed and unexposed children.

INTRODUCTION: Whole blood is used for diagnosis of lead exposure. A non-invasive method to obtain samples for the biomonitoring of lead contamination has become a necessity. This study 1) compares the lead content in whole saliva samples (Pb-saliva) of children from a city with no reported lead contamination (Ribeirão Preto, São Paulo State, Brazil) and children of a region notoriously contaminated with lead (Bauru, São Paulo State, Brazil), and 2) correlates Pb-saliva with the lead content in the enamel microbiopsy samples (Pb-enamel) in the case of these two populations. METHODS: From a population of our previous study that had included 247 children (4- to 6-year-old) from Ribeirão Preto, and 26 children from Bauru, Pb-saliva was analyzed in 125 children from Ribeirão Preto and 19 children from Bauru by inductively coupled plasma mass spectrometry (ICPMS). To correlate Pb-saliva with Pb-enamel, we used Pb-enamel data obtained in our previous study. The Mann-Whitney test was employed to compare the Pb-saliva data of the two cities. Pb-saliva and Pb-enamel values were then Log10 transformed to normalize data, and Pb-saliva and Pb-enamel were correlated using Pearson's correlation coefficient. RESULTS: Median Pb-saliva from the Ribeirão Preto population (1.64 microg/L) and the Bauru population (5.85 microg/L) were statistically different (p<0.0001). Pearson's correlation coefficient for Log10 Pb-saliva versus Log10 Pb-enamel was 0.15 (p=0.08) for Ribeirão Preto and 0.38 (p=0.11) for Bauru. CONCLUSIONS: A clear relationship between Pb-saliva and environmental contamination by lead is shown. Further studies on Pb-saliva should be undertaken to elucidate the usefulness of saliva as a biomarker of lead exposure, particularly in children.

A plateau detected in lead accumulation in subsurface deciduous enamel from individuals exposed to lead may be useful to identify children and regions exposed to higher levels of lead.

In a previous study, we showed 4 times more lead in surface deciduous enamel (1.9-5.9 microm) of a notoriously contaminated area (Bauru, São Paulo State, Brazil) in comparison to samples from a region with no lead contamination described (Ribeirão Preto, São Paulo State, Brazil). The samples from the more superficial enamel (1.9-3.18 microm) showed higher amounts of lead and the highest variability, while in the subsurface enamel (3.18-5.9 microm) a plateau in lead content was detected in children living in the contaminated environment (around 600 microg/g). Here we expand our previous study, and use only samples obtained from subsurface enamel (Ribeirão Preto, n=186; Bauru, n=20). We tried to distinguish regions with more children with lead above the threshold of 600 microg/g of lead in enamel. We tested whether differences in the percentage of children with ''high" lead (600 microg/g) could be observed among the different Kindergartens studied in Ribeirão Preto. We also tested whether these results were different from the ones provided by conventional comparison of the data. Ribeirão Preto showed almost 4 times less lead than Bauru (p<0.0001), and a statistically significant difference was found only in Ribeirão Preto between Kindergarten 2 and 5 (p<0.01). Twelve percent of the children from Ribeirão Preto had "high" lead, while 55% of the children from Bauru did so. However, when we looked at the percentages of children with "high" lead in each Kindergarten, and compared them, a whole new picture emerged, in which we could see children with "high" lead concentrated mainly in 3 Kindergartens from Ribeirão Preto, with Kindergarten 5 with 33% of the children with "high" lead, being statistically different from all Kindergartens, except 4 and 6. The threshold of 600 microg/g of lead in subsurface enamel was tentatively settled here after the plateau seen in exposed children, and enabled us to identify locations with more children exposed to a higher amount of lead.

Quantitative study of the proportion of the pore volume of human fluorotic enamel filled by resin infiltrant.

AIM: Capillarity theory predicts that the pore volume infiltrated by a liquid in a body with tubular capillaries is directly proportional to the capillary radius. The expected volume available for infiltration is the loosely bound water volume, which can be related to the capillary radii. We tested the hypothesis that the proportion of the pore volume infiltrated by resin infiltrant (Vratioresin) is correlated and agrees with the proportion of the pore volume with loosely bound water ( [Formula: see text] ). DESIGN: Seven human fluorotic third molars (4 unerupted and 3 erupted; TF scores 4 to 7; fluoride content of inner coronal dentin ranged from 143 to 934µg Fluoride/g) were prepared and resin infiltration was performed during 10min in fluorotic enamel ground sections. Penetration depths were measured (polarizing microscopy and CLSM) and mineral volume and non-mineral volumes were measured at histological points (n=92) along transversal lines traced from the enamel surface to the enamel-dentin junction. RESULTS: No well-mineralized surface layer was found. Infiltration depths ranged from 250µm to 900µm. Vratioresin ranged from 1.8 to 17.7% (mean of 10.13%±4.1%), was lower than [Formula: see text] (p<0.00001 Hedge's g=1.51, 95% CI: 1.18/1.83), and correlated positively with [Formula: see text] (R=0.684; 95% CI: 0.557/0.780) and negatively with the air volume remained after infiltration (R=-0.79; 95% CI: -0.698/-0.780). [Formula: see text] exceeded Vratioresin in 5% (1/4 of [Formula: see text] ) on average. CONCLUSION: [Formula: see text] and Vratioresin correlated well, but lacked good agreement. Organic matter, firmly bound water and air remained in enamel pores after resin infiltration.

Dental enamel as biomarker for environmental contaminants in relevant industrialized estuary areas in São Paulo, Brazil.

Heavy metal contamination is a long-standing and very well-known public health problem, and its exposure can cause damage to several organs of human body, especially on the central nervous system of young children and teenagers. The aim of this article is to evaluate lead, cadmium, and manganese contamination in 125 children from 6 to 13 years old living in contaminated areas during the period from 2006 to 2009 (São Vicente, Cubatão Downtown, Bertioga and Cubatão Pilões/Água Fria). This estuary area is the most important example of environmental degradation by chemicals from industrial sources. This is a cross-sectional study through clinical examinations and dental enamel tests. All mothers from these children lived in the area since before the pregnancy. Lead, cadmium, and manganese levels (µg/g) were measured on dental enamel samples through graphite furnace atomic absorption spectrometry, searching for the occurrence of heavy metals. The mean lead concentrations were 139.48 µg/g in Cubatão Pilões/Água Fria, 170.45 µg/g in Cubatão Downtown, 213.52 µg/g in São Vicente, and 151.89 µg/g in Bertioga. The mean cadmium concentrations were 10.83 µg/g in Cubatão Pilões/Água Fria, 12.58 µg/g in Cubatão Downtown, 10.92 µg/g in São Vicente, and 14.57 µg/g in Bertioga. The mean manganese concentrations were 23.49 µg/g in Cubatão Pilões/Água Fria, 30.90 µg/g in Cubatão Downtown, 41.46 µg/g in São Vicente, and 42.00 µg/g in Bertioga. Dental surface enamel may be used as an efficient biomarker of past environmental exposure to lead, manganese, and cadmium which are associated to well-known sources of heavy metal contamination. The results suggest that the evaluated children were exposed to sources of lead, cadmium, and manganese since before their conceptions. Although Bertioga initially was chosen as a control area of this study, it was also was verified to have heavy metal contamination on examined children.

A critical review of biomarkers used for monitoring human exposure to lead: advantages, limitations, and future needs.

Lead concentration in whole blood (BPb) is the primary biomarker used to monitor exposure to this metallic element. The U.S. Centers for Disease Control and Prevention and the World Health Organization define a BPb of 10 microg/dL (0.48 micromol/L) as the threshold of concern in young children. However, recent studies have reported the possibility of adverse health effects, including intellectual impairment in young children, at BPb levels < 10 microg/dL, suggesting that there is no safe level of exposure. It appears impossible to differentiate between low-level chronic Pb exposure and a high-level short Pb exposure based on a single BPb measurement; therefore, serial BPb measurements offer a better estimation of possible health outcomes. The difficulty in assessing the exact nature of Pb exposure is dependent not so much on problems with current analytical methodologies, but rather on the complex toxicokinetics of Pb within various body compartments (i.e., cycling of Pb between bone, blood, and soft tissues). If we are to differentiate more effectively between Pb stored in the body for years and Pb from recent exposure, information on other biomarkers of exposure may be needed. None of the current biomarkers of internal Pb dose have yet been accepted by the scientific community as a reliable substitute for a BPb measurement. This review focuses on the limitations of biomarkers of Pb exposure and the need to improve the accuracy of their measurement. We present here only the traditional analytical protocols in current use, and we attempt to assess the influence of confounding variables on BPb levels. Finally, we discuss the interpretation of BPb data with respect to both external and endogenous Pb exposure, past or recent exposure, as well as the significance of Pb determinations in human specimens including hair, nails, saliva, bone, blood (plasma, whole blood), urine, feces, and exfoliated teeth.

In vivo studies on lead content of deciduous teeth superficial enamel of preschool children.

The amount of lead in the superficial enamel of deciduous teeth from 4- and 5-year-old children was determined by means of an enamel biopsy followed by lead analysis by graphite furnace atomic absorption spectrometry. Children examined in this study (n=329) attended public preschools in the city of Piracicaba, SP, Brazil. The selection of preschools was based on geographical location: some were located in an industrial area of the city, whereas others were located in an area devoid of industries. The objectives of this study were (1) to test whether enamel biopsies were an appropriate method for lead content population screening in young children; (2) to verify the lead content of superficial enamel of preschool children's deciduous teeth and to compare the results obtained from children exposed to different environments; (3) to analyze how biopsy depth correlated with lead content and affected comparisons between groups. An adhesive tape with a central hole (diameter=1.6 mm) was placed on one of the upper central incisors. Five microliters of 1.6 M l(-1) HCl in 70% glycerol (v/v) were applied to this area for 20 s, followed by a rinse with 5-microl ultrapurified water for 10 s. It was shown that the technique was feasible for population studies on young children. The lead concentration in enamel biopsies from the industrial area children (median: 169 ppm Pb) was significantly higher than that from the children living far from industries (118.1 ppm Pb) (P<0.0001). Analysis of the data showed that biopsies deeper than 3.9 microm gave more reliable results for comparisons between groups. To our knowledge, this study is the first to use an enamel microbiopsy to characterize a large sample of young children in vivo for lead monitoring in teeth.

The use of a DNA stabilizer in human dental tissues stored under different temperature conditions and time intervals.

OBJECTIVE: The present study evaluated the use of a reagent to stabilize the DNA extracted from human dental tissues stored under different temperature conditions and time intervals. MATERIAL AND METHODS: A total of 161 teeth were divided into two distinct groups: intact teeth and isolated dental pulp tissue. The samples were stored with or without the product at different time intervals and temperature. After storage, DNA extraction and genomic DNA quantification were performed using real-time PCR; the fragments of the 32 samples that represented each possible condition were analyzed to find the four pre-selected markers in STR analysis. RESULTS: The results of the quantification showed values ranging from 0.01 to 10,246.88 ng/µL of DNA. The statistical difference in the quantity of DNA was observed when the factors related to the time and temperature of storage were analyzed. In relation to the use of the specific reagent, its use was relevant in the group of intact teeth when they were at room temperature for 30 and 180 days. The analysis of the fragments in the 32 selected samples was possible irrespective of the amount of DNA, confirming that the STR analysis using an automated method yields good results. CONCLUSIONS: The use of a specific reagent showed a significant difference in stabilizing DNA in samples of intact human teeth stored at room temperature for 30 and 180 days, while the results showed no justification for using the product under the other conditions tested.

Anatomical variations in primary teeth microelements with known differences in lead content by micro-Synchrotron Radiation X-Ray Fluorescence (µ-SRXRF) - A preliminary study.

Shed teeth have been proposed as trace element biomarkers. This study determined variations in the spatial distribution of Ca, K, Zn, Pb, Mn, Cu, and Sr in four anatomical locations: superficial enamel (SE, 0-10µm), subsuperficial enamel (SSE, 10-30µm), primary dentin (PD), and secondary dentin (SD). Five primary incisors were analyzed by micro Synchrotron Radiation X-Ray Fluorescence (µ-SRXRF). Two teeth had low concentrations of lead in the SE (<250µg/g), while three contained very high lead concentrations in the SE (>2000µg/g). Teeth were sliced, and five spot measurements (20µm beam diameter) were accomplished in each location. The data are shown as absolute values and as the ratio between the different elements and Ca. The distribution of K was close to that of Ca. Zn was the third most abundant element, with the highest levels being found in the SE and SD and low levels detected in the PD. Increasing Sr levels were found progressing from the enamel to the dentin, with the highest levels being found in the SD, a distribution that was unique. Pb, Mn, and Cu exhibited a similar trend, with higher signals for these elements detected in the SE. This study provides preliminary data on the heterogeneous distribution of different elements in the tooth, highlighting the importance of the first 10µm of the SE for determination of some elements, such as Zn, Pb, Mn, and Cu.