Lead Toxicity and Maternal Exposure: Characterisation of Alveolar Bone Changes on Offspring Rats.

Lead poisoning is a global public health concern. Maternal exposure during intrauterine and lactational periods can present a higher susceptibility of harm to the offspring. Thus, pregnant female Wistar rats (Rattus norvegicus) were randomly divided in two experimental groups: control group and Lead group. The animals were exposed to 50 mg/kg of Lead Acetate daily for 42 days (21 days of gestational period + 21 days of lactational period). After the exposure period, the mandibles of the offspring were collected for lead quantification, Raman spectroscopy analysis, micro-CT, morphometric e histochemical analysis. Lead exposure altered the physical-chemical composition of alveolar bone and caused histological damage associated with a reduction in osteocyte density and collagen area fraction, increase in collagen maturity, as well as a reduction in bone volume fraction. An increase in trabecular spaces with anatomical compromise of the vertical dimensions of the bone was observed. Thus, the results suggest that developing alveolar bone is susceptible to toxic effects of lead when organisms are exposed during intrauterine and lactation periods.

Biological action of bleaching agents on tooth structure: A review.

The use of bleaching agents to remove stains is one of the main dental procedures to improve the aesthetics of teeth. This review presents the main agents used for tooth whitening, existing clinical protocols, and the structural changes that may occur through their use. The main bleaching agents consist of hydrogen peroxide and carbamide peroxide, which are used in bleaching techniques for vital teeth. These techniques can be performed in the office by a professional or by the individual in a home en-vironment under professional guidance. Bleaching agents come in a variety of concentrations and there are over-the-counter products available on the market with lower concentrations of hydrogen peroxide. Due to the chemical characteristics of the agents, changes in the organic and inorganic content of the tooth structure can be observed. These changes are related to morphological changes characterized by in-creased permeability and surface roughness, such changes compromise the mechanical resistance of the tooth. Furthermore, bleaching agents can promote molecular changes after reaching the dental pulp, resulting in oxidative stress of pulp cells and the release of pro-inflammatory mediators. Despite the bleaching effectiveness, tooth sensitivity is considered the main side effect of use. Therefore, among the heterogeneity of protocols, those that used the bleaching agent for a prolonged time and in lower con-centrations presented more harmful effects on the tooth structure.

Maternal methylmercury exposure during early-life periods adversely affects mature enamel structure of offspring rats at human exposure levels: a concern for oral health.

Although there are many studies on the health effects of methylmercury (MeHg) toxicity during in utero and early development, little is known about its effects on mineralized tissues present in the oral cavity, such as enamel structure. Therefore, this study evaluated the effects of MeHg exposure on the physico-chemical, ultrastructural and functional properties of mature tooth enamel. Specifically, we studied offspring of mothers exposed to MeHg during the prenatal and postnatal periods which are the developmental stages associated with tooth enamel formation. Female rats were exposed to MeHg at a dose of 40 µg/kg/day for 42 days of pregnancy and lactation. The enamel of offspring was analyzed by (1) Fourier Transform Infrared Spectroscopy and Raman to assess physicochemical composition, (2) Scanning Electron Microscopy for ultrastructural evaluation, (3) Transmitted Polarizing Light Microscopy for analysis of the enamel extracellular matrix, and (4) resistance and hardness were evaluated by microhardness. The results showed that MeHg exposure during this sensitive enamel formation period induced changes in inorganic and organic content and enamel prisms ultrastructure alterations and disturbed the organic extracellular matrix due to a decreased enamel strength. These novel findings establish for the first time that maternal exposure to MeHg pre and postnatal promoted relevant changes in mature enamel of their offspring rats.

A non-destructive X-ray fluorescence method of analysis of formalin fixed-paraffin embedded biopsied samples for biomarkers for breast and colon cancer.

In this work we present a methodology for the non-destructive elemental determination of formalin-fixed paraffin-embedded (FFPE) human tissue samples based on the Fundamental Parameters method for the quantification of micro Energy Dispersive X Ray Fluorescence (micro-EDXRF) area scans. This methodology intended to overcome two major constraints in the analysis of paraffin embedded tissue samples - retrieval of optimal region of analysis of the tissue within the paraffin block and the determination of the dark matrix composition of the biopsied sample. This way, an image treatment algorithm, based on R® tool to select the regions of the micro-EDXRF area scans was developed. Also, different dark matrix compositions were evaluated using varying combinations of H, C, N and O until the most accurate matrix was found: 8% H, 15% C, 1% N and 60% O for breast FFPE samples and 8% H, 23% C, 2% N and 55% O for colon. The developed methodology was applied to paired normal-tumour samples of breast and colon biopsied tissues in order to gauge potential elemental biomarkers for carcinogenesis in these tissues. The obtained results showed distinctive biomarkers for breast and for colon: there was a significant increase of P, S, K and Fe in both tissues, while a significant increase of Ca an Zn concentrations was also determined for breast tumour samples.

Evaluation of the Efficacy of CPP-ACP Remineralizing Mousse in Molar-Incisor Hypomineralized Teeth Using Polarized Raman and Scanning Electron Microscopy-An In Vitro Study.

Remineralization of tooth enamel can be achieved by applying a complex of casein phosphopeptides and amorphous calcium phosphate (CPP-ACP). However, the efficacy and optimization of this agent in molar−incisor hypomineralization (MIH) lacks evidence. The purpose of this study is to evaluate the efficacy of CPP-ACP tooth mousse in remineralizing MIH-affected enamel in an optimized 28-day protocol using polarized Raman microscopy and scanning electron microscopy. The protocol was applied to two types of MIH opacities, white and yellow, and compared against sound enamel specimens before and after treatment. Data was analyzed using a one-way ANOVA and LSD post hoc multiple comparisons test (p < 0.05) for the Raman analysis. Hypomineralized enamel showed an improvement of its structure after CPP-ACP supplementation. In addition, Raman spectroscopy results showed a decrease in the depolarization ratio of the symmetric stretching band of phosphate (p < 0.05 for both groups). In conclusion, there was an improvement in mineral density and organization of the hypomineralized enamel after treatment with CPP-ACP tooth mousse.

Evaluation of the Efficacy of CPP-ACP Remineralizing Mousse in MIH White and Yellow Opacities-In Vitro Vickers Microhardness Analysis.

Remineralization of tooth enamel can be partially achieved by the application of a casein phosphopeptides and amorphous phosphate (CPP-ACP) complex. However, evidence to support its effectiveness in Molar-incisor-hypomineralization (MIH)-affected teeth is scarce. The study's aim is to evaluate the efficacy of CPP-ACP mousse in remineralizing MIH-affected enamel using a Vickers microhardness test. Two groups of enamel opacities of hypomineralized permanent teeth, white (group A) and yellow (group B) lesions (n = 14), went through a 28-day treatment protocol with GC Tooth Mousse. Before and after treatment, microhardness was measured in three different areas of each tooth (hypomineralized, transition, and outside the hypomineralized area). Data were analyzed using parametric and non-parametric tests with a significance of p < 0.05. The mean microhardness values increased in the hypomineralized and transition areas in both groups after the treatment protocol (in group A, 105.38 ± 11.70 to 158.26 ± 37.34; 123.04 ± 22.84 to 156.33 ± 35.70; in group B, 108.63 ± 14.66 to 143.06 ± 22.81; 132.55 ± 20.66 to 146.00 ± 12.88) and the differences pre/post-treatment were statistically significant within each group (p < 0.001 for both groups). Between groups, there was a statistically significant difference in the same areas (hypomineralized: p = 0.003; transition: p = 0.008) with a higher improvement in enamel hardness in group A. Topical application of CPP-ACP showed an increase in the physical strength of the hypomineralized and transition areas of MIH-affected enamel, likely due to an increase in mineral content.

Fluoride exposure duringintrauterine and lactation periods promotes changes in the offspring rats' alveolar bone.

The importance of fluoride (F) for oral health is well established in the literature. However, evidence suggests that excessive exposure to this mineral is associated with adverse effects at different life stages and may affect many biological systems, especially mineralized tissues. The purpose of this study was to investigate the effects of F exposure during pregnancy and breastfeeding on the alveolar bone of the offspring since the alveolar bone is one of the supporting components of the dental elements. For this, the progeny rats were divided into three groups: control, 10 mg F/L, and 50 mg F/L for 42 (gestational and lactation periods). Analysis of the quantification of F levels in the alveolar bone by particle-induced gamma emission; Raman spectroscopy to investigate the physicochemical aspects and mineral components; computed microtomography to evaluate the alveolar bone microstructure and analyses were performed to evaluate osteocyte density and collagen quantification using polarized light microscopy. The results showed an increase in F levels in the alveolar bone, promoted changes in the chemical components in the bone of the 50 mg F/L animals (p < 0.001), and had repercussions on the microstructure of the alveolar bone, evidenced in the 10 mg F/L and 50 mg F/L groups (p < 0.001). Furthermore, F was able to modulate the content of organic bone matrix, mainly collagen; thus, this damage possibly reduced the amount of bone tissue and consequently increased the root exposure area of the exposed groups in comparison to a control group (p < 0.001). Our findings reveal that Fcan modulate the physicochemical and microstructural dimensions and reduction of alveolar bone height, increasing the exposed root region of the offspring during the prenatal and postnatal period. These findings suggest that F can modulate alveolar bone mechanical strength and force dissipation functionality.

Effects of inorganic mercury exposure in the alveolar bone of rats: an approach of qualitative and morphological aspects.

BACKGROUND: In comparison to organic mercury (MeHg), the environmental inorganic mercury (IHg) can be found in some skin-lightening cosmestics were considered "harmless" for a long time. However, recent studies have shown that long-term exposure to low doses of IHg may affect biological systems. Therefore, this study investigated the effects of IHg long-term exposure to the alveolar bone of adult rats. METHODS: Adult Wistar rats were distributed in control and HgCl2 exposed (0.375 mg/kg/day). After 45 days, the rats were euthanized and both blood and hemimandibles were collected. Total blood Hg levels were measured and both inorganic and organic components of the alveolar bone were determined through XRD and ATR-FTIR. The microstructure of the alveolar bone was assessed by using micro-CT and the morphometric analysis was performed by using stereomicroscopy. RESULTS: Alterations in the physicochemical components of the alveolar bone of exposed animals were observed. The bone changes represented a tissue reaction at the microstructural level, such as bone volume increase. However, no significant dimensional changes (bone height) were observed. CONCLUSION: Exposure to IHg at this dose can promote microstructural changes and alteration in the organic and inorganic components in the alveolar bone.

Analysis of human tissues using Energy Dispersive X Ray Fluorescence - Dark matrix determination for the application to cancer research.

BACKGROUND: X ray Fluorescence has been essayed as a suitable technique for the elemental quantification of trace element in human tissues, namely comparison of normal and cancerous tissue. However, accurate results depend on a robust quantification approach, namely correct evaluation of the samples' dark matrix. METHODS: In order to determine the most suitable dark matrix composition for the quantification of such samples using the Fundamental Parameter approach, we have measured several Certified Reference Materials and essayed different dark matrix compositions to achieve the most accurate results. The resulting dark matrix was then applied to normal and tumor ovarian and prostate tissue samples, and the obtained results were compared with the ones obtained with a comparative method using external standard calibration curves. RESULTS: Using a dark matrix composed of 10 % - H, 22 % - C, 3 % - N and 60 % - O yielded the best compromise in accuracy for the light and heavy elements. For the reduced sample size and conditions of this study, for both organs, the concentrations of transition metals decrease in tumor tissues, while the concentration of lighter elements, P and Cl, increases. On the other hand, there are elements that showed different behavior between the two types of tissue, namely Zn and S, that increase in prostate tumor tissue and decrease in ovarian tissue. CONCLUSION: An increase in precision was one of the improvements found with the newly developed method, as the FP-approach contemplates matrix effects and the influence of other elements in the analytes' quantification. Additionally, the determined dark matrix can be employed in any tissue analysis application by means of EDXRF.

Long-term exposure to low doses of aluminum affects mineral content and microarchitecture of rats alveolar bone.

Aluminum (Al) is one of the most found elements in nature in many forms, and human exposure can be quite common. Therefore, it is important to investigate the effects of exposure to Al mainly at low doses and for a prolonged period, in order to simulate human exposure in the periodontium, an important structure for support and protection of the teeth. This investigation aimed to study the aluminum chloride (AlCl3) toxicological effects in the mineral composition and micromorphology of the alveolar bone of rats. Two groups of eight male Wistar rats were used for the experiment. AlCl3 group was exposed to AlCl3 orally at a dose of 8.3 mg/kg/day for 60 days, while the control group received only distilled water. After that, the mandibles were collected and submitted to the following analyses: Fourier transform infrared spectroscopy, Raman spectroscopy, and X-ray microtomography analysis; blood was also collected for determination of Al circulating levels. Our data showed that AlCl3 was capable of increasing Al circulating levels in blood. It was able to promote changes in the mineral content and triggers significant changes in the mineralized bone microstructure, such as number and thickness of trabeculae, being associated with alveolar bone-loss.

Determination of gold leaf thickness using X-ray fluorescence spectrometry: Accuracy comparison using analytical methodology and Monte Carlo simulations.

In this work, we compared the accuracy of gold leaves thickness determination using X-ray Fluorescence (XRF) setups for different X-ray excitation sources. The sources used consisted on: a) direct Bremsstrahlung from an Amptek Mini-X X-ray tube with Rh target, b) partially monochromatized radiation of the same X-ray tube using combination of Al and Ag filters and, c) monochromatic radiation using secondary target, in a portable setup with triaxial geometry. The performance of the used setups was compared using model samples made with gold foil standard reference materials of known thicknesses (1, 2 and 2.5 µm). Thickness determination was based on the selective attenuation that the different energies of a given element of an under-layer undergo in the gilded layer. Ideally, as it can be expected from physics fundamentals, monochromatic radiation is needed for improved accuracy. However, considering the difficulties in using monochromatic radiation in portable instrumentation applicable in Cultural Heritage studies, filtered radiation can also be used for reliable results. Moreover, Monte Carlo simulations of the portable setup with conventional planar geometry were performed for comparison and for future application in more complex samples.

Assessment of Toxic Metals and Hazardous Substances in Tattoo Inks Using Sy-XRF, AAS, and Raman Spectroscopy.

Synchrotron radiation X-ray fluorescence spectroscopy, in conjunction with atomic absorption and Raman spectroscopy, was used to analyze a set of top brand tattoo inks to investigate the presence of toxic elements and hazardous substances. The Cr, Cu, and Pb contents were found to be above the maximum allowed levels established by the Council of Europe through the resolution ResAP(2008)1 on requirements and criteria for the safety of tattoos and permanent makeup. Raman analysis has revealed the presence of a set of prohibited substances mentioned in ResAP(2008)1, among which are the pigments Blue 15, Green 7, and Violet 23. Other pigments that were identified in white, black, red, and yellow inks are the Pigment White 6, Carbon Black, Pigment Red 8, and a diazo yellow, respectively. The present results show the importance of regulating tattoo ink composition.

Quantitative analysis of human remains from 18(th)-19(th) centuries using X-ray fluorescence techniques: The mysterious high content of mercury in hair.

In this work, we report the unusual concentration of mercury in the hair of an individual buried in the 18th to mid-19th centuries and the comparison with the elemental composition of other remains from the same individual. Two energy dispersive X-ray fluorescence (EDXRF) setups, one with tri-axial geometry and the second one with micro-beam capabilities and a vacuum system, for light elements detection, have been used. Quantitative evaluation of the obtained spectra were made by fundamental parameters and winAXIL program by compare mode method. The levels of Hg in the hair of buried samples presented a concentration over 5% (w/w), a significantly lower presence of this element in the cranium, and no Hg in the remaining organs. Furthermore, there was no evidence of Hg in the burial soil, which has been also analyzed. From this result, we could conclude that the possibility of post-mortem contamination from the burial surroundings is very unlikely. The obtained results are indicative of the apparent use of a mercury-based compound for medical purposes, most likely lice infestation.

Microscopy and X-ray spectroscopy analyses for assessment of gilding and silvering techniques of Portuguese illuminated manuscripts.

The objects of this study are various local charters (cartas de foral, in Portuguese) granted by Dom Manuel I, King of Portugal (1495-1521), which substituted for medieval ones and were intended to achieve an administrative unification. These are luxuriously illuminated manuscripts, and our study aims at obtaining a better understanding of the gilding and silvering techniques applied to the parchments, in which the forais were written, between 1500 and 1520. The combined use of microscopy and X-ray spectroscopy analyses allowed us to identify the vestigial materials used for making the parchments, including products such as salt (NaCl), lime (CaO), pumice stone (SiO2+Al2O3), and chalk (CaCO3). Chalk was employed as a whitening agent to give the parchment its final color and opacity. Shell-gold and shell-silver mixed in with animal glue or gum binding media were directly applied on type 1 and 3 forais, while very thin gold leaves (<1 µm) were applied over lead-based tempera grounds (50-180 µm thick) in type 2 forais. Silver was always employed in its finest form without a further protective layer (thus its recursive state of corrosion), while gold was used in various alloy grades.

Micro-analytical study of a rare papier-mâché sculpture.

The analysis of a Portuguese "papier-mâché" sculpture depicting Saint Anthony is presented in this case study. Several questions were addressed such as the characteristics of the support, pigments used, and artistic technique in order to establish a possible timeline for its production. Qualitative analyses of the cross-sections and of the paper support were performed by optical microscopy using reflected light. Two polychrome layers from different periods and a rag pulped support were identified on the sculpture. The use of micro X-ray fluorescence and Raman microscopy techniques enabled the differentiation of coloring materials used in both polychromies. Semi-quantitative analyses of the gilded samples were also performed by scanning electron microscopy in combination with energy-dispersive spectroscopy allowing the determination of a common Au-Ag-Cu alloy with differences in the purity of the gold. The identified coloring materials lead us to believe that the sculpture was produced in the 19th century, being overpainted in the first half of the 20th century.

Spectroscopic characterization of a masterpiece: the Manueline foral charter of Sintra.

The foral charter attributed by D. Manuel I of Portugal, in 1514, to the village of Sintra was studied using Energy Dispersive X-ray Fluorescence spectrometry, Raman and Infrared micro-spectroscopies. A complete characterization of the pictorial materials used in the production of this masterpiece allowed the identification of iron gall inks used in the written text; pigments such as malachite, azurite, lead white, cinnabar, yellow ochre, gold, silver and carbon black in the illuminations and letterings; filler and binder used in the production of coloring materials and inks. Gum and calcium carbonate were the most recurrent binder and filler identified in this study. Silvering and gilding were mostly obtained by applying ground silver and gold on parchment.

Study of air-induced paper discolorations by infrared spectroscopy, X-ray fluorescence, and scanning electron microscopy.

Air-induced paper discoloration is described as being different from other discoloration morphologies. It seems to be the result of prolonged exposure to air in a humid and polluted environment without appropriate protecting coverage. In this work, three folios from the same eighteenth century book, presenting three degrees of discoloration and opacity and subjected to different environmental conditions, were examined and compared. Samples were analyzed and compared by three different instrumental techniques, mid-infrared spectroscopy, X-ray fluorescence (XRF), and scanning electron microscopy (SEM). Chemical and physical changes were confirmed from the data collected by these techniques. The absence of the secondary amide band characteristic of proteins in the infrared spectra of the two discolored folios, accompanied by the appearance and increase of white mineral-like deposits visible in the SEM micrographs, support the idea that oxidation reactions occurred and that these two folios were subjected to more severe degradation hazard.

Analysis of lead content in automotive shredder residue (ASR).

Automotive shredder residue (ASR) is a very heterogeneous waste, which could have a very high metal content on finest fractions phi<6 mm produced by the shredding of end of live vehicles. The approval of Directive 2000/53/EC and its transposition to the European Union member states requires an analytical technique for in-situ checking of the content of some metals in ASR wastes. The objective of this study is the evaluation of total Pb content in the different fractions using a rapid measurement method to easily accomplish the current legislation. An experimental Energy Dispersive X-ray Fluorescence (EDXRF) spectrometer with tri-axial geometry was used to analyse the ASR in order to test the feasibility of this analytical technique. Likewise, a correction of the intensities by the incoherent scattering (Compton) radiation was made to compensate the matrix effects. The results show that values in the smaller fractions are bigger (11,600 mg kg(-1) in the fraction <125 microm) than in the coarser fractions (4600 mg kg(-1) in the fraction between 2 and 6 mm) and that such type of instrumentation enables a fast measurement with a limit of detection of 1.1 mg kg(-1) for 1000 s measurement).