The effects of mercury exposure on Amazonian fishes: An investigation of potential biomarkers.

Exposure to mercury can interfere with the expression of proteins and enzymes, compromise important pathways, such as apoptosis and glucose metabolism, and even induce the expression of metallothioneins. In this study, analytical techniques were used to determine the concentration of total mercury (THg) in muscle and liver tissue, protein pellets, and spots [using graphite furnace atomic absorption spectrometry (GFAAS)], and molecular techniques were used to identify metalloproteins present in mercury-associated protein spots. Thirty individuals from three different fish species, Cichla sp. (n = 10), Brachyplatystoma filamentosum (n = 10), and Semaprochilodus sp. (n = 10) from the Brazilian Amazon were used. Oxidative stress indicators [such as glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), a marker of lipid peroxidation (LPO)] and the possible expression of metallothioneins in muscle and liver tissues were investigated. The two piscivorous species, Cichla sp. and B. filamentosum, presented the highest concentrations of mercury in their hepatic tissue, 1219 ± 15.00 and 1044 ± 13.6 µg kg-1, respectively, and in their muscle tissue, 101 ± 1.30 µg kg-1 and 87.4 ± 0.900 µg kg-1, respectively. The non-carnivorous species Semaprochilodus sp. had comparatively low concentrations of mercury in both its hepatic (852 ± 11.1 µg kg-1) and muscle (71.4 ± 0.930 µg kg-1) tissues. The presence of mercury was identified in 24 protein spots using GFAAS; concentrations ranged from 11.5 to 787 µg kg-1, and mass spectrometry identified 21 metal-binding proteins. The activities of GSH-Px, CAT, and SOD, related to oxidative stress, decreased proportionally as tissue Hg concentrations increased, while the levels of LPO markers increased, indicating the presence of stress. Our study results demonstrate possible mercury interference in oxidative stress markers (GSH-Px, CAT, SOD, and LPO), in addition to the identification of 21 metal-binding proteins as possible biomarkers of mercury exposure in fish.

Proteomic analysis of serum samples of paracoccidioidomycosis patients with severe pulmonary sequel.

BACKGROUND: Pulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification. METHODOLOGY/PRINCIPAL FINDINGS: This non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, data are available via ProteomeXchange with identifier PXD026906. Serum differentially expressed proteins were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 15 proteins as candidate biomarkers for severe PS. CONCLUSIONS/SIGNIFICANCE: Development of severe PS is related to increased expression of proteins involved in glycolytic pathway and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers.

Author Correction: Modification of the head proteome of nurse honeybees (Apis mellifera) exposed to field-relevant doses of pesticides.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Modification of the head proteome of nurse honeybees (Apis mellifera) exposed to field-relevant doses of pesticides.

Understanding the effect of pesticides on the survival of honeybee colonies is important because these pollinators are reportedly declining globally. In the present study, we examined the changes in the head proteome of nurse honeybees exposed to individual and combined pesticides (the fungicide pyraclostrobin and the insecticide fipronil) at field-relevant doses (850 and 2.5 ppb, respectively). The head proteomes of bees exposed to pesticides were compared with those of bees that were not exposed, and proteins with differences in expression were identified by mass spectrometry. The exposure of nurse bees to pesticides reduced the expression of four of the major royal jelly proteins (MRJP1, MRJP2, MRJP4, and MRJP5) and also several proteins associated with carbohydrate metabolism and energy synthesis, the antioxidant system, detoxification, biosynthesis, amino acid metabolism, transcription and translation, protein folding and binding, olfaction, and learning and memory. Overall, when pyraclostrobin and fipronil were combined, the changes in protein expression were exacerbated. Our results demonstrate that vital proteins and metabolic processes are impaired in nurse honeybees exposed to pesticides in doses close to those experienced by these insects in the field, increasing their susceptibility to stressors and affecting the nutrition and maintenance of both managed and natural colonies.

Proposed mechanism for understanding the dose- and time-dependency of the effects of fluoride in the liver.

Fluoride (F) can induce changes in the expression of several liver proteins. It is suggested that these changes are dose- and time-dependent. The objective of this study was to analyze the effect of different F concentrations and exposure times to this ion on the pattern of protein expression in the liver of rats. Thirt-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the treatment duration (20 or 60 days). Each of these groups was then divided into 3 subgroups (n = 6) according to the concentration of F administered in drinking water, as follows: 0 mg/L (control), 15 mg/L or 50 mg/L. After the experiment periods, the animals were anesthetized and the liver and blood were collected. F was analyzed in plasma and liver. Part of the liver was fixed for histological analysis. Liver proteins were extracted and prepared for quantitative label-free mass spectrometry analysis. F concentrations in plasma and liver were significantly higher in the group treated with 50 mg /L in comparison with control, regardless the time of exposure. Histological alterations in the liver were more evident in the subgroups treated for 20 days. The proteomic analysis revealed changes in proteins related to endoplasmic reticulum and mitochondrial oxidative stress, mitochondrial alteration, apoptosis and cellular respiration upon exposure to F. The results reinforce previous findings showing that the effects of F in the liver are dose- and time-dependent and provide the molecular basis for understanding the evolution of these effects.

Fluoride affects bone repair differently in mice models with distinct bone densities.

We grouped mice [strains: C57BL/6J (n=32) and C3H/HeJ (n=32)] to address the influence of bone density on fluoride's (F's) biological effects. These animals received low-fluoride food and water containing 0 (control group) or 50ppm of F for up to 28days. The upper left central incisor was extracted, and the left maxilla was collected at 7, 14, 21, and 28days for histological and histomorphometric analysis to estimate bone neoformation. Our results showed bone neoformation in all of the evaluated groups, with the presence of bone islets invading the center of the alveoli when replacing the existing connective tissue. Curiously, this biological phenomenon was more evident in the C57BL/6J strain. The histomorphometric analysis confirmed the histological findings in relation to the amount of new bone tissue and showed a decrease in C3H/HeJ mice (control group). Altogether, our results showed differential effects of fluoride bone metabolism, confirming a genetic component in susceptibility to the effects of fluoride.

Effects of fluoride in bone repair: an evaluation of RANKL, OPG and TRAP expression.

The objective of this study was to evaluate comparatively the effect of fluoride in the expression of the receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP) in alveolar bone repair in rats. We used 3 groups of male Wistar rats (n = 5/group), which received drinking water containing different doses of F (NaF): 0, 5 and 50 ppm, for 60 days before the incisors extraction. The upper incisors were extracted and the animals were killed 7, 14, 21 and 30 days after extraction. The hemi-maxillae were collected for microscopic examination (histomorphometric and immunostaining for RANKL, OPG and TRAP). Histomorphometric analysis confirmed an increase in the volume density of neoformed bone between 7 and 30 days for groups control, 5 and 50 ppm of F, with a concomitant decrease in the volume density of connective tissue and blood clot. Higher blood clot for groups 5 and 50 ppm of F at 30 days was observed. The RANKL and OPG expressions were not changed by chronic exposure to fluoride in the drinking water during the studied periods; on the other hand, TRAP expression was changed (at 7 days) by chronic exposure to fluoride (p < 0.05). It was concluded that F in high concentrations can slow the blood clot remission and bone repair, and alter the TRAP expression in the beginning of the bone tissue repair. However, a better understanding about this blood clot remission phenomenon is required.

Fluoride effect on the process of alveolar bone repair in rats: evaluation of activity of MMP-2 and 9

Objective: The aim of this study was to evaluate comparatively the effect of fluoride (F) on the activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) involved in process of alveolar bone repair. Material and methods: This study used 4 groups of Wistar rats with 80 days of life (n = 160) which received drinking water containing different doses of fluoride (NaF): 5, 15, 50 ppm and deionized water (control) throughout the experiment. These animals had their right upper incisors extracted. After extraction, the animals were euthanized at 7, 14, 21 and 30 days and the hemi-maxillae were collected for microscopic analysis (Hematoxylin and Eosin and immunohistochemistry for MMP-9) and zymography (MMP-2 and 9).Results: Microscopically the process of bone repair was similar in all groups, being noted only a delay of the blood clot resorption and bone formation in the group of 50 ppm F. The expression for MMP-9 showed differences betweengroups only during the initial repair (7 days). However, the zymography showed no significant differences between treated and control groups. Conclusion: Ours results suggest an effect of fluoride on the activity of MMPs 2 and 9 at the initial period of alveolar repair which could be associated to the process of blood clot remission and delay in bone repair. Further studies are needed to establish the relationship between the initial process of resorption of the blood clot, and the involvement of MMPs 2 and 9 and its regulators/tissue inhibitors.

Alkaline phosphatase activity in plasma and liver of rats submitted to chronic exposure to fluoride

The aim of this study was to compare the effect of fluoride (F) on alkaline phosphatase activity in the liver and plasma of the rats. Four groups of male Wistar rats (n=6), which received drinking water containing 5, 15 or 50 ppm F or deionized water (control) throughout the experiment were included in the study. The animals were euthanized and had their tissues and blood plasma collected for the analysis of fluoride and alkaline phosphatase. There was an increase in F concentration in most tissues in the animals treated with higher F concentrations, except for the heart. The alkaline phosphatase assay showed an increase in the activity in the liver and blood plasma of the animals treated with fluoride concentrations of 15 and 50 ppm (p<0.05). This study suggested that F at a concentration of 50 ppm in drinking water promotes increased the activity of alkaline phosphatase in the liver and blood plasma.

Avaliação da influência do fluoreto no reparo ósseo de alvéolos de ratos. Estudo dos processos de reabsorção e neoformação óssea / Evaluation of the influence of fluoride in bone repair in dental alveoli in rats. Analysis of the reabsortion and neoformation of the bone

O processo de reparo do tecido ósseo tem sido alvo de estudos com o intuito de promover uma melhora na qualidade e tempo de reparo. Um elemento que tem sido estudado por alguns grupos é o fluoreto, pois tem sido proposto como terapêutico para osteoporose (Europa). Deste modo, o objetivo deste trabalho foi avaliar comparativamente o efeito do fluoreto administrado na água de beber sobre o reparo ósseo alveolar de ratos. Neste estudo foram utilizados 4 grupos com ratos Wistar de 80 dias de vida (n=200), os quais receberam água de beber contendo 5, 15 e 50 ppm de fluoreto e água deionizada (controle) durante todo experimento. Esses animais tiveram o incisivo superior direito extraído. Os animais foram eutanasiados 0 hora, 7, 14, 21 e 30 e 60 dias após a extração. Amostras de plasma sanguíneo foram obtidas para análise de concentração de flúor, e a região do alvéolo dental foi coletada para as análises de microscopia (Hematoxilina Eosina e imunoistoquímica) e zimografia (metaloproteinases de matriz 2 e 9 MMP-2 e 9). A análise de concentração de flúor no plasma sangüíneo mostrou maior presença desse elemento no grupo tratado com 50 ppm de F nos períodos de 21 e 30 dias. Na análise histológica foi detectado osso neoformado em todos os animais até 60 dias, porém o grupo de 50 ppm de F apresentou menor formação óssea nos períodos de 14, 21 e 30dias. A análise morfométrica confirmou um aumento na densidade de volume de osso neoformado, entre 7 e 60 dias, nos grupos controle, 5 ppm, 15 ppm e 50 ppm de F, com concomitante diminuição na densidade de volume de coágulo sangüíneo. A expressão de RANK-L e OPG não foi alterada pela exposição crônica ao fluoreto na água de beber durante os períodos estudados (p<0,05). A atividade da MMP-2 foi mais acentuada no período inicial, enquanto a MMP-9 teve maior atividade no período final. Concluiu-se que o F, em altas concentrações pode alterar a dinâmica do reparo ósseo alveolar diminuindo a formação de novo tecido ósseo, associado...

The repair of bone has been investigated with the aim of promoting an improvement in the quality and time of the repair process. One element that has been studied by some groups is fluoride, it has been proposed as a treatment for osteoporosis (Europe). Thus, the objective was to comparatively evaluate the effect of fluoride administered in drinking water on alveolar bone repair in rats. This study used 4 groups of rats of 80 days (n=200), which received drinking water containing 5, 15, and 50 ppm of fluoride, and deionized water (control) throughout the experiment. These animals had their upper right incisor extracted. The animals were euthanized 0 hours, 7, 14, 21 and 30 and 60 days after extraction. Samples of blood plasma were obtained for analysis of fluoride concentration, and the region of the alveolus was collected for microscopic analysis (Hematoxylin eosin and Immunohistochemistry) and zymography (metalloproteinases 2 and 9 MMP-2 and 9). Analysis of fluoride concentration in blood plasma showed a greater presence of this element in the group treated with 50 ppm of F in periods of 21 and 30 days. Histological analysis detected a new bone formed in all animals up to 60 days, but the group of 50 ppm F showed lower bone formation at 14, 21 and 30 days. Morphometric analysis confirmed an increase in the volume density of newly formed bone, between 7 and 60 days in control groups, 5 ppm, 15 ppm and 50 ppm F, with a concomitant decrease in the volume density of blood clot. The expression of RANK-L and OPG was not altered by chronic exposure to fluoride in drinking water during the study period (p<0.05). The activity of MMP-2 was more pronounced in the early period, while MMP-9 had higher activity in the final period. It was conclude that the F, in high concentrations change the dynamics of the alveolar bone repair by decreasing the formation of new bone, associated with a delay in treating the blood clot. Proteins as RNK-L, OPG, MMP-2 and MMP-9 may also...

Absence of DNA damage in multiple organs (blood, liver, kidney, thyroid gland and urinary bladder) after acute fluoride exposure in rats.

Fluoride has been widely used in dentistry as a caries prophylactic agent. However, there has been some speculation that excess fluoride could cause an impact on genome integrity. In the current study, the potential DNA damage associated with exposure to fluoride was assessed in cells of blood, liver, kidney, thyroid gland and urinary bladder by the single cell gel (comet) assay. Male Wistar rats aging 75 days were distributed into seven groups: Groups 1 (control), 2, 3, 4, 5, 6 and 7 received 0 (deionized water), 10, 20, 40, 60, 80 and 100 mgF/Kg body weight from sodium fluoride (NaF), respectively, by gastrogavage. These groups were killed at 2 h after the administration of the fluoride doses. The level of DNA strand breaks did not increase in all organs evaluated and at all doses of NaF tested, as depicted by the mean tail moment. Taken together, our results suggest that oral exposure to NaF did not result in systemic genotoxic effect in multiple organs related to fluoride toxicity. Since DNA damage is an important step in events leading to carcinogenesis, this study represents a relevant contribution to the correct evaluation of the potential health risk associated with chemical exposure.