Oxidative Stress Induced by 30 Days of Mercury Exposure Accelerates Hypertension Development in Prehypertensive Young SHRs.

Mercury is considered a risk factor for the development of hypertension and other cardiovascular diseases. We investigated whether the effects of mercury exposure on haemodynamic parameters of young Wistar rats and prehypertensive SHRs might alter the time course of hypertension development. Young (4 weeks) male Wistar rats and SHRs were randomly assigned to four groups: untreated Wistar rats (Wistar Ct), Wistar rats exposed to mercury chloride for 30 days (Wistar Hg), untreated SHRs (SHR Ct) and SHRs exposed to mercury chloride (SHR Hg) for 30 days. Non-invasive and invasive arterial pressures were measured to investigate pressure reactivity; nitrite/nitrate levels, ACE activity, and lipid peroxidation were measured in plasma. The systolic blood pressure (SBP) of the Wistar rat groups did not change but increased in the SHRs from the second week to the last week. Hg exposure accelerated the increase in the SBP of SHRs. L-NAME administration increased SBP and diastolic blood pressure (DBP) in all groups, but this increase was smaller in SHRs exposed to Hg. A decrease in plasma nitrite and nitrate levels in the SHR Hg group suggested that mercury reduced NO bioavailability. Tempol-reduced blood pressure suggesting that the superoxide anion played a role in the marked increase in this parameter. These findings provide evidence that Hg exposure might activate mechanisms to accelerate hypertension development, including a reduction in NO bioavailability. Therefore, predisposed individuals under mercury exposure are at greater risk from an enhanced development of hypertension.

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.

DNA Damage and Proteomic Profile Changes in Rat Salivary Glands After Chronic Exposure to Inorganic Mercury.

Mercury (Hg) is a toxic metal that became a public health problem due to environmental contamination caused by anthropogenic activity. In this sense, oral homeostasis can undergo changes due to the toxic effects of metal on the salivary glands. Therefore, our objective was to investigate the proteomic and genotoxic changes in salivary glands after exposure to inorganic mercury (IHg). Forty Wistar rats that were divided into a control group, which received distilled water, and an exposed group, which received 0.375 mg/kg of mercury chloride for 45 days via orogastric gavage. After that, the animals were euthanized, and the parotid and submandibular glands were collected for analysis of the genotoxic effects, using the comet assay and proteome global profile assessment. The results showed that IHg promoted damage to cellular DNA associated with proteomic changes that showed events such as oxidative stress, mitochondrial dysfunction, changes in the cytoskeleton, and apoptosis. Therefore, these findings show a profile of molecular changes due to the interactions of IHg with several proteins and mechanisms inherent to the cell, which consequently may result in dysfunction of the salivary glands and impaired homeostasis of the oral cavity.

Chronic mercury exposure induces oxidative stress in female rats by endothelial nitric oxide synthase uncoupling and cyclooxygenase-2 activation, without affecting oestrogen receptor function.

Mercury has been shown to be a significant health risk factor and is positively associated with cardiovascular diseases. Evidence reveals that men are more likely to develop cardiovascular diseases than women during reproductive age. However, the effects of mercury in females remain poorly investigated, despite the finding that female hormones demonstrate a cardioprotective role. In the present study, we evaluated whether chronic mercury chloride exposure could alter blood pressure and vascular function of the female rat aorta. Ten-week-old female Wistar rats were divided into two groups: control (vehicle) and mercury treated (first dose of 4.6 µg/kg, subsequent daily doses of 0.07 µg/kg), im. Mercury treatment did not modify systolic blood pressure (SBP) but increased vascular reactivity due to the reduction of nitric oxide bioavailability associated with the increase in reactive oxygen species from endothelial nitric oxide synthase (eNOS) uncoupling. Furthermore, increased participation of the cyclooxygenase-2 pathway occurred through an imbalance in thromboxane 2 and prostacyclin 2. However, the oestrogen signalling pathway was not altered in either group. These results demonstrated that chronic exposure to mercury in females induced endothelial dysfunction and, consequently, increased aortic vascular reactivity, causing vascular damage to the female rat aorta and representing a risk of cardiovascular diseases.

Spinal cord neurodegeneration after inorganic mercury long-term exposure in adult rats: Ultrastructural, proteomic and biochemical damages associated with reduced neuronal density.

Mercury chloride (HgCl2) is a chemical pollutant widely found in the environment. This form of mercury is able to promote several damages to the Central Nervous System (CNS), however the effects of HgCl2 on the spinal cord, an important pathway for the communication between the CNS and the periphery, are still poorly understood. The aim of this work was to investigate the effects of HgCl2 exposure on spinal cord of adult rats. For this, animals were exposed to a dose of 0.375 mg/kg/day, for 45 days. Then, they were euthanized, the spinal cord collected and we investigated the mercury concentrations in medullary parenchyma and the effects on oxidative biochemistry, proteomic profile and tissue structures. Our results showed that exposure to this metal promoted increased levels of Hg in the spinal cord, impaired oxidative biochemistry by triggering oxidative stress, mudulated antioxidant system proteins, energy metabolism and myelin structure; as well as caused disruption in the myelin sheath and reduction in neuronal density. Despite the low dose, we conclude that prolonged exposure to HgCl2 triggers biochemical changes and modulates the expression of several proteins, resulting in damage to the myelin sheath and reduced neuronal density in the spinal cord.

Chronic Mercury Exposure in Prehypertensive SHRs Accelerates Hypertension Development and Activates Vasoprotective Mechanisms by Increasing NO and H2O2 Production.

Mercury is a heavy metal associated with cardiovascular diseases. Studies have reported increased vascular reactivity without changes in systolic blood pressure (SBP) after chronic mercury chloride (HgCl2) exposure, an inorganic form of the metal, in normotensive rats. However, we do not know whether individuals in the prehypertensive phase, such as young spontaneously hypertensive rats (SHRs), are susceptible to increased arterial blood pressure. We investigated whether chronic HgCl2 exposure in young SHRs accelerates hypertension development by studying the vascular function of mesenteric resistance arteries (MRAs) and SBP in young SHRs during the prehypertensive phase. Four-week-old male SHRs were divided into two groups: the SHR control group (vehicle) and the SHR HgCl2 group (4 weeks of exposure). The results showed that HgCl2 treatment accelerated the development of hypertension; reduced vascular reactivity to phenylephrine in MRAs; increased nitric oxide (NO) generation; promoted vascular dysfunction by increasing the production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2); increased Gp91Phox protein levels and in situ levels of superoxide anion (O2·-); and reduced vasoconstrictor prostanoid production compared to vehicle treatment. Although HgCl2 accelerated the development of hypertension, the HgCl2-exposed animals also exhibited a vasoprotective mechanism to counterbalance the rapid increase in SBP by decreasing vascular reactivity through H2O2 and NO overproduction. Our results suggest that HgCl2 exposure potentiates this vasoprotective mechanism against the early establishment of hypertension. Therefore, we are concluding that chronic exposure to HgCl2 in prehypertensive animals could enhance the risk for cardiovascular diseases.

Mercury chloride exposure induces DNA damage, reduces fertility, and alters somatic and germline cells in Drosophila melanogaster ovaries.

Mercury exposure has been shown to affect the reproductive system in many organisms, although the molecular mechanisms are still elusive. In the present study, we exposed Drosophila melanogaster Canton-S adult females to concentrations of 0 mM, 0.1 mM, 0.3 mM, 3 mM, and 30 mM of mercury chloride (HgCl2) for 24 h, 48 h, or 72 h to determine how mercury could affect fertility. Alkaline assays performed on dissected ovaries showed that mercury induced DNA damage that is not only dose-dependent but also time-dependent. All ovaries treated for 72 h have incorporated mercury and exhibit size reduction. Females treated with 30 mM HgCl2, the highest dose, had atrophied ovaries and exhibited a drastic 7-fold reduction in egg laying. Confocal microscopy analysis revealed that exposure to HgCl2 disrupts germinal and somatic cell organization in the germarium and leads to the aberrant expression of a germline-specific gene in somatic follicle cells in developing egg chambers. Together, these results highlight the potential long-term impact of mercury on germline and ovarian cells that might involve gene deregulation.

Neurochemical dysfunction in motor cortex and hippocampus impairs the behavioral performance of rats chronically exposed to inorganic mercury.

Chronic exposure to mercury chloride (HgCl2) has been shown to promote oxidative stress and cell death in the central nervous system of adult rats displaying motor and cognitive impairments. However, there are no investigations about neurochemical function after this type of exposure in rodents that may be associated with those behavioral changes already reported. Thus, the aim of this study was to analyze glutamatergic and GABAergic dysfunctions in the motor cortex and hippocampus of adult rats, in a model of chronic exposure to HgCl2 in. Twenty rats were exposed to a daily dose of 0.375 mg/kg for 45 days. After this period, they were submitted to motor and cognitive functions tests and euthanized to collect the motor cortex and hippocampus for measurement of mercury (Hg) levels in the parenchyma and neurochemical assays for analysis of glutamatergic and GABAergic functions. It was observed that chronic exposure to HgCl2 promoted increase in total Hg levels in these two brain areas, with changes in glutamatergic transport, but without changes in GABAergic transport. Functionally this model of exposure caused the decrease of the spontaneous motor locomotion and in the process of learning and memory. In this way, our results provide evidences that glutamatergic neurochemical dysfunction can be pointed out as a strong causal factor of motor and cognitive deficits observed in rats exposed to this HgCl2.

Phytotoxicity reduction of the mercury chloride effect by natural products from Eugenia jambolana Lam.: A new strategy against the toxic metal pollution.

The objective of this work was to evaluate the antioxidant, metal chelating and cytoprotective activity of the Eugenia jambolana Lam. extract, as well as of its flavonoid and tannic fractions, against the action of Mercury Chloride (HgCl2). Flavonoids were quantified and an LC-MS chromatographic analysis was performed to identify secondary metabolites. Fe2+ and Fe3+ chelation tests and antioxidant activity were carried out using the FRAP method. Microbiological tests were performed by microdilution to determine the Minimum Inhibitory Concentration (MIC). From these results the Minimum Bactericidal (MBC) and Minimum Fungicide Concentration (MFC) were evaluated. The allelopathy and cytoprotection assays were performed using eukaryotic and prokaryotic models. The results revealed the presence of phenolic acids and flavonoids in the E. jambolana extract and fractions. The sub-allelopathic concentration (64 µg/mL) was used and the results demonstrated the E. jambolana potential cytoprotective effect against mercury chloride.

Exposure to Inorganic Mercury Causes Oxidative Stress, Cell Death, and Functional Deficits in the Motor Cortex.

Mercury is a toxic metal that can be found in the environment in three different forms - elemental, organic and inorganic. Inorganic mercury has a lower liposolubility, which results in a lower organism absorption and reduced passage through the blood-brain barrier. For this reason, exposure models that use inorganic mercury in rats in order to evaluate its effects on the central nervous system are rare, especially in adult subjects. This study investigated if a chronic exposure to low doses of mercury chloride (HgCl2), an inorganic form of mercury, is capable of promoting motor alterations and neurodegenerative in the motor cortex of adult rats. Forty animals were exposed to a dose of 0.375 mg/kg/day, for 45 days. They were then submitted to motor evaluation and euthanized to collect the motor cortex. Measurement of mercury deposited in the brain parenchyma, evaluation of oxidative balance, quantification of cellular cytotoxicity and apoptosis and density of mature neurons and astrocytes of the motor cortex were performed. It was observed that chronic exposure to inorganic mercury caused a decrease in balance and fine motor coordination, formation of mercury deposits and oxidative stress verified by the increase of lipoperoxidation and nitrite concentration and a decrease of the total antioxidant capacity. In addition, we found that this model of exposure to inorganic mercury caused cell death by cytotoxicity and induction of apoptosis with a decreased number of neurons and astrocytes in the motor cortex. Our results provide evidence that exposure to inorganic mercury in low doses, even in spite of its poor ability to cross biological barriers, is still capable of inducing motor deficits, cell death by cytotoxicity and apoptosis, and oxidative stress in the motor cortex of adult rats.

Xanthine Oxidase Activation Modulates the Endothelial (Vascular) Dysfunction Related to HgCl2 Exposure Plus Myocardial Infarction in Rats.

Heavy metal exposure is associated with cardiovascular diseases such as myocardial infarction (MI). Vascular dysfunction is related to both the causes and the consequences of MI. We investigated whether chronic exposure to low doses of mercury chloride (HgCl2) worsens MI-induced endothelial dysfunction 7 days after MI. Male Wistar rats were divided into four groups: Control (vehicle), HgCl2 (4 weeks of exposure), surgically induced MI and combined HgCl2-MI. Morphological and hemodynamic measurements were used to characterize the MI model 7 days after the insult. Vascular reactivity was evaluated in aortic rings. Chronic HgCl2 exposure did not cause more heart injury than MI alone in terms of the morphological or hemodynamic parameters. Vascular reactivity increased in all groups, but the combination of HgCl2-MI increased the vasorelaxation induced by ACh compared with the HgCl2 and MI groups. Results showed reduced endothelial nitric oxide synthase (eNOS) protein expression in the MI group; increased iNOS activity in the HgCl2-MI group, although without enough magnitude to reverse the reduction in NO bioavailability; and increased phenylephrine response in the HgCl2-MI group due to an increase in ROS production, notably via xanthine oxidase (XO). Results suggest that the combination of 1 month pre-exposure of HgCl2 before MI changed the endothelial generation of oxidative stress induced by mercury exposure from NADPH oxidase pathway to XO (xanthine oxidase)-dependent ROS production.

Chemical Characterization and Cytoprotective Effect of the Hydroethanol Extract from Annona coriacea Mart. (Araticum).

INTRODUCTION: Annona coriacea Mart. (araticum) is a widely distributed tree in the cerrado. Its value is attributed principally to the consumption of its fruit which possesses a large nutritive potential. The objective was to identify the chemical profile and evaluate the antimicrobial and cytoprotective activity of the hydroethanol extract of A. coriacea Mart. (HEAC) leaves against the toxicity of mercury chloride. MATERIALS AND METHODS: The characterization of components was carried out using high-performance liquid chromatography (HPLC). The minimum inhibitory concentration (MIC) was determined by microdilution method in broth with strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. For evaluation of the modulatory and cytoprotective activity of aminoglycoside antibiotics (gentamicin and amikacin) and mercury chloride (HgCl2), the substances were associated with the HEAC at subinhibitory concentrations (MIC/8). RESULTS AND DISCUSSION: The HPLC analysis revealed the presence of flavonoids such as Luteolin (1.84%) and Quercetin (1.19%) in elevated concentrations. The HEAC presented an MIC ≥512 µg/mL and significant antagonistic action in aminoglycosides modulation, and it also showed cytoprotective activity to S. aureus (significance P < 0.0001) and E. coli (significance P < 0.05) bacteria against the mercury chloride heavy metal with significance, this action being attributed to the chelating properties of the flavonoids found in the chemical identification. CONCLUSIONS: The results acquired in this study show that the HEAC presents cytoprotective activity over the tested strains in vitro and can also present antagonistic effect when associated with aminoglycosides, reinforcing the necessity of taking caution when combining natural and pharmaceutical products. SUMMARY: The hydroalcoholic extract of A. coriacea Mart. presents in vitro cytoprotective activity against the toxic effect of Hg. Abbreviations Used: HPLC-DAD: High-performance liquid chromatography with a diode array detector; MIC: Minimum inhibitory concentration; DMSO: Dimethyl sulfoxide.

Ação protetora de Duguetia furfuracea (A. St.-Hil.) Saff. contra a toxicidade do cloreto de mercúrio em Escherichia coli / Protective action of Duguetia furfuracea (A. St.-Hil.) Saff. against toxicity due to mercury chloride in Escherichia coli / Acción protectora de Duguetia furfuracea (A. St.-Hil.) Saff. contra la toxicidad por cloruro de mercurio en Escherichia coli

Introdução: o mercúrio constitui um dos metais mais tóxicos e sua contaminação tem sido alvo de muitos estudos que buscam desvendar os mecanismos envolvidos em sua toxicidade e seus efeitos deletérios para os seres vivos. A espécie Duguetia furfuracea (A. St.-Hil.) Saff., conhecida popularmente como araticum-bravo, ata-brava e ata de lobo, tem sido utilizada na medicina popular como anti-reumáticas, para o tratamento de disfunções renais, dores na coluna e no estômago, e contra pediculose. Objetivo: avaliar o efeito citoprotetor do extrato etanólico e frações de D. furfuracea frente ao metal pesado cloreto de mercúrio (HgCl 2). Métodos: a caracterização dos metabólitos secundários foi realizada através de prospecção fitoquímica, na qual se observam a alteração de cor ou formação de precipitados após adição de reagentes específicos. A Concentração Inibitória Mínima (CIM) foi determinada pelo método de microdiluição em caldo e a Concentração Bactericida Mínima (CBM) verificada utilizando placas de petri com HIA (Heart Infusion Agar) para transferência das soluções incubadas em placas de microdiluição. Resultados: a prospecção fitoquímica revelou a presença de taninos, flavonoides e alcaloides. Todas as amostras apresentaram um CIM ≥ 1024 µg/mL. De acordo com os resultados obtidos na CBM, o extrato etanólico e as frações de D. furfuracea, exceto a fração hexânica, apresentaram atividade citoprotetora à bactéria E. coli frente ao metal pesado cloreto de mercúrio, sendo essa ação atribuída às propriedades quelantes dos flavonoides. Conclusões: os dados obtidos indicam a espécie D. furfuracea como uma fonte promissora no combate a metais pesados, apresentando-se como protetora de seres procariontes. Este é o primeiro relato do uso de plantas medicinais como agente citoprotetor em modelo bacteriano.

Introduction: mercury is one of the most toxic metals. Contamination by mercury has been the object of many studies aimed at unraveling the mechanisms involved in its toxicity and its harmful effects on living beings. The species Duguetia furfuracea (A. St.-Hil.) Saff., popularly known asaraticum-bravo’, ‘ata-brava’ and ‘ata de lobo’, has been used in folk medicine as antirheumatic and for the treatment of renal dysfunction, spinal pain, stomach ache and pediculosis. Objective: evaluate the cytoprotective effect of the ethanolic extract and fractions of D. furfuracea against the heavy metal mercury chloride (HgCl 2). Methods: characterization of secondary metabolites was performed by phytochemical screening, in which color change and precipitate formation are examined after the addition of specific reagents. Minimum inhibitory concentration (MIC) was determined by broth microdilution, and minimum bactericidal concentration (MBC) was verified using petri plates with HIA (heart infusion agar) to transfer the solutions incubated on microdilution plates. Results: phytochemical screening revealed the presence of tannins, flavonoids an alkaloids. All samples exhibited a MIC ≥ 1024 µg/mL. MBC results showed that the ethanolic extract and all fractions of D. furfuracea except for the hexane fraction have cytoprotective activity by the bacterium E. coli against mercury chloride, an action attributed to the chelating properties of flavonoids. Conclusions: data point to the promising value of the species D. furfuracea against heavy metals, presenting as protectors of procaryote beings. This is the first report on the use of medicinal plants as cytoprotective agents on the bacterial model.

Introducción: el mercurio es uno de los metales más tóxicos y su contaminación ha sido objeto de muchos estudios que tratan de desentrañar los mecanismos implicados en su toxicidad y sus efectos nocivos en los seres vivos. La especie Duguetia furfuracea (A. St.-Hil.) Saff., popularmente conocido como ‘araticum-bravo’, ‘ata-brava’ y ‘ata de lobo’, se ha utilizado en la medicina popular como un anti-reumático para el tratamiento de la disfunción renal, dolor vertebral y de estómago, y contra la pediculosis. Objetivo: evaluar el efecto citoprotector del extracto de etanol y fracciones de D. furfuracea al metal pesado cloruro de mercurio (HgCl2). Métodos: la caracterización de metabolitos secundarios se realizó mediante la prospección fitoquímica, en la que se observa el cambio de color o la formación de precipitados después de la adición de los reactivos específicos. La Concentración Inhibitoria Mínima (CIM) se determinó mediante microdilución en caldo y la Concentración Bactericida Mínima (CBM) verificada usando placas de petri con HIA ( Heart Infusion Agar) para transferencia de las soluciones incubadas en placas de microdilución. Resultados: la prospección fitoquímica reveló la presencia de taninos, flavonoides y alcaloides. Todas las muestras presentaron una CIM ≥ 1024 µg/mL. De acuerdo con los resultados obtenidos en la CBM, el extracto de etanol y fracciones de D. furfuracea, excepto la fracción de hexano, mostraron actividad citoprotectora de la bacteria E. coli delante del cloruro de mercurio, siendo esa acción atribuida a las propiedades quelantes de los flavonoides. Conclusiones: los datos obtenidos indican que la especie D. furfuracea como una fuente prometedora para combatir los metales pesados, que se presentan como protectores de seres procariotas. Este es el primer informe del uso de plantas medicinales como agente citoprotector en el modelo bacteriano.

Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats

The present study was carried out in order to compare the effects of administration of organic (methylmercury, MeHg) and inorganic (mercury chloride, HgCl 2 ) forms of mercury on in vivo dopamine (DA) release from rat striatum. Experiments were performed in conscious and freely moving female adult Sprague-Dawley (230-280 g) rats using brain microdialysis coupled to HPLC with electrochemical detection. Perfusion of different concentrations of MeHg or HgCl 2 (2 muL/min for 1 h, N = 5-7/group) into the striatum produced significant increases in the levels of DA. Infusion of 40 muM, 400 muM, or 4 mM MeHg increased DA levels to 907 ± 31, 2324 ± 156, and 9032 ± 70 percent of basal levels, respectively. The same concentrations of HgCl 2 increased DA levels to 1240 ± 66, 2500 ± 424, and 2658 ± 337 percent of basal levels, respectively. These increases were associated with significant decreases in levels of dihydroxyphenylacetic acid and homovallinic acid. Intrastriatal administration of MeHg induced a sharp concentration-dependent increase in DA levels with a peak 30 min after injection, whereas HgCl 2 induced a gradual, lower (for 4 mM) and delayed increase in DA levels (75 min after the beginning of perfusion). Comparing the neurochemical profile of the two mercury derivatives to induce increases in DA levels, we observed that the time-course of these increases induced by both mercurials was different and the effect produced by HgCl 2 was not concentration-dependent (the effect was the same for the concentrations of 400 muM and 4 mM HgCl 2 ). These results indicate that HgCl 2 produces increases in extracellular DA levels by a mechanism differing from that of MeHg.