In vivo chronic exposure to inorganic mercury worsens hypercholesterolemia, oxidative stress and atherosclerosis in the LDL receptor knockout mice.

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Consequences of thimerosal on human erythrocyte hemoglobin: Assessing functional and structural protein changes induced by an organic mercury compound.

BACKGROUND: Thimerosal (TM) is an organic mercury compound used as a preservative in many pharmacological inputs. Mercury toxicity is related to structural and functional changes in macromolecules such as hemoglobin (Hb) in erythrocytes (Ery). METHOD: Human Hb and Ery were used to evaluate O2 uptake based on the TM concentration, incubation time, and temperature. The influence of TM on the sulfhydryl content, production of reactive oxygen species (ROS), and membrane fragility was also evaluated. Raman spectra and atomic force microscopy (AFM) profiles for Ery in the presence and absence of TM were calculated, and docking studies were performed. RESULTS: At 37 °C, with 2.50 µM TM (higher concentration) and after 5 min of incubation in Hb and Ery, we observed a reduction in O2 uptake of up to 50 %, while HgCl2, which was used as a positive control, showed a reduction of at least 62 %. Total thiol assays in the presence of NEM (thiol blocker) quantified the preservation of almost 60 % of free SH in Ery. Based on the Raman spectrum profile from Ery-TM, structural differences in the porphyrinic ring and the membrane lipid content were confirmed. Finally, studies using AFM showed changes in the morphology and biomechanical properties of Ery. Theoretical studies confirmed these experimental results and showed that the cysteine (Cys) residues present in Hb are involved in the binding of TM. CONCLUSION: Our results show that TM binds to human Hb via free Cys residues, causing conformation changes and leading to harmful effects associated with O2 transport.

Impact of environmental mercury exposure on the blood cells oxidative status of fishermen living around Mundaú lagoon in Maceió - Alagoas (AL), Brazil.

Mercury in the aquatic environment can lead to exposure of the human population and is a known toxic metal due to its capacity for accumulation in organs. We aimed to evaluate the mercury level in the blood and urine of fishermen and correlate it with the level of oxidative stress in blood cells. We show in this case-control study that the fishermen of the exposed group (case) of Mundaú Lagoon (Maceió - Alagoas, Brazil) have higher concentrations of total mercury in the blood (0.73-48.38 µg L-1) and urine (0.430-10.2 µg L-1) than the total mercury concentrations in blood (0.29-17.30 µg L-1) and urine (0.210-2.65 µg L-1) of the control group. In the blood cells of fishermen, we observed that the lymphomononuclear cells produced high levels of reactive oxygen species (61.7%), and the erythrocytes presented increased lipid peroxidation (151%) and protein oxidation (41.0%) and a decrease in total thiol (36.5%), GSH and the REDOX state (16.5%). The activity of antioxidant system enzymes (SOD, GPx, and GST) was also reduced in the exposed group by 26.9%, 28.3%, and 19.0%, respectively. Furthermore, hemoglobin oxygen uptake was decreased in the exposed group (40.0%), and the membrane of cells presented increased osmotic fragility (154%) compared to those in the control group. These results suggest that mercury in the blood of fishermen can be responsible for causing impairments in the oxidative status of blood cells and is probably the cause of the reduction in oxygen uptake capacity and damage to the membranes of erythrocytes.

Evaluation of gold nanorods toxicity on isolated mitochondria.

Gold nanorods (AuNRs) have been studied extensively in biomedicine due to their biocompatibility and their unique properties. Some studies reported that AuNRs selectively accumulate on cancer cell mitochondria causing its death. However, the immediate effects of this accumulation needed further investigations. In this context, we evaluated the effect of AuNRs on the mitochondrial integrity of isolated rat liver mitochondria. We verified that AuNRs decreased the mitochondrial respiratory ratio by decreasing the phosphorylation and maximal states. Additionally, AuNRs caused a decrease in the production of mitochondrial ROS and a delay in mitochondrial swelling. Moreover, even with cyclosporine A treatment, AuNRs disrupted the mitochondrial potential. With the highest concentration of AuNRs studied, disorganized mitochondrial crests and intermembrane separation were observed in TEM images. These results indicate that AuNRs can interact with mitochondria, disrupting the electron transport chain. This study provides new evidence of the immediate effects of AuNRs on mitochondrial bioenergetics.

Spontaneous experimental atherosclerosis in hypercholesterolemic mice advances with ageing and correlates with mitochondrial reactive oxygen species.

Ageing and atherosclerosis are associated with oxidative stress. Mitochondrial redox function declines with ageing. Here we tested whether ageing LDL receptor knockout mice (LDLr-/-) develop spontaneous atherosclerosis and whether mitochondrial reactive oxygen species (mtROS) correlate with atherosclerosis. Compared with young mice, aged LDLr-/- mice exhibited 20-fold larger aortic lesion size, although the plasma cholesterol levels did not vary between age groups. The lesion sizes increased exponentially from 3 to 24months of age (r=0.92, p=0.0001) and were correlated with mtROS across the age range (r=0.81, p=0.0001). Thus, LDLr-/- mice develop spontaneous diet-independent atherosclerosis, that advances exponentially with ageing. We propose that age related increases in mtROS contribute to accelerate atherosclerosis development in hypercholesterolemic mice.

The effect of maternal low-protein diet on the heart of adult offspring: role of mitochondria and oxidative stress.

Protein restriction during perinatal and early postnatal development is associated with a greater incidence of disease in the adult, such arterial hypertension. The aim in the present study was to investigate the effect of maternal low-protein diet on mitochondrial oxidative phosphorylation capacity, mitochondrial reactive oxygen species (ROS) formation, antioxidant levels (enzymatic and nonenzymatic), and oxidative stress levels on the heart of the adult offspring. Pregnant Wistar rats received either 17% casein (normal protein, NP) or 8% casein (low protein, LP) throughout pregnancy and lactation. After weaning male progeny of these NP or LP fed rats, females were maintained on commercial chow (Labina-Purina). At 100 days post-birth, the male rats were sacrificed and heart tissue was harvested and stored at -80 °C. Our results show that restricting protein consumption in pregnant females induced decreased mitochondrial oxidative phosphorylation capacity (51% reduction in ADP-stimulated oxygen consumption and 49.5% reduction in respiratory control ratio) in their progeny when compared with NP group. In addition, maternal low-protein diet induced a significant decrease in enzymatic antioxidant capacity (37.8% decrease in superoxide dismutase activity; 42% decrease in catalase activity; 44.8% decrease in glutathione-S-transferase activity; 47.9% decrease in glutathione reductase; 25.7% decrease in glucose-6 phosphate dehydrogenase) and glutathione level (34.8% decrease) when compared with control. From these findings, we hypothesize that an increased production of ROS and decrease in antioxidant activity levels induced by protein restriction during development could potentiate the progression of metabolic and cardiac diseases in adulthood.

Mitochondria generated nitric oxide protects against permeability transition via formation of membrane protein S-nitrosothiols.

Mitochondria generated nitric oxide (NO) regulates several cell functions including energy metabolism, cell cycling, and cell death. Here we report that the NO synthase inhibitors (L-NAME, L-NNA and L-NMMA) administered either in vitro or in vivo induce Ca2+-dependent mitochondrial permeability transition (MPT) in rat liver mitochondria via a mechanism independent on changes in the energy state of the organelle. MPT was determined by the occurrence of cyclosporin A sensitive mitochondrial membrane potential disruption followed by mitochondrial swelling and Ca2+ release. In in vitro experiments, the effect of NOS inhibitors was dose-dependent (1 to 50 microM). In addition to cyclosporin A, L-NAME-induced MPT was sensitive to Mg2+ plus ATP, EGTA, and to a lower degree, to catalase and dithiothreitol. In contrast to L-NAME, its isomer D-NAME did not induce MPT. L-NAME-induced MPT was associated with a significant decrease in both the rate of NO generation and the content of mitochondrial S-nitrosothiol. Acute and chronic in vivo treatment with L-NAME also promoted MPT and decreased the content of mitochondrial S-nitrosothiol. SNAP (a NO donor) prevented L-NAME mediated MPT and reversed the decrease in the rate of NO generation and in the content of S-nitrosothiol. We propose that S-nitrosylation of critical membrane protein thiols by NO protects against MPT.