From Molecules to Behavior in Long-Term Inorganic Mercury Intoxication: Unraveling Proteomic Features in Cerebellar Neurodegeneration of Rats.

Mercury is a severe environmental pollutant with neurotoxic effects, especially when exposed for long periods. Although there are several evidences regarding mercury toxicity, little is known about inorganic mercury (IHg) species and cerebellum, one of the main targets of mercury associated with the neurological symptomatology of mercurial poisoning. Besides that, the global proteomic profile assessment is a valuable tool to screen possible biomarkers and elucidate molecular targets of mercury neurotoxicity; however, the literature is still scarce. Thus, this study aimed to investigate the effects of long-term exposure to IHg in adult rats' cerebellum and explore the modulation of the cerebellar proteome associated with biochemical and functional outcomes, providing evidence, in a translational perspective, of new mercury toxicity targets and possible biomarkers. Fifty-four adult rats were exposed to 0.375 mg/kg of HgCl2 or distilled water for 45 days using intragastric gavage. Then, the motor functions were evaluated by rotarod and inclined plane. The cerebellum was collected to quantify mercury levels, to assess the antioxidant activity against peroxyl radicals (ACAPs), the lipid peroxidation (LPO), the proteomic profile, the cell death nature by cytotoxicity and apoptosis, and the Purkinje cells density. The IHg exposure increased mercury levels in the cerebellum, reducing ACAP and increasing LPO. The proteomic approach revealed a total 419 proteins with different statuses of regulation, associated with different biological processes, such as synaptic signaling, energy metabolism and nervous system development, e.g., all these molecular changes are associated with increased cytotoxicity and apoptosis, with a neurodegenerative pattern on Purkinje cells layer and poor motor coordination and balance. In conclusion, all these findings feature a neurodegenerative process triggered by IHg in the cerebellum that culminated into motor functions deficits, which are associated with several molecular features and may be related to the clinical outcomes of people exposed to the toxicant.

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.

Low-level fluoride exposure reduces glycemia in NOD mice.

Water fluoridation is the most widespread measure to prevent dental caries but its relationship with the development of type-1 diabetes (T1D), which has been increasing by 2-5% worldwide, is not quite well understood. AIM: This study evaluated if fluoride (F) administered in the drinking water can prevent or reduce the development of T1D in non-obese diabetic (NOD) mice, as well as to explore the underlying mechanisms. MATERIALS AND METHODS: Twenty-four weaning NOD mice received water containing 0, 10 or 50 ppm F for 21 days. Plasma glucose and insulin were analyzed. Quantitative proteomic analysis was conducted in the liver and gastrocnemius muscle. RESULTS: Animals treated with 10 ppm F had significantly lower glucose levels than the control group, but there was no significant difference among the groups in relation to insulin. The % of ß-cell function was significantly higher in the 10 ppm F group. Changes in the proteomic profile of muscle and liver were seen among the groups. In the muscle, the 10 ppm F group presented, when compared with control, increased expression of proteins involved in energy metabolism. The 50 ppm F group, compared with control, presented increased expression of proteins related to muscle contraction, differentiation of brown adipose tissue and apoptosis. For the liver, the 10 ppm F group had increase in proteins involved in energy metabolism and protein synthesis, in respect to control. There was also an increase in isoforms of Glutathione S transferase, which was confirmed by Western blotting. In the group treated with 50 ppm F, proteins related to ROS metabolism and energetic metabolism were altered. CONCLUSION: Increased expression of antioxidant proteins by treatment with low F concentration may possibly help to explain protection against the development of T1D, which should be better explored in future mechanistic studies.

Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis.

The effect of duration of chronic treatment with fluoride (F, 50 mg/L as NaF) on the lipid profile, lipid droplets and triglycerides (TG) in liver was evaluated in mice with nonalcoholic fatty liver disease (NAFLD) previously induced by hyperlipidic diet and in animals fed normocaloric diet. In addition, the effect of F administered for a short period (20 days) was evaluated on de novo lipogenesis, by nuclear magnetic resonance. GRP78, Apo-E, and sterol regulatory element-binding protein (SREBP) were quantified by Western blotting. Our data indicate that F interferes in lipid metabolism and lipid droplets, having a different action depending on the exposure time and type of diet administered. F improved lipid parameters and reduced steatosis only when administered for a short period of time (up to 20 days) to animals fed normocaloric diet. However, when NAFLD was already installed, lipid parameters were only slightly improved at 20 days of treatment, but no effect was observed on the degree of steatosis. In addition, lipid profile was in general impaired when the animals were treated with F for 30 days, regardless of the diet. Moreover, F did not alter de novo lipogenesis in animals with installed NAFLD. Furthermore, hyperlipidic diet increased F accumulation in the body. GRP78 increased, while Apo-E and SREBP decreased in the F-treated groups. Our results provide new insights on how F affects lipid metabolism depending on the available energy source.

Fluoride Intensifies Hypercaloric Diet-Induced ER Oxidative Stress and Alters Lipid Metabolism.

BACKGROUND: Here, we evaluated the relationship of diet and F-induced oxidative stress to lipid metabolism in the liver of rats eating normocaloric or hypercaloric diets for two time periods (20 or 60 days). METHODS: Seventy-two 21-day-old Wistar rats were divided into 2 groups (n = 36) based on the type of diet they were eating; each of these groups was then further divided into another two groups (n = 18) based on the time periods of either 20 or 60 days, for a total of four groups. Each of these was divided into 3 subgroups (n = 6 animals/subgroup), dependent on the dose of F administered in the drinking water (0 mg/L(control), 15 mg/L or 50 mg/L). After the experimental period, blood samples and the liver were collected. Plasma samples were analyzed for HDL, cholesterol and triglycerides. Western blots were performed to probe for GRP78, Erp29, SOD2, Apo-E and SREBP in hepatic tissues. RESULTS: As expected,the expression of target proteins involved in oxidative stress increased in the F-treated groups, especially in liver tissue obtained from animals eating a hypercaloric diet. Most changes in the lipid levels and pathological conditions were seen earlier in the time period, at day 20. The morphometric analyses showed a reduction in steatosis in groups on ahypercaloric diet and treated with 50 mg F/L compared to the control, while no changes were obtained in normocaloric-fed rats. Accordingly, plasma TG was reduced in the F-treated group. The reduced expression of Apo-E in a time- and diet-dependent pattern may account for the particular decrease in steatosis in hypercaloric-fed F-treated rats. CONCLUSIONS: These results suggest that F changes liver lipid homeostasis, possibly because of the induction of oxidative stress, which seems to be higher in animals fed hypercaloric diets.