Vanda Andrade

The inhibitory effect of manganese on acetylcholinesterase activity enhances oxidative stress and neuroinflammation in the rat brain

BACKGROUND Manganese (Mn) is a naturally occurring element and an essential nutrient for humans and animals. However, exposure to high levels of Mn may cause neurotoxic effects. The pathological mechanisms associated with Mn neurotoxicity are poorly understood, but several reports have established it is mediated, at least in part, by oxidative stress. OBJECTIVES The present study was undertaken to test the hypothesis that a decrease in acetylcholinesterase (AChE) activity mediates Mn-induced neurotoxicity. METHODS Groups of 6 rats received 4 or 8 intraperitoneal (i.p.) injections of 25mg MnCl(2)/kg/day, every 48 h. Twenty-four hours after the last injection, brain AChE activity and the levels of F(2)-isoprostanes (F(2)-IsoPs) and F(4)-neuroprostanes (F(4)-NPs) (biomarkers of oxidative stress), as well as prostaglandin E(2) (PGE(2)) (biomarker of neuroinflammation) were analyzed. RESULTS The results showed that after either 4 or 8 Mn doses, brain AChE activity was significantly decreased (p<0.05), to 60 ± 16% and 55 ± 13% of control levels, respectively. Both treated groups exhibited clear signs of neurobehavioral toxicity, characterized by a significant (p<0.001) decrease in ambulation and rearings in open-field. Furthermore, Mn treatment caused a significant increase (p<0.05) in brain F(2)-IsoPs and PGE(2) levels, but only after 8 doses. In rats treated with 4 Mn doses, a significant increase (p<0.05) in brain F(4)-NPs levels was found. To evaluate cellular responses to oxidative stress, we assessed brain nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and Mn-superoxide dismutase (Mn-SOD, SOD2) protein expression levels. A significant increase in Mn-SOD protein expression (p<0.05) and a trend towards increased Nrf2 protein expression was noted in rat brains after 4 Mn doses vs. the control group, but the expression of these proteins was decreased after 8 Mn doses. Taken together, these results suggest that the inhibitory effect of Mn on AChE activity promotes increased levels of neuronal oxidative stress and neuroinflammatory biomarkers.

Protective effects of ebselen (Ebs) and para-aminosalicylic acid (PAS) against manganese (Mn)-induced neurotoxicity

Chronic, excessive exposure to manganese (Mn) may induce neurotoxicity and cause an irreversible brain disease, referred to as manganism. Efficacious therapies for the treatment of Mn are lacking, mandating the development of new interventions. The purpose of the present study was to investigate the efficacy of ebselen (Ebs) and para-aminosalicylic acid (PAS) in attenuating the neurotoxic effects of Mn in an in vivo rat model. Exposure biomarkers, inflammatory and oxidative stress biomarkers, as well as behavioral parameters were evaluated. Co-treatment with Mn plus Ebs or Mn plus PAS caused a significant decrease in blood and brain Mn concentrations (compared to rats treated with Mn alone), concomitant with reduced brain E₂ prostaglandin (PGE₂) and enhanced brain glutathione (GSH) levels, decreased serum prolactin (PRL) levels, and increased ambulation and rearing activities. Taken together, these results establish that both PAS and Ebs are efficacious in reducing Mn body burden, neuroinflammation, oxidative stress and locomotor activity impairments in a rat model of Mn-induced toxicity.

Urinary delta-ALA: a potential biomarker of exposure and neurotoxic effect in rats co-treated with a mixture of lead, arsenic and manganese.

Lead (Pb), arsenic (As) and manganese (Mn) are neurotoxic elements that often occur in mixtures for which practically no information is available on biomarkers (BMs) for the evaluation of exposure/effects. Exposures to these metals may increase delta-aminolevulinic acid (delta-ALA), which in itself may potentiate neurotoxicity. The objective of this study was to investigate the utility of urinary delta-ALA (delta-ALA-U) levels as BM of exposure and/or neurotoxic effects induced by this mixture. Five groups of Wistar rats were treated for 8 days with Pb (5mg/kg), As (60mg/L), Mn (10mg/kg), the 3-metal mixture (same doses of the single metals), and control group. Motor activity was evaluated and 24-h urine collected before and after the treatment. 24-hours (h) after the last dose, the rats were sacrificed and the brains removed for analyses. Delta-ALA and metal levels were determined in brain and urine. Co-treated rats showed a significant (p<0.05) correlation between increased Pb, As, Mn and delta-ALA levels in the brain and decreased motor activity. Delta-ALA-U concentrations were higher in the mixture-treated group than the sum of the delta-ALA-U levels in each single-treated groups and discriminated (p<0.05) between the mixture and untreated rats. Moreover, delta-ALA-U was correlated (p<0.05) with brain delta-ALA levels. These results establish that treatments with this metal mixture exacerbate behavioral dysfunction, increasing most prominently brain Pb levels. This study is the first to establish that delta-ALA-U levels represent a sensitive BM of exposure/neurotoxic effect to this metal mixture.

Changes in rat urinary porphyrin profiles predict the magnitude of the neurotoxic effects induced by a mixture of lead, arsenic and manganese.

The neurotoxic metals lead (Pb), arsenic (As) and manganese (Mn) are ubiquitous contaminants occurring as mixtures in environmental settings. The three metals may interfere with enzymes of the heme bioshyntetic pathway, leading to excessive porphyrin accumulation, which per se may trigger neurotoxicity. Given the multi-mechanisms associated with metal toxicity, we posited that a single biomarker is unlikely to predict neurotoxicity that is induced by a mixture of metals. Our objective was to evaluate the ability of a combination of urinary porphyrins to predict the magnitude of motor activity impairment induced by a mixture of Pb/As/Mn. Five groups of Wistar rats were treated for 8 days with Pb (5mg/kg), As (60 mg/L) or Mn (10mg/kg), and the 3-metal mixture (same doses as the single metals) along with a control group. Motor activity was evaluated after the administration of the last dose and 24-hour (h) urine was also collected after the treatments. Porphyrin profiles were determined both in the urine and brain. Rats treated with the metal-mixture showed a significant decrease in motor parameters compared with controls and the single metal-treated groups. Both brain and urinary porphyrin levels, when combined and analyzed by multiple linear regressions, were predictable of motor activity (p<0.05). The magnitude of change in urinary porphyrin profiles was consistent with the greatest impairments in motor activity as determined by receiver operating characteristic (ROC) curves, with a sensitivity of 88% and a specificity of 96%. Our work strongly suggests that the use of a linear combination of urinary prophyrin levels accurately predicts the magnitude of motor impairments in rats that is induced by a mixture of Pb, As and Mn.

Role of N-acetylcysteine in protecting against 2,5-hexanedione neurotoxicity in a rat model: Changes in urinary pyrroles levels and motor activity performance

The interference of N-acetylcysteine (NAC) on 2,5-hexanedione (2,5-HD) neurotoxicity was evaluated through behavioral assays and the analysis of urinary 2,5-HD, dimethylpyrrole norleucine (DMPN), and cysteine-pyrrole conjugate (DMPN NAC), by ESI-LC-MS/MS, in rats exposed to 2,5-HD and co-exposed to 2,5-HD and NAC. Wistar rats were treated with 4 doses of: 400mg 2,5-HD/kg bw (group I), 400mg 2,5-HD/kg bw+200mg NAC/kg bw (group II), 200mg NAC/kg bw (group III) and with saline (group IV). The results show a significant decrease (p<0.01) in urinary DMPN and free 2,5-HD, a significant increase (p<0.01) in DMPN NAC excretion, and a significant recovery (p<0.01) on motor activity in rats co-exposed to 2,5-HD+NAC, as compared with rats exposed to 2,5-HD alone. Taken together, our findings suggest that at the studied conditions NAC protects against 2,5-HD neurotoxicity and DMPN may be proposed as a new sensitive and specific biomarker of 2,5-HD neurotoxicity in animals treated with a toxic amount of 2,5-hexanedione.

Biomarkers of exposure and effect in a working population exposed to lead, manganese and arsenic

ABSTRACT Lead (Pb), manganese (Mn) and arsenic (As) are among the major toxicants in mining environments. Miners are commonly and repeatedly exposed to this toxic mixture. Some adverse effects may appear at concentrations below environmental quality guidelines for individual mixture components. Further, Pb, Mn, and As induce common adverse outcomes, such as interferences in the cholinergic system and heme synthesis. It is thus vital to monitor miners through biomarkers (BM), such that subclinical effects may be identified at an early stage. The main objectives of this study were to evaluate the exposure of a mining population to these three metals and determine alterations in cholinergic and heme synthesis parameters. Blood and urine samples of workers (n = 60) were obtained from a Portuguese mining industry and compared with a control population (n = 80). The levels of the metals were determined in biological samples, as well as urinary heme precursor levels, delta aminolevulinic acid (ALA) and porphyrins, and blood acetylcholinesterase (AChE) activity. The miners exhibited significantly higher values of Pb and As in blood and urine compared to control. In the case of Mn near or slightly higher than limit values were found. Our data show that heme precursors may be used simultaneously with metal levels as BMs for multiple metal exposures on an individual basis, resulting in 94.3% and 95.7% accuracy, respectively, in blood and urine, for subjects correctly identified with respect to occupation. This study also revealed that biological monitoring of this working population regarding metal body burden and heme precursor accumulation is advisable.