Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese.

Pb, As and Mn are neurotoxic metals, present as mixtures at various settings. All metals are known to interfere with cholinergic/dopaminergic neurotransmission and motor function. The main objective of this work was to assess metal mixture effects of lead (Pb), arsenic (As) and manganese (Mn) on motor activity, and to evaluate the role of each mixture component as well as their additive/synergic interactions on dopaminergic and cholinergic neurotransmission. Wistar rats were treated with 8 doses of each single metal, Pb, As and Mn, or a triple metal mixture. Motor activity was evaluated along with cholinergic/dopaminergic neurotransmission, using brain acetylcholinesterase (AChE-Br) activity and serum prolactin (PRL-S) levels, respectively. Brain concentrations of Pb, As, Mn were also quantified. The metal mixture induced decreased motor activity relative to all other groups with factor analysis revealing close proximity between AChE-Br and motor activity. Pb brain levels increased significantly as compared to all the other groups, while ß coefficients of multiple regression showed that this metal was the most effective in changing AChE-Br. Significant effects of interactions among the three metals on the activity of this enzyme were also noted for the metal mixture. In conclusion, co-exposure to Pb, As and Mn mixture alters the cholinergic system and motor activity to a greater extent than the dopaminergic system. Additive/synergic interactions between Pb, As and Mn may have a relevant role in mediating these events.

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.

Alternative biomarkers of n-hexane exposure: characterization of aminoderived pyrroles and thiol-pyrrole conjugates in urine of rats exposed to 2,5-hexanedione.

The identification of pyrrole derivatives in urine of rats exposed to 2,5-hexanedione (2,5-HD), was performed to select an adequate peripheral biomarker predictive of 2,5-HD neurotoxicity. Studies on molecular mechanism of 2,5-HD neurotoxicity have revealed that 2,5-hexanedione reacts with free amino groups of lysine in proteins forming primary pyrrole adducts, which may autoxidize and form pyrrole dimers, responsible for protein crosslinking in neurofilaments, or react with sulfhydryl groups of cysteine in peptides and proteins, forming secondary pyrrole adducts, which probably may inhibit the process responsible by 2,5-HD neurotoxicity. In this work, the analysis of excreted 2,5-HD and pyr-role derivatives in urine of rats i.p. treated with 3 doses of 2,5-HD (400 mg/kg bw/48 h) was performed using ESI-LC-MS/MS. Several pyrrole compounds were identified, namely dimethylpyrrole norleucine(DMPN), cysteine-pyrrole conjugate (DMPN NAC), glutathione-pyrrole conjugate (DMPN GSH) and 2,5-dimethylpyrrole (2,5-DMP). Additionally, free and total 2,5-HD, DMPN and DMPN NAC were quantified. The observed results suggest that DMPN is a sensitive and specific indicator of repeated exposure to 2,5-HD.

Evaluation of neurobehavioral and neuroinflammatory end-points in the post-exposure period in rats sub-acutely exposed to manganese.

Manganese (Mn) can cause manganism, a neurological disorder similar to Parkinson' Disease (PD). The neurobehavioral and neuroinflammatory end-points in the Mn post exposure period have not been studied yet. Rats were injected on alternate days with 8 doses of MnCl2 (25mg/kg) or saline, then euthanized 1, 10, 30 or 70 days following the last dose. Whole-blood (WB) (p<0.05), urine (p<0.05) and brain cortical (p<0.0001) Mn levels were significantly increased 24h after the last dose. Decreases in the rats' ambulation were noted 1, 10 and 30 days after the last Mn dose (p<0.001; p<0.05; p<0.001, respectively) and also in the rearing activity at the four time-points (p<0.05). Cortical glial fibrillary acid protein immunoreactivity (GFAP-ir) was significantly increased at 1, 10, 30 (p<0.0001) and 70 (p<0.001) days after the last Mn dose, as well as tumor necrosis α (TNF-α) levels (p<0.05) but just on day 1. Taken together, the results show that, during the 70-day clearance phase of Mn, the recovery is not immediate as behavioral alterations and neuroinflammation persist long after Mn is cleared from the cortical brain compartment.

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.

Comparison between 5-aminosalicylic acid (5-ASA) and para-aminosalicylic acid (4-PAS) as potential protectors against Mn-induced neurotoxicity.

Manganese (Mn) is an essential metal for biological systems; however, occupational or clinical exposure to high levels of Mn can produce a neurological disorder called manganism. Oxidative stress and neuroinflammation play major roles in the Mn-induced neurodegeneration leading to dysfunction of the basal ganglia. We investigated the toxic effects of MnCl2 in an immortalized rat brain endothelial cell line (RBE4) and the protective effects of the radical scavenging aminosalicylic acids, 5-aminosalicylic acid (5-ASA) and 4-aminosalicylic acid (4-PAS). Mn cytotoxicity was determined with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) activity. A significant decrease in MTT reduction concomitant with increased LDH release was noted in RBE4 cells exposed for 24 h to MnCl2 (600 and 800 µM; p < 0.0001). Our results establish that compared to 4-PAS, 5-ASA has greater efficacy in protecting RBE4 cells from Mn-induced neurotoxicity after preexposure to MnCl2 800 µM (p < 0.0001).

Manganese alters rat brain amino acids levels.

Manganese (Mn) is an essential element and it acts as a cofactor for a number of enzymatic reactions, including those involved in amino acid, lipid, protein, and carbohydrate metabolism. Excessive exposure to Mn can lead to poisoning, characterized by psychiatric disturbances and an extrapyramidal disorder. Mn-induced neuronal degeneration is associated with alterations in amino acids metabolism. In the present study, we analyzed whole rat brain amino acid content subsequent to four or eight intraperitoneal injections, with 25 mg MnCl2/kg/day, at 48-h intervals. We noted a significant increase in glycine brain levels after four or eight Mn injections (p < 0.05 and p < 0.01, respectively) and arginine also after four or eight injections (p < 0.001). Significant increases were also noted in brain proline (p < 0.01), cysteine (p < 0.05), phenylalanine (p < 0.01), and tyrosine (p < 0.01) levels after eight Mn injections vs. the control group. These findings suggest that Mn-induced alterations in amino acid levels secondary to Mn affect the neurochemical milieu.

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.

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.

Antioxidants prevent the cytotoxicity of manganese in RBE4 cells.

Manganese (Mn) is an essential trace element required for ubiquitous enzymatic reactions. Chronic overexposure to this metal may, however, promote potent neurotoxic effects. The mechanism of Mn toxicity is not well established, but several studies indicate that oxidative stress and mitochondria play major roles in the Mn-induced neurodegenerative processes that lead to dysfunction in the basal ganglia. The aim of this study was to address the toxic effects of MnCl2 and MnSO4 on the immortalized rat brain microvessel endothelial cell line (RBE4) and to characterize toxic mechanism associated with exposure to Mn. The cytotoxicity of Mn in RBE4 cells was evaluated using the LDH and the MTT assays. A significant increase was noted in LDH release from RBE4 cells exposed for 24 h to MnCl2 at concentrations of 800 microM and MnSO4 at concentrations > or = 400 microM (p < 0.05) when compared with control unexposed cells. The MTT assay established significant decrease in cellular viability upon exposure to MnCl2 at concentrations > or = 100 microM and to MnSO4 at concentrations > or = 50 microM (p < 0.05). Thus, the cytotoxicity assays showed that the MTT assay was more sensitive than the LDH assay, suggesting that mitochondrial changes precede other toxic effects of Mn. In addition, upon exposure to MnCl2 (200 and 800 microM), intracellular reduced glutathione (GSH) levels in RBE4 cells decreased as Mn exposure concentrations increased (p < 0.05). To confirm the oxidative hypothesis of Mn cytotoxicity, co-exposure of MnCl2 with antioxidant agents (N-acetylcysteine [NAC] or Trolox) were carried out. The cellular viability was evaluated using the MTT assay. A significant decrease in Mn cytotoxicity was observed in co-exposed cells confirming that (1) oxidative stress plays a critical role in the mechanism of Mn toxicity, and (2) antioxidants may offer a useful therapeutic modality to reverse the aberrant effects of Mn.

Biomarkers of exposure and effect as indicators of the interference of selenomethionine on methylmercury toxicity.

The present study was conducted to clarify the interference of selenomethionine (SeMet) on methylmercury (MeHg) toxicity through the evaluation of changes in biomarkers of exposure and effect in rats exposed to MeHg and co-exposed to MeHg and SeMet. Male Wistar rats received two intraperitoneally (i.p.) administrations, either MeHg (1.5mg/kg body weight), SeMet alone (1mg/kg body weight) or combined MeHg and SeMet, followed by 3 weeks of rat urine collection and neurobehavioural assays. The effects of different administrations were investigated by the quantification of total mercury in kidney and brain, analysis of urinary porphyrins, determination of hepatic GSH and evaluation of motor activity functions (rearing and ambulation). MeHg exposure resulted in a significant increase of urinary porphyrins during the 3 weeks of rat urine collection, where as it caused a significant decrease in motor activity only at the first day after cessation of rat exposure. Additionally, SeMet co-exposure was able to normalize the porphyrins excretion, and a tendency to restore rat motor activity was observed, on the first day after cessation of exposure. Brain and kidney mercury levels increased significantly in rats exposed to MeHg; however, in co-exposed rats to SeMet no significant changes in Hg levels were found as compared to rats exposed to MeHg alone. Hence, the present study shows that urinary porphyrins are sensitive and persistent indicators of MeHg toxicity and demonstrates for the first time that SeMet reduces its formation. Finally, these results confirm that the mechanism of interaction between SeMet and MeHg cannot be explained by the reduction of Hg levels in target organs and suggestions are made to clarify the interference of SeMet on MeHg toxicity.

Evidence of zinc protection against 2,5-hexanedione neurotoxicity: correlation of neurobehavioral testing with biomarkers of excretion.

Risk prevention of human exposure against n-hexane neurotoxicity is relevant towards the protective measures to be proposed in occupational toxicology. Metabolic studies have identified 2,5-hexanedione (2,5-HD) as the main neurotoxic metabolite of n-hexane, which reacts with amino groups of lysine in axonal neurofilaments forming 2,5-dimethylpyrrole adducts, which are responsible for n-hexane neurotoxicity. In the present study, we have investigated the interaction of zinc with 2,5-HD, by correlating the decrease of pyrrole derivatives excretion with changes of neurobehavioral effects. Two subchronic experiments (11 and 8 weeks of exposure) were performed in Wistar rats exposed to different doses of 2,5-HD (200, 400 mg/kg per day) and to the mixture of 2,5-HD + zinc acetate (200 + 300 mg/kg per day) and (400 + 500 mg/kg per day). The results obtained show a significant increase in the excretion of pyrroles in the groups exposed to 2,5-HD alone as compared to controls, and a significant decrease in the excretion of pyrrole derivatives in the groups of rats co-exposed to 2,5-HD + zinc acetate when compared to the rats exposed to 2,5-HD alone. These biochemical changes were immediately evident after the first day of exposure. Simultaneously, neurobehavioral testing (rearing and ambulation in open field) was performed weekly in the same groups of rats. The results demonstrated a significant decrease in neurobehavioral dysfunction in rats co-exposed to 2,5-HD and zinc acetate. At the end of the exposure period, pyrroles levels returned to control values progressively, and the recovery of the neurotoxic effects was gradually established depending on the dose of exposure. The results suggest that zinc is a potential chemo-protector against 2,5-HD neurotoxicity which was identified by neurobehavioral testing. Moreover, pyrrole derivatives are good predictive biochemical biomarkers of 2,5-HD exposure and could be used as a complementary tool to characterize its neurotoxic effects.