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

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.

Carcinogenic Risk from Lead and Cadmium Contaminating Cow Milk and Soya Beverage Brands Available in the Portuguese Market.

Our previous work demonstrated the presence of lead (Pb) and cadmium (Cd) contamination in cow milk (CM) and soy beverages (SBs) in Portugal. These metals share carcinogenic mechanisms, suggesting at least additive effects. Our goals were to assess carcinogenic risks from Pb and Cd intake detected in various CM and SB brands on the Portuguese market and to determine the relative contributions of Pb and Cd. Furthermore, we modeled different consumption scenarios for various age/body weight groups to estimate cumulative Excess Lifetime Carcinogenic Risk (ELCR). ELCR was computed by multiplying chronic daily intake by a cancer slope factor for each metal, with an ELCR > 1 × 10-4 indicating carcinogenic risk. Five CM and three SB brands posed cancer risks in children, with the highest values at 1.75 × 10-4 and 9.12 × 10-5, respectively; Pb had mean relative contributions of 87.8 ± 3.1% in CM and 54.9 ± 12.1% in SB. Carcinogenic risks were observed for children, adolescents, and adults in several CM or SB consumption scenarios, albeit at levels above typical Portuguese intakes. Strict monitoring of metal levels, such as Pb and Cd, is advised because CM is a component of many foods, including baby food.

Exploring the Relationship between Mediterranean Diet Adherence and Subjective Well-Being among Greek and Cypriot Adults.

Associations between subjective well-being (SWB) and dietary habits, employment status, and habitual activities are increasingly capturing the focus of researchers as well as policymakers worldwide. This study aimed to explore these associations in a sample of the population in Greece and Cyprus via an online survey. In total, 936 questionnaires (470: Cyprus, 466: Greece) were analyzed to study the associations between the Mediterranean Diet (MD) (using the 14-item MEDAS score, (14-MEDAS)), subjective well-being (SWB), and several socioeconomic factors. Key remarks of this survey highlight the positive impact of MD adherence on some well-being items. Namely, statistically significant differences were found on the following items: Satisfied with life (p < 0.001), Life worthwhile (p < 0.001), Feeling happy (p < 0.001), worried (p = 0.005), and depressed (p = 0.001), when comparing Low MD adherence (14-MEDAS < 5) to High MD adherence (14-MEDAS > 10). Other lifestyle habits such as spending time with friends and family, spending time in nature, and habitual physical activity were associated with aspects of SWB such as Life satisfaction, Life worthwhile, Feeling happy, and energetic. The findings support adherence to the MD, since it is associated with higher life satisfaction and self-reported happiness in this sample and should be considered when developing health policies on well-being.

Effects of Insect Consumption on Human Health: A Systematic Review of Human Studies.

Insects have been consumed as food in diverse cultures worldwide, gaining recognition as a sustainable and nutritious food source. This systematic review aims to update information on the impact of insect consumption on human health based on human randomized controlled trials (RCTs) and allergenicity assessment studies. Following PRISMA guidelines, studies published in the last 10 years were analyzed. From one-thousand and sixty-three retrieved references, nine RCTs and five allergenicity studies were analyzed. Post-prandial amino acid levels increased after insect protein consumption. In comparison with other protein sources, insect protein showed no significant differences in the area under the curve (AUC) values for essential amino acids but tended to have lower peaks and peak later. In terms of muscle protein synthesis, there were no significant differences between insect protein and other protein sources. Glucose levels did not differ; however, insulin levels were lower after the consumption of insect-based products. The effects on inflammatory markers and microbiota composition were inconclusive and the studies did not show significant effects on appetite regulation. Allergenicity assessments revealed a sensitisation and cross-reactivity between insect allergens and known allergens. A partial reduction of cross-allergenicity was observed via thermal processing. Insect protein is an adequate protein source with promising health benefits; however, further research is needed to fully understand its potential and optimise its inclusion into the human diet.

Metals in Cow Milk and Soy Beverages: Is There a Concern?

Nowadays, there is an increased consumption of plant-based protein beverages like soy beverages (SBs) as substitutes for cow milk (CM). Both accumulate toxic metals like lead (Pb), cadmium (Cd), and manganese (Mn), which, although essential, are neurotoxic at high levels. Metals can also perturb the normal development of children. This work aimed to evaluate these metal concentrations in CM and SB purchased on the Portuguese market. After validation of the method, linearity of calibration curves, work range, detection and quantification limits, and selectivity, metals were determined in 14 CM and 14 SB brands using atomic absorption spectrometry. The values were compared between CM and SB and with permissible limit values. Soy beverages had significantly (p < 0.05) higher concentrations of Cd (5.6 ± 4.2 µg/L) and Mn (117.4 ± 30.3) µg/L) than CM (2.15 ± 1.84 µg/L and 5.93 ± 1.21 µg/L, respectively); the Pb concentrations in CM (19.3 ± 12.1 µg/L) were not significantly (p > 0.05) higher than in SB (13.4 ± 9.6 µg/L). These values were similar to other studies and close to but under permissible limit values. Nevertheless, due to the toxicity and bioaccumulation of metals, the fact that these foods are routinely ingested by all ages, mainly children, and represent key ingredients in many processed foods, including baby foods, we suggest strict surveying of metal levels in CM and SBs.

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.

Contribution of cereals and cows' milk consumption to the exposure to mycotoxins: a study with Portuguese children.

The ubiquitous occurrence of mycotoxins in the environment results in unavoidable and repeated human exposure to mixtures of mycotoxins, the main exposure being through the consumption of contaminated foods, such as cereals and milk. Considering the frequency of contamination of these foods with mycotoxins, this study aimed to evaluate the risk of exposure to aflatoxins, ochratoxin A, deoxynivalenol and zearalenone in a Portuguese population under 17 years old through the consumption of these foods. To assess mycotoxin exposure, food contamination data was provided by the Official National Plan to Control Food and the food consumption information from the National Food, Nutrition, and Physical Activity Survey of the Portuguese General Population (2015-2016); risk assessment calculations were performed through the Monte Carlo probabilistic method. In view of the results obtained for aflatoxins, ochratoxin A, deoxynivalenol and zearalenone, and considering the legislation in force, the levels observed were below the maximum levels. However, there was a risk in deoxynivalenol exposure for children from 0 to 9 years old (average and high consumers), and for high consumers from 10 to 17 years old. Given the potential adverse effects of these mycotoxins, their co-existence in the same foods and being a priority issue defined by the European Food Safety Authority, tight control should be carried out, in addition to re-evaluation of the maximum levels of these mycotoxins.

Exploring Hedonic and Eudaimonic Items of Well-Being in Mediterranean and Non-Mediterranean Countries: Influence of Sociodemographic and Lifestyle Factors.

Increased understanding of subjective well-being (SWB), as well as factors that influence it, are essential to enhance well-being at the individual and national level. We have applied a hedonic and eudaimonic 9-item composed tool (SWB score) to measure SWB across several Mediterranean (MED) and non-Mediterranean (non-MED) countries, and to explore the association between the SWB score and a range of sociodemographic, health and Mediterranean lifestyle factors. A specifically designed web-based questionnaire was distributed to adult participants (N = 2400) from Spain, Italy, Portugal, Bulgaria and Republic of North Macedonia. Results showed that the SWB score was significantly different across the examined countries with the MED participants displaying slightly higher average scores than the non-MED ones (6.3 ± 1.5 vs. 6.1 ± 1.6, p = 0.002). Several sociodemographic, health status and lifestyle factors displayed a significant but limited association with the 9-item SWB score, with a multiple regression model explaining around 17% of the variance. Nevertheless, our results support that a closer adherence to Mediterranean lifestyle habits-the Mediterranean Diet, spending time with friends, family, and in nature, being active, and getting adequate rest at night-has a positive influence on the 9-item SWB score. Further research is needed to advance the understanding of the measuring and differentiating of SWB across different populations and to establish all the factors that influence it.

NEUROTOXICITY OF METAL MIXTURES.

Environmental exposures and/or alterations in the homeostasis of essential transition metals (ETM), such as Fe, Cu, Zn or Mn, are known to contribute to neurodegenerative diseases (ND), such as Alzheimer's Disease (AD) and Parkinson's Disease (PD). Aberrant ETM homeostasis leads to altered distributions, as significant amounts may accumulate in specific brain areas, while causing metal deficiency in others. The disruption of processes reliant on the interplay between these ETM, may lead to loss of metal balance and the ensuing neurotoxicity via shared mechanisms, such as the induction of oxidative stress (OS). Both ETM imbalance and OS may play a role, via complex positive loop processes, in primary neuropathological signatures of AD, such as the accumulation of amyloid plaques and neurofibrillary tangles (NTF), and in PD, α-Syn aggregation and loss of dopamine(DA)rgic neurons. The association between ETM imbalance and ND is rarely approached under the view that metals such as Fe, Cu, Zn and Mn, can act as dangerous endogenous neurotoxic mixtures when their control mechanisms became disrupted. In fact, their presence as mixtures implies intricacies, which should be kept in mind when developing therapies for complex disorders of metal dyshomeostasis, which commonly occur in ND.

Persistent Moderate-to-Weak Mediterranean Diet Adherence and Low Scoring for Plant-Based Foods across Several Southern European Countries: Are We Overlooking the Mediterranean Diet Recommendations?

The Mediterranean diet (MD) has been sponsored worldwide as a healthy and sustainable diet. Our aim was to update and compare MD adherence and food choices across several Southern European countries: Spain (SP), Portugal (PT), Italy (IT), Greece (GR), and Cyprus (CY) (MED, Mediterranean), and Bulgaria (BG) and the Republic of North Macedonia (NMK) (non-MED, non-Mediterranean). Participants (N = 3145, ≥18 y) completed a survey (MeDiWeB) with sociodemographic, anthropometric, and food questions (14-item Mediterranean Diet Adherence Screener, 14-MEDAS). The MED and non-MED populations showed moderate (7.08 ± 1.96) and weak (5.58 ± 1.82) MD adherence, respectively, with significant yet small differences across countries (SP > PT > GR > IT > CY > BG > NMK, p-value < 0.001). The MED participants scored higher than the non-MED ones for most of the Mediterranean-typical foods, with the greatest differences found for olive oil (OO) and white meat preference. In most countries, ≥70% of the participants reported quantities of red meat, butter, sweet drinks, and desserts below the recommended cutoff points, whereas <50% achieved the targets for plant-based foods, OO, fish, and wine. Being a woman and increasing age were associated with superior adherence (p-value < 0.001), but differences were rather small. Our results suggest that the campaigns carried out to support and reinforce the MD and to promote plant-based foods have limited success across Southern Europe, and that more hard-hitting strategies are needed.

Metal environmental contamination within different human exposure context- specific and non-specific biomarkers.

Exposure to high levels of persistent pollutants, such as metal mixtures, is commonly encountered by the general population especially in industrialized countries. The aim of this work was to evaluate how metal pollution in contaminated areas is reflected in terms of biomarkers (BMs) of exposure and effect in human sub-populations living in distinct non-occupational environmental contexts. Thus, four Portuguese sub-populations living in different areas of Portugal were studied: i- the exposure of each member of these sub-populations to lead (Pb), manganese (Mn) and arsenic (As) was evaluated by determining metal levels in urine; ii- biochemical changes were assessed, establishing the levels of urinary metabolites of heme biosynthesis; iii- the ability of combinations of these BMs to predict the context of exposure of each subject was tested, as to develop a tool to identify adverse health effects in these environmentally exposed populations. Concerning the combinations of BMs, heme precursors in urine (delta-aminolevulinic acid and porphyrins), were predictive of contexts of environmental exposures, with 94.2% of the studied subjects correctly identified as to their sub-population origin. The use of non-specific BMs may affirm the exposure to Pb, Mn and As, also reflecting health effects induced by a chemical environmental mixture. Our studies affirm the difficulty in establishing a metal reference population.

Exploring the Validity of the 14-Item Mediterranean Diet Adherence Screener (MEDAS): A Cross-National Study in Seven European Countries around the Mediterranean Region.

This study provides comprehensive validation of the 14-item Mediterranean Diet Adherence Screener (14-MEDAS) in an adult population from Greece (GR), Portugal (PT), Italy (IT), Spain (SP), Cyprus (CY), Republic of North Macedonia (NMK), and Bulgaria (BG). A moderate association between the 14-MEDAS and the reference food diary was estimated for the entire population (Pearson r = 0.573, p-value < 0.001; Intraclass Correlation Coefficient (ICC) = 0.692, p-value < 0.001) with the strongest correlation found in GR, followed by PT, IT, SP, and CY. These results were supported by kappa statistics in GR, PT, IT, and SP with ≥50% of food items exhibiting a fair or better agreement. Bland-Altman analyses showed an overestimation of the 14-MEDAS score in the whole population (0.79 ± 1.81, 95%Confidence Interval (CI) 0.61, 0.96), but this value was variable across countries, with GR, NMK, and BG exhibiting the lowest bias. Taking all analyses together, the validation achieved slightly better results in the Mediterranean countries but a definitive validation ranking order was not evident. Considering growing evidence of the shift from Mediterranean Diet (MD) adherence and of the importance of culture in making food choices it is crucial that we further improve validation protocols with specific applications to compare MD adherence across countries.

Mediterranean Diet Adherence and Subjective Well-Being in a Sample of Portuguese Adults.

The Mediterranean diet (MD) and other lifestyle characteristics have been associated with well-being, a broad multiparameter concept that includes individual's subjective assessment of their own well-being (SWB). Some studies have suggested that diet influences SWB, thus, this work aimed to add novel information on the association of MD and SWB in a sample of Portuguese adults. Data on sociodemographic, economic, lifestyle, diet, and SWB were collected through a self-filled online questionnaire. MD adherence was assessed by the Mediterranean Diet Adherence Screener (MEDAS) score ]. Results showed a moderate adherence to the MD in 490 Portuguese adults (mean MEDAS of 7.4 ± 2.1). A higher MD adherence was found to be significantly positively associated with women, employed individuals, a higher number of meals per day, and those with frequent contact with nature (p-value < 0.0025, using Bonferroni adjustment). As a novelty, this study divided the participants into low SWB, medium SWB, and medium to high SWB profiles (3.9 ± 1.0; 6.2 ± 1.0; 8.2 ± 1.3, respectively; p-value < 0.05), which reported significantly increasing MEDAS scores (6.5 ± 2.1; 7.3 ± 2.1; 7.8 ± 1.9; respectively, p-value < 0.05).

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.

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.

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.

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.