Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with ß-mercaptoethanol (BME) for 24 h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5 days. The cells were exposed to different lead acetate concentrations (0.1-100 µm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100 µg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100 µm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100 µm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

Distribution of Pb and Se in mouse brain following subchronic Pb exposure by using synchrotron X-ray fluorescence.

Lead (Pb) is a well-known neurotoxicant and environmental hazard. Recent experimental evidence has linked Pb exposure with neurological deterioration leading to neurodegenerative diseases, such as Alzheimer's disease. To understand brain regional distribution of Pb and its interaction with other metal ions, we used synchrotron micro-x-ray fluorescence technique (µ-XRF) to map the metal distribution pattern and to quantify metal concentrations in mouse brains. Lead-exposed mice received oral gavage of Pb acetate once daily for 4 weeks; the control mice received sodium acetate. Brain tissues were cut into slices and subjected for analysis. Synchrotron µ-XRF scans were run on the PETRA III P06 beamline (DESY). Coarse scans of the entire brain were performed to locate the cortex and hippocampus, after which scans with higher resolution were run in these areas. The results showed that: a) the total Pb intensity in Pb-exposed brain slices was significantly higher than in control brain; b) Pb typically deposited in localized particles of <10 um2 in both the Pb-exposed and control brain slices, with more of these particles in Pb-exposed samples; c) selenium (Se) was significantly correlated with Pb in these particles in the cortex and hippocampus/corpus callosum regions in the Pb-exposed samples, and the molar ratio of the Se and Pb in these particles is close to 1:1. These results indicated that Se may play a crucial role in Pb-induced neurotoxicity. Our findings call for further studies to investigate the relationship between Pb exposure and possible Se detoxification responses, and the implication in the etiology of Alzheimer's disease.

Chronic developmental lead exposure increases µ-opiate receptor levels in the adolescent rat brain.

Childhood lead (Pb2+) intoxication is a global public health problem best known for producing deficits in learning and poor school performance. Human and preclinical studies have suggested an association between childhood Pb2+ intoxication and proclivity to substance abuse and delinquent behavior. While environmental factors have been implicated in opioid addiction, less is known about the role of exposure to environmental pollutants on the brain opioid system. Opioid receptors are involved in the biological effects of opioids and other drugs of abuse. In this study, we examine the effect of chronic developmental Pb2+ exposure (1500 ppm in the diet) on µ-opioid receptor (MOR) levels in the rat brain using [3H]-d-Ala2-MePhe4-Gly-ol5 enkephalin ([3H]-DAMGO) quantitative receptor autoradiography at different developmental stages (juvenile, early-adolescent, late adolescent and adult) in male and female rats. Our results indicate that chronic developmental Pb2+ exposure increases the levels of [3H]-DAMGO specific binding to MOR in juvenile and early adolescent Pb2+-exposed male and female rat brain with no changes in late-adolescent (PN50) and minor changes in Pb2+-exposed adult male rats (PN120). Specifically, at PN14, Pb2+-exposed males had an increase in MOR binding in the lateral posthalamic nuclei (LPTN), and Pb2+-exposed females had increased MOR binding in LPTN, medial thalamus, and hypothalamus. At PN28, Pb2+-exposed males had increased MOR levels in the striatum, stria medullaris of the thalamus, LPTN, medial thalamus, and basolateral amygdala, while Pb2+-exposed females showed an increase in nucleus accumbens core, LPTN, and medial thalamus. No changes were detected in any brain region of male and female rats at PN50, and at PN120 there was a decrease in MOR binding of Pb2+-exposed males in the medial thalamus. Our findings demonstrate age and gender specific effects of MOR levels in the rat brain as a result of chronic developmental Pb2+ exposure. These results indicate that the major changes in brain MOR levels were during pre-adolescence and early adolescence, a developmental period in which there is higher engagement in reward and drug-seeking behaviors in humans. In summary, we show that chronic exposure to Pb2+, an ubiquitous and well-known environmental contaminant and neurotoxicant, alters MOR levels in brain regions associated with addiction circuits in the adolescent period, these findings have important implications for opioid drug use and abuse.

Amyotrophic lateral sclerosis and lead: A systematic update.

Heavy metals are considered to be among the leading environmental factors that trigger amyotrophic lateral sclerosis (ALS). However, no convincing biopathological mechanism and therapeutic clinical implication of such metals in ALS pathogenesis have been established. This is partly attributable to the technical and scientific difficulties in demonstrating a direct and causative role of heavy metals in the onset of ALS in patients. However, a body of epidemiological, clinical and experimental evidences suggest that lead (Pb), more than other metals, could actually play a major role in the onset and progression of ALS. Here, to clarify the nature of the association and the causative role of Pb in ALS, we comprehensively reviewed the scientific literature of the last decade with objective database searches and the methods typically adopted in systematic reviews, critically analysing and summarising the various scientifically sound evidence on the relationship between ALS and Pb. From these tasks, we noted a number of multidisciplinary associations between ALS and Pb, and specifically the importance of occupational exposure to Pb in ALS development and/or progression. We also report the possible involvement of TAR DNA binding protein (TDP-43)-based molecular mechanism in Pb-mediated ALS, although these data rely on a single study, which included both in vitro experiments and an animal model, and are therefore still preliminary. Finally, we briefly examined whether this knowledge could inspire new targeted therapies and policies in the fight against ALS.

Mitigation of lead neurotoxicity by the ethanolic extract of Laurus leaf in rats.

The present study was conducted in order to assess the chemical composition of Laurus, its antioxidant activities, and benefit from the Laurus extract effect on neurotoxicity caused by lead acetate (Pb). Chemical profile was assayed by using liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS). In this study, 40 male rats were divided into four groups (10 rats per each group): (1) control group, (2) Laurus group: rats treated with 250 mg/kg b. wt. of Laurus leaves extract, (3) Pb group: rats treated with 100 mg/kg b. wt. of lead acetate, (4) Pb + Laurus group: rats treated with 250 mg/kg b. wt. of Laurus leaves extract in addition to lead acetate for 30 days. At the end of experiment, some estimates were calculated from blood samples, brain tissue, and histological examination. The results showed that the extract is highly affluent in total flavonoids, total phenolic, and also has antioxidant activity. The LC-MS appeared a wide range of compounds in the extract. The oxidative stress resulted from exposure to lead acetate has been reported to cause reduction in body and brain weights, levels of RBCs, acetylcholinesterase (AChE), GSH, SOD, and CAT in addition to increase in levels of WBCs and MAD. Moreover, Laurus leaves extract notably lessened the biochemical changes caused by lead acetate in the blood, homogenate, and brain tissue (P < 0.05). The current study indicates the antioxidant activity of Laurus leaves extract and assumes that it has a defensive role against the oxidative damage caused by lead in a rat's brain.

Brain ethanol-metabolizing enzymes are differentially expressed in lead-exposed animals after voluntary ethanol consumption: Pharmacological approaches.

Developmentally-lead (Pb)-exposed rats showed an enhanced vulnerability to the stimulating and motivational effects of ethanol (EtOH). This is accompanied by differential activity of the brain EtOH-metabolizing enzymes catalase (CAT) and mitochondrial aldehyde dehydrogenase (ALDH2). Based on the theory that brain acetaldehyde accumulation is associated with the reinforcing properties of EtOH, this study sought to determine brain CAT and ALDH2 expression in limbic areas of control and Pb-exposed animals after voluntary EtOH intake. Thirty-five-day-old rats perinatally exposed to 220 ppm Pb were offered with water or increasing EtOH solutions (2-10% v/v) during 28 days until postnatal day (PND) 63. Once intake was stable, the animals were administered: 1) saline (SAL; test days 21-24 or 21-28, as corresponds), or 2) a CAT inhibitor: 3-amine 1, 2, 4-triazole (AT; 250 mg/kg intraperitoneally [i.p.], 5 h before the last eight EtOH intake sessions -test days 21-24 and 25-28), or 3) a CAT booster: 3-nitropropionic acid (3NPA; 20 mg/kg subcutaneously [s.c.], 45 min before the last four EtOH intake sessions -test days 25-28). Two additional groups were centrally-administered cyanamide (CY, an ALDH2 inhibitor, 0.3 mg i.c.v. immediately before the last four EtOH sessions, test days 25-28) or its corresponding vehicle (VEH). Lead exposure increased EtOH intake, an effect potentiated in both groups by 3NPA or CY pretreatments and reduced by AT, albeit selectivity in the Pb group. Catalase abundance in limbic areas parallels these observations in the Pb group, showing higher CAT expression in all areas after EtOH consumption respect to the controls, an effect prevented by AT administration. In contrast, ALDH2 expression was reduced in the Pb animals after EtOH intake, with CY potentiating this effect in all brain areas under study. Based on these results and on previous evidences, we suggest that Pb exposure promotes acetaldehyde accumulation in limbic regions, providing some insights into the mechanism of action that underlies the vulnerability to the excessive EtOH consumption reported in these animals.

Pb exposure reduces the expression of SNX6 and Homer1 in offspring rats and PC12 cells.

Lead (Pb) is a widespread environmental heavy metal toxicant and chronic Pb exposure can have irreversible effects on memory and cognitive function, which is closely related to dendritic spines. Studies have shown that SNX6 and Homer1 can regulate the growth of dendritic spines. We aimed to investigate the effect of Pb exposure on the dendritic spines in hippocampus, the expression of SNX6 and Homer1 in rats and PC12 cells. The animals were randomly divided to three groups: control group, low lead group and high lead group. PC12 cells were divided into 3 groups: 0 µM, 1 µM and 100 µM Pb acetate. The results showed that the Pb levels in blood and hippocampus of all exposure groups were significantly higher than that of the control group. The morphology of dendritic spines in hippocampus after Pb treatment was changed and the density of dendritic spines was reduced. The expression of SNX6 and Homer1 was decreased in Pb exposed groups compared with the control group. Furthermore, up-regulation of SNX6 expression could reverse the down-regulation of Pb exposure on Homer1. These results indicate that Pb exposure can reduce the expression of SNX6 and lead to a decrease in Homer1 expression, which affects the changes in dendritic spines causing learning and memory impairment.

Differential protein expression of hippocampal cells associated with heavy metals (Pb, As, and MeHg) neurotoxicity: Deepening into the molecular mechanism of neurodegenerative diseases.

Chronic exposure to heavy metals such as Pb, As, and MeHg can be associated with an increased risk of developing neurodegenerative diseases. Our in vitro bioassays results showed the potency of heavy metals in the order of Pb < As < MeHg on hippocampal cells. The main objective of this study was combining in vitro label free proteomics and systems biology approach for elucidating patterns of biological response, discovering underlying mechanisms of Pb, As, and MeHg toxicity in hippocampal cells. The omics data was refined by using different filters and normalization and multilevel analysis tools were employed to explore the data visualization. The functional and pathway visualization was performed by using Gene ontology and PathVisio tools. Using these all integrated approaches, we identified significant proteins across treatments within the mitochondrial dysfunction, oxidative stress, ubiquitin proteome dysfunction, and mRNA splicing related to neurodegenerative diseases. The systems biology analysis revealed significant alterations in proteins implicated in Parkinson's disease (PD) and Alzheimer's disease (AD). The current proteomics analysis of three metals support the insight into the proteins involved in neurodegeneration and the altered proteins can be useful for metal-specific biomarkers of exposure and its adverse effects. SIGNIFICANCE: The proteomics techniques have been claimed to be more sensitive than the conventional toxicological assays, facilitating the measurement of responses to heavy metals (Pb, As, and MeHg) exposure before obvious harm has occurred demonstrating their predictive value. Also, proteomics allows for the comparison of responses between Pb, As, and MeHg metals, permitting the evaluation of potency differences hippocampal cells of the brain. Hereby, the molecular information provided by pathway and gene functional analysis can be used to develop a more thorough understanding of each metal mechanism at the protein level for different neurological adverse outcomes (e.g. Parkinson's disease, Alzheimer's diseases). Efforts are put into developing proteomics based toxicity testing methods using in vitro models for improving human risk assessment. Some of the key proteins identified can also potentially be used as biomarkers in epidemiologic studies. These heavy metal response patterns shed new light on the mechanisms of mRNA splicing, ubiquitin pathway role in neurodegeneration, and can be useful for the development of molecular biomarkers of heavy metals exposure.

Hemin provides protection against lead neurotoxicity through heme oxygenase 1/carbon monoxide activation.

The neurotoxicity of lead (Pb) is well established, and oxidative stress is strongly associated with Pb-induced neurotoxicity. Heme oxygenase 1 (HO-1) is an important antioxidative enzyme for protection against oxidative stress in many disease models. In this study, we applied hemin, the substrate and a well-known inducer of HO-1, to investigate the possible role of HO-1 in protecting against Pb neurotoxicity. Hemin can significantly attenuate Pb acetate-induced cell death and oxidative stress in the hippocampus and frontal cortex of developmental rats. Consistent with in vivo results, the protective effects of hemin were also observed in SH-SY5Y cells after inducing cell survival and maintaining redox balance. However, knocking down HO-1 could significantly abolish the cytoprotective action of hemin against Pb toxicity, confirming HO-1 contributed to the protection. Finally, the HO-1-derived production of carbon monoxide, but not of bilirubin or Fe2+ , mediated the protective effects of HO-1 activation induced by hemin treatment against Pb-induced cell death and oxidative stress in SHSY5Y cells. Overall, this study showed that hemin provided protection against Pb neurotoxicity by HO-1/carbon monoxide activation.

Lead exposure inhibits expression of SV2C through NRSF.

Lead (Pb) exposure has been shown to affect presynaptic neurotransmitter release in the animal and cell models. The mechanism by which Pb exposure impairs neurotransmitter release remains unknown. In this study, we aimed to investigate the effect of Pb exposure on synaptic vesicle protein 2C (SV2C) and its molecular mechanism. SV2C promoter region contains a neuron-restrictive silencer element (NRSE) binding motif. Neuron-restrictive silencer factor (NRSF) is a transcription repressor that regulates gene expression by binding to NRSE. We also observed whether Pb exposure regulates the transcriptional level of SV2C by influencing the expression of NRSF. Pregnant female rats were exposed to 0, 0.5 and 2.0 g/L lead acetate (PbAc) via drinking water from the first day of gestation until postnatal week 3. Neuro-2a (N2a) cells were divided into 3 groups: 0 µM (control group), 1 µM and 100 µM PbAc. Our data revealed that the ability of learning and memory in Pb-exposed rats were decreased, Pb exposure decreased SV2C expression and increased NRSF expression in the rat hippocampus and N2a cell. Silencing NRSF can reverse the down-regulation of Pb exposure on SV2C. These results indicate that Pb exposure can inhibit the transcription level of SV2C by up regulating the expression of NRSF. Decreased expression of SV2C can affect neurotransmitter release and synaptic transmission, which affect synaptic plasticity and then result in impairment of learning and memory.

Neuroprotective effect of morin on lead acetate- induced apoptosis by preventing cytochrome c translocation via regulation of Bax/Bcl-2 ratio.

Lead (Pb) intoxication is a prevalent type of environmental toxicity as well as minimal amount of lead exposure is liable for neurobehavioral or perhaps intelligence defects. The present study was undertaken to investigate the beneficial effects of morin in protecting the lead acetate (PbAc)-induced oxidative stress in rat brain. PbAc intoxication resulted in motor deficit, memory impairment and oxidative stress Further, PbAc administration alters Bax/Bcl-2 expression thereby increases cytochrome c release from the mitochondria. Treatment with morin at a dose of 40 mg/kg b.wt. significantly restored back the abnormal changes that were noticed in PbAc intoxicated rats. Histopathological sections of cortex, cerebellum and hippocampus showed the extent of neuronal loss in PbAc induced rats and its restoration upon administration of morin. These outcomes imply that morin might be employed therapeutically to chelate toxic metals like Pb, thus possibly lowering PbAc-induced neurotoxicity and tissue damage.

Ascorbic Acid Ameliorates Gestational Lead Exposure-Induced Developmental Alteration in GAD67 and c-Kit Expression in the Rat Cerebellar Cortex.

In the present study, we investigated the effects of ascorbic acid on lead-exposed developing cerebellum. Female rats were divided into the following three groups: control (distilled water), lead (0.2% lead acetate), and lead plus ascorbic acid (100 mg/kg/day, 10% solution). To evaluate the effect of lead exposure and ascorbic acid treatment accurately on the cerebellar development for the gestational period, we halted further treatment with lead and ascorbic acid in the dams after delivery of the pups. Although the ascorbic acid slightly decreased the lead level in pups, lead level was still high in the group treated with lead plus ascorbic acid group compared with the control group. The blood lead levels indicated that the ascorbic acid could facilitate both the excretion and transfer of lead from a dam to its pups via milk. At postnatal day 21, lead exposure significantly reduced the number of Purkinje cells in the cerebellar cortex of pups. Additionally, lead treatment induced degenerative changes such as reduction of glutamic acid decarboxylase (GAD67) and c-kit expressions are observed in the developing cerebellar cortex. In the cerebellum of the pups from the lead plus ascorbic acid group, reduction of the number of Purkinje cells, GAD67 expression, and c-kit immunopositivity were remarkably restored compared with the lead group. Our present results suggested that ascorbic acid treatment to lead-exposed dam exerted protective effects on the developing cerebellum against lead-induced neurotoxicity.

Metals and Paraoxonases.

The paraoxonases (PONs) are a three-gene family which includes PON1, PON2, and PON3. PON1 and PON3 are synthesized primarily in the liver and a portion is secreted in the plasma, where they are associated with high-density lipoproteins (HDLs), while PON2 is an intracellular enzyme, expressed in most tissues and organs, including the brain. PON1 received its name from its ability to hydrolyze paraoxon, the active metabolite of the organophosphorus (OP) insecticide parathion, and also more efficiently hydrolyzes the active metabolites of several other OPs. PON2 and PON3 do not have OP-esterase activity, but all PONs are lactonases and are capable of hydrolyzing a variety of lactones, including certain drugs, endogenous compounds, and quorum-sensing signals of pathogenic bacteria. In addition, all PONs exert potent antioxidant effects. PONs play important roles in cardiovascular diseases and other oxidative stress-related diseases, modulate susceptibility to infection, and may provide neuroprotection (PON2). Hence, significant attention has been devoted to their modulation by a variety of dietary, pharmacological, lifestyle, or environmental factors. A number of metals have been shown in in vitro, animal, and human studies to mostly negatively modulate expression of PONs, particularly PON1, the most studied in this regard. In addition, different levels of expression of PONs may affect susceptibility to toxicity and neurotoxicity of metals due to their aforementioned antioxidant properties.

Occupational Metal Exposure and Parkinsonism.

Parkinsonism is comprised of a host of neurological disorders with an underlying clinical feature of movement disorder, which includes many shared features of bradykinesia, tremor, and rigidity. These clinical outcomes occur subsequent to pathological deficits focused on degeneration or dysfunction of the nigrostriatal dopamine system and accompanying pathological inclusions of alpha-synuclein and tau. The heterogeneity of parkinsonism is equally matched with the complex etiology of this syndrome. While a small percentage can be attributed to genetic alterations, the majority arise from an environmental exposure, generally composed of pesticides, industrial compounds, as well as metals. Of these, metals have received significant attention given their propensity to accumulate in the basal ganglia and participate in neurotoxic cascades, through the generation of reactive oxygen species as well as their pathogenic interaction with intracellular targets in the dopamine neuron. The association between metals and parkinsonism is of critical concern to subsets of the population that are occupationally exposed to metals, both through current practices, such as mining, and emerging settings, like E-waste and the manufacture of metal nanoparticles. This review will explore our current understanding of the molecular and pathological targets that mediate metal neurotoxicity and lead to parkinsonism and will highlight areas of critical research interests that need to be addressed.

Neurotoxicity of Metal Mixtures.

Metals are the oldest toxins known to humans. Metals differ from other toxic substances in that they are neither created nor destroyed by humans (Casarett and Doull's, Toxicology: the basic science of poisons, 8th edn. McGraw-Hill, London, 2013). Metals are of great importance in our daily life and their frequent use makes their omnipresence and a constant source of human exposure. Metals such as arsenic [As], lead [Pb], mercury [Hg], aluminum [Al] and cadmium [Cd] do not have any specific role in an organism and can be toxic even at low levels. The Substance Priority List of Agency for Toxic Substances and Disease Registry (ATSDR) ranked substances based on a combination of their frequency, toxicity, and potential for human exposure. In this list, As, Pb, Hg, and Cd occupy the first, second, third, and seventh positions, respectively (ATSDR, Priority list of hazardous substances. U.S. Department of Health and Human Services, Public Health Service, Atlanta, 2016). Besides existing individually, these metals are also (or mainly) found as mixtures in various parts of the ecosystem (Cobbina SJ, Chen Y, Zhou Z, Wub X, Feng W, Wang W, Mao G, Xu H, Zhang Z, Wua X, Yang L, Chemosphere 132:79-86, 2015). Interactions among components of a mixture may change toxicokinetics and toxicodynamics (Spurgeon DJ, Jones OAH, Dorne J-L, Svendsen C, Swain S, Stürzenbaum SR, Sci Total Environ 408:3725-3734, 2010) and may result in greater (synergistic) toxicity (Lister LJ, Svendsen C, Wright J, Hooper HL, Spurgeon DJ, Environ Int 37:663-670, 2011). This is particularly worrisome when the components of the mixture individually attack the same organs. On the other hand, metals such as manganese [Mn], iron [Fe], copper [Cu], and zinc [Zn] are essential metals, and their presence in the body below or above homeostatic levels can also lead to disease states (Annangi B, Bonassi S, Marcos R, Hernández A, Mutat Res 770(Pt A):140-161, 2016). Pb, As, Cd, and Hg can induce Fe, Cu, and Zn dyshomeostasis, potentially triggering neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Additionally, changes in heme synthesis have been associated with neurodegeneration, supported by evidence that a decline in heme levels might explain the age-associated loss of Fe homeostasis (Atamna H, Killile DK, Killile NB, Ames BN, Proc Natl Acad Sci U S A 99(23):14807-14812, 2002).The sources, disposition, transport to the brain, mechanisms of toxicity, and effects in the central nervous system (CNS) and in the hematopoietic system of each one of these metals will be described. More detailed information on Pb, Mn, Al, Hg, Cu, and Zn is available in other chapters. A major focus of the chapter will be on Pb toxicity and its interaction with other metals.

Effects of Zinc and N-Acetylcysteine in Damage Caused by Lead Exposure in Young Rats.

This study investigated the toxicity of rats exposed to lead acetate (AcPb) during the second phase of brain development (8-12 days postnatal) in hematological and cerebral parameters. Moreover, the preventive effect of zinc chloride (ZnCl2) and N-acetylcysteine (NAC) was investigated. Pups were injected subcutaneously with saline (0.9% NaCl solution), ZnCl2 (27 mg/kg/day), NAC (5 mg/kg/day) or ZnCl2 plus NAC for 5 days (3rd-7th postnatal days), and with saline (0.9% NaCl solution) or AcPb (7 mg/kg/day) in the five subsequent days (8th-12th postnatal days). Animals were sacrificed 21 days after the last AcPb exposure. Pups exposed to AcPb presented inhibition of blood porphobilinogen-synthase (PBG-synthase) activity without changes in hemoglobin content. ZnCl2 pre-exposure partially prevented PBG-synthase inhibition. Regarding neurotoxicity biomarkers, animals exposed to AcPb presented a decrease in cerebrum acetylcholinesterase (AChE) activity and an increase in Pb accumulation in blood and cerebrum. These changes were prevented by pre-treatment with ZnCl2, NAC, and ZnCl2 plus NAC. AcPb exposure caused no alteration in behavioral tasks. In short, results show that AcPb inhibited the activity of two important enzymatic biomarkers up to 21 days after the end of the exposure. Moreover, ZnCl2 and NAC prevented the alterations induced by AcPb.

Lead-Induced ERK Activation Is Mediated by GluR2 Non-containing AMPA Receptor in Cortical Neurons.

Lead is a persistent environmental pollutant and exposure to high environmental levels causes various deleterious toxicities, especially to the central nervous system (CNS). The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor that is devoid of the glutamate receptor 2 (GluR2) subunit is Ca2+-permeable, which increases the neuronal vulnerability to excitotoxicity. We have previously reported that long-term exposure of rat cortical neurons to lead acetate induces decrease of GluR2 expression. However, it is not clarified whether lead-induced GluR2 decrease is involved in neurotoxicity. Therefore, we investigated the contribution of GluR2 non-containing AMPA receptor to lead-induced neurotoxic events. Although the expression of four AMPA receptor subunits (GluR1, GluR2, GluR3, and GluR4) was decreased by lead exposure, the decrease in GluR2 expression was remarkable among four subunits. Lead-induced neuronal cell death was rescued by three glutamate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a non-selective AMPA receptor blocker), MK-801 (N-methyl-D-aspartate (NMDA) receptor blocker), and 1-naphthyl acetyl spermine (NAS, a specific Ca2+-permeable AMPA receptor blocker). Lead exposure activated extracellular signal-regulated protein kinase (ERK) 1/2, which was significantly ameliorated by CNQX. In addition, lead exposure activated p38 mitogen-activated protein kinase (MAPK p38), and protein kinase C (PKC), which was partially ameliorated by CNQX. Our findings indicate that Ca2+-permeable AMPA receptors resulting from GluR2 decrease may be involved in lead-induced neurotoxicity.

GRIN2A polymorphisms and expression levels are associated with lead-induced neurotoxicity.

Lead acts as an antagonist of the N-methyl-d-aspartate receptor (NMDAR). GRIN2A encodes an important subunit of NMDARs and may be a critical factor in the mechanism of lead neurotoxicity. Changes in GRIN2A expression levels or gene variants may be mechanisms of lead-induced neurotoxicity. In this study, we hypothesized that GRIN2A might contribute to lead-induced neurotoxicity. A preliminary HEK293 cell experiment was performed to analyze the association between GRIN2A expression and lead exposure. In addition, in a population-based study, serum GRIN2A levels were measured in both lead-exposed and control populations. To detect further the influence of GRIN2A gene single nucleotide polymorphisms (SNPs) in lead-induced neurotoxicity, 3 tag SNPs (rs2650429, rs6497540, and rs9302415) were genotyped in a case-control study that included 399 lead-exposed subjects and 398 controls. Lead exposure decreased GRIN2A expression levels in HEK293 cells ( p < 0.001) compared with lead-free cells. Lead-exposed individuals had lower serum GRIN2A levels compared with controls ( p < 0.001), and we found a trend of decreasing GRIN2A level with an increase in blood lead level ( p < 0.001). In addition, we found a significant association between rs2650429 CT and TT genotypes and risk of lead poisoning compared with the rs2650429 CC genotype (adjusted odds ratio = 1.42, 95% confidence interval = 1.01-2.00]. Therefore, changes in GRIN2A expression levels and variants may be important mechanisms in the development of lead-induced neurotoxicity.

Original Research: The expression of MMP2 and MMP9 in the hippocampus and cerebral cortex of newborn mice under maternal lead exposure.

The current study focused on the MMP2 and MMP9 expression in cerebral cortex and hippocampus of newborn mice under maternal lead exposure. Lead exposure was initiated from gestation to weaning. Lead acetate was dissolved in deionized water with concentration of 0.1, 0.2, and 0.5% and was absorbed through daily drinking. On day 21 after birth, lead in blood and tissue levels was examined by Graphite Furnace Atomic Absorption Spectrum (GFAAS). The protein expressions of MMP2 and MMP9 in hippocampus and cerebral cortex tissues were tested by western blotting and immunohistochemistry. Compared to the control group, blood, cerebral cortex, and hippocampus lead levels of newborn mice in 0.1, 0.2, and 0.5% lead exposure groups were markedly high (P < 0.05), and mice within the 0.2 and 0.5% lead exposure groups performed much worse than that of the control group in Water Maze test (P < 0.05). Compared with the control group, MMP2 and MMP9 expressions in hippocampus were up-regulated in the lead exposure groups (P < 0.05), and the MMP2 and MMP9 expressions in cerebral cortex were also higher (P < 0.05). The increased expression of MMP2 and MMP9 in the hippocampus and cerebral cortex may lead to the neurotoxicity in the context of maternal lead exposure.

Evidence of a subcommissural organ involvement in the brain response to lead exposure and a modulatory potential of curcumin.

Substantial evidence supports the neurochemical vulnerability to lead (Pb) as one of the most potent neurotoxic heavy metals. In the present study, we aimed to assess: (i) The subcommissural organ (SCO) responsiveness as a secretory circumventricular organ to chronic and acute Pb intoxication together with its serotoninergic innervation. (ii) The possible restorative effect of curcumin against Pb intoxication under the same pathological conditions. We used immunohistochemistry with antibodies against Reissner's fiber and serotonin [5-hydroxytryptophan (5-HT)] in Wistar rats following chronic as well as acute Pb administration, respectively, at 25 mg/kg intraperitoneally for 3 days and 0.3% in drinking water from the intrauterine stage until 2 months of adult age. Our data showed a significant decrease in Reissner's fiber material immunoreactivity concomitant with an overall increased 5-HT innervation of the SCO and the ventricular borders. Coadministration of curcumin (50 mg/kg body weight) restores this impairment by reversing the effect of chronic and acute Pb on the secretory activity and the 5-HTergic innervation of the SCO. The investigation showed, on the one hand, the involvement of the SCO in the response to heavy metals, especially Pb, and on the other, the beneficial corrector role of curcumin. As a part of the circumventricular organ, known as a privileged area of brain-blood exchanges, the SCO may play a key role in the mechanism of brain defense against heavy metal neurotoxicity in rats.