Empirical analysis of lead neurotoxicity mode of action and its application in health risk assessment.

The mode of action (MOA) framework is proposed to inform a biological link between chemical exposures and adverse health effects. Despite a significant increase in knowledge and awareness, the application of MOA in human health risk assessment (RA) remains limited. This study aims to discuss the adoption of MOA for health RA within a regulatory context, taking our previously proposed but not yet validated MOA for lead neurotoxicity as an example. We first conducted a quantitative weight of evidence (qWOE) assessment, which revealed that the MOA has a moderate confidence. Then, targeted bioassays were performed within an in vitro blood-brain barrier (BBB) model to quantitatively validate the scientific validity of key events (KEs) in terms of essentiality and concordance of empirical support (dose/temporal concordance), which increases confidence in utilizing the MOA for RA. Building upon the quantitative validation data, we further conducted benchmark dose (BMD) analysis to map dose-response relationships for the critical toxicity pathways, and the lower limit of BMD at a 5% response (BMDL5) was identified as the point of departure (POD) value for adverse health effects. Notably, perturbation of the Aryl Hydrocarbon Receptor (AHR) signaling pathway exhibited the lowest POD value, measured at 0.0062 µM. Considering bioavailability, we further calculated a provisional health-based guidance value (HBGV) for children's lead intake, determining it to be 2.56 µg/day. Finally, the health risk associated with the HBGV was assessed using the hazard quotient (HQ) approach, which indicated that the HBGV established in this study is a relative safe reference value for lead intake. In summary, our study described the procedure for utilizing MOA in health RA and set an example for MOA-based human health risk regulation.

Lead Disrupts Mitochondrial Morphology and Function through Induction of ER Stress in Model of Neurotoxicity.

Lead exposure may weaken the ability of learning and memory in the nervous system through mitochondrial paramorphia and dysfunction. However, the underlying mechanism has not been fully elucidated. In our works, with SD rats, primary culture of hippocampal neuron and PC12 cell line model were built up and behavioral tests were performed to determine the learning and memory insults; Western blot, immunological staining, and electron microscope were then conducted to determine endoplasmic reticulum stress and mitochondrial paramorphia and dysfunction. Co-immunoprecipitation were performed to investigate potential protein-protein interaction. The results show that lead exposure may cripple rats' learning and memory capability by inducing endoplasmic reticulum stress and mitochondrial paramorphia and dysfunction. Furthermore, we clarify that enhanced MFN2 ubiquitination degradation mediated by PINK1 may account for mitochondrial paramorphia and endoplasmic reticulum stress. Our work may provide important clues for research on the mechanism of how Pb exposure leads to nervous system damage.

Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?

A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3.

Neuroprotective Actions of Clinoptilolite and Ethylenediaminetetraacetic Acid Against Lead-induced Toxicity in Mice Mus musculus.

OBJECTIVES: Oxidative stress is considered as a possible molecular mechanism involved in lead (Pb(2+)) neurotoxicity. Very few studies have been investigated on the occurrence of oxidative stress in developing animals due to Pb(2+) exposure. Considering the vulnerability of the developing brain to Pb(2+), this study was carried out to investigate the effects of Pb(2+) exposure in brain regions especially on antioxidant enzyme activities along with ameliorative effects of ethylenediaminetetraacetic acid (EDTA) and clinoptilolite. METHODS: Three-week old developing Swiss mice Mus musculus were intraperitoneally administered with Pb(2+) acetate in water (w/v) (100 mg/kg body weight/day) for 21 days and control group was given distilled water. Further Pb(2+)-toxicated mice were made into two subgroups and separately supplemented with EDTA and clinoptilolite (100 mg/kg body weight) for 2 weeks. RESULTS: In Pb(2+)-exposed mice, in addition to increased lipid peroxidation, the activity levels of catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH) found to decrease in all regions of brain indicating, existence of severe oxidative stress due to decreased antioxidant function. Treatment of Pb(2+)-exposed mice with EDTA and clinoptilolite lowered the lipid peroxidation (LPO) levels revealing their antioxidant potential to prevent oxidative stress. Similarly their administration led to recover the level of catalase, SOD, and GPx enzymes affected during Pb(2+) toxicity in different regions of brain. CONCLUSIONS: The protection of brain tissue against Pb(2+)-induced toxicity by clinoptilolite and EDTA in the present experiment might be due to their ability to react faster with peroxyl radicals there by reducing the severity of biochemical variable indicative of oxidative damage. Thus, the results of present study indicate the neuroprotective potential of clinoptilolite and EDTA against Pb(2+) toxicity.

Adansonia digitata L. fruit shell extract alleviates lead-induced neurotoxicity in mice via modulation of oxidative stress and a possible chelating activity.

BACKGROUND: Lead is a ubiquitous environmental heavy metal known to induce neurotoxicity. It has been postulated that substance with high antioxidant capacity could alleviate lead-induced neurotoxicity. Adansonia digitata fruit shell extract (ADFS) has been reported to have high phenolic contents and exerts antioxidant activity. This study investigated the effects of Adansonia digitata fruit shell extract on lead-induced neurotoxicity in mice. METHODS: Male balb/c mice (n = 7) were administered with Pb-acetate (50 mg/kg) 30 mins before ADFS (250 mg/kg and 500 mg/kg) or succimer (50 mg/kg) per orally for 28 days. Motor activities were evaluated on days 29 and 30 through horizontal bar and open field tests respectively. Further, spectrophotometry, atomic absorption spectrophotometry and haematoxylin and eosin staining were carried-out to determine the expression of oxidative stress biomarkers, level of lead concentration in the brain and histology of the cerebellum respectively. RESULTS: Lead acetate exposure significantly (p < 0.05) induced motor deficits in horizontal bar test and open field test, caused oxidative stress, high concentration of lead in the brain as well as histological aberration in the cerebellum. ADFS significantly (p < 0.05) reversed the motor deficits evident by increased muscle strength and number of lines crossed. Further, ADFS significantly reversed oxidative stress evident by increased levels of SOD, CAT and GSH and decreased level of MDA. There was also significant (p < 0.05) decrease in brain lead concentration as well as reduced cerebellar cells death. CONCLUSION: Findings suggest that ADFS attenuated motor deficits via inhibition of oxidative stress and chelating activity which is comparable to succimer. Hence, ADFS should be explored for possible development of chelating agent against lead and other heavy metals toxicity.

Hydrogen sulfide protects hippocampal CA1 neurons against lead mediated neuronal damage via reduction oxidative stress in male rats.

H2S plays vital roles in modulation brain function. It is associated with antioxidant and anti-inflammatory properties. We assessed the H2S impact on spatial learning and memory deficit and cell death due to lead exposure, and probable mechanisms of action. The 36 male Wistar rats that (200-220 g), were in random assigned to 3 groups, control group (12 rats), lead acetate group (12 rats), and lead acetate +H2S groups (NaHS as a H2S donor; 5/6 mg/kg; 12 rats). Administration of lead to rats was performed through acute lead poisoning (25 mg/kg of lead acetate, IP through 3 days). Using male Morris water maze, their spatial learning and memory function were measured. We carried out ELISA method to calculate TNF-α and antioxidant enzymes level. Immunohistochemical staining was applied for evaluating the caspase-3 expression levels. Treatment with H2S improved learning and memory impairment in Pb-exposed rats (P<0.05). H2S treatment suppressed Pb-related apoptosis in the hippocampal CA1 subfield (P<0.01). Also, the TNF-α over-expression in the CA1 region of hippocampus due to lead exposure showed a significant reduction (P<0.05) after administrating H2S. Simultaneously, H2S treatment reduced the MDA levels, enhanced SOD, GSH level than the Pb-exposed group in hippocampus (P<0.05). H2S was able to significantly improve Pb-related spatial learning and memory deficit, and neuronal cell death in the CA1 region of hippocampus in the male rats at least partly by reducing oxidative stress and TNF.

Crocus sativus restores dopaminergic and noradrenergic damages induced by lead in Meriones shawi: A possible link with Parkinson's disease.

Lead (Pb) is a metal element released into the atmosphere and a major source of environmental contamination. The accumulation and concentration of this metal in a food web may lead to the intoxication of the body, more precisely, the nervous system (NS). In addition, Pb-exposure can cause structural and functional disruption of the NS. Studies have shown that Pb-exposure could be a risk factor in the development of Parkinson's disease (PD). The latter is related to dopaminergic deficiency that may be triggered by genetic and environmental factors such as Pb intoxication. In this study, we have evaluated, in one hand, the neurotoxic effect of Pb (25 mg / kg B.W i.p) for three consecutive days on dopaminergic system and locomotor performance in Merione shawi. In the other hand, the possible restorative potential of C. sativus (CS) (50 mg / kg BW) by oral gavage. The immunohistochemical approach has revealed that Pb-intoxicated Meriones show a significant increase of Tyrosine Hydroxylase (TH) levels within the Substantia Nigra compacta (SNc), Ventral Tegmental Area (VTA), Locus Coeruleus (LC), Dorsal Striatum (DS) and Medial Forebrain Bundle (MFB), unlike the control meriones, a group intoxicated and treated with Crocus sativus hydroethanolic extract (CSHEE) and treated group by CSHEE. Treatment with CSHEE, has shown a real potential to prevent all Pb-induced damages. In fact, restores the TH levels by 92%, 90%, 88%, 90% and 93% in SNc, VTA, LC, DS and MFB respectively, similarly, locomotor activity dysfunction in Pb-intoxicaed meriones was reinstated by 90%. In this study, we have revealed a new pharmacological potential of Crocus sativus that can be used as a neuroprotective product for neurodegenerative disorders, especially, which implying dopaminergic and noradrenergic injuries, like PD, trigged by heavy metals.

Altered nigrostriatal dopaminergic and noradrenergic system prompted by systemic lead toxicity versus a treatment by curcumin-III in the desert rodent Meriones shawi.

Exposure to lead is a threat factor for neurodegenerative disorders progress as it could trigger dopaminergic deficiency. We aimed herein to assess the effect of acute lead exposure (25mg/kg B.W i.p.) during three continuous days on the dopaminergic and noradrenergic systems together with locomotor performance in Meriones shawi (M. shawi), then the neuroprotective potential of curcumin-III (30mg/kg B.W) by oral gavage. Pb-exposed M. shawi exhibited increased tyrosine hydroxylase (TH) immunoreactivity in substantia nigra compacta (SNc), ventral tegmental area (VTA), locus coeruleus (LC), and dorsal striatum (DS), unlike the controls. This was correlated with decreased locomotor performance. A noticeable protective effect by co-treatment with curcumin-III was observed; in consequence, TH-immunoreactivity and locomotor disturbance were restored in Pb-treated Meriones. Our data results proved, on the one hand, an evident neurotoxic effect of acute Pb exposure and, on the other hand, a potent therapeutic effect of curcumin-III. Thereby, this compound may be recommended as a neuroprotective molecule for neurodegenerative disorders involving catecholaminergic impairment initiated by metallic elements.

Both physiology and epidemiology support zero tolerable blood lead levels.

Inorganic lead is one of the most common causes of environmental metal poisonings, and its adverse effects on multiple body systems are of great concern. The brain, along with the kidneys, are critically susceptible to lead toxicity for their hosting of high affinity lead binding proteins, and very sensitive physiology. Prolonged low-lead exposure frequently remains unrecognized, causes subtle changes in these organ systems, and manifests later at an irreversible stage. With the repeated documentation of "no safe blood lead level", the pernicious effects of lead at any measurable concentration need to be emphasized. In this review, we surveyed articles on chronic low-level lead exposures with a blood lead concentrations <10µg/dL and the development of neurobehavioral or renal disorders. The negative impacts of lead on both nervous and renal systems were obvious at a blood lead concentration of 2µg/dL, with the absence of any detectable threshold. The deleterious effect of lead on two different organ systems at such low concentrations drew our attention to the various extracellular and intracellular events that might be affected by minimal concentration of body lead, especially blood lead. Is there a true common ground between low-level lead toxicity in both the nervous system and the kidney?

Perinatal exposure to lead (Pb) promotes Tau phosphorylation in the rat brain in a GSK-3ß and CDK5 dependent manner: Relevance to neurological disorders.

Hyperphosphorylation of Tau is involved in the pathomechanism of neurological disorders such as Alzheimer's, Parkinson's diseases as well as Autism. Epidemiological data suggest the significance of early life exposure to lead (Pb) in etiology of disorders affecting brain function. However, the precise mechanisms by which Pb exerts neurotoxic effects are not fully elucidated. The purpose of this study was to evaluate the effect of perinatal exposure to low dose of Pb on the Tau pathology in the developing rat brain. Furthermore, the involvement of two major Tau-kinases: glycogen synthase kinase-3 beta (GSK-3ß) and cyclin-dependent kinase 5 (CDK5) in Pb-induced Tau modification was evaluated. Pregnant female rats were divided into control and Pb-treated group. The control animals were maintained on drinking water while females from the Pb-treated group received 0.1% lead acetate (PbAc) in drinking water, starting from the first day of gestation until weaning of the offspring. During the feeding of pups, mothers from the Pb-treated group were still receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and Tau mRNA and protein level as well as Tau phosphorylation were analyzed in forebrain cortex (FC), cerebellum (C) and hippocampus (H). Concomitantly, we examined the effect of Pb exposure on GSK-3ß and CDK5 activation. Our data revealed that pre- and neonatal exposure to Pb (concentration of Pb in whole blood below 10µg/dL, considered safe for humans) caused significant increase in the phosphorylation of Tau at Ser396 and Ser199/202 with parallel rise in the level of total Tau protein in FC and C. Tau hyperphosphorylation in Pb-treated animals was accompanied by elevated activity of GSK-3ß and CDK5. Western blot analysis revealed activation of GSK-3ß in FC and C as well as CDK5 in C, via increased phosphorylation of Tyr-216 and calpain-dependent p25 formation, respectively. In conclusion, perinatal exposure to Pb up-regulates Tau protein level and induces Tau hyperphosphorylation in the rat brain cortex and cerebellum. We suggest that neurotoxic effect of Pb might be mediated, at least in part, by GSK-3ß and CDK5-dependent Tau hyperphosphorylation, which may lead to the impairment of cytoskeleton stability and neuronal dysfunction.

Ferulic Acid Protects Against Lead Acetate-Induced Inhibition of Neurite Outgrowth by Upregulating HO-1 in PC12 Cells: Involvement of ERK1/2-Nrf2 Pathway.

Prenatal lead exposure is associated with poor intellectual development in children. However, there are few breakthroughs in therapeutic intervention of developmental lead neurotoxicity. The aim of this study is to evaluate the hypothesis that ferulic acid-mediated promotion of neurite outgrowth following lead exposure might mainly result from its antioxidant capability by extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Exposure of PC12 cells to lead acetate inhibits neurite outgrowth and causes oxidative stress as measured by ROS, LPO, GSH/GSSG, and NAD+/NADH. FA treatment significantly, although not completely, protected the cells against lead acetate-induced neurite outgrowth inhibition. The effects of FA could be blocked by PD98059, zinc protoporphyrin (Zn-PP), and Nrf2 shRNA. In addition, FA induced heme oxygenase 1 (HO-1) gene expression, enhanced antioxidant response element (ARE) promoter activity, promoted ERK1/2 phosphorylation, and Nrf2 translocation in PC12 cells exposed to lead acetate. ERK1/2 locate upstream of Nrf2 and regulate Nrf2-dependent HO-1 expression in antioxidative effects of FA. Our results suggest that FA is a promising candidate for treatment of developmental lead neurotoxicity. These promising findings warrant future investigation evaluating the FA-mediated potentiation of neurite outgrowth following lead exposure in vivo.

Reversal effect of monoisoamyl dimercaptosuccinic acid (MiADMSA) for arsenic and lead induced perturbations in apoptosis and antioxidant enzymes in developing rat brain.

Oxidative stress (OS) has been implicated in the pathophysiology of many neurodegenerative disorders. Several studies have shown that exposure to arsenic (As) and lead (Pb) produces oxidative stress, one of the most noted molecular mechanisms for the neurotoxicity of these metals. In the present study, we examined the effect of combined exposure to these metals (As and Pb) on the activity levels of antioxidant enzymes and apoptotic marker enzymes in brain regions (cerebral cortex, hippocampus and cerebellum) of rats at postnatal day (PND) 21, 28 and 3 months age and compared the toxicity levels with individual metals (As or Pb). Further, we also evaluated the therapeutic efficacy of a chelating agent, monoisoamyl dimercaptosuccinic acid (MiADMSA) against arsenic and lead induced developmental neurotoxicity. Pregnant rats were exposed to sodium meta-arsenite (50 ppm) and lead acetate (0.2%) individually, and in combination (As=25 ppm+Pb=0.1%) via drinking water throughout perinatal period (GD 6 to PND 21). MiADMSA (50 mg/kg, orally through gavage) was given for three consecutive days to the PND 18 pups (i.e., PND 18 to PND 20). Exposure to metal mixture resulted in a significant decrease in the activity levels of antioxidant enzymes such as manganese-superoxide dismutase (Mn-SOD), Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) while the malondialdehyde (MDA) levels and mRNA expression levels of caspase-3 and caspase-9 were significantly increased in all the three brain regions. The observed alterations were greater with exposure to metal mixture than individual metals (As or Pb) and the changes were more prominent at PND 28 and greater in cerebral cortex than hippocampus and cerebellum. Interestingly, chelation therapy with MiADMSA showed significant recovery in antioxidant enzymes, lipid peroxidation and gene expression levels of caspase-3 and caspase-9. From these findings, it can be concluded that combined exposure to As and Pb showed an additive effect on antioxidant enzymes than individual metal exposure and chelation therapy with MiADMSA significantly reversed the As and Pb induced apoptosis and oxidative stress, a major contributing factor to neurotoxicity.