Joseph P. Bressler

MECHANISMS OF LEAD NEUROTOXICITY

During the past several years, there has been a renewed interest in the mechanisms by which lead poisoning disrupts brain function. In part, this is related to clinical observations that imply an absence of threshold for toxicity in the immature brain. Many of the neurotoxic effects of lead appear related to the ability of lead to mimic or in some cases inhibit the action of calcium as a regulator of cell function. At a neuronal level, exposure to lead alters the release of neurotransmitter from presynaptic nerve endings. Spontaneous release is enhanced and evoked release is inhibited. The former may be due to activation of protein kinases in the nerve endings and the latter to blockade of voltage-dependent calcium channels. This disruption of neuronal activity may, in turn, alter the developmental processes of synapse formation and result in a less efficient brain with cognitive deficits. Brain homeostatic mechanisms are disrupted by exposure to higher levels of lead. The final pathway appears to be a breakdown in the blood-brain barrier. Again, the ability of lead to mimic or mobilize calcium and activate protein kinases may alter the behavior of endothelial cells in immature brain and disrupt the barrier. In addition to a direct toxic effect upon the endothelial cells, lead may alter indirectly the microvasculature by damaging the astrocytes that provide signals for the maintenance of blood-brain barrier integrity.

Molecular mechanisms of lead neurotoxicity.

Epidemiological studies have shown a strong relationship between the level of lead in blood and bone as assessed by performance on IQ tests and other psychometric tests. Approximately 1 out of 10 children in the United States have blood lead levels above 10μg/dl, which has been established as the level of concern. Studies on experimental animals exposed to lead after birth have shown learning deficits at similar blood lead levels. Since learning requires the remodeling of synapses in the brain, lead may specifically affect synaptic transmission. Although the molecular targets for lead are unknown, a vast amount of evidence accumulated over many years has shown that lead disrupts processes that are regulated by calcium. Our laboratory has been studying the effect of lead on protein kinase C, a family of isozymes some of which require calcium for activity. We and others have shown that picomolar concentrations of lead can replace micromolar concentrations of calcium in a protein kinase C enzyme assay. Furthermore, lead activates protein kinase C in intact cells and induces the expression of new genes by a mechanism dependent on protein kinase C. We propose that the learning deficits caused by lead are due to events regulated by protein kinase C that most likely occur at the synapse.

Divalent metal transporter 1 in lead and cadmium transport.

Abstract: The effect of exposure to cadmium (Cd) and lead (Pb) on human health has been recognized for many years and recent information suggests that minimal exposure levels are themselves too high. Common scenarios for Pb exposure include occupational, residential, and/or behavioral (hand‐to‐mouth activity) settings. The main source of Cd exposure for nonsmokers is dietary, through plants or animals that accumulate the metal. Specific cellular importers for Pb and Cd are unlikely as these metals are nonessential and toxic. Accordingly, in the intestine, the operational mechanism is assumed to be inadvertent uptake through pathways intended for essential nutrients such as iron. Results from experimental and epidemiological studies indicated that diets low in iron (Fe) result in increased absorption of Pb and Cd, suggesting common molecular mechanisms of Cd and Pb transport. Indeed, recent mechanistic studies found that the intestinal transporter for nonheme iron, divalent metal transporter 1 (DMT1), mediates the transport of Pb and Cd. DMT1 is regulated, in part, by dietary iron, and chemical species of Cd and Pb that are transported by DMT1 would be made available through digestion and are also found in plasma. Accordingly, the involvement of DMT1 in metal uptake offers a mechanistic explanation for why an iron‐deficient diet is a risk factor for Pb and Cd poisoning. It also suggests that diets rich in iron‐containing food could be protective against heavy metal poisoning.

Metal transporters in intestine and brain: their involvement in metal-associated neurotoxicities

The transport of essential metals and other nutrients across tightmembrane barriers such as the gastrointestinal tract and blood-brain barrier is mediated by specific transport mechanisms. Specific transporters take up metals at the apical surface and export them at the basolateral surface, and are involved in their intracellular distribution. Transporters for each of the major essential metals, calcium, iron and zinc, have been identified. These transporters also mediate the transport of non-essential metals across tight membrane barriers. For example, the intestinal iron transporter divalent metal transporter 1 mediates the uptake of lead and cadmium. The levels of essential metals are strictly regulated by transporters. When dietary levels of essential metals are low, levels of the corresponding transporters increase in the intestine, after which there is a greater potential for increased transport of toxic metals. In the brain, the strict regulation of metals prevents injury that potentially would result from oxidative damage induced by the essential metals iron, copper and zinc. Indeed, the oxidative damage found in neurodegenerative diseases is likely to be due to higher levels of these metals. Involvement of intracellular transporters for copper and zinc has been shown in animal models of Alzheimers disease, raising the possibility that higher levels of iron, zinc and copper might be due to a disruption in the activity of transporters. Accordingly, exposure to toxicants that affect the activity of transporters potentially could contribute to the aetiology/progression of neurodegenerative diseases.

Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice.

We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice.

Hydroquinone Increases 5-Hydroxymethylcytosine Formation through Ten Eleven Translocation 1 (TET1) 5-Methylcytosine Dioxygenase

Background: Hydroquinone is a benzene metabolite shown to lead to decreased DNA methylation. Results: Hydroquinone exposure increases Ten Eleven Translocation 1 methylcytosine dioxygenase activity and 5-hydroxymethylcytosine levels and decreases DNA methylation. Conclusion: Hydroquinone leads to DNA demethylation through a Ten Eleven Translocation 1-dependent mechanism. Significance: This mechanism may explain observations of decreased DNA methylation and cytotoxicity following exposure to benzene and hydroquinone. DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase.

γ-Glutamyl Transpeptidase Activity in Brain Microvessels Exhibits Regional Heterogeneity

Abstract: Brain microvessels form a tight blood‐tissue permeability barrier and express high levels of specific enzymes, including γ‐Glutamyl transpeptidase (GGTP). This differentiation is thought to be induced by perivascular astrocytes. By using histochemical methods, we found that the percentage of GGTP‐positive vessels varied considerably in different areas of rat brain. Enzyme activity was not found in the pineal gland or the median eminence, where the blood‐brain barrier is not expressed. In areas where the blood‐brain barrier is expressed, the percentage of GGTP‐positive vessels varied from 8% in the optic nerve to 100% in the anterior commissure. The neocortex showed a lower percentage of GGTP‐positive vessels (2‐15%) than anterior olfactory nucleus (42%), subiculum (70%), hippocampus (48%), and striatum (50‐58%). Alkaline phosphatase, another brain microvessel‐enriched enzyme, did not show these marked regional differences. The morphometric histochemical results were verified by enzymatic assays in homogenates of different regions from rat and bovine brain and in microvessel preparations of bovine putamen and neocortex. During the postnatal development of rat brain, the difference between neocortex and striatum appeared after day 20. The regional heterogeneity of brain microvessels may be caused by astrocytic heterogeneity and reflect regional heterogeneity in microvascular function.

Glutathione pathway gene variation and risk of autism spectrum disorders

Despite evidence that autism is highly heritable with estimates of 15 or more genes involved, few studies have directly examined associations of multiple gene interactions. Since inability to effectively combat oxidative stress has been suggested as a mechanism of autism, we examined genetic variation 42 genes (308 single-nucleotide polymorphisms (SNPs)) related to glutathione, the most important antioxidant in the brain, for both marginal association and multi-gene interaction among 318 case–parent trios from The Autism Genetic Resource Exchange. Models of multi-SNP interactions were estimated using the trio Logic Regression method. A three-SNP joint effect was observed for genotype combinations of SNPs in glutaredoxin, glutaredoxin 3 (GLRX3), and cystathione gamma lyase (CTH); OR = 3.78, 95% CI: 2.36, 6.04. Marginal associations were observed for four genes including two involved in the three-way interaction: CTH, alcohol dehydrogenase 5, gamma-glutamylcysteine synthetase, catalytic subunit and GLRX3. These results suggest that variation in genes involved in counterbalancing oxidative stress may contribute to autism, though replication is necessary.

Toward a 3D model of human brain development for studying gene/environment interactions

This project aims to establish and characterize an in vitro model of the developing human brain for the purpose of testing drugs and chemicals. To accurately assess risk, a model needs to recapitulate the complex interactions between different types of glial cells and neurons in a three-dimensional platform. Moreover, human cells are preferred over cells from rodents to eliminate cross-species differences in sensitivity to chemicals. Previously, we established conditions to culture rat primary cells as three-dimensional aggregates, which will be humanized and evaluated here with induced pluripotent stem cells (iPSCs). The use of iPSCs allows us to address gene/environment interactions as well as the potential of chemicals to interfere with epigenetic mechanisms. Additionally, iPSCs afford us the opportunity to study the effect of chemicals during very early stages of brain development. It is well recognized that assays for testing toxicity in the developing brain must consider differences in sensitivity and susceptibility that arise depending on the time of exposure. This model will reflect critical developmental processes such as proliferation, differentiation, lineage specification, migration, axonal growth, dendritic arborization and synaptogenesis, which will probably display differences in sensitivity to different types of chemicals. Functional endpoints will evaluate the complex cell-to-cell interactions that are affected in neurodevelopment through chemical perturbation, and the efficacy of drug intervention to prevent or reverse phenotypes. The model described is designed to assess developmental neurotoxicity effects on unique processes occurring during human brain development by leveraging human iPSCs from diverse genetic backgrounds, which can be differentiated into different cell types of the central nervous system. Our goal is to demonstrate the feasibility of the personalized model using iPSCs derived from individuals with neurodevelopmental disorders caused by known mutations and chromosomal aberrations. Notably, such a human brain model will be a versatile tool for more complex testing platforms and strategies as well as research into central nervous system physiology and pathology.

Temporal and Regional Alterations in NMDA Receptor Expression in Mecp2-Null Mice

Our previous postmortem study of girls with Rett Syndrome (RTT), a development disorder caused by MECP2 mutations, found increases in the density of N‐Methyl‐D‐aspartate (NMDA) receptors in the prefrontal cortex of 2–8‐year‐old girls, whereas girls older than 10 years had reductions in NMDA receptors compared with age‐matched controls (Blue et al., Ann Neurol 1999b;45:541–545). Using [3H]‐CGP to label NMDA‐type glutamate receptors in 2‐ and 7‐week old wild‐type (WT), Mecp2‐null, and Mecp2‐heterozygous (HET) mice (Bird model), we found that frontal areas of the brain also exhibited a bimodal pattern in NMDA expression, with increased densities of NMDA receptors in Mecp2‐null mice at 2 weeks of age but decreased densities at 7 weeks of age. Visual cortex showed a similar pattern, while other cortical regions only exhibited changes in NMDA receptor densities at 2 weeks (retrosplenial granular) or 7 weeks (somatosensory). In thalamus of null mice, NMDA receptors were increased at 2 and 7 weeks. No significant differences in density were found between HET and WT mice at both ages. Western blots for NMDAR1 expression in frontal brain showed higher levels of expression in Mecp2‐null mice at 2 weeks of age but not at 1 or 7 weeks of age. Our mouse data support the notion that deficient MeCP2 function is the primary cause of the NMDA receptor changes we observed in RTT. Furthermore, the findings of regional and temporal differences in NMDA expression illustrate the importance of age and brain region in evaluating different genotypes of mice. Anat Rec, 2011.